Anti-cd25 antibodies and uses thereof

Abstract

The application provides an anti-CD25 antibody or an antigen binding fragment thereof and application thereof. The antibody or the antigen binding fragment thereof contains CDR sequences selected from at least one of the following: heavy chain variable region CDR1-3 sequences: SEQ ID NO: 1-3; and light chain variable region CDR1-3 sequences: SEQ ID NO: 4-6. The antibody of the application has enhanced CD25 binding properties, can effectively block the combination of IL-2 and CD25, and further inhibit IL-2-induced downstream signals, inhibit T cell proliferation, promote PBMC killing CD25 positive T cells, and can be used for preventing and / or treating CD25-mediated diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, this invention relates to an anti-CD25 antibody or its antigen-binding fragment and its applications. Background Technology

[0002] CD25, or interleukin-2 receptor α chain, is a type I transmembrane protein mainly expressed on the surface of Treg cells and activated T cells, and is highly associated with cell survival, proliferation, and activation. There are two types of IL-2 receptors on the surface of T cells: low-affinity IL-2Rβ / IL-2Rγ and high-affinity IL-2Rα / IL-2Rβ / IL-2Rγ. Resting T cells express the low-affinity IL-2Rβ / IL-2Rγ receptor. Upon activation, IL-2Rα (CD25) expression is upregulated, forming the high-affinity IL-2Rα / IL-2Rβ / IL-2Rγ receptor. Treg cells highly express IL-2Rα (CD25), and its expression abundance is much higher than that of other immune cells. Targeting CD25 to eliminate Treg cells can reverse tumor immunosuppression. The surface of Treg cells is dominated by high-affinity IL-2 receptors IL-2Rα / IL-2Rβ / IL-2Rγ. Studies have shown that Treg cells are much more dependent on IL-2 than T cells. Blocking IL-2 signaling can inhibit Treg survival, reverse the immunosuppressive function of Treg cells, and restore anti-cancer immunity.

[0003] Currently, CD25-targeted therapies are used in autoimmune diseases and immune-related diseases such as cancer. In autoimmune diseases, activated autoantigen-specific T cells attack normal tissue cells, causing tissue damage. These abnormally activated T cells express CD25 on their surface. The monoclonal antibody Daclizumab is a known IgG1 anti-human CD25 antibody that inhibits the binding of IL-2 to CD25, suppressing CD25-mediated IL-2 immune activation and has been developed to reduce effector T cell activation. Daclizumab is currently approved for the treatment of multiple sclerosis. In tumor tissues, Treg cells suppress the immune function of anti-tumor immune cells, leading to tumor development and progression. Clearing Treg cells with anti-CD25 antibodies can delay tumor development and progression. Roche and Tusk Therapeutics have jointly developed the CD25-targeting antibody RG6292, which is currently undergoing a Phase I clinical trial to investigate its safety and efficacy in solid tumors.

[0004] However, the development of anti-CD25 antibodies with stronger CD25 binding and the ability to effectively block the binding of IL-2 to CD25 or inhibit IL-2-induced downstream signaling remains a pressing need in this field. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems existing in the prior art. Therefore, one object of this invention is to provide an anti-CD25 antibody or its antigen-binding fragment and conjugates thereof. Another object of this invention is to provide pharmaceutical compositions prepared using said anti-CD25 antibody or its antigen-binding fragment and conjugates thereof, which can be used to eliminate CD25-positive T cells, block the binding of IL-2 to CD25, or prevent and / or treat CD25-mediated diseases. Yet another object of this invention is to provide applications of said anti-CD25 antibody or its antigen-binding fragment and conjugates thereof, etc.

[0006] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0007] The inventors' in-depth research revealed that Daclizumab, as an IL-2 signaling inhibitory antibody, can only partially inhibit the binding of CD25 to IL-2, lacking complete blocking function and exhibiting weak inhibitory effect on IL-2 signaling. In contrast, antibody RG6292 is a non-inhibitory antibody that cannot block the binding of CD25 to IL-2 and does not affect IL-2 signal transduction.

[0008] Therefore, in a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof. According to embodiments of the present invention, the anti-CD25 antibody or its antigen-binding fragment comprises: heavy chain variable regions (CDRs) and light chain variable regions (CDRs), wherein...

[0009] Heavy chain variable regions (CDRs) have amino acid sequences as shown in SEQ ID NO:1-3 or their conserved modified forms;

[0010] The light chain variable regions (CDRs) have amino acid sequences as shown in SEQ ID NO:4-6 or their conserved modified forms.

[0011] The antibody or its antigen-binding fragment according to embodiments of the present invention possesses enhanced CD25 binding properties, effectively blocking the binding of IL-2 to CD25, thereby inhibiting IL-2-induced downstream signaling, suppressing T cell proliferation, and promoting PBMC-mediated killing of CD25-positive T cells. This antibody or its antigen-binding fragment also exhibits weaker antibody endocytosis activity, making it suitable for the development of monoclonal and bispecific antibody drugs. Furthermore, drugs prepared using the antibody or its antigen-binding fragment possess excellent properties of clearing or killing CD25-positive T cells, blocking the binding of IL-2 to CD25, or inhibiting IL-2-induced downstream signaling, and can be used for the prevention and / or treatment of CD25-mediated diseases. Therefore, the anti-CD25 antibody or its antigen-binding fragment of the present invention has good clinical application value and drug development value.

[0012] In a second aspect, the present invention provides a conjugate. According to embodiments of the present invention, the conjugate comprises: the antibody or its antigen-binding fragment described in the first aspect of the present invention. As previously described, the antibody or its antigen-binding fragment of the embodiments of the present invention can bind to CD25 and effectively block the binding of IL-2 to CD25, thereby inhibiting IL-2-induced downstream signaling, inhibiting T cell proliferation, promoting PBMC killing of CD25-positive T cells; and exhibiting weaker antibody endocytosis activity, suitable for the development of monoclonal and bispecific antibody drugs. Therefore, the conjugate comprising the anti-CD25 antibody or its antigen-binding fragment can be further used to clear or kill CD25-positive T cells, block the binding of IL-2 to CD25 or inhibit IL-2-induced downstream signaling, or prevent and / or treat CD25-mediated diseases. Thus, the conjugate comprising the anti-CD25 antibody or its antigen-binding fragment of the first aspect of the present invention has good clinical application value and drug development value.

[0013] In a third aspect, the present invention provides a nucleic acid. According to embodiments of the present invention, the nucleic acid encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention. The antibody or antigen-binding fragment thereof encoded by the nucleic acid according to embodiments of the present invention can bind to CD25 and effectively block the binding of IL-2 to CD25, thereby inhibiting IL-2-induced downstream signaling, inhibiting T cell proliferation, promoting PBMC killing of CD25-positive T cells; and exhibiting weaker antibody endocytosis activity, suitable for the development of monoclonal and bispecific antibody drugs. Furthermore, the protein encoded by the nucleic acid has the properties of clearing or killing CD25-positive T cells, blocking the binding of IL-2 to CD25 or inhibiting IL-2-induced downstream signaling, and has good preventive and / or therapeutic effects on CD25-mediated diseases.

[0014] In a fourth aspect, the present invention provides a vector or transformant. According to embodiments of the present invention, the vector or transformant contains the nucleic acid described in the third aspect of the present invention. Thus, using the constructed vector or transformant, the antibody or antigen-binding fragment of the first aspect of the present invention can be effectively expressed, and the conjugate of the second aspect of the present invention can be obtained.

[0015] In a fifth aspect, the present invention provides a cell. According to embodiments of the invention, the cell carries the nucleic acid described in the third aspect of the invention or the vector or transformant described in the fourth aspect of the invention, or expresses the antibody or its antigen-binding fragment described in the first aspect of the invention. According to embodiments of the invention, the cell can efficiently express the antibody or its antigen-binding fragment of the first aspect of the invention under suitable conditions, and further, obtains an antibody or its antigen-binding fragment capable of binding to CD25 and effectively blocking the binding of IL-2 to CD25, thereby inhibiting IL-2-induced downstream signaling, inhibiting T cell proliferation, promoting PBMC killing of CD25-positive T cells, and exhibiting weaker antibody endocytosis activity.

[0016] In a sixth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, a conjugate according to the second aspect of the present invention, a nucleic acid according to the third aspect of the present invention, or a carrier or transformant according to the fourth aspect of the present invention. The resulting drug can be further used to eliminate or kill CD25-positive T cells, block IL-2 binding to CD25 or inhibit IL-2-induced downstream signaling, or prevent and / or treat CD25-mediated diseases.

[0017] In a seventh aspect, the present invention provides a kit. According to embodiments of the invention, the kit comprises an antibody or an antigen-binding fragment thereof from the first aspect of the invention. Thus, the obtained kit can be used for CD25-related studies, such as for detecting and / or enriching and / or isolating and purifying CD25 in humans or other mammals.

[0018] In an eighth aspect, the present invention provides for the use of the antibody or antigen-binding fragment thereof from the first aspect of the invention in the preparation of a kit for detecting and / or enriching and / or isolating and purifying CD25. Those skilled in the art will understand that the features and advantages of the antibody or antigen-binding fragment thereof described above also apply to this use, and will not be repeated here.

[0019] In a ninth aspect of the invention, a method for detecting, enriching, or purifying CD25 is provided. According to an embodiment of the invention, the method comprises: contacting a biological sample containing CD25 with an antibody or antigen-binding fragment thereof from the first aspect of the invention or an antibody or antigen-binding fragment thereof from the kit of the seventh aspect of the invention. Utilizing the strong binding properties of the antibody or antigen-binding fragment thereof described in the first aspect of the invention to CD25 and its ability to block the binding of CD25 and IL-2, contacting the biological sample with the antibody or antigen-binding fragment thereof allows for the detection, enrichment, or purification of CD25 through specific binding to CD25.

[0020] In a tenth aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment thereof of the first aspect of the invention, the conjugate thereof of the second aspect of the invention, the nucleic acid thereof of the third aspect of the invention, the carrier or transformant thereof of the fourth aspect of the invention, or the pharmaceutical composition thereof of the sixth aspect of the invention in the preparation of a medicament for the purpose of eliminating CD25-positive T cells, blocking the binding of IL-2 to CD25, or preventing and / or treating CD25-mediated diseases. The antibody or antigen-binding fragment thereof of the invention and its corresponding conjugate, nucleic acid, carrier or transformant, or pharmaceutical composition can be further prepared into a medicament that can be used clinically for the prevention or treatment of diseases.

[0021] Those skilled in the art will understand that the features and advantages described above for antibodies or their antigen-binding fragments, conjugates, nucleic acid molecules, vectors or transformants and pharmaceutical compositions also apply to this use and will not be repeated here.

[0022] Beneficial effects:

[0023] (1) The antibody of the present invention can bind to human and monkey CD25, block the binding of CD25 and IL-2, and inhibit downstream IL-2 signaling; the antibody of the present invention has an ADCC effect and can promote the clearance of CD25 positive T cells. Therefore, the antibody or its antigen-binding fragment of the present invention can be further used for the prevention and / or treatment of immune-related diseases such as autoimmune diseases, transplant rejection and cancer.

[0024] (2) The antibodies of the present invention have weaker endocytic activity and are more suitable for the development of monoclonal and bispecific antibodies, and have good prospects for drug development.

[0025] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0027] Figure 1 This is an ELISA result of the binding of the mouse CD25 antibody 54A7 to human CD25 protein in Example 2 of the present invention;

[0028] Figure 2 This is a flow cytometry result of the binding of CD25 murine antibody 54A7 to human peripheral blood T cells in Example 3 of the present invention.

[0029] Figure 3 This is an ELISA result diagram of the binding of the CD25 chimeric antibody 54A7-hIgG1 to human and monkey CD25 proteins in Example 4 of the present invention;

[0030] Figure 4 This is a flow cytometry result of the binding of the CD25 chimeric antibody 54A7-hIgG1 to human peripheral blood T cells in Example 5 of the present invention.

[0031] Figure 5 This is a flow cytometry result of the binding of the CD25 chimeric antibody 54A7-hIgG1 to CHO-K1-human CD25 cells in Example 6 of the present invention.

[0032] Figure 6 This is a flow cytometry result of the binding of the CD25 chimeric antibody 54A7-hIgG1 to CHO-K1-cynomolgus CD25 cells in Example 6 of the present invention.

[0033] Figure 7 This is an ELISA result diagram of the CD25 chimeric antibody 54A7-hIgG1 blocking CD25 binding to IL-2-Biotin in Example 7 of the present invention;

[0034] Figure 8 This is a flow cytometry result of the CD25 chimeric antibody 54A7-hIgG1 inhibiting the downstream signal pSTAT5 of IL-2 in Example 8 of the present invention;

[0035] Figure 9 This is a flow cytometry result of the CD25 chimeric antibody 54A7-hIgG1 inhibiting T cell proliferation in Example 9 of the present invention;

[0036] Figure 10 This is a diagram showing the results of the CD25 chimeric antibody 54A7-hIgG1 promoting PBMC killing of CD25-positive T cells in Example 10 of the present invention.

[0037] Figure 11 This is a flow cytometry result of the endocytic activity of the CD25 chimeric antibody 54A7-hIgG1 in Example 11 of the present invention. Detailed Implementation

[0038] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0039] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0040] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0041] Terms and Definitions

[0042] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0043] In this article, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 48544861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases.

[0044] In this paper, certain regions within the variable region exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the location where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk). J. Mol. Biol. 196:901-917 (1987)).

[0045] In this article, the term "CD25" refers to the interleukin-2 receptor α chain, or interleukin-2 receptor subunit α, equivalent to "p55," "IL2RA," "IMD41," "TCGFR," and "IDDM10." The interleukin-2 (IL2) receptor α (IL2RA) and β (IL2RB) chains, along with the common γ chain (IL2RG), constitute the high-affinity IL2 receptor. The homodimeric α chain (IL2RA) produces the low-affinity receptor, while the homodimeric β (IL2RB) chain produces the intermediate-affinity receptor.

[0046] In this article, the term "anti-CD25 antibody" refers to an antibody that can bind to CD25. This antibody is also referred to as "CD25-binding antibody" in this article.

[0047] In this document, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment," and can include a portion of a complete antibody, generally an antigen-binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated," "PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol).

[0048] In this article, the term "chimeric antibody" refers to a recombinant antibody obtained by replacing the constant region amino acid sequence of a monoclonal antibody from one species (such as a mouse) with the constant region of an antibody from another species (such as a human) using recombinant DNA technology.

[0049] In this article, the term "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv backbone region (FR)) amino acid sequences in the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with non-CDR amino acid sequences in the constant and variable regions of an antibody from another species (e.g., human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, while when all non-CDR amino acid sequences in both the constant and variable regions are humanized, it is called a humanized antibody. The humanization methods can be performed using conventional antibody engineering techniques and will not be elaborated upon here.

[0050] For nucleotides, the term "homology" is used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0051] For peptides, the term "(substantial) homology" is used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0052] In this document, the term "conservatively modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing that amino acid sequence. Such modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0053] In this article, the term "conjugate" is understood in context to refer to an antibody or its antigen-binding fragment that is conjugated to a coupling part such as a carrier substance, drug, toxin, cytokine, protein tag, modifier, therapeutic agent, or chemotherapeutic agent using any covalent or non-covalent biological conjugation strategy.

[0054] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0055] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0056] In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0057] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0058] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0059] In this document, the term "treatment" means used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0060] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0061] This invention proposes an anti-CD25 antibody or its antigen-binding fragment and its conjugate, corresponding nucleic acid molecule, vector or transformant, cell, pharmaceutical composition, kit and its application, which will be described in detail below.

[0062] Anti-CD25 antibody or its antigen-binding fragment

[0063] This invention provides an anti-CD25 antibody or its antigen-binding fragment. The anti-CD25 antibody or its antigen-binding fragment comprises: heavy chain variable regions (CDRs) and light chain variable regions (CDRs), wherein...

[0064] Heavy chain variable regions (CDRs) have amino acid sequences as shown in SEQ ID NO:1-3 or their conserved modified forms;

[0065] The light chain variable regions (CDRs) have amino acid sequences as shown in SEQ ID NO:4-6 or their conserved modified forms.

[0066] The antibody or its antigen-binding fragment of the present invention possesses enhanced CD25 binding properties, effectively blocking the binding of IL-2 to CD25, thereby inhibiting IL-2-induced downstream signaling, suppressing T cell proliferation, and promoting PBMC-mediated killing of CD25-positive T cells. This antibody or its antigen-binding fragment also exhibits weaker antibody endocytosis activity, making it suitable for the development of monoclonal and bispecific antibody drugs. Furthermore, drugs prepared using the antibody or its antigen-binding fragment possess excellent properties of clearing or killing CD25-positive T cells, blocking the binding of IL-2 to CD25, or inhibiting IL-2-induced downstream signaling, and can be used for the prevention and / or treatment of CD25-mediated diseases. Therefore, the anti-CD25 antibody or its antigen-binding fragment of the present invention has good clinical application and drug development value.

[0067] It should be noted that one or more amino acid residues in the CDR region of the antibody or its antigen-binding fragment of the present invention may be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0068] According to embodiments of the present invention, the antibody or its antigen-binding fragment comprises: a heavy chain variable region CDR1 of the amino acid sequence shown in SEQ ID NO:1, a heavy chain variable region CDR2 of the amino acid sequence shown in SEQ ID NO:2, a heavy chain variable region CDR3 of the amino acid sequence shown in SEQ ID NO:3, a light chain variable region CDR1 of the amino acid sequence shown in SEQ ID NO:4, a light chain variable region CDR2 of the amino acid sequence shown in SEQ ID NO:5, and a light chain variable region CDR3 of the amino acid sequence shown in SEQ ID NO:6.

[0069] It should be noted that the antigen-binding fragment will consist of a partial sequence of the heavy chain variable region or the light chain variable region of its source antibody, or contain partial sequences of the light and heavy chain variable regions, wherein the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as its source antibody.

[0070] According to embodiments of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

[0071] According to embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0072] According to embodiments of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of mouse antibodies, human antibodies, and primate antibodies.

[0073] It should be noted that, in order to further improve the bioacceptability of the antibody, the antibody can also be humanized, that is, the antibody is a chimeric antibody or a humanized antibody.

[0074] As previously described, one or more amino acid residues in the heavy or light chain variable region of the antibody or its antigen-binding fragment of the present invention may be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody may be tested using the functional assay methods described herein.

[0075] According to embodiments of the present invention, the antibody or its antigen-binding fragment comprises: a heavy chain variable region of an amino acid sequence as shown in SEQ ID NO:7 or a conserved modified form of an amino acid sequence thereof; and / or, a light chain variable region of an amino acid sequence as shown in SEQ ID NO:8 or a conserved modified form of an amino acid sequence thereof.

[0076] It should be noted that, without substantially affecting the CD25 binding activity of the antibody or its antigen-binding fragment (retaining at least 95% activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the heavy or light chain variable region of the antibody or its antigen-binding fragment to obtain sequences of the antibody or its antigen-binding fragment. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the heavy or light chain variable region. The sequences of the above-mentioned variants of this invention can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN.

[0077] According to an embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:7; and / or, the light chain variable region comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:8.

[0078] According to an embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a constant region; wherein the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

[0079] According to embodiments of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0080] It should be noted that the immunoglobulins discussed herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc moieties that provide reduced or enhanced effector cell activity. Immunoglobulins can be derived from any species.

[0081] According to embodiments of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and / or, the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0082] In some specific implementations, the heavy chain constant region includes a heavy chain constant region selected from human IgG, IgA, IgM, IgE or IgD, such as human IgG1, human IgG2, human IgG3, human IgG4, human IgA, human IgM, human IgE or human IgD.

[0083] In some specific implementations, the heavy chain constant region includes a heavy chain constant region selected from mouse IgG, IgA, IgM, IgE or IgD, such as mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG2c, mouse IgG3, mouse IgA, mouse IgM, mouse IgE or mouse IgD.

[0084] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse antibodies or their mutants, or human antibodies or their mutants.

[0085] It should be noted that the amino acid sequences mentioned in this invention are shown from the N-terminus to the C-terminus.

[0086] According to an embodiment of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region; and / or the N end of the light chain constant region is connected to the N end of the light chain variable region.

[0087] As previously stated, without substantially affecting the CD25 binding activity of the antibody or its antigen-binding fragment (retaining at least 95% activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the constant region of the heavy or light chain of the antibody or its antigen-binding fragment to obtain sequences of the antibody or its antigen-binding fragment. These variations are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties may be substituted in the constant region of the heavy or light chain. The sequences of the above-described variations of this invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence.

[0088] According to an embodiment of the present invention, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO:21 or 23 or an amino acid sequence having at least 80% identity with it; and / or the light chain constant region has an amino acid sequence as shown in SEQ ID NO:22 or 24 or an amino acid sequence having at least 80% identity with it.

[0089] According to embodiments of the present invention, the heavy chain constant region and the light chain constant region have amino acid sequences as shown in SEQ ID NO:21 and SEQ ID NO:22; and / or the heavy chain constant region and the light chain constant region have amino acid sequences as shown in SEQ ID NO:23 and SEQ ID NO:24.

[0090] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain of an amino acid sequence as shown in SEQ ID NO:10 or 12 or an amino acid sequence having at least 90% homology therewith; and / or a light chain of an amino acid sequence as shown in SEQ ID NO:11 or 13 or an amino acid sequence having at least 90% homology therewith.

[0091] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain of an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology therewith, and a light chain of an amino acid sequence as shown in any one of SEQ ID NO:11 or an amino acid sequence having at least 80% homology therewith; or a heavy chain of an amino acid sequence as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% homology therewith, and a light chain of an amino acid sequence as shown in any one of SEQ ID NO:13 or an amino acid sequence having at least 80% homology therewith.

[0092] As previously described, the antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or contain only the antigen-binding portion (e.g., Fab, Fab', Fab'-SH, F(ab')2, or Fv, scFv fragments), or may be modified to affect function. The present invention includes anti-CD25 antibodies with modified glycosylation patterns. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or to antibodies lacking a fucose moiety on the oligosaccharide chain to, for example, enhance antibody-dependent cytotoxicity (ADCC) function. In other embodiments, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0093] According to embodiments of the present invention, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH, or F(ab')2; the antigen-binding fragment of the antibody includes at least one selected from F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab)2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, and a minimum recognition unit.

[0094] Conjugate

[0095] This invention provides a conjugate. The conjugate comprises: the aforementioned antibody or its antigen-binding fragment; and a conjugated portion, wherein the antibody or its antigen-binding fragment is linked to the conjugated portion. As previously described, the conjugate comprising the aforementioned anti-CD25 antibody or its antigen-binding fragment can be further used to eliminate or kill CD25-positive T cells, block IL-2 binding to CD25 or inhibit IL-2-induced downstream signaling, or prevent and / or treat CD25-mediated diseases. Therefore, the conjugate comprising the anti-CD25 antibody or its antigen-binding fragment of the first aspect of this invention has good clinical application value and drug development value.

[0096] According to embodiments of the present invention, the coupling portion includes at least one selected from carriers, drugs, toxins, cytokines, protein tags, modifiers, therapeutic agents, and chemotherapeutic agents.

[0097] Nucleic acid

[0098] This invention provides a nucleic acid. The nucleic acid encodes the aforementioned antibody or its antigen-binding fragment. As previously described, the antibody or its antigen-binding fragment encoded by the nucleic acid has the properties of clearing or killing CD25-positive T cells, blocking IL-2 binding to CD25, or inhibiting IL-2-induced downstream signaling, and has good preventive and / or therapeutic effects against CD25-mediated diseases.

[0099] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the molecular sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0100] According to embodiments of the present invention, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:25, 26.

[0101] According to embodiments of the present invention, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:27, 28.

[0102] Vector or transformant

[0103] This invention provides a vector or transformant. The vector or transformant contains the aforementioned nucleic acid. The vector or transformant may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The vector or transformant constructed thereby can effectively express the antibody or antigen-binding fragment of the first aspect of this invention, and obtain the conjugate of the second aspect of this invention.

[0104] When linking the aforementioned nucleic acid molecules to the vector or transformant, such as an expression vector, the nucleic acid molecules can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. Of course, these control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. The nucleic acid molecules and control elements simply need to be operatively linked.

[0105] According to embodiments of the present invention, the vector may refer to a cloning vector or an expression vector, and can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this invention is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0106] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the proteins encoded by the nucleic acid molecules.

[0107] According to an embodiment of the present invention, the carrier is a eukaryotic carrier or a prokaryotic carrier.

[0108] According to embodiments of the present invention, the vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0109] cell

[0110] This invention provides a cell. The cell carries the aforementioned nucleic acid, the aforementioned vector or transformant, or expresses the aforementioned antibody or its antigen-binding fragment. Thus, the obtained cell, under suitable conditions, can efficiently express the antibody or its antigen-binding fragment of the first aspect of this invention. Furthermore, it yields an antibody or its antigen-binding fragment capable of binding to CD25 and effectively blocking the binding of IL-2 to CD25, thereby inhibiting IL-2-induced downstream signaling, inhibiting T cell proliferation, promoting PBMC killing of CD25-positive T cells, and exhibiting weaker antibody endocytosis activity.

[0111] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell, or a bacteriophage.

[0112] According to an embodiment of the present invention, the prokaryotic cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0113] According to an embodiment of the present invention, the eukaryotic cell is a fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0114] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces cerevisiae, or Trichoderma.

[0115] According to an embodiment of the present invention, the insect cell is a grass armyworm cell; according to an embodiment of the present invention, the plant cell is a tobacco plant cell; according to an embodiment of the present invention, the mammalian cell is a BHK cell, a CHO cell, a COS cell, a myeloma cell, or a human embryonic kidney 293 cell; and does not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0116] According to an embodiment of the present invention, the cell is a mammalian cell.

[0117] According to an embodiment of the present invention, the cells are BHK cells, CHO cells, COS cells, or NSO cells.

[0118] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibody or its antigen-binding fragment described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the antibody or its antigen-binding fragment include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the recombinant antibody based on the specific environment of their laboratory.

[0119] Pharmaceutical Composition

[0120] This invention provides a pharmaceutical composition. The pharmaceutical composition comprises: the antibody or its antigen-binding fragment described above, the conjugate described above, the nucleic acid described above, or the carrier or transformant described above. The resulting drug can be further used to eliminate or kill CD25-positive T cells, block the binding of IL-2 to CD25 or inhibit IL-2-induced downstream signaling, or prevent and / or treat CD25-mediated diseases, with significant effects.

[0121] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0122] According to embodiments of the present invention, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0123] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0124] It should be noted that the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0125] The medicament of this invention contains a safe and effective amount of the active ingredient of this invention and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage forms of the medicament of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The medicament is preferably manufactured under aseptic conditions.

[0126] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0127] The pharmaceutically acceptable excipients described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0128] Reagent test kit

[0129] This invention provides a kit. The kit contains the antibody or its antigen-binding fragment described above. As previously stated, the antibody or its antigen-binding fragment of this invention can effectively bind specifically to CD25. The CD25-related kit developed using this characteristic can be used for CD25-related research, such as for detecting and / or enriching and / or isolating and purifying CD25 in humans or other mammals.

[0130] The kit can effectively detect, enrich, or purify CD25 in biological samples for further scientific research, such as qualitative or quantitative detection of CD25 protein molecules in biological samples. More specifically, it can be used in kits involving the detection of CD25 and antibody-specific binding properties, such as immunoblotting and immunoprecipitation. These kits may contain any one or more of the following: antagonists, CD25 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. CD25 antibodies can be used in various types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for CD25-related diseases by detecting the serum or blood of a subject.

[0131] Uses in the preparation kit

[0132] This invention provides the use of the above-described antibody or its antigen-binding fragment in the preparation of a kit for detecting and / or enriching and / or isolating and purifying CD25.

[0133] According to embodiments of the present invention, the kit detects CD25 by ELISA, flow cytometry, Western blotting, or immunoprecipitation.

[0134] According to an embodiment of the present invention, the kit enriches and / or purifies CD25 by affinity chromatography.

[0135] As previously described, the antibodies or antigen-binding fragments of the present invention can specifically bind to CD25; therefore, the antibodies or antigen-binding fragments can be used to detect CD25. Furthermore, they can be used to prepare CD25-related kits for scientific research, such as for the qualitative or quantitative detection of CD25 protein molecules in biological samples. More specifically, they can be used in kits involving the specific binding properties of CD25 and antibodies or their binding fragments, such as immunoblotting and immunoprecipitation. These kits may contain any one or more of the following: antagonists, CD25 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. CD25 antibodies or antigen fragments can be used in different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, CD25-related diseases can be tested by examining the serum or blood of a subject.

[0136] method

[0137] This invention provides a method for detecting, enriching, or isolating and purifying CD25. The method comprises contacting a biological sample containing CD25 with the antibody or its antigen-binding fragment described above, or with the antibody or its antigen-binding fragment from the kit described above. As previously stated, the antibody or its antigen-binding fragment of this invention can specifically bind to CD25. CD25 research methods developed using this characteristic, such as methods for detecting and / or enriching and / or isolating and purifying human or other mammalian CD25, can be further used for CD25-related research, such as for detecting and / or enriching and / or isolating and purifying human or other mammalian CD25.

[0138] Uses in drug preparation

[0139] The present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned conjugate, the above-mentioned nucleic acid, the above-mentioned carrier or transformant, or the above-mentioned pharmaceutical composition in the preparation of a drug for the purpose of eliminating CD25-positive T cells, blocking the binding of IL-2 to CD25, or preventing and / or treating CD25-mediated diseases.

[0140] According to an embodiment of the present invention, the CD25-mediated disease is an inflammatory disease or an immune-related disease;

[0141] According to embodiments of the present invention, the immune-related diseases include transplant rejection, autoimmune diseases, infectious diseases, cancer, or tumors.

[0142] According to an embodiment of the present invention, transplant rejection is allogeneic or xenograft rejection.

[0143] According to embodiments of the present invention, the inflammatory disease or immune-related disease is selected from at least one of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, type 1 diabetes, insulin-dependent type 2 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, polymyositis cutanea, Sjögren's syndrome, arteritis (including giant cell arteritis), aplastic anemia, asthma, scleroderma, uveitis, psoriasis, palmoplantar pustulosis, corrosive lichen planus, bullous pemphigus, bullous epidermolysis bullosa, contact dermatitis, and atopic dermatitis.

[0144] According to embodiments of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0145] In some specific implementations, the tumors include, but are not limited to, carcinoma, lymphoma, leukemia, germ cell tumors, and sarcomas. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (intestinal) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

[0146] In some specific implementations, the cancer or tumor can be a solid tumor, including but not limited to sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue), carcinomas (including tumors arising from epithelial cells), mesotheliomas, neuroblastomas, retinoblastomas, etc. Cancers involving solid tumors include, but are not limited to, brain cancer, lung cancer, stomach cancer, duodenal cancer, esophageal cancer, breast cancer, colon and rectal cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, cervical cancer, lymphoma, etc.

[0147] In some specific implementations, the cancer involves tumors that express CD25, including but not limited to lymphomas such as Hodgkin's lymphoma and lymphocytic leukemias such as chronic lymphocytic leukemia (CLL).

[0148] Disease treatment methods

[0149] This invention provides a method for preventing and / or treating CD25-mediated diseases. According to embodiments of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned antibody or its antigen-binding fragment, the aforementioned conjugate, the aforementioned nucleic acid, the aforementioned carrier or transformant, or the aforementioned pharmaceutical composition.

[0150] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0151] The effective amount of the antibody or its antigen-binding fragment, conjugate, nucleic acid, carrier, transformant, or pharmaceutical composition described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0152] In some specific implementations, the transplant rejection of the aforementioned subjects manifests as allogeneic or xenograft rejection, occurring in patients who are undergoing or have undergone organ or tissue transplantation, such as heart, lung, combined heart and lung, trachea, kidney, liver, pancreas, esophagus, intestine, skin, limb, umbilical cord, stem cell, islet cell transplantation, etc.

[0153] In some specific implementations, the subjects' conditions are selected from inflammatory diseases, immune diseases, or autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, type 1 diabetes, insulin-dependent type 2 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, polymyositis cutanea, Sjögren's syndrome, arteritis (including giant cell arteritis), aplastic anemia, asthma, scleroderma, and uveitis; inflammatory or hyperproliferative skin diseases, such as psoriasis, including plaque psoriasis, palmoplantar pustulosis (PPP), corrosive lichen planus, bullous pemphigus, epidermolysis bullosa, contact dermatitis, and atopic dermatitis.

[0154] In some specific implementations, the aforementioned subjects' conditions are proliferative disorders (e.g., cancer or tumors) or they have proliferative disorders (e.g., cancer or tumors). These tumors include, but are not limited to, carcinoma, lymphoma, leukemia, germ cell tumors, and sarcomas. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (intestinal) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

[0155] In some specific implementations, the cancer or tumor can be a solid tumor, including but not limited to sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue), carcinomas (including tumors arising from epithelial cells), mesotheliomas, neuroblastomas, retinoblastomas, etc. Cancers involving solid tumors include, but are not limited to, brain cancer, lung cancer, stomach cancer, duodenal cancer, esophageal cancer, breast cancer, colon and rectal cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, cervical cancer, lymphoma, etc.

[0156] In some specific implementations, the cancer involves tumors that express CD25, including but not limited to lymphomas such as Hodgkin's lymphoma and lymphocytic leukemias such as chronic lymphocytic leukemia (CLL).

[0157] The sequence descriptions involved in this invention are detailed in Table 1.

[0158] Table 1. Amino Acid / Nucleotide Sequence Description

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] The present invention will be described in detail below through examples. In the examples or test cases, experimental methods without specific conditions are performed under conventional conditions.

[0166] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0167] Example 1: Preparation of the anti-CD25 antibody of the present invention

[0168] Hybridoma technology is used to screen for and obtain murine monoclonal antibodies against the target protein CD25. Gene recombination technology is used to obtain CD25 chimeric antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or their mutants. Exemplarily, the screening and preparation methods for CD25 murine monoclonal antibody (CD25 murine antibody 54A7) and the preparation method for CD25 chimeric antibody (CD25 chimeric antibody 54A7-hI gG1) in this embodiment are as follows.

[0169] 1. Screening of CD25 murine antibody 54A7 and preparation of the anti-CD25 antibody of this invention

[0170] Screening method for CD25 murine antibodies: A murine monoclonal antibody against human CD25 was generated. Then, purified recombinant CD25 extracellular His-tagged fusion protein (CD25-His) (purchased from Acro) was used as the antigen to immunize Balb / c mice (9 weeks old, purchased from Shanghai Lexco, weighing approximately 20g). The immunized mice were immunized three times with the purified antigen and complete Freund's adjuvant, and incomplete Freund's adjuvant. Immune response was detected after blood collection via tail vein. Serum was screened by ELISA and flow cytometry to obtain mice with anti-human CD25 immunoglobulin. Spleen cells from mice with the highest anti-CD25 immunoglobulin levels were fused with murine myeloma cells SP2 / 0 (ATCC number CRL-1581). The fused hybridoma cells were screened for antibodies to obtain the CD25 murine antibody, namely CD25 murine antibody 54A7.

[0171] CD25 murine 54A7 antibody preparation method using gene recombination technology: The total number of candidate hybridoma cells is cultured to 10... 6Cells were collected by centrifugation at 800 rpm for 10 minutes, and total RNA was extracted using the Trizol kit (Invitrogen). Using the total RNA as a template, a cDNA library was synthesized by reverse transcription (Invitrogen), and the corresponding variable region nucleic acid sequence of hybridoma cells was amplified by PCR using the cDNA as a template. The primer sequences used in the PCR amplification reaction were complementary to the first frame region or signal peptide region and constant region of the antibody variable region (Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). In a 50 μl reaction system, 2 μl of cDNA, 5 μl of 10×PCR buffer, 2 μl (5 μmol) of upstream and downstream primers, 2 μl of dNTPs, 1 μl of Taq enzyme (Takara, Ex Taq), and 38 μl of H2O were added. The mixture was pre-denatured at 95 °C for 5 min, followed by temperature cycling for PCR amplification. The reaction conditions were: denaturation at 94 °C for 30 s, annealing at 58 °C for 45 s, extension at 72 °C for 50 s, for a total of 32 cycles, followed by a final extension at 72 °C for 7 min. Sequencing of the amplified products yielded the heavy and light chain variable region sequences of the mouse 54A7 antibody (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8), and the heavy and light chain sequences of the CD25 mouse antibody 54A7 were obtained (the amino acid sequence of the heavy chain is shown in SEQ ID NO:10, and the amino acid sequence of the light chain is shown in SEQ ID NO:11).

[0172] Based on the variable region sequence of the light chain and the amino acid sequence of the variable region of the CD25 murine antibody 54A7, the CD25 chimeric antibody 54A7-hIgG1 of this invention was designed and prepared. The amino acid sequence of its heavy chain is shown in SEQ ID NO:12, and the amino acid sequence of its light chain is shown in SEQ ID NO:13. Specific preparation methods can be performed using conventional antibody engineering techniques and will not be elaborated here.

[0173] 2. Antibody production, the specific experimental operation is as follows: (1) Use ExpiCHO Expression Medium (purchased from Thermo Fisher) to culture ExpiCHO cells (purchased from Thermo Fisher), and adjust the cell concentration to 6×10 6(2) Add pcDNA3.4 vector (synthesized by Nanjing GenScript) containing nucleic acid sequences encoding antibody heavy and light chains to 2 mL OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1 to obtain solution A. (3) Add 160 μL of ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2 mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution B. (4) Then mix solution A and solution B to obtain transfection mixture, and add the entire transfection mixture to 50 mL ExpiCHO cell solution within 5 minutes. (5) After culturing at 37℃ and 5% CO2 for 1 day, add 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher), and culture at 32℃ and 5% CO2 for 9 days. Harvest the culture supernatant, and add 8 mL of Feed on day 5. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nanomicro).

[0174] Example 2: CD25 mouse antibody ELISA binding assay of the present invention

[0175] ELISA assays were used to detect the binding properties of CD25 antibodies. In this embodiment, the His-tagged fusion protein of the extracellular region of CD25 was coated into a 96-well plate, and the intensity of the signal after antibody addition was used to determine the binding properties between the antibody and the CD25 protein.

[0176] Human CD25-His protein (purchased from Acro) was diluted to 2 μg / ml with PBS buffer and added to 96-well plates at a volume of 100 μl / well. The plates were incubated overnight at 4°C. The PBS buffer was removed from the 96-well plates, and the plates were washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer. Then, 200 μl / well of PBS / 10% BSA was added, and the plates were incubated at 37°C for 2 h for blocking. The blocking buffer was removed, and the plates were washed 6 times with PBST. Then, 100 μl / well of the CD25 mouse 54A7 antibody obtained in Example 1 of this invention (heavy chain amino acid sequence as shown in SEQ ID NO: 10, and light chain amino acid sequence as shown in SEQ ID NO: 11) serially diluted with PBST / 0.05% BSA was added, and the plates were incubated at 37°C for 1 h. Remove the reaction mixture, wash the plate 6 times with PBST, and then dilute HRP (horseradish peroxidase)-labeled anti-mouse IgG antibody secondary antibody (purchased from Jacksonlab) with PBST / 0.05% BSA at 100 μl / well. Incubate at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μl / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then stop the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0177] The results are as follows Figure 1 As shown. Experimental results indicate that the CD25 murine antibody 54A7 of this invention can bind to the CD25 protein.

[0178] Example 3: Flow cytometry binding experiment of the CD25 mouse antibody of the present invention

[0179] Flow cytometry was used to detect the binding properties of murine CD25 antibodies. In this embodiment, CD25 antibodies were added to human peripheral blood T cells, and the strength of the signal after antibody addition was used to determine the binding properties between the antibody and CD25.

[0180] Human peripheral blood T cells were diluted to 2 × 10⁻⁶ using PBS. 6 / ml (purchased from Selene Biotech), add 100μl / tube of goat serum to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add CD25 mouse antibody 54A7 (heavy chain amino acid sequence as shown in SEQ ID NO:10, and light chain amino acid sequence as shown in SEQ ID NO:11) and mIgG1 (purchased from Sinocare) of different concentration gradients obtained in Example 1 of this invention, and incubate at 4℃ for 30min. Add 1ml of PBS to the EP tube, centrifuge at 3500rpm for 5min at 4℃, discard the supernatant, and wash once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100μl / tube of PBS, add 1μl / tube of Alexa-647-labeled goat anti-mouse IgG antibody secondary antibody (purchased from Biolegend), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge, and discard the supernatant. Cells were resuspended in 200 μl / tube of PBS and analyzed by flow cytometry. Results are as follows: Figure 2 As shown.

[0181] Further experimental results show that the CD25 murine antibody 54A7 of the present invention can bind to T cells.

[0182] Example 4: CD25 chimeric antibody ELISA binding assay of the present invention

[0183] ELISA assays were used to detect the binding properties of CD25 antibodies. In this embodiment, a His-tagged fusion protein of the extracellular region of human and monkey CD25 was coated into a 96-well plate, and the intensity of the signal after antibody addition was used to determine the binding properties between the antibody and the CD25 protein.

[0184] Human and monkey CD25-His proteins (purchased from Acro; the amino acid sequence of human CD25 is shown in SEQ ID NO:9, and the amino acid sequence of Cynomolgus CD25 is shown in SEQ ID NO:16) were diluted to 2 μg / ml with PBS buffer and added to 100 μl / well of a 96-well plate. The plate was incubated overnight at 4°C. The PBS buffer in the 96-well plate was removed, and the plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer. Then, 200 μl / well of PBS / 10% BSA was added, and the plate was blocked by incubation at 37°C for 2 h. After removing the blocking buffer and washing the plate 6 times with PBST, add 100 μl / well of the following: the CD25 chimeric antibody 54A7-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:12 and light chain amino acid sequence as shown in SEQ ID NO:13), the control CD25 antibody AB917-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:14 and light chain amino acid sequence as shown in SEQ ID NO:15), and hIgG1 (purchased from Baiying Biotechnology), which are serially diluted with PBST / 0.05% BSA. Incubate at 37°C for 1 h. After removing the reaction system and washing the plate 6 times with PBST, add 100 μl / well of HRP (horseradish peroxidase)-labeled anti-human IgG antibody secondary antibody (purchased from Southern Biotech) diluted with PBST / 0.05% BSA. Incubate at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μl / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then stop the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader. Results are as follows: Figure 3 As shown.

[0185] Experimental results show that the CD25 chimeric antibody 54A7-hIgG1 of the present invention can bind to human and monkey CD25 proteins.

[0186] Example 5: CD25 binding assay using flow cytometry with the CD25 chimeric antibody of the present invention.

[0187] Flow cytometry was used to detect the binding properties of CD25 antibodies. In this embodiment, CD25 antibodies were added to human peripheral blood T cells, and the strength of the signal after antibody addition was used to determine the binding properties between the antibody and CD25.

[0188] Human peripheral blood T cells were diluted to 2 × 10⁻⁶ using PBS. 6 / ml (purchased from Selene Biotech), add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted CD25 chimeric antibody 54A7-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:12, and light chain amino acid sequence as shown in SEQ ID NO:13), control CD25 antibody AB917-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:14, and light chain amino acid sequence as shown in SEQ ID NO:15, preparation method see WO2019 / 175216, performance is closest to Roche's CD25 antibody RG6292 in Phase I clinical trial), and hIgG1 (purchased from Baiying Biotech), and incubate at 4℃ for 30min. Add 1ml of PBS to the EP tube, centrifuge at 3500rpm for 5min at 4℃, discard the supernatant, and wash once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100 μl / tube of PBS. 1 μl / tube of Alexa-647-labeled anti-human IgG antibody secondary antibody (purchased from Jacksonlab) was added, and the cells were incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μl / tube of PBS and analyzed by flow cytometry. Results are as follows: Figure 4 As shown.

[0189] Further experimental results show that the CD25 chimeric antibody 54A7-hIgG1 of the present invention can bind to T cells, and the binding strength is higher than that of the control CD25 antibody AB917-hIgG1.

[0190] Example 6: Flow cytometry experiment of the CD25 chimeric antibody of the present invention on human and monkey CD25 binding.

[0191] Flow cytometry was used to detect the binding properties of CD25 antibodies. In this embodiment, antibodies were further added to CHO-K1-human CD25 and CHO-K1-cynomolgus CD25 cells, and the strength of the signal after antibody addition was used to determine the binding properties of the chimeric antibody to human and monkey CD25.

[0192] HEK293T cells were processed at a rate of 5 × 10 5Cells were seeded into 6-well plates and cultured overnight in DMEM medium without antibiotics. Before transfection, the medium was discarded, and 1 ml of fresh DMEM medium without antibiotics was added. The coding sequence (nucleotide sequence as shown in SEQ ID NO:19) of human CD25 protein (amino acid sequence as shown in SEQ ID NO:9) was inserted between the EcoRI and BamHI restriction sites of the pLVX-EF1a-IRES-puro vector, or the coding sequence (nucleotide sequence as shown in SEQ ID NO:16) of monkey CD25 protein (amino acid sequence as shown in SEQ ID NO:16) was inserted between the EcoRI and BamHI restriction sites of the pLVX-EF1a-IRES-puro vector. As shown in NO:20), pMD2G and psPAX2 vector (3 μg total) were added to 200 μl of serum-free DMEM medium in a 2:1:1 ratio, along with 12 μg of polyetherimide (PEI, purchased from Polysciences). After mixing, the mixture was allowed to stand for 16 min, and then all the liquid was added to a six-well plate containing HEK293T cells. After culturing for 6 h, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (purchased from Millipore) to obtain the viral supernatant. All of the viral supernatant was added to a medium containing 1×10⁻⁶ cells. 4 In a 6-well plate containing CHO-K1 cells, polybrene (purchased from Sigma) was added to a final concentration of 4 μg / ml and the cells were cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were CHO-K1-human CD25 and CHO-K1-cynomo lgus CD25 cells.

[0193] CHO-K1-human CD25 and CHO-K1-cynomolgus CD25 cells were diluted to 2 × 10⁻⁶ with PBS. 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted CD25 chimeric antibody 54A7-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:12, and light chain amino acid sequence as shown in SEQ ID NO:13), control CD25 antibody AB917-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:14, and light chain amino acid sequence as shown in SEQ ID NO:15), control Daclizumab (heavy chain amino acid sequence as shown in SEQ ID NO:17, and light chain amino acid sequence as shown in SEQ ID NO:18) or hIgG1 (purchased from Baiying Biotechnology), and incubate at 4℃ for 30min. Add 1ml of PBS to the EP tube, centrifuge at 3500rpm for 5min at 4℃, discard the supernatant, and wash once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100 μl / tube of PBS. 1 μl / tube of Alexa-647-labeled anti-human Fc antibody secondary antibody (purchased from Jackson LASB) was added, and the cells were incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μl / tube of PBS and analyzed by flow cytometry. Results are as follows: Figure 5 , Figure 6 As shown.

[0194] Further experimental results show that the CD25 chimeric antibody 54A7-hIgG1 of the present invention can bind to human and monkey CD25, and its affinity for binding to human CD25 is stronger than that of the control CD25 antibody AB917-hIgG1 and stronger than that of the control Daclizumab.

[0195] Example 7: Investigation of the blocking ability of the CD25 chimeric antibody of the present invention

[0196] ELISA assays were used to detect the blocking properties of chimeric CD25 antibodies. In this embodiment, the human CD25 extracellular His-tagged fusion protein was coated into a 96-well plate, and the intensity of the signal after the addition of the antibody and IL-2-Biotin was used to determine the effect of the antibody on CD25 binding to IL-2.

[0197] Human CD25-His protein (purchased from Acro) was diluted to 2 μg / ml with PBS buffer and added to 96-well plates at a volume of 100 μl / well. The plates were incubated overnight at 4°C. The PBS buffer in the 96-well plates was then removed, and the plates were washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer. Finally, 200 μl / well of PBS / 10% BSA was added, and the plates were incubated at 37°C for 2 hours for blocking. After removing the blocking buffer and washing the plate 6 times with PBST, add 50 μl / well of IL-2-Biotin (purchased from Acro) diluted to an appropriate concentration with PBST / 0.05% BSA and 50 μl / well of serially diluted CD25 chimeric antibody 54A7-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:12 and light chain amino acid sequence as shown in SEQ ID NO:13), control CD25 antibody AB917-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:14 and light chain amino acid sequence as shown in SEQ ID NO:15), Daclizumab (heavy chain amino acid sequence as shown in SEQ ID NO:17 and light chain amino acid sequence as shown in SEQ ID NO:18), and hIgG1 (purchased from Baiying Biotechnology). Incubate at 37°C for 1 h. After removing the reaction mixture, wash the plate 6 times with PBST. Dilute HRP (horseradish peroxidase)-labeled anti-Streptavidin secondary antibody (purchased from Southern Biotech) with PBST / 0.05% BSA at 100 μl / well and incubate at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μl / well of TMB (tetramethylbenzidine) and incubate at room temperature for 3 min. Terminate the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader. Results are as follows: Figure 7 As shown.

[0198] Experimental results show that the CD25 chimeric antibody 54A7-hIgG1 of the present invention can block the binding of CD25 to IL-2, and is stronger than the control CD25 antibody AB917-hIgG1 and the control Daclizumab.

[0199] Example 8: Performance evaluation of the CD25 chimeric antibody of the present invention in inhibiting downstream IL-2 signaling.

[0200] Flow cytometry was used to detect the inhibitory effect of chimeric CD25 antibodies on downstream IL-2 signaling. In this embodiment, IL-2 and CD25 antibodies were added to human peripheral blood T cells, and the intensity of the pSTAT5 signal after antibody addition was used to determine the effect of the chimeric antibody on downstream IL-2 signaling.

[0201] Human peripheral blood T cells were diluted to 2 × 10⁶ cells using complete RPMI 1640 medium. 6 / ml (purchased from Selex Biotech), 100μl / tube was added to each well of a 96-well plate. 10U / ml IL-2 and serially diluted CD25 chimeric antibody 54A7-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:12, and light chain amino acid sequence as shown in SEQ ID NO:13), control CD25 antibody AB917-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:14, and light chain amino acid sequence as shown in SEQ ID NO:15), Daclizumab (heavy chain amino acid sequence as shown in SEQ ID NO:17, and light chain amino acid sequence as shown in SEQ ID NO:18), and hIgG1 (purchased from Bio-Legend Biotech) were added. The plates were incubated at 37°C for 1 h. pSTAT5 fluorescent antibody (purchased from Biolegend) was then labeled, and the cells were resuspended in 200μl / tube of PBS for flow cytometry analysis. Results are as follows: Figure 8 As shown.

[0202] Experimental results show that the CD25 chimeric antibody 54A7-hIgG1 of the present invention can inhibit STAT5 phosphorylation, that is, it can inhibit IL-2-induced downstream signaling, which is stronger than the control Daclizumab, while the CD25 antibody AB917-hIgG1 antibody does not have this function.

[0203] Example 9: Investigation of the inhibitory effect of the CD25 chimeric antibody of the present invention on T cell proliferation.

[0204] Flow cytometry was used to detect the inhibitory effect of CD25 antibodies on T cell proliferation. In this embodiment, human peripheral blood T cells were labeled with CFSE, and CD3 and CD28 antibodies were added. After culturing for 72 hours, the strength of the CFSE signal was used to determine the effect of CD25 antibodies on T cell proliferation.

[0205] T cells were labeled with 5 μM CFSE. After labeling, human peripheral blood T cells were diluted to 2 × 10⁶ cells / mL with complete RPMI 1640 medium. 5 / ml (purchased from Selene Biotechnology), 100μl / tube volume was added to a 96-well plate, along with 2μg / ml CD3 and CD28 antibodies, the test CD25 chimeric antibody 54A7-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:12, and light chain amino acid sequence as shown in SEQ ID NO:13) obtained in Example 1 of this invention, the control CD25 antibody AB917-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:14, and light chain amino acid sequence as shown in SEQ ID NO:15), and hIgG1 (purchased from Baiying Biotechnology), to make the final antibody concentration 100μg / ml, and incubated at 37℃ for 72h. Then, the results were detected by flow cytometry, as shown in the figure. Figure 9 As shown.

[0206] Experimental results show that the CD25 chimeric antibody 54A7-hIgG1 of the present invention can inhibit T cell proliferation, while the control antibody AB917 does not affect T cell proliferation.

[0207] Example 10: Performance evaluation of the CD25 chimeric antibody of the present invention in promoting PBMC killing of CD25-positive T cells

[0208] Flow cytometry was used to detect the properties of CD25 antibodies in promoting the killing of CD25-positive T cells by PBMCs.

[0209] T cells were labeled with 5 μM CTV. After labeling, human peripheral blood T cells were diluted to 1 × 10⁻⁶ cells using complete RPMI 1640 medium. 5 / ml (purchased from Selene Biotechnology), 100μl / tube was added to each well of a 96-well plate as target cells; PBMCs were diluted to 2.5×10 using complete RPMI 1640 cells. 6 / ml (purchased from Selene Biotechnology), 80μl / tube was added to each well of a 96-well plate as effector cells; 20μl / tube was added to each well of the CD25 chimeric antibody 54A7-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:12, and light chain amino acid sequence as shown in SEQ ID NO:13) or hIgG1 (purchased from Baiying Biotechnology) diluted to an appropriate concentration, and incubated at 37°C for 4 h. 7-AAD was added, and the results were analyzed by flow cytometry. The results are as follows: Figure 10 As shown.

[0210] Experimental results show that the CD25 chimeric antibody 54A7-hIgG1 of the present invention can promote PBMC killing of CD25-positive T cells and has ADCC activity.

[0211] Example 11: Detection of CD25 chimeric antibody endocytosis activity

[0212] Flow cytometry was used to detect the endocytic properties of chimeric CD25 antibodies.

[0213] CHO-K1-human CD25 cells were diluted to 1×10⁻⁶ using complete DMEM medium. 5 / ml, 100μl / well was added to a 96-well plate; the CD25 chimeric antibody 54A7-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:12, and light chain amino acid sequence as shown in SEQ ID NO:13), the control CD25 antibody AB917-hIgG1 (heavy chain amino acid sequence as shown in SEQ ID NO:14, and light chain amino acid sequence as shown in SEQ ID NO:15) or hIgG1 (purchased from Baiying Biotechnology) was mixed 1:1 with pHrodo-labeled anti-human IgG secondary antibody (purchased from Jacksonlab), and added to the cells to make the final CD25 antibody concentration 20μg / ml, and incubated at 37℃ for 24h. The results were detected by flow cytometry. The results are as follows: Figure 11 As shown.

[0214] Experimental results show that the CD25 chimeric antibody 54A7-hIgG1 of the present invention has weaker endocytic activity and is more suitable for the development of monoclonal and bispecific antibodies.

[0215] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0216] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment comprises: The heavy chain variable region CDR1 of the amino acid sequence shown in SEQ ID NO: 1, The heavy chain variable region CDR2 of the amino acid sequence shown in SEQ ID NO: 2, The heavy chain variable region CDR3 of the amino acid sequence shown in SEQ ID NO: 3, The light chain variable region CDR1 of the amino acid sequence shown in SEQ ID NO: 4, The light chain variable region CDR2 of the amino acid sequence shown in SEQ ID NO: 5, The light chain variable region CDR3 of the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain framework region and a light chain framework region.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, At least a portion of the heavy chain framework region and the light chain framework region are derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy cow antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

4. The antibody or its antigen-binding fragment according to claim 2, characterized in that, At least a portion of the heavy chain framework region and the light chain framework region are derived from at least one of mouse antibodies, human antibodies, and primate antibodies.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises: The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 7; and, The light chain variable region of the amino acid sequence shown in SEQ ID NO:

8.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a constant region; The constant region includes a heavy chain constant region and a light chain constant region.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The heavy chain constant region and the light chain constant region are derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

8. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; and, The light chain constant region includes light chain constant regions selected from κ-type or λ-type.

9. The antibody or its antigen-binding fragment according to claim 6, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse antibodies or human antibodies.

10. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The N-end of the constant region of the heavy chain is connected to the C-end of the variable region of the heavy chain; and The N-end of the constant region of the light chain is connected to the N-end of the variable region of the light chain.

11. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that, The heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 21 or 23; and The light chain constant region has an amino acid sequence as shown in SEQ ID NO: 22 or 24.

12. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The heavy chain constant region and the light chain constant region have amino acid sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; and The heavy chain constant region and the light chain constant region have amino acid sequences as shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

13. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment comprises: Heavy chains of amino acid sequences as shown in SEQ ID NO: 10 or 12; and Light chains of amino acid sequences as shown in SEQ ID NO:11 or 13.

14. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment includes: The heavy chain of the amino acid sequence shown in SEQ ID NO:10, and the light chain of the amino acid sequence shown in any one of SEQ ID NO:11; or The heavy chain of the amino acid sequence shown in SEQ ID NO:12, and the light chain of the amino acid sequence shown in any one of SEQ ID NO:

13.

15. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH or F(ab')2; The antigen-binding fragment of the antibody includes at least one selected from the following: F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab)2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, and scFv-Fv fusion protein.

16. A nucleic acid, characterized in that, Encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 15.

17. The nucleic acid according to claim 16, characterized in that, The nucleotide sequences of the nucleic acids are shown in SEQ ID NO: 25 and 26, respectively.

18. The nucleic acid according to claim 16, characterized in that, The nucleotide sequences of the nucleic acids are shown in SEQ ID NO: 27 and 28, respectively.

19. A vector or transformant, characterized in that, It contains the nucleic acid as described in any one of claims 16 to 18.

20. The carrier or transformant according to claim 19, characterized in that, The vector is a eukaryotic vector or a prokaryotic vector.

21. The carrier or transformant according to claim 19, characterized in that, The vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

22. A cell characterized in that, It carries the nucleic acid according to any one of claims 16 to 18, the vector or transformant according to any one of claims 19 to 21, or expresses the antibody or its antigen-binding fragment according to any one of claims 1 to 15.

23. The cell according to claim 22, characterized in that, The cells are prokaryotic cells, eukaryotic cells, or bacteriophages.

24. The cell according to claim 23, characterized in that, The prokaryotic cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

25. The cell according to claim 23, characterized in that, The eukaryotic cells are fungal, insect, plant, or mammalian cells.

26. The cell according to claim 25, characterized in that, The fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces or Trichoderma.

27. The cell according to claim 25, characterized in that, The insect cells were grass armyworm cells.

28. The cell according to claim 25, characterized in that, The plant cells mentioned are tobacco plant cells.

29. The cell according to claim 25, characterized in that, The mammalian cells are BHK cells, CHO cells, COS cells, myeloma cells, or human embryonic kidney 293 cells.

30. The cell according to claim 22, characterized in that, The cells in question are mammalian cells.

31. The cell according to claim 22, characterized in that, The cells are BHK cells, CHO cells, COS cells, or NSO cells.

32. A pharmaceutical composition, characterized in that, It comprises: the antibody or its antigen-binding fragment as described in any one of claims 1 to 15, the nucleic acid as described in any one of claims 16 to 18, or the vector or transformant as described in any one of claims 19 to 21.

33. The pharmaceutical composition according to claim 32, characterized in that, This further includes pharmaceutically acceptable excipients.

34. The pharmaceutical composition according to claim 33, characterized in that, The excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

35. The pharmaceutical composition according to claim 32, characterized in that, The pharmaceutical composition is an injectable preparation.

36. A reagent kit, characterized in that, It includes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 15.

37. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 in the preparation of a kit for detecting and / or enriching and / or isolating and purifying CD25.

38. The use according to claim 37, characterized in that, The kit detects CD25 by ELISA, flow cytometry, Western blotting, or immunoprecipitation.

39. The use according to claim 37, characterized in that, The kit is used to enrich and / or purify CD25 by affinity chromatography.

40. A method for detecting CD25 for non-diagnostic and non-therapeutic purposes, characterized in that, Include: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 15, or the antibody or antigen-binding fragment thereof in the kit as described in claim 37, is used to contact an in vitro biological sample containing CD25.

41. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 in the preparation of a medicament for treating CD25-mediated autoimmune diseases.

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