Anti-human IL2RA monoclonal antibody and application thereof

Abstract

The invention provides a monoclonal antibody for resisting human IL2RA (interleukin-2 receptor A) and application of the monoclonal antibody. The provided antibody comprises an HCDR1 sequence, an HCDR2 sequence and an HCDR3 sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an LCDR1 sequence, an LCDR2 sequence and an LCDR3 sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; or comprises HCDR1, HCDR2 and HCDR3 sequences as shown in SEQ ID NO: 7, 8 and 9 and LCDR1, LCDR2 and LCDR3 sequences as shown in SEQ ID NO: 10, 11 and 12. The antibody provided by the invention can be specifically combined with IL2RA, can be used for preparing a kit, and is strong in specificity and high in sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and specifically relates to a monoclonal antibody against human IL2RA and its application. Background Art

[0002] Interleukin-2 receptor (IL-2R) is a heterotrimer composed of three chains, α, β and γ. It is a transmembrane glycoprotein receptor that plays a vital role in the immune response. It can bind to IL-2 and trigger a series of biological events, ultimately leading to the activation and proliferation of immune cells.

[0003] The interleukin-2 receptor alpha subunit (IL2RA, or IL2Rα), also known as the Tac antigen, P55, or CD25, is a 55kDa type I transmembrane protein. IL2RA is primarily expressed in tissues such as the spleen and lymph nodes, as well as on the surfaces of cells such as Treg cells, basophils, and activated T cells. As a cell surface protein, IL2RA participates in signaling between immune cells, mediating the binding of IL-2 and enhancing the receptor's affinity for cytokines.

[0004] The IL2RA protein plays a crucial role in the immune system, extensively involved in T cell activation and proliferation. Its abnormal expression is closely associated with a variety of immune-related diseases. IL2RA is highly expressed on the surface of regulatory cells and is also upregulated on some activated effector cells and immune cell subsets. In research on related diseases, abnormal IL2RA protein expression and function are closely associated with the development and progression of diseases such as rheumatoid arthritis and type 1 diabetes.

[0005] Therefore, it is very necessary to develop a human IL2RA diagnostic kit with excellent performance. Summary of the Invention

[0006] The present invention aims to at least partially address one of the technical problems in the related art. The present invention provides an anti-human IL2RA monoclonal antibody that can be used to prepare a kit or a medicament. The provided anti-human IL2RA monoclonal antibody exhibits excellent performance, specifically binding to human IL2RA with high sensitivity, accuracy, and specificity. The provided anti-human IL2RA monoclonal antibody can be used to develop a high-performance diagnostic kit for diagnosing the content of human IL2RA in a variety of samples, thereby assisting in clinical diagnosis.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] A first aspect of the present invention provides an anti-human IL2RA monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment thereof is selected from at least one of the following:

[0009] (1) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 1, 2 and 3;

[0010] and LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NOs: 4, 5, and 6, or sequences having one or two conservative amino acid mutations compared to the sequences as shown in SEQ ID NOs: 4, 5, and 6;

[0011] (2) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 7, 8 and 9, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 7, 8 and 9;

[0012] and LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NOs: 10, 11, and 12, or sequences having one or two conservative amino acid mutations compared to the sequences as shown in SEQ ID NOs: 10, 11, and 12;

[0013] The HCDR1-HCDR3 and LCDR1-LCDR3 sequences were obtained according to the Kabat definition scheme.

[0014] A second aspect of the present invention provides an anti-human IL2RA monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment thereof is selected from at least one of the following:

[0015] (a) the HCDR1, HCDR, and HCDR3 sequences of the heavy chain variable region set forth in SEQ ID NO: 13, and the LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region set forth in SEQ ID NO: 14; or

[0016] (b) The HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 15, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO: 16.

[0017] According to an embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof provided above, the sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 13 or 15;

[0018] The sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO:14 or 16.

[0019] According to a specific embodiment of the present invention, the sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the sequence of the light chain variable region is shown in SEQ ID NO: 14;

[0020] Alternatively, the sequence of the heavy chain variable region is shown as SEQ ID NO: 15, and the sequence of the light chain variable region is shown as SEQ ID NO: 16.

[0021] The third aspect of the present invention provides a polynucleotide encoding the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0022] According to an embodiment of the present invention, the polynucleotide comprises the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18;

[0023] Or include the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20.

[0024] The fourth aspect of the present invention provides a construct comprising the polynucleotide described in the third aspect.

[0025] The fifth aspect of the present invention provides a host cell containing the polynucleotide described in the third aspect or the construct described in the fourth aspect.

[0026] A sixth aspect of the present invention provides a kit comprising the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to the first or second aspect.

[0027] The provided kit can be used for in vitro quantitative analysis of the content of human IL2RA protein in human serum, plasma, cell lysate, cell culture supernatant, and urine.

[0028] According to an embodiment of the present invention, the kit includes a first antibody and a second antibody, wherein the first antibody includes the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 4, 5, and 6; and the second antibody includes the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 10, 11, and 12. The provided kit can include both the first and second antibodies, and, for example, a double-antibody sandwich enzyme-linked immunosorbent assay can be used to detect a sample. One of the antibodies is used as a coating antibody, and the other is used as a detection antibody.

[0029] A seventh aspect of the present invention provides a method for detecting the content of anti-human IL2RA in a biological sample, comprising:

[0030] Incubating the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof described in the first aspect or the second aspect with a biological sample, and measuring the OD value of the reaction product at a predetermined wavelength;

[0031] Based on the OD value, the IL2RA content in the biological sample is determined.

[0032] An eighth aspect of the present invention provides a pharmaceutical composition comprising the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to the first or second aspect, and a pharmaceutically acceptable carrier.

[0033] A ninth aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof described in the first or second aspect in preparing a human IL2RA protein detection kit or drug.

[0034] The beneficial effects achieved by the present invention are at least:

[0035] The present invention provides a monoclonal antibody against human IL2RA, which can be used to prepare a diagnostic kit. In particular, a kit prepared by combining the provided monoclonal antibodies has the characteristics of specificity and high affinity for detecting human IL2RA protein. Also provided are methods for preparing a detection kit based on the above-mentioned antibodies, thereby improving the specificity of the diagnostic reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The following are the ELISA test results of the antibodies provided in the embodiments of the present invention.

[0037] Figure 2 The figure is a standard curve diagram of the kit provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0038] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0039] As used in this specification and the claims, "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0040] As used herein, the term "antibody" is used in the broadest sense to refer to a protein or polypeptide containing an antigen-binding site, antigen-binding fragment, or antigen-binding portion, encompassing natural and artificial antibodies of various structures, including but not limited to intact antibody forms or antigen-binding fragments of antibodies. Depending on the embodiment, the antibody referred to is a monoclonal antibody. A "complete antibody" is typically a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH), which includes a heavy chain constant domain (CH1), a heavy chain constant domain (CH2), and a heavy chain constant domain (CH3). Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant domain (CL). The VH and VL regions can be further divided into complementarity determining regions (also called hypervariable regions or hypervariable regions, abbreviated as CDRs), which are separated by conserved framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) in the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2, and HCDR3, respectively, starting from the amino terminus, and the CDRs of the light chain variable region are referred to as LCDR1, LCDR2, and LCDR3, respectively, starting from the amino terminus.

[0041] An "antigen-binding fragment" refers to a portion of a full-length antibody that exhibits the property or function of binding to an antigen. The term "variable region" or "variable domain" refers to the domain involved in antigen binding to the heavy or light chain of an antibody. As mentioned above, the hypervariable or hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen. Examples of antigen-binding fragments or antigen-binding portions include, but are not limited to, Fab, Fab', F(ab')2, bispecific Fab' and Fv fragments (variable regions), linear antibodies, single-chain antibodies, single-domain antibodies, and the like. Papain digestion of an intact antibody produces two identical antigen-binding fragments, called Fab fragments, each containing the heavy and light chain variable regions and the light chain constant domain and heavy chain constant domain CH1. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxyl terminus of the heavy chain constant domain CH1, including one or more cysteines from the antibody hinge region. Pepsin digestion of an intact antibody produces F(ab')2 fragments. F(ab')2 fragments contain two antigen-binding F(ab) moieties linked by disulfide bonds, making them bivalent antibodies. Single-chain antibodies are fusion proteins composed of the variable regions of the heavy and light chains of antibodies, linked by a flexible peptide of approximately 10-25 amino acids. Single-domain antibodies are antibody fragments composed of the variable regions of a single monomer. Because single-domain antibodies are typically derived from the variable regions of the heavy chains of camelid or shark antibodies, they are often referred to as nanobodies. Nanobodies contain only the heavy chain CDR regions and are the smallest fully functional antigen-binding fragments. Nanobodies consist of three CDR regions (CDR1-CDR3) and four framework regions (FR1-FR4). The framework regions FR1, FR2, FR3, and FR4 are separated by complementarity determining regions (CDR1, CDR2, and CDR3), respectively. Antigen-binding fragments can be obtained recombinantly. For example, a nucleic acid encoding the desired antigen-binding fragment can be constructed, introduced into an expression vector, and expressed in a suitable host to produce the antigen-binding fragment.

[0042] The term "comprising" or "including" means including the elements or steps mentioned, but does not exclude other elements or steps. Of course, unless otherwise specified, "comprising" or "including" also covers the situation where it consists of the elements or steps mentioned. For example, when referring to an antibody variable region comprising a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0043] The "affinity" or "binding affinity" mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and / or stability of the binding sites between an antigen and an antibody or antigen-binding fragment.

[0044] "Specific binding" or "specifically binds to", "binds to", "specifically targets" a specific antigen or epitope, or "has specificity" or "can bind to" a specific antigen or epitope means to distinguish it from non-specific interactions, and this specific binding can be measured by some methods commonly used in the art. The ability of an antibody to bind to an antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, cells carrying an antigen can be detected by flow cytometry, and the competitive binding between the test antibody and the labeled antibody can be detected by measuring the positive rate index of the cells. Since the spatial structure of the antigen on the cell surface is closer to the form existing in the body, this method can better reflect the real situation.

[0045] Whether provided herein as an antibody or antigen-binding fragment, or a polynucleotide, it is generally separable or recombinant. "Separable" refers to being able to identify and separate and / or recycle from cells or cell cultures expressing a polypeptide or protein. Typically, the isolated polypeptide will be prepared by at least one purification step. "Isolated antibody" refers to being substantially free of other antibodies or their antigen-binding fragments that have different antigenic specificities. "Recombinant" means that the antibody can be produced in an exogenous host cell using genetic recombination techniques.

[0046] The CDR sequences of the antibodies shown herein can be obtained by combining existing database analysis. The CDR sequences of the antibodies provided are obtained by combining the Kabat definition scheme (for example, see US Patent No. 1983, “Sequences of Proteins of Immunological Interest”). They can also be obtained by using definition schemes such as IMGT (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38: D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6: 737-749 (2011)) and Chothia (for example, see J. Mol. Biol. 196: 901-917 (1987)). It is understood by those skilled in the art that differences in CDRs due to differences in definition methods are also included in the scope of protection of the present invention.

[0047] Antibodies or antigen-binding fragments thereof

[0048] The present invention provides an anti-human IL2RA antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment thereof is selected from at least one of the following:

[0049] (1) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 1, 2 and 3;

[0050] and LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NOs: 4, 5, and 6, or sequences having one or two conservative amino acid mutations compared to the sequences as shown in SEQ ID NOs: 4, 5, and 6;

[0051] (2) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 7, 8 and 9, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 7, 8 and 9;

[0052] and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one or two conservative amino acid mutations compared to the sequences shown in SEQ ID NOs: 10, 11 and 12.

[0053] As used herein, a "conservative amino acid mutation" refers to an amino acid mutation that does not cause a significant change in the conformational structure of a protein or polypeptide, retaining its biological activity. The conservative amino acid mutations mentioned herein may be mutations between different amino acids with similar physiological and biochemical properties. For example, after conservative amino acid mutations, the provided antibody still retains specific binding activity to the IL2RA protein. Depending on the embodiment, the conservative amino acid mutations mentioned herein may be substitutions, deletions, or additions of conservative amino acids. Depending on the embodiment, a conservative amino acid deletion is a deletion of one, two, or three amino acids based on the listed CDR sequences. Depending on the embodiment, a conservative amino acid addition is an addition of one, two, or three amino acids based on the listed CDR sequences. Depending on the embodiment, a "conservative amino acid substitution" means replacing an amino acid residue with a different amino acid residue having a side chain with similar physiological and biochemical properties. For example, conservative amino acid substitutions can occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative amino acid substitutions generally do not cause significant changes in the conformational structure of the protein, and thus can retain the biological activity of the protein. The conservative amino acid substitutions mentioned can be one conservative amino acid substitution, two conservative amino acid substitutions, three conservative amino acid substitutions, etc. The names of the amino acids used herein are represented by standard single-letter or three-letter codes commonly used in the art. In some embodiments, the HCDR sequences provided have one conservative amino acid substitution compared to the HCDR sequences shown. In some embodiments, the HCDR sequences provided have one conservative amino acid deletion from the HCDR sequences shown. In some embodiments, the HCDR sequences provided have one conservative amino acid addition from the HCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid substitution from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid deletion from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid addition from the LCDR sequences shown.

[0054] The present invention also provides a monoclonal antibody or antigen-binding fragment thereof, comprising the HCDR1, HCDR2, and HCDR3 sequences from the heavy chain variable region of SEQ ID NO: 13, and the LCDR1, LCDR2, and LCDR3 sequences from the light chain variable region of SEQ ID NO: 14; or comprising the HCDR1, HCDR2, and HCDR3 sequences from the heavy chain variable region of SEQ ID NO: 15, and the LCDR1, LCDR2, and LCDR3 sequences from the light chain variable region of SEQ ID NO: 16. The HCDR sequences and LCDR sequences from these heavy chain variable regions and light chain variable regions may differ slightly according to different definition schemes, but are all included within the scope of protection of the present invention.

[0055]

[0056]

[0057]

[0058]

[0059] According to a specific embodiment, the present invention provides an anti-human IL2RA monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 13, and the sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 14. According to a specific embodiment, the present invention provides an anti-human IL2RA monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 15, and the sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 16.

[0060] The "sequence identity", "sequence homology" and "sequence similarity" mentioned herein refer to the degree of sequence identity when two polypeptides, proteins or nucleotide sequences are compared. In order to determine the ratio of sequence identity, it can be achieved in a variety of ways known in the art. For example, it can be obtained using public software such as BLAST, ALIGN, BLAST-2 and the like. In some specific embodiments, the sequence identity is due to conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity with the above-mentioned heavy chain variable region or light chain variable region may be due to 1 conservative amino acid mutation, 2 conservative amino acid mutations, 3 conservative amino acid mutations, 4 conservative amino acid mutations, 5 conservative amino acid mutations, 6 conservative amino acid mutations, 7 conservative amino acid mutations, 8 conservative amino acid mutations, 9 conservative amino acid mutations or 10 conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity with the above-mentioned heavy chain variable region or light chain variable region differs in the framework region.

[0061] According to a preferred embodiment, the present invention provides an anti-human IL2RA monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the sequence of the heavy chain variable region being shown in SEQ ID NO: 13, and the sequence of the light chain variable region being shown in SEQ ID NO: 14. According to a preferred embodiment, the present invention provides an anti-human IL2RA monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the sequence of the heavy chain variable region being shown in SEQ ID NO: 15, and the sequence of the light chain variable region being shown in SEQ ID NO: 16.

[0062] According to a more preferred embodiment, the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof provided comprises the heavy chain set forth in SEQ ID NO: 22 and the light chain set forth in SEQ ID NO: 23. According to a more preferred embodiment, the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof provided comprises the heavy chain set forth in SEQ ID NO: 24 and the light chain set forth in SEQ ID NO: 25.

[0063] polynucleotides

[0064] The present invention also provides a polynucleotide encoding the anti-human IL2RA antibody or antigen-binding fragment thereof. The polynucleotide is isolatable, including but not limited to DNA, RNA, or cDNA. Conventional methods in the art can be used to obtain isolated polynucleotide sequences to encode the monoclonal antibody, antibody, or antigen-binding fragment. According to specific embodiments, the polynucleotides provided encode the sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18, which encode the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 14. According to specific embodiments, the polynucleotides provided encode the sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, which encode the heavy chain variable region set forth in SEQ ID NO: 15 and the light chain variable region set forth in SEQ ID NO: 16.

[0065] As used herein, the term "polynucleotide" refers to a polynucleotide that does not exist naturally in nature, including polynucleotides isolated from nature (including organisms) through biological techniques, as well as artificially synthesized polynucleotides. The isolated polynucleotide can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or a combination thereof. Provided herein are multiple nucleotide sequences encoding the heavy chain variable region and light chain variable region of an anti-human IL2RA monoclonal antibody. It should be noted that, based on the amino acid sequences of the heavy chain variable region and light chain variable region provided herein, one skilled in the art can design nucleotide sequences that are not completely identical to the nucleotide sequences provided above, but all encode the same amino acid sequence, based on codon degeneracy. These modified nucleotide sequences are also included within the scope of the present invention.

[0066] Construct

[0067] To prepare the antibodies described herein, the polynucleotide sequence encoding the antibody can be inserted into a replicable expression vector and expressed in a host cell or cell-free expression system. The present invention also provides a construct comprising the polynucleotide described above. Constructs can be obtained using a variety of methods commonly used in the art, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. For example, the polynucleotide can be inserted into the multiple cloning site of an expression vector to form a construct. The construct can contain various operators, such as promoters, terminators, and marker genes, as needed, operably linked to the polynucleotide. Promoters typically provide a signal to initiate transcription; promoters can include the lactose promoter (Lac), Trp promoter, Tac promoter, or phage PL and PR promoters. Terminators provide a signal to terminate transcription during the transcription process, and marker genes in the construct are often used for screening. Enhancers can also be included as needed to enhance protein expression. The expression vector is not particularly limited and can be commercially available or artificially modified, such as plasmids, phages, or viruses. Viruses can include plant cell viruses and mammalian cell viruses. The construct can express antibodies or proteins in vitro or be transferred into cells to express antibodies or proteins.

[0068] host cells

[0069] The present invention also provides a host cell containing the aforementioned polynucleotide or construct. Any cell suitable for expressing an antibody or protein using a polynucleotide or construct can be used as a host cell. The host cell can be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a yeast cell or a mammalian cell. Commonly used host cells include yeast cells, CHO cells, HEK-293 cells, COS cells, and Drosophila S2 or Sf9 insect cells. Host cells containing the polynucleotide or construct can be obtained using methods commonly used in the art, such as microinjection, electroporation, chemical transfection, and viral-mediated transformation.

[0070] Reagent test kit

[0071] The present invention also provides a kit comprising the above-mentioned monoclonal antibody or antigen-binding fragment. The antibody or antigen-binding fragment can be present in the kit as a reagent.

[0072] The provided kit includes at least one of the aforementioned antibodies. When containing only one antibody, biological samples can be directly tested (e.g., by directly incubating the antibody with the sample and then detecting it with a labeled secondary antibody to determine the level of human IL2RA in the biological sample). When containing two antibodies, biological samples can be tested using a double antibody sandwich method (i.e., using two specific antibodies to capture and detect the target antigen in the biological sample in a sandwich-like manner). The double antibody sandwich method, because it uses two specific antibodies, can effectively capture and detect the target antigen, avoiding false positives and false negatives. Although it requires two incubations, it is simple to use and can reduce the impact of human error.

[0073] According to a specific embodiment, the provided kit includes a first antibody and a second antibody, wherein the first antibody includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6; and the second antibody includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12. According to a specific embodiment, the first antibody includes a heavy chain variable region set forth in SEQ ID NO: 13 and a light chain variable region set forth in SEQ ID NO: 14. According to a specific embodiment, the second antibody includes a heavy chain variable region set forth in SEQ ID NO: 15 and a light chain variable region set forth in SEQ ID NO: 16.

[0074] The first antibody mentioned above can be used as a coating antibody, and the second antibody can be used as a detection antibody; or the first antibody can be used as a detection antibody, and the second antibody can be used as a coating antibody.

[0075] According to a specific embodiment, the kit comprises a first antibody composition and a second antibody composition,

[0076] The first antibody composition includes a first antibody and a 0.1 M PBS buffer;

[0077] The second antibody composition comprises a biotin-labeled second antibody, 0.1 M MES buffer, 0.1% to 0.5% v / v Tween-20, 0.5% to 1.0% w / v NaCl, 1% to 3% w / v BSA, and 0.1% to 0.5% v / v Proclin-300. According to a preferred embodiment, the second antibody composition comprises a biotin-labeled second antibody, 0.1 M MES buffer, 0.2% Tween-20, 0.85% NaCl, 2% BSA, and 0.2% Proclin-300.

[0078] The kits may also include a human IL2RA standard, comprising human IL2RA, the sequence of which is set forth in SEQ ID NO:21. The purity of the human IL2RA standard may meet certain requirements, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc. The kits may also include containers, buffers, and the like as needed. For example, they may include PBS and 1% bovine serum albumin. These kits can be used in a variety of experimental techniques, including but not limited to flow cytometry, immunofluorescence, immunohistochemistry, and Western blot. Accordingly, the kits may also include instructions for use to facilitate operation and use by those skilled in the art.

[0079] To this end, the present invention also provides a method for detecting the content of human IL2RA in a biological sample, comprising:

[0080] Incubating the above-mentioned anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof with a biological sample, and measuring the OD value of the reaction product at a predetermined wavelength;

[0081] Based on the OD value, the IL2RA content in the biological sample is determined.

[0082] According to a specific embodiment, the provided method includes:

[0083] 1) Coat the plate with the primary antibody and block with blocking solution;

[0084] 2) Add human IL2RA standard or biological sample, incubate at 37°C, and rinse the plate;

[0085] 3) Continue adding biotin-labeled secondary antibody, incubate at 37°C, and then rinse the plate;

[0086] 4) Add horseradish peroxidase-labeled streptavidin, incubate at 37°C, and rinse the plate.

[0087] 5) Continue adding colorimetric reagent to develop color, then add stop solution to terminate the reaction. The reaction product is detected at wavelengths of 450 nm and 630 nm, and the OD value is determined based on the difference between the two wavelengths.

[0088] 6) preparing a standard curve based on the OD value of the human IL2RA standard, and determining the IL2RA content in the biological sample based on the standard curve.

[0089] The provided kit can refer to this method for the detection of human IL2RA.

[0090] As used herein, biological samples include, but are not limited to, serum, plasma, cell lysate, cell culture supernatant, urine, and the like. The biological samples referred to herein are ex vivo samples. According to specific embodiments, the provided methods for detecting the level of human IL2RA in biological samples are for non-disease diagnosis purposes.

[0091] Methods for producing antibodies

[0092] The present invention further provides a method for producing the above-mentioned antibody or antigen-binding fragment, comprising: culturing the above-mentioned host cell, and collecting the antibody or antigen-binding fragment from the culture.

[0093] The antibodies or antigen-binding fragments collected from the culture can be purified to obtain a substantially pure product. "Substantially pure" means that the purity of the antibody or antigen-binding fragment is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or even greater than 99.5%, 99.6%, 99.7%, or 99.8%.

[0094] The technical scheme of the present invention is described in detail below with reference to Examples. It should be noted that these Examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. The embodiments of the present invention are described in detail below. The described embodiments are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention. Unless otherwise stated, the methods or reagents used in the examples are methods or reagents commonly used in the art.

[0095] Example 1

[0096] Example 1 Human IL2RA was identified, produced, and purified by the following method to obtain the corresponding protein.

[0097] First, the human IL2RA amino acid sequence (as shown in SEQ ID NO: 21) was searched on the NCBI official website. A histidine tag was added to the C-terminus of the protein amino acid. Codon optimization software was used to optimize the codons in a mammalian cell expression system. The gene was synthesized into an expression plasmid, and the gene sequence was verified by sequencing.

[0098] Then, human IL2RA expression and production were performed:

[0099] One day before transfection, cells were cultured at 0.8 × 10 6Inoculate 100 mL of the IL-2RA vector into a cell culture flask and culture in OPM-293CD03 Medium at 37°C, 5% CO2. Transfection is performed when the cells reach 80%-90% confluency and viability is greater than 95%. Mix the IL2RA expression vector with the transfection reagent PEI and incubate at room temperature for 15-30 minutes. Then slowly add the vector to the HEK293 cells, add the enhancer and excipients, and continue culturing at 37°C, 5% CO2. When cell viability is <70%, harvest the cell culture, centrifuge at 8000g for 10 minutes, collect the supernatant, and then centrifuge at 12000g at 4°C for 30 minutes.

[0100] The collected cell supernatant was then affinity purified:

[0101] The cell supernatant was filtered through a 0.22 μm filter and affinity purified using a Ni TED-4FF bead-prepacked gravity column. Washing was performed with 20 mM PB, 300 mM NaCl, 10% glycerol, 50 mM imidazole, pH 8.0. Elution was performed with 20 mM PB, 300 mM NaCl, 10% glycerol, 250 mM imidazole, pH 8.0. Protein purity was assessed by SDS-PAGE, which showed >85%. The protein was dialyzed into 20 mM PB, 300 mM NaCl, 10% glycerol, pH 8.0 buffer and sterile filtered through a 0.22 μm filter. Protein concentration was determined by BCA assay and stored at -20°C at 1 mg / mL until use. The purified antigen protein had a purity of at least 80%.

[0102] Example 2

[0103] Example 2: A monoclonal antibody against human IL2RA was prepared by the following method, including:

[0104] After affinity-purified human IL2RA protein was mixed and emulsified with equal volumes of Freund's adjuvant, rats were immunized at a dose of 200 μg / rat. Three weeks later, the immunization was boosted with a dose of 100 μg / rat, and the booster immunization was repeated twice, with a two-week interval between each. Blood was collected from the rats to separate serum, and the serum antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). Rats with good immunity were selected for hybridoma fusion experiments. After screening and culture in HAT screening medium, human IL2RA was used as the antigen coated on an ELISA plate, and the hybridoma monoclonal antibody supernatant was collected for ELISA to identify high-quality positive cell lines. Two rounds of cell subcloning were performed to clone monoclonal cell lines, named 1A5 and 4E10. The antibodies produced are denoted as 1A5 and 4E10 antibodies.

[0105] Total RNA from B lymphocytes was extracted using the TRIzol method and reverse transcribed into cDNA by RT-PCR. The antibody heavy and light chains were amplified and sequenced using nested PCR. The nucleic acid sequences encoding the heavy and light chains were then ligated into the pCDNA3.4 vector and transiently transfected into HEK293F cells. The cell culture supernatant was purified using a Protein A column to obtain an antibody that recognizes human IL2RA.

[0106] After sequencing, the heavy chain sequence of the 1A5 antibody is shown in SEQ ID NO:22, the light chain sequence is shown in SEQ ID NO:23, the heavy chain variable region sequence is shown in SEQ ID NO:13, and the light chain variable region sequence is shown in SEQ ID NO:14. The heavy chain sequence of the 4E10 antibody is shown in SEQ ID NO:24, the light chain sequence is shown in SEQ ID NO:25, the heavy chain variable region sequence is shown in SEQ ID NO:15, and the light chain variable region sequence is shown in SEQ ID NO:16. The 1A5 antibody showed a purity of >95% on SDS-PAGE, and the 4E10 antibody showed a purity of >95% on SDS-PAGE.

[0107] Example 3

[0108] Example 3 studied the affinity of the 1A5 and 4E10 antibodies prepared in Example 2 for antigens, including:

[0109] The human IL2RA protein prepared in Example 1 was used as the coating antigen and incubated at 4°C overnight. The titers of the two antibodies were then tested as follows:

[0110] Antigen was diluted to 1 μg / mL and coated onto ELISA plates at 50 μL / well, followed by overnight incubation at 4°C. 1A5 and 4E10 antibodies were added at varying dilutions, with PBS added to the control group, and incubated at 37°C for 30 minutes. After washing, horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody was added at 50 μL / well and incubated at 37°C for 30 minutes. The plates were then washed, developed, and read.

[0111] like Figure 1 As shown: the EC50 of the 1A5 antibody is 0.33 nM, and the EC50 of the 4E10 antibody is 0.22 nM.

[0112] The results are shown above, indicating that both the 1A5 antibody and the 4E10 antibody have good affinity with the antigen.

[0113] Example 4 Double Antibody Sandwich ELISA for Detecting IL2RA Concentration

[0114] Example 4: Using the 1A5 antibody and the 4E10 antibody obtained in Example 2, a kit was prepared based on the double antibody sandwich method. The kit includes:

[0115] (1) Using 1A5 antibody as coating antibody

[0116] The 1A5 antibody prepared in Example 2 was transferred to a 14 kDa dialysis bag and dialyzed against PBS buffer, and then adjusted to a concentration of 2 mg / mL for later use. The antibody storage solution was 0.1 M PBS.

[0117] (2) Biotin-labeled 4E10 antibody as detection antibody

[0118] Labeling reaction was performed at room temperature for 1-2 hours with 10mM biotin and 4E10 antibody at a molecular molar ratio of 20:1. After the reaction, the labeled solution was transferred to a 14kDa dialysis bag and dialyzed against 1× PBS by rotation, changing the solution 6 times for 1-2 hours each time. After overnight dialysis, the labeled solution was removed and the protein content in the labeled solution was measured using a spectrophotometer. The protein content was calculated and protective solution was added. After thorough mixing, the solution was stored at 18-20°C. For testing, the protein concentration was diluted to 0.5ug / mL using the labeled preservation solution.

[0119] The labeling preservation solution used had a formulation of 0.1 M MES buffer, 0.2% v / v Tween-20, 0.85% w / v NaCl, 2% w / v BSA, and 0.2% v / v Proclin-300.

[0120] (3) Preparation of standard products

[0121] Use antigen diluent to dilute human IL2RA antigen to 0, 1.37, 4.1, 12.3, 37, 111, 333, and 1000 pg / mL, respectively, and dispense into calibrator tubes.

[0122] The diluent used for antigen dilution was 1×PBS and 1% w / v BSA.

[0123] (4) ELISA test method evaluation

[0124] Coating: Dilute the coating antibody to the working concentration (2 μg / mL) with PBS buffer (pH 9.6), add 100 μL / well to the ELISA plate, and incubate overnight at 4°C.

[0125] Blocking: The next day, wash the coated microplate 4 times, pat dry on absorbent paper, add 10% newborn calf serum, 400 μL / well, incubate at 37°C for 1 hour, and then pat dry on absorbent paper.

[0126] To add standards: Add 100 μL of protein or serum sample (diluted serially with 1% bovine serum albumin at the specified concentration). Add 100 μL of 2% newborn calf serum to the blank wells. Incubate at 37°C for 1 hour, then pat dry on absorbent paper. Wash the plate four times with TBST, patting dry on absorbent paper after each wash.

[0127] Add detection antibody: Add 100 μL of detection antibody diluted with 1% bovine serum albumin to each well and incubate at 37°C for 1 hour. Discard the liquid and wash the plate twice by adding 300 μL of TBST wash buffer to each well. Pat the plate dry on absorbent paper after each wash.

[0128] Add enzyme: Add 100 μL of diluted horseradish peroxidase-conjugated streptavidin (1:4000 dilution) to each well and incubate at 37°C for 45 minutes. Discard the liquid and wash the plate six times with 300 μL of TBST wash buffer per well. Pat the plate dry on absorbent paper after each wash. For optimal assay performance, remove any residual liquid completely.

[0129] Add substrate for color development: Add 50 μL of A solution and 50 μL of B solution to each well. Incubate at room temperature for 40 minutes in the dark. Then, add 50 μL of stop solution to each well. The color will change from blue to yellow. If the color appears green or the color change is obviously uneven, gently tap the plate frame to mix thoroughly.

[0130] Reading: Within 30 minutes, measure the OD value at either the 450nm maximum absorbance wavelength or the 630nm reference wavelength using a microplate reader. The calibrated OD value is the 450nm value minus the 630nm value. Using only the 450nm wavelength will result in an inflated OD value and reduced accuracy.

[0131] Figure 2 This is the OD result of the double antibody sandwich method for detecting different concentrations of IL2RA in Example 4 of the present invention. Figure 2 It can be seen that the concentration range of the anti-IL2RA monoclonal antibodies provided by the present invention that can be used in pairs to detect the recombinant protein IL2RA using the double antibody sandwich method is 1.37-1000 pg / mL.

[0132] In the description of this specification, the reference terms "one embodiment", "some embodiments", "specific implementation methods", etc. mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and are not to be construed as limiting the present invention. Those skilled in the art may make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-human IL2RA monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that: The antibody or antigen-binding fragment thereof is selected from at least one of the following: (1) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 1, 2 and 3; and LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NOs: 4, 5, and 6, or sequences having one or two conservative amino acid mutations compared to the sequences as shown in SEQ ID NOs: 4, 5, and 6; (2) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 7, 8 and 9, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 7, 8 and 9; and LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NOs: 10, 11, and 12, or sequences having one or two conservative amino acid mutations compared to the sequences as shown in SEQ ID NOs: 10, 11, and 12; The HCDR1-HCDR3 sequences and LCDR1-LCDR3 sequences were obtained according to the Kabat definition scheme.

2. An anti-human IL2RA monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that: The antibody or antigen-binding fragment thereof is selected from at least one of the following: (a) the HCDR1, HCDR, and HCDR3 sequences of the heavy chain variable region set forth in SEQ ID NO: 13, and the LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region set forth in SEQ ID NO: 14; or (b) The HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 15, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO:

16.

3. The anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The heavy chain variable region sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 13 or 15; The sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 14 or 16; Optionally, the sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the sequence of the light chain variable region is shown in SEQ ID NO: 14; or the sequence of the heavy chain variable region is as shown in SEQ ID NO: 15, and the sequence of the light chain variable region is as shown in SEQ ID NO: 16; Optionally, the antibody or antigen-binding fragment thereof comprises the heavy chain shown in SEQ ID NO: 22 and the light chain shown in SEQ ID NO: 23; or comprises the heavy chain shown in SEQ ID NO: 24 and the light chain shown in SEQ ID NO:

25.

4. A polynucleotide, characterized in that The polynucleotide encodes the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Optionally, the polynucleotide comprises the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18; Or include the sequences shown in SEQ ID NO: 19 and SEQ ID NO:

20.

5. A construct, characterized in that Comprising the polynucleotide according to claim 4.

6. A host cell, characterized in that Containing the polynucleotide according to claim 4 or the construct according to claim 5.

7. A kit, characterized in that An anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Optionally, the kit comprises a first antibody and a second antibody, wherein the first antibody comprises the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3, and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6; The second antibody comprises the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 7, 8 and 9, and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 10, 11 and 12; Optionally, the first antibody is a coating antibody and the second antibody is a detection antibody; Optionally, the kit comprises a first antibody composition and a second antibody composition, The first antibody composition includes a first antibody and a 0.1 M PBS buffer; The second antibody composition comprises a biotin-labeled second antibody, 0.1 M MES buffer, 0.1% to 0.5% v / v Tween-20, 0.5% to 1.0% w / v NaCl, 1% to 3% w / v BSA and 0.1% to 0.5% v / v Proclin-300; Optionally, the kit further comprises a human IL2RA standard, wherein the human IL2RA standard comprises human IL2RA, and the sequence of the human IL2RA is shown in SEQ ID NO: 21; Optionally, the purity of the human IL2RA standard is at least 80%; Optionally, the kit further comprises PBS and 1% bovine serum albumin.

8. A method for detecting the content of human IL2RA in a biological sample, characterized in that: include: incubating the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 with a biological sample, and measuring the OD value of the reaction product at a predetermined wavelength; determining the IL2RA content in the biological sample based on the OD value; Optionally, include: 1) Coat the plate with the primary antibody and block with blocking solution; 2) Add human IL2RA standard or biological sample, incubate at 37°C, and rinse the plate; 3) Continue adding biotin-labeled secondary antibody, incubate at 37°C, and then rinse the plate; 4) Add horseradish peroxidase-labeled streptavidin, incubate at 37°C, and rinse the plate. 5) Continue adding colorimetric reagent to develop color, then add stop solution to terminate the reaction. The reaction product is detected at wavelengths of 450 nm and 630 nm, and the OD value is determined based on the difference between the two wavelengths. 6) preparing a standard curve based on the OD value of the human IL2RA standard, and determining the content of IL2RA in the biological sample based on the standard curve; Optionally, the biological sample is selected from at least one of serum, plasma, cell lysate, cell culture supernatant, and urine.

9. A pharmaceutical composition, characterized in that It comprises the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

10. Use of the anti-human IL2RA monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a human IL2RA protein detection kit or a drug.

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