Anti-CD24 antibody and application thereof

Abstract

The invention discloses an anti-CD24 antibody and an application thereof. Specifically disclosed is an anti-CD24 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (SEQ ID NO: 1, 5, 9 or 13) and a light chain variable region (SEQ ID NO: 3, 7, 11 or 15). The anti-CD24 antibody (including a mouse monoclonal antibody and three humanized monoclonal antibodies) is obtained through a hybridoma technology and a CDR transplantation method. The anti-CD24 antibody has high affinity, can significantly inhibit the growth of tumors, has a tumor inhibition rate of 82.3%, has a significant anti-tumor curative effect, has a strong binding capacity with various tumor cells and tissues, does not bind with normal cells, and has good tumor selectivity. The antibody can be prepared into prevention and treatment drugs, diagnosis drugs, detection kits and the like for CD24 target related diseases, and has very wide clinical application prospects in the fields of tumor treatment and diagnosis and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-CD24 antibody and an application thereof. Background Art

[0002] Phagocytes play an important role in anti-tumor immunity. Macrophages are the main phagocytes that can directly eliminate tumor cells. Phagocytosis is regulated by a variety of signaling molecules. Tumor cells can generate "don't eat me" signals and induce immune escape by expressing anti-phagocytic proteins such as CD47, PD-L1, and CD24.

[0003] CD24 (differentiation antigen cluster 24) is a highly glycosylated glycosylphosphatidylinositol-anchored membrane protein and an innate immune checkpoint molecule. Studies have shown that CD24 can interact with Siglec-10 (sialic acid-binding immunoglobulin-like lectin 10) receptors on the surface of various immune cells to inhibit inflammatory responses. The intracellular region of Siglec-10 contains an immune receptor tyrosine inhibitory motif, which can regulate intracellular signals through the participation of SHP1 and SHP2 phosphatases, inhibit TLR (Toll-like receptor)-mediated inflammation and cytoskeleton rearrangement, thereby inhibiting macrophage phagocytosis and promoting tumor immune escape. CD24 is highly expressed in a variety of tumors, and is significantly higher than CD47 and PD-L1. Macrophages in the tumor microenvironment highly express Siglec-10 molecules. Knocking out CD24 can enhance macrophage phagocytosis of tumor cells and inhibit immune escape. The high expression of CD24 is most obvious in ovarian cancer and triple-negative breast cancer. The high expression of CD24 is also closely related to the poor prognosis of ovarian cancer and breast cancer.

[0004] As an important immune checkpoint molecule, CD24 has great potential in the treatment of tumors and other immune system diseases. However, CD24 is a very small and highly glycosylated protein, which poses a great challenge to the development of antibody drugs. Currently, research in this field is still in its infancy. In addition to being widely expressed in cancer cells, CD24 is also expressed in normal cells such as some epithelial cells. In view of this, the development of new CD24 antibodies with high affinity, strong anti-tumor ability, and good tumor selectivity is of great significance for accurately targeting tumor cells, reducing damage to normal cells, improving treatment effects, and reducing side effects. It can provide patients with safer and more effective treatment options and will have broad clinical application value in the field of tumor treatment. Summary of the invention

[0005] One of the purposes of the present invention is to provide an anti-CD24 antibody and its application. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] To achieve the above-mentioned object, the present invention first provides an anti-CD24 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions whose amino acid sequences are respectively positions 31-35 of SEQ ID NO:1, positions 50-66 of SEQ ID NO:1 and positions 99-102 of SEQ ID NO:1; and the light chain variable region comprises three complementary determining regions whose amino acid sequences are respectively positions 24-39 of SEQ ID NO:3, positions 55-61 of SEQ ID NO:3 and positions 94-102 of SEQ ID NO:3.

[0007] The antigen binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide bond-stabilized Fv (dsFv), single-chain antibody (ScFv), single-domain antibody (sdAb, i.e., nanoantibody), minibody and minimum recognition unit (MRU).

[0008] The sequences of the complementarity determining regions (CDRs) can be defined according to the Kabat numbering system.

[0009] The heavy chain variable region and the light chain variable region both contain a framework region (FR, the region outside the CDR in the variable region). The framework region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, camels, etc.).

[0010] Furthermore, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from any one of the following A1)-A4):

[0011] A1) the amino acid sequence of the heavy chain variable region may be SEQ ID NO: 1, or an amino acid sequence having more than 80% identity with SEQ ID NO: 1 obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region may be SEQ ID NO: 3, or an amino acid sequence having more than 80% identity with SEQ ID NO: 3 obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 3;

[0012] A2) the amino acid sequence of the heavy chain variable region may be SEQ ID NO: 5, or an amino acid sequence having more than 80% identity with SEQ ID NO: 5 obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence of the light chain variable region may be SEQ ID NO: 7, or an amino acid sequence having more than 80% identity with SEQ ID NO: 7 obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 7;

[0013] A3) the amino acid sequence of the heavy chain variable region may be SEQ ID NO: 9, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 9 and having an amino acid sequence with more than 80% identity with SEQ ID NO: 9; the amino acid sequence of the light chain variable region may be SEQ ID NO: 11, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 11 and having an amino acid sequence with more than 80% identity with SEQ ID NO: 11;

[0014] A4) The amino acid sequence of the heavy chain variable region may be SEQ ID NO: 13, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 13, thereby obtaining an amino acid sequence with more than 80% identity with SEQ ID NO: 13; the amino acid sequence of the light chain variable region may be SEQ ID NO: 15, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 15, thereby obtaining an amino acid sequence with more than 80% identity with SEQ ID NO: 15.

[0015] The antibody containing the heavy chain variable region and the light chain variable region described in A1) may be a murine monoclonal antibody and may be named IMB4H7.

[0016] The antibody containing the heavy chain variable region and the light chain variable region described in A2) may be a humanized antibody and may be named IMB4H7h1.

[0017] The antibody containing the heavy chain variable region and light chain variable region described in A3) may be a humanized antibody and may be named IMB4H7h2.

[0018] The antibody containing the heavy chain variable region and light chain variable region described in A4) may be a humanized antibody and may be named IMB4H7h3.

[0019] Furthermore, the substitution may be a conservative substitution. The inconsistency of the amino acid sequence may be in the framework region, for example, there is one or more amino acid substitutions, deletions or additions in the framework region. The number of substitutions may be 2-10 or more.

[0020] Further, the antibody or its antigen-binding fragment may also include a heavy chain constant region (CH) and a light chain constant region (CL). The heavy chain constant region may be selected from the heavy chain constant region of IgG, IgA, IgM, IgD or IgE. The heavy chain constant region may also be selected from CH1, Fc and CH3 domains. The light chain constant region may be selected from Kappa (κ) or lambda (λ) type light chain constant region. The heavy chain constant region and light chain constant region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, camels, etc.).

[0021] Further, the heavy chain constant region can be selected from the heavy chain constant region of human IgG subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant region can also be selected from the heavy chain constant region of mouse IgG subclasses such as IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgG5 and IgG6.

[0022] Furthermore, the heavy chain constant region may be the heavy chain constant region of human IgG1, mouse IgG1 or mouse IgG2b.

[0023] Furthermore, the light chain constant region may be a light chain constant region of human kappa or mouse kappa.

[0024] Specifically, the nucleotide sequence of the heavy chain constant region may be SEQ ID NO: 18 or SEQ ID NO: 20. The nucleotide sequence of the light chain constant region may be SEQ ID NO: 19 or SEQ ID NO: 21.

[0025] The present invention also provides a biomaterial, which may be any of the following:

[0026] B1) nucleic acid molecules encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof;

[0027] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0028] B3) a recombinant vector containing the nucleic acid molecule described in B1);

[0029] B4) a recombinant microorganism containing the nucleic acid molecule described in B1);

[0030] B5) A recombinant host cell containing the nucleic acid molecule described in B1).

[0031] In the above biological material, the recombinant vector can be a cloning vector or an expression vector.

[0032] Further, the recombinant vector may be a recombinant expression vector obtained by cloning a nucleic acid molecule encoding the antibody or its antigen-binding fragment into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector, and a viral expression vector). Although the expression vector used in the embodiments provided by the present invention is a pcMV3 vector, the present invention is not limited to this specific vector. Those skilled in the art may use other suitable vectors as long as the vector can express a nucleic acid molecule encoding the antibody or its antigen-binding fragment.

[0033] The prokaryotic expression vector can be selected from Escherichia coli expression vectors (e.g., pET series vectors, etc.). The eukaryotic expression vector can be selected from yeast expression vectors (e.g., pYES2, pPICZaA, etc.), insect cell expression vectors (e.g., pFastBac1, pMT-Bip-V5-HisA, pAc5.1, etc.), and mammalian cell expression vectors (e.g., pCMV3, pcDNA3.1, etc.). The viral expression vector can be selected from adeno-associated virus (AAV) vectors, adenovirus vectors, herpes simplex virus (HSV) vectors, lentivirus (LV) vectors, poxvirus vectors, retrovirus vectors, rhabdovirus (baculovirus) vectors, papillomavirus vectors, Sendai virus vectors, and simian virus (Simian virus) expression vectors.

[0034] In the above biological material, the nucleic acid molecule in B1) can be any of the following:

[0035] C1) a DNA molecule whose coding sequence is SEQ ID NO: 2 and SEQ ID NO: 4;

[0036] C2) a DNA molecule whose coding sequence is SEQ ID NO: 6 and SEQ ID NO: 8;

[0037] C3) a DNA molecule whose coding sequence is SEQ ID NO: 10 and SEQ ID NO: 12;

[0038] C4) The coding sequence of the DNA molecule is SEQ ID NO:14 and SEQ ID NO:16.

[0039] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16. Considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression of specific species as needed.

[0040] Those skilled in the art can easily adopt known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis and cassette mutagenesis, etc.) or directed evolution (including error-prone PCR, DNA shuffling and in vitro random priming recombination, etc.) to mutate the nucleotide sequence encoding any of the antibodies or antigen-binding fragments described herein. Those artificially modified nucleotide sequences that have more than 75% identity with the nucleotide sequence encoding any of the antibodies or antigen-binding fragments described herein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode any of the antibodies or antigen-binding fragments described herein.

[0041] In certain embodiments, substitutions, insertions or deletions may occur in one or more complementary determining regions or framework regions of any of the antibodies or antigen-binding fragments thereof of the present invention, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as is well known to those skilled in the art, conservative substitutions of amino acids do not change the properties and functions of proteins) may be made to the complementary determining regions and / or framework regions, which do not substantially reduce binding affinity. For example, such changes may be outside the antigen contact residues in the complementary determining regions.

[0042] The present invention also provides the use of the antibody or antigen-binding fragment thereof, or the biomaterial in any of the following:

[0043] D1) Use in the preparation of products for preventing and / or treating tumors;

[0044] D2) Use in the preparation of products for preventing and / or treating CD24 target related diseases;

[0045] D3) Use in the preparation of a product for inhibiting the proliferation of CD24-positive tumor cells;

[0046] D4) Use in the preparation of a product for inhibiting the growth of CD24-positive tumors;

[0047] D5) Use in the preparation of products for diagnosis, auxiliary diagnosis or screening of CD24 target related diseases;

[0048] D6) Use in the preparation of a product for detecting CD24 protein or cells expressing CD24;

[0049] D7) Use in the preparation of a product for binding to CD24 protein.

[0050] In the above application, the CD24 target-related diseases include breast cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, lung cancer, rectal cancer, colon cancer, pancreatic cancer, nasopharyngeal cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, head and neck cancer, bile duct cancer, kidney cancer, thyroid cancer, testicular cancer, melanoma, brain tumors, head and neck tumors, oral tumors, lymphomas, soft tissue tumors and leukemia, but are not limited thereto.

[0051] The breast cancer includes triple negative breast cancer. The lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC).

[0052] The tumor in D1) or the CD24 target-related disease in D2) may be a CD24-positive tumor.

[0053] Furthermore, the CD24-positive tumor may be a CD24-positive cancer, including early-stage cancer, mid-stage cancer, mid-late stage cancer, late stage cancer, recurrent cancer, metastatic cancer, and refractory cancer.

[0054] D5) The application may include using the antibody or antigen-binding fragment of the present invention to detect the expression level of CD24 protein based on the principle of antigen-antibody specific reaction, and then used for screening and diagnosis (including prognostic diagnosis, assessment of tumor malignancy, etc.) of CD24 target-related diseases (such as triple-negative breast cancer, ovarian cancer, liver cancer, colorectal cancer, etc. with high expression of CD24). For example, any antibody or antigen-binding fragment described herein can be prepared into an immunohistochemistry kit for screening or diagnosis of CD24-positive tumor patients, and further guiding medication and treatment.

[0055] The detection of CD24 protein or cells expressing CD24 in D6) includes any in vivo or in vitro detection of CD24 protein based on the principle of antigen-antibody specific reaction. The detection of CD24 protein can be to detect whether the sample to be tested contains CD24 protein and / or to detect the content of CD24 protein in the sample to be tested. The cells expressing CD24 can further be tumor cells expressing CD24.

[0056] D7) The products for binding CD24 protein include CD24 protein inhibitors, products for separating or purifying CD24 protein and in vivo imaging products of CD24 protein, but are not limited thereto. For example, the antibody or antigen-binding fragment thereof of the present invention can be prepared into an immunoaffinity chromatography column, and the CD24 protein can be screened and separated based on the principle that the antigen can be captured by the antibody when passing through the chromatography column, and the antigen is dissociated from the antigen by changing environmental conditions such as pH value. The antibody or antigen-binding fragment thereof of the present invention can also be coupled with a molecule having an imaging function (including but not limited to radionuclides, near-infrared dyes, luciferase, nanoparticles for magnetic resonance imaging, and quantum dots for magnetic resonance imaging). After being injected into the patient's body, based on the targeting function of the antibody, it can reach the corresponding tissue site by itself, and perform immunoimaging, thereby achieving in vivo imaging for CD24 protein. In addition, it is well known to those skilled in the art that antibodies are commonly used protein inhibitors that can inhibit the activity of their ligands, so the antibody or antigen-binding fragment thereof of the present invention can be made into a CD24 inhibitor or a signaling pathway inhibitor involved in CD24 (such as a CD24-Siglec10 signaling pathway inhibitor).

[0057] The products described herein include, but are not limited to, reagents, kits (such as treatment kits or detection kits), preparations, drugs, pharmaceutical compositions, chips, test strips, test cards, immunosensors, and the like.

[0058] The present invention also provides an antibody conjugate, comprising an antibody portion and a conjugated portion, wherein the antibody portion comprises the antibody or an antigen-binding fragment thereof.

[0059] Furthermore, the antibody portion and the coupling portion can be directly linked, or covalently linked via a linker (eg, a hydrazone bond, a disulfide bond, a thioether bond, a peptide bond).

[0060] Furthermore, the coupling moiety can be selected from chemical drugs, cytotoxins and detectable labels.

[0061] Furthermore, the chemical drugs can be chemical drugs for preventing or treating diseases, including chemotherapeutic drugs, anti-tumor drugs, anti-inflammatory drugs, etc., such as cytokines, apoptosis inducers (Bcl-xL inhibitors), nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, anti-tumor antibiotics, immunomodulators, anti-angiogenic agents, antimetabolites, boron drugs, alkylating agents, hormones and anti-hormonal drugs, corticosteroids, photodynamic therapy drugs, etc.

[0062] Furthermore, the cytotoxin can be a substance that inhibits cell proliferation or induces cell apoptosis, including microtubulin inhibitors (such as maytansine, auristatins (MMAE and MMAF), docetaxel, paclitaxel, vincristine, colchicine), DNA damaging agents (such as ozogamicin, calicheamicin, dukamycin, camptothecin, duocartoxin, mitomycin C (MMC), cisplatin, carboplatin, nedaplatin, oxaliplatin, isoproplatin), RNA synthesis inhibitors (such as amatoxin, talanstatin and its analogs, carmustine) or protein synthesis inhibitors (such as ricin).

[0063] Furthermore, the detectable markers include enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, etc.), chemiluminescent agents (such as acridinium ester compounds, acridinium sulfonamide compounds, luminol and its derivatives, ruthenium derivatives, etc.), fluorescent dyes (such as AMCA, FITC, CFSE, GFP, DAPI, 7-AAD, Hoechst 33342, Pacific Blue, PE, PE-TR, PE-Cy7, PE-Cy5, PI, PerCP-Cy5.5, APC, APC-CY7, APC-H7, V500, Alexa The invention relates to nanoparticles for magnetic resonance imaging (MRI), nanoparticles for magnetic resonance imaging (MRI), quantum dots for magnetic resonance imaging (MRI), magnetic substances (such as magnetic beads, nanoparticles containing gadolinium complexes, superparamagnetic iron oxide nanoparticles), and colloidal gold, but are not limited thereto.

[0064] The linker (connector) includes a non-cleavable linker and a cleavable linker. The linker can be selected from N-succinyl-4-(maleimido) cyclohexane-1-carboxylic acid (SMCC), N-ε-maleimidocaproic acid hydrazide (EMCH), succinimide 3-(2-pyridyldisulfide)-propionate (SPDP), Val-Cit (valine-citrulline) dipeptide, GGFG tetrapeptide, phosphatase and pyrophosphatase linker, β-galactosidase linker and sulfatase linker.

[0065] Furthermore, the antibody conjugate may be an antibody drug conjugate (ADC).

[0066] Further, the coupling part can also be a label. In order to facilitate the separation, purification, detection and / or positioning of the antibody or its antigen-binding fragment of the present invention, a label protein can be connected to the amino terminal or carboxyl terminal of the antibody or its antigen-binding fragment. The label includes but is not limited to: GST (glutathione sulfhydryl transferase) label protein, Trx (thioredoxin) label protein, nitrogen utilization substrate A (NusA) label protein, His label protein (His-tag), MBP (maltose binding protein) label protein, Flag label protein, SUMO label protein, HA (influenza hemagglutinin) label protein, Myc label protein, LacZ label protein, CBD (cellulose binding domain) label protein, bacteriophage T7 protein kinase (T7PK) label protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag label protein. Those skilled in the art know how to select a suitable label protein according to the desired purpose. The use of tags does not change the function of the antibody, and its purpose is to separate, purify, detect or trace, so the tagged proteins suitable for this application are not limited to specific types. The tag can be separated from the antibody by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site to use TEV protease to cleave and remove the tag).

[0067] The present invention also provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof, the biological material or the antibody conjugate, and one or more pharmaceutically acceptable carriers.

[0068] The pharmaceutical composition may have at least one of the following uses:

[0069] (1) Used for preventing and / or treating tumors; (2) Used for preventing and / or treating CD24 target related diseases; (3) Used for inhibiting the proliferation of CD24 positive tumor cells; (4) Used for inhibiting the growth of CD24 positive tumors; (5) Used for diagnosing, assisting in the diagnosis or screening of CD24 target related diseases.

[0070] Furthermore, the pharmaceutical composition also includes one or more pharmaceutically acceptable carriers.

[0071] The pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, cosolvents, diluents (such as water, saline, PBS (phosphate buffer), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), adhesives (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), penetration enhancers (such as Brij-78), pH regulator, stabilizer (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactant (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption promoter (such as chitosan), thickener (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidant (such as sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium thiosulfate, vitamin C, etc.), plasticizer (such as glycerol, sorbitol, phthalate, etc.), propellant (such as hydrofluoroalkane, dimethyl ether, etc.), aerosolizer, suspending agent, dispersant, colorant (such as TiO2, pigment, etc.) and flavoring agent. Those skilled in the art know that a carrier usually has multiple functions, for example, starch can be used as both a disintegrant and a binder. Those skilled in the art can make conventional selections of the above carriers according to the properties of the drug and the route of administration.

[0072] Excipients are usually used in medicines to shape the medicines, change the physical state of the medicines, and play a supporting role. Excipients include, but are not limited to: (1) Excipients for injections: such as solvent water, alcohols, ethers, amides, sulfoxides, esters, etc.; (2) Excipients for injection powders: such as sucrose, lactose, mannitol, etc.; (3) Excipients for sprays: such as soy lecithin, propylene glycol, borneol, ethanol, phenol, etc.; (4) Excipients for tablets: such as starch, sucrose, dextrin, methyl cellulose, gelatin, polyethylene glycol, tartaric acid, boric acid, etc.; (5) Excipients for eye drops: such as sodium hyaluronate, disodium ethylenediaminetetraacetic acid (EDTA-Na2), etc.; (6) Excipients for suppositories: such as cocoa butter, semi-synthetic or fully synthetic fatty acid glycerides, glycerol gelatin, polyethylene glycol, etc.; (7) Excipients for granules: such as corn flour, bentonite, zeolite powder, etc.; (8) Excipients for capsules: such as gelatin, etc.; (9) Excipients for gels: such as gelatin, pectin, gum arabic, etc.; (10) Excipients for ointments: such as vaseline, paraffin, liquid paraffin, lanolin, lanolin alcohol, beeswax, lard, vegetable oil, silicone oil, silicone, soaps, higher fatty alcohols, fatty alcohol sulfates, polyols, polyethylene glycol, FAPG, etc.; (11) Excipients for patches: such as ethylene-vinyl acetate copolymer (EVA), pressure-sensitive adhesive (PSA), etc.; (12) Excipients for films: such as gelatin, shellac, gum arabic, polyvinyl alcohol compounds, acrylic acid copolymers, etc.

[0073] Biological drug preparations are easily contaminated by exogenous microorganisms during production, transportation and storage, which may affect the quality of the drug. Therefore, preservatives are usually added to inhibit the growth and reproduction of microorganisms. The preservatives include but are not limited to thimerosal, formaldehyde, phenol and m-cresol.

[0074] Protective agents generally refer to any substance that can protect the activity of drugs, including freeze-drying protective agents. Freeze-drying protective agents can be used to change the physical and chemical environment of biological samples during freeze-drying, reduce cell damage, maintain original biological activity, improve the stability of proteins and cells, etc. The freeze-drying protective agents include but are not limited to sugars / polyols (such as sucrose, trehalose, lactose, glucose, maltose, maltodextrin, mannitol, sorbitol, etc.), polymers (such as HES, PVP, PEG, dextran, albumin, etc.), anhydrous solvents (such as polyethylene glycol, ethylene glycol, glycerol, DMSO, DMF, etc.), surfactants (such as Tween-80, etc.), amino acids (such as L-serine, sodium glutamate, alanine, glycine, sarcosine, acetyltryptophan, etc.), and salts and amines (such as phosphates, acetates, citrates, etc.).

[0075] The dosage form of the pharmaceutical composition includes, but is not limited to, injection (including injection solution and powder for injection), gel, eye drops (including eye drops and intraocular injection solution), oral solution, suppository, effervescent tablet, capsule, ointment, cream, spray, aerosol, external solution, tablet, powder, pill, powder, scarification agent, granule, drop, coating, patch and long-acting sustained-release preparation. Those skilled in the art know that the above-mentioned various dosage forms can be prepared by using active components (such as the antibody or antigen-binding fragment thereof of the present invention) with suitable pharmaceutically acceptable carriers according to conventional preparation processes of preparations.

[0076] Furthermore, the pharmaceutical composition may be in the form of an injection preparation.

[0077] Injection generally refers to a solution or emulsion for injection into the body made from extracted and purified medicinal materials, as well as a sterile powder prepared into a solution before use, which includes injection and powder injection for injection. The preparation method of injection is well known to those skilled in the art, for example, powder injection for injection can be prepared by vacuum freeze drying technology, spray drying technology, spray freeze drying technology. It is also possible to prepare a solution by a concentrated preparation method or a diluted preparation method by mixing the drug with a suitable diluent, cosolvent and / or wetting agent, and then preparing the injection by filtering (such as surface filtration and / or deep filtration), encapsulation, sterilization and other steps.

[0078] In order to prepare the pharmaceutical composition into an injection preparation, such as a solution, emulsion, lyophilized powder injection and suspension, a commonly used diluent in the art, such as water, physiological saline, PBS (phosphate buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc., can be used as a solvent to prepare an injection. In addition, in order to prepare an isotonic injection, an appropriate amount of a carrier such as sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, a conventional cosolvent, buffer, pH adjuster and other carriers can also be added.

[0079] The administration methods of the pharmaceutical composition include, but are not limited to, injection (such as administration in the form of injection), mucosal administration (such as administration in the form of sprays, aerosols, tablets, eye drops, suppositories, granules, capsules, etc.) and transdermal administration (such as administration in the form of gels, ointments, patches, films, etc.).

[0080] The administration methods of the pharmaceutical composition include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumor injection, intracranial injection, mucosal administration, oral administration, skin application, oral and nasal spraying, aerosol inhalation, in vivo implantation, and in vitro device administration.

[0081] The active ingredient of the pharmaceutical composition may be any antibody or antigen-binding fragment thereof described herein.

[0082] The present invention also provides a kit, which contains the antibody or the antigen-binding fragment thereof.

[0083] The kit may have at least one of the following uses: (1) diagnosis, auxiliary diagnosis or screening of CD24 target-related diseases; (2) detection of CD24 protein or cells expressing CD24; (3) inhibition or binding of CD24 protein (such as separation, purification and in vivo imaging of CD24 protein).

[0084] Furthermore, the kit can be used to detect tumor cells expressing CD24, or to detect whether CD24-positive cancer exists in a subject.

[0085] The test sample of the kit can be a blood sample (such as whole blood, plasma, serum), a tissue sample, a cell sample, etc., but is not limited thereto.

[0086] The kit may be a chemiluminescent immunoassay kit, an enzyme-linked immunosorbent assay kit, an immunoprecipitation assay kit, an immunoblotting assay kit, an immunochromatography assay kit, a flow cytometry assay kit, an immunohistochemistry assay kit, a colloidal gold immunoassay kit or a fluorescent immunoassay kit, but is not limited thereto.

[0087] Furthermore, the kit may also include reagents required for immunoassay, such as labeled antibodies or antigens, magnetic particles, blocking solution, diluent, washing solution, color developing solution, stop solution, etc., but not limited thereto.

[0088] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0089] The components of the kit may be provided in the form of a solution, such as an aqueous solution. In the case of being in the form of an aqueous solution, the concentration or content of these components can be easily determined by those skilled in the art according to different needs. For example, for storage purposes, the concentration of the components may be present in a higher form, and when in working state or use, the concentration may be reduced to the working concentration by diluting the above-mentioned higher concentration solution.

[0090] The present invention also provides a method for preparing an anti-CD24 antibody or an antigen-binding fragment thereof, which comprises expressing the antibody or the antigen-binding fragment thereof in a host cell, and recovering or isolating the antibody or the antigen-binding fragment thereof.

[0091] Furthermore, the preparation method may include the following steps: cloning a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector and a viral expression vector) to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell to obtain a recombinant host cell expressing the antibody or antigen-binding fragment thereof; culturing the recombinant host cell, and recovering or isolating the antibody or antigen-binding fragment thereof from the cultured recombinant host cell culture.

[0092] Furthermore, the recovery or separation can be separated from the culture (including all substances in the culture container) by precipitation methods (such as salting out, organic solvent precipitation, caprylic acid-saturated ammonium sulfate precipitation, isoelectric precipitation) or chromatography techniques (such as ion exchange chromatography, gel filtration chromatography, affinity chromatography).

[0093] The introduction method may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation or metal cation-mediated transformation) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria by phage transduction. (3) directly transferring the target gene into plant receptor cells by physical or chemical methods, such as chemical stimulation, electric shock, liposome-mediated method, microinjection, gene gun method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method and vortex method. (4) transferring the target gene into plant receptor cells by vector, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method. (5) Introducing the target gene into in vitro animal cells (transfection) through calcium phosphate co-precipitation, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation (i.e. electrotransfection method), microinjection, gene gun method or virus-mediated method (such as adenovirus infection method, lentivirus infection method), etc.

[0094] The antibody of the present invention may be a monoclonal antibody or a humanized antibody (including a chimeric antibody, a CDR-grafted antibody and an SDR-grafted antibody).

[0095] CD24 is widely expressed in cancer cells, but it is also expressed in normal cells such as some epithelial cells. Therefore, there is a need in the art for a method that can distinguish tumor cells from normal cells. The degree of glycosylation of CD24 expressed by tumor cells and normal cells is different. The present invention uses hybridoma technology and a method of subcutaneous immunization + cell shock immunization (CHO-hCD24 cells) of KLH-coupled CD24 antigen polypeptide to successfully screen a monoclonal antibody (IMB4H7) with high affinity for non-glycosylated modified CD24. The present invention further uses the CDR transplantation method to humanize the obtained mouse monoclonal antibody IMB4H7, and obtains three humanized antibodies: IMB4H7h1, IMB4H7h2 and IMB4H7h3.

[0096] Compared with the commercial CD24 antibody SN3, the anti-CD24 antibodies (IMB4H7, IMB4H7h1, IMB4H7h2 and IMB4H7h3) of the present invention have a significantly improved affinity for tumor cells, and bind strongly to a variety of tumor cells and tumor tissues, but bind weakly to normal cells. The anti-CD24 antibodies of the present invention have significantly enhanced binding to deglycosylated CD24, indicating that the binding of the antibody to CD24 may be blocked by glycosylation in normal cells rather than cancer cells, so that the antibody has good tumor selectivity. The results of in vivo treatment experiments show that the anti-CD24 antibodies of the present invention have significant anti-tumor efficacy and can significantly inhibit tumor growth, with a tumor inhibition rate of up to 82.3%.

[0097] The anti-CD24 antibody of the present invention can be expressed and produced in prokaryotic cells, eukaryotic cells and various recombinant systems, and can be made into clinically used preventive and / or therapeutic drugs, diagnostic drugs, CD24 protein detection kits or CD24 protein in vivo imaging products, etc. for CD24 target-related diseases. It can be used alone or in combination with other therapeutic drugs, or as a drug delivery system to carry related drugs for targeted delivery, and has very broad clinical application prospects in the fields of tumor treatment and diagnosis.

[0098] Definition of terms

[0099] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0100] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in the alignment, and is generally expressed as a percentage. Identity as described herein may refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies with exactly the same sequence have 100% identity. It is known to those skilled in the art that the identity of an amino acid sequence or a nucleotide sequence can be determined using an identity search site on the Internet, such as the BLAST webpage of the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of the amino acid sequence, then the value (%) of identity can be obtained. In addition, sequence analysis software (such as CLC Main Workbench and MegAlignTM) can be used for determination, such as computer programs BLAST using default parameters, especially BLASTP or TBLASTN. The 75% or more identity described herein can be at least 75%, 80%, 85%, 90% or 95% or more identity. The 80% or more identity described herein can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity.

[0101] The term "conservative substitution" generally refers to replacing an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions usually do not cause significant changes in the conformational structure of the protein and do not substantially change the biological activity of the protein. Conservative substitutions in a protein sequence that are expected to have little or no effect on the protein structure or function can be easily designed by a person of ordinary skill in the art.

[0102] The term "antibody conjugate" herein includes not only antibody-drug conjugates (ADCs) formed by coupling an antibody to a drug (a substance for preventing, treating, or diagnosing a disease) via a linker, but also conjugates obtained by coupling an antibody to a detectable marker or label.

[0103] The term "CD24-positive tumor" generally refers to a tumor that expresses CD24 protein. Methods for detecting tumor CD24 protein expression are well known to those skilled in the art, for example, the expression of CD24 protein can be determined by immunological detection methods based on antigen-antibody specific reactions (such as immunohistochemistry, FACS, etc.), or the expression of CD24 mRNA can be determined by real-time fluorescence quantitative PCR, Northern blotting or RNA in situ hybridization.

[0104] The term "antigen binding fragment" generally refers to an antigen binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen binding region or variable region (e.g., one or more CDRs) of a parental antibody. The antigen binding fragment retains at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target. It is well known to those skilled in the art that the antigen binding fragment can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies to produce antigen binding fragments of antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 The SDS-PAGE electrophoresis diagram of the purified antibody.

[0106] Figure 2 Flow cytometry was used to detect the subtypes of IMB4H7 antibodies.

[0107] Figure 3 Flow cytometry was used to detect the binding of IMB4H7 and SN3 to CHO-hCD24 and CHO-K1 cells, and mouse IgG was used as an isotype control.

[0108] Figure 4 Flow cytometry was used to detect the binding of IMB4H7g1 to CHO-hCD24 and CHO-K1 cells, and mouse IgG was used as an isotype control.

[0109] Figure 5 Flow cytometry was used to detect the binding of IMB4H7 and SN3 to CD24 depleted of N-glycans (SKOV3+PNGase F) and sialic acid (SKOV3+sialic acid).

[0110] Figure 6 Flow cytometry was used to compare the binding activities of humanized antibodies IMB4H7h1, IMB4H7h2 and IMB4H7h3 with SN3 on CHO-hCD24 and CHO-K1 cells.

[0111] Figure 7 Flow cytometry was used to detect the half effective binding concentration of humanized antibodies IMB4H7h1, IMB4H7h2, IMB4H7h3 and mouse antibody IMB4H7.

[0112] Figure 8 Flow cytometry was used to detect the binding of IMB4H7h2 antibody to various tumor cells.

[0113] Fig. 9 Flow cytometry was used to compare the differences in binding of humanized antibodies to normal human cells and cancer cells.

[0114] Fig.10 Tissue microarray was used to detect the binding of IMB4H7 to different tumor tissues (mean ± SEM).

[0115] Fig.11 The distribution of IMB4H7h2 antibody in bilateral tumor-bearing mice.

[0116] Fig.12 The in vivo antitumor activity of IMB4H7.

[0117] Fig.13 The effect of IMB4H7 on the body weight of mice. DETAILED DESCRIPTION

[0118] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0119] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0120] Example 1. Preparation of CD24 Antibody and Binding Activity Detection

[0121] The present invention selects a human CD24 extracellular region fragment (SETTTGTSSNSSQSTSNSGLAP, SEQ ID NO: 17) to inoculate mice, takes serum from the mice, and screens out mice with high titers. The CD24 antibody is prepared by hybridoma technology, and the specific steps are as follows:

[0122] 1. Construction of Chinese hamster ovarian cancer cells with high expression of human CD24

[0123] The gene encoding human CD24 (hCD24, Uniprot: P25063) was cloned into the PHBLV lentiviral vector (prepared by Hangzhou Laibo Biotechnology Co., Ltd.) and placed under the promoter of EF1α (EF-1α) to obtain a recombinant vector. The recombinant vector was transfected into CHO-K1 cells ((Wuhan Pronosai Life Science Technology Co., Ltd., Catalog No. CL-0062) and a Chinese hamster ovarian cancer cell line highly expressing human CD24 was obtained by limiting dilution method and named CHO-hCD24 cells. The above cells were cultured in Ham's F-12K medium supplemented with 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin solution.

[0124] 2. Mouse immunization

[0125] The SETTTGTSSNSSQSTSNSGLAPC-KLH peptide was synthesized through commercial channels, and each BALB / c mouse (6-8 weeks old) was immunized subcutaneously with 30 μg of KLH-coupled peptide, emulsified with Freund's complete adjuvant before immunization, once every two weeks for a total of three times; two weeks later, 1×10 7 CHO-hCD24 cells were pulsed and immunized once.

[0126] 3. Cell fusion

[0127] After the above immunization, the blood of mice was collected and antibody titer was tested using CHO-hCD24 cells. The two mice with the highest positive rate were killed, and spleen cells were fused with SP2 / 0 cells (Hangzhou Laibo Biotechnology Co., Ltd.). The fused cells were titered, positive fused cells were screened, and monoclones were picked using the limiting dilution method.

[0128] 4. Hybridoma cell screening

[0129] Flow cytometry was used to select clones with high binding activity to CHO-hCD24 cells. The flow cytometry assay steps were as follows: 1×10 CHO-hCD24 cells in logarithmic growth phase were collected. 6After centrifugation at 300g for 5 minutes, the precipitate was collected, and the cells were resuspended in a staining solution (precooled PBS containing 1% BSA), and each cell was dispensed into a flow detection tube at a volume of 100 μL per tube. The antibody to be detected (i.e., hybridoma cell culture supernatant) diluted in different proportions was added, incubated at 4°C for 30 minutes, washed 3 times with the staining solution, and incubated at 4°C for 30 minutes with a FITC-labeled anti-mouse IgG secondary antibody, washed 3 times with the staining solution, and used for flow cytometry to detect the fluorescence intensity. The clones selected by flow cytometry were then screened for subclones, and finally a positive monoclonal clone with strong binding activity to CHO-hCD24 cells was obtained. A hybridoma cell line with the strongest binding activity to CHO-hCD24 cells and capable of secreting CD24 monoclonal antibodies was named 4H7 cells. The monoclonal antibody secreted by the hybridoma cell line was named IMB4H7.

[0130] The obtained hybridoma cell line 4H7 was injected into the peritoneal cavity of 8-12 week-old BalB / c mice at a cell concentration of 1×10 6 cells / mouse. After 7-10 days, the ascites containing monoclonal antibodies was collected. The antibodies in the ascites were purified using a Protein A affinity column. The purification method is as follows: (1) 20mM PB, pH 7.0 equilibrated on a 1mL-Protein A affinity column at 1.0mL / min; (2) 50mL of culture medium was loaded at 1mL / min and equilibrated with equilibrated solution; (3) 0.1M glycine-HCl, pH 2.7 was eluted at 1mL / min and the 280nm absorption peak was collected; (4) 20-50mM PB, 150mM NaCl, pH 6.7 was equilibrated on a Superdex 200Increase10 / 300GL column at 1mL / min; (5) 2mL of the 280nm absorption peak was collected. The mouse anti-CD24 monoclonal antibody IMB4H7 was obtained. After 10% SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, the antibody heavy and light chains were visible and there were no impurity bands ( Figure 1 ).

[0131] 5. Determination of monoclonal antibody sequence and subtype

[0132] Subtype identification and sequencing analysis of monoclonal antibodies were performed. The steps are as follows: 1) Aliquot an appropriate amount of hybridoma cells into 1.5mL Eppendorf tubes, centrifuge at 4000rpm for 5 minutes, and discard the supernatant. 2) Resuspend the above cells in 50μL of Goat anti-mouseIgG-FITC, Goat anti-mouseIgG1-FITC, Goat anti-mouseIgG2a-FITC, Goat anti-mouseIgG2b-FITC (Hangzhou Lai Bo Biotechnology Co., Ltd.) diluted with PBS 1:500, and let stand at 4°C for 15 minutes. 3) 4000rpm, 5 minutes, discard the supernatant. 6) Resuspend the cells in 400μL PBS and analyze by flow cytometry. The results are as follows. Figure 2 As shown, the IMB4H7 antibody subtype is IgG2b.

[0133] On the basis of the nucleotide sequence obtained by Sanger sequencing, the corresponding amino acid sequence was obtained using the rule that a triplet codon encodes one amino acid. The amino acid sequence of the heavy chain variable region of the monoclonal antibody IMB4H7 was finally determined to be as shown in SEQ ID NO: 1, and the nucleotide sequence of the heavy chain variable region encoding gene was as shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region of the monoclonal antibody IMB4H7 was as shown in SEQ ID NO: 3, and the nucleotide sequence of the light chain variable region encoding gene was as shown in SEQ ID NO: 4.

[0134] in:

[0135] The amino acid sequence of the heavy chain variable region CDR1 of the monoclonal antibody IMB4H7 is shown at positions 31-35 of SEQ ID NO: 1;

[0136] The amino acid sequence of the heavy chain variable region CDR2 of the monoclonal antibody IMB4H7 is shown at positions 50-66 of SEQ ID NO: 1;

[0137] The amino acid sequence of the heavy chain variable region CDR3 of the monoclonal antibody IMB4H7 is shown at positions 99-102 of SEQ ID NO: 1;

[0138] The amino acid sequence of the light chain variable region CDR1 of the monoclonal antibody IMB4H7 is shown at positions 24-39 of SEQ ID NO: 3;

[0139] The amino acid sequence of the light chain variable region CDR2 of the monoclonal antibody IMB4H7 is shown at positions 55-61 of SEQ ID NO: 3;

[0140] The amino acid sequence of the light chain variable region CDR3 of the monoclonal antibody IMB4H7 is shown at positions 94-102 of SEQ ID NO:3.

[0141] 6. Detection of monoclonal antibody binding activity

[0142] Flow cytometry was used to detect the binding of monoclonal antibody IMB4H7 and commercial CD24 antibody SN3 (Invitrogen, Catalog No. 11-0247-42) to CHO-K1 cells and CHO-hCD24 cells, and mouse IgG was used as an isotype control (Invitrogen, Catalog No. 02-6502). The test results are shown in Figure 3 As shown, IMB4H7 showed stronger binding to CHO-hCD24 cells than SN3, and both antibodies bound weakly to CHO-K1 cells.

[0143] 7. Genetic engineering methods to prepare antibodies

[0144] When the antibody is prepared by genetic engineering method, the heavy chain constant region of IMB4H7 antibody is replaced with the heavy chain constant region of mouse IgG1. The antibody is named IMB4H7g1, and the heavy chain expression vector and the light chain expression vector are prepared separately: the nucleotide sequence of the heavy chain gene of monoclonal antibody IMB4H7g1 is obtained by directly connecting the nucleotide sequence of the IMB4H7 heavy chain variable region encoding gene (SEQ ID NO: 2) and the nucleotide sequence of the mouse IgG1 heavy chain constant region encoding gene (SEQ ID NO: 18).

[0145] The nucleotide sequence of the monoclonal antibody IMB4H7g1 light chain gene is obtained by directly connecting the nucleotide sequence of the IMB4H7 light chain variable region encoding gene (SEQ ID NO: 4) and the nucleotide sequence of the mouse kappa light chain constant region encoding gene (SEQ ID NO: 19).

[0146] The heavy chain gene and light chain gene of monoclonal antibody IMB4H7g1 were cloned into pcMV3 vector (Beijing Sino Biological Science and Technology Co., Ltd.) to obtain heavy chain expression vector and light chain expression vector.

[0147] HEK293 cells were transfected with the heavy chain expression vector and the light chain expression vector, and the cells were cultured in serum-free medium at 37°C for 6 days. The culture supernatant was collected and purified using a Protein A affinity column in the same manner as step 4 in Example 1. The binding of IMB4H7g1 (4 μg / mL) to CHO-K1 and CHO-hCD24 was detected by flow cytometry in the same manner as step 4 in Example 1, with mouse IgG used as an isotype control. The results showed that IMB4H7g1 had good binding activity to CHO-hCD24 cells, but weak binding to CHO-K1 cells ( Figure 4 ).

[0148] Example 2: Monoclonal antibody IMB4H7 can bind to hypoglycosylated CD24

[0149] Sialidases (NEU1, NEU3) and CD24 are highly expressed in ovarian cancer (Nomura H et al., 2006), suggesting that CD24 may be hypoglycosylated in tumors. Sialidases (NEW ENGLAND Biolabs, catalog number P0722S) and PNGase F (peptide N-glycosidase F, NEW ENGLAND Biolabs, catalog number P0704S) were used to treat ovarian cancer SKOV3 cells (Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CL-0215), and the binding of IMB4H7 and SN3 antibodies to cells was detected by flow cytometry. The experiment was carried out according to the instructions, and the specific steps are as follows: SKOV3 cells were collected by trypsin digestion and washed twice with PBS; divided into 3 EP tubes, 1×10 6 cells; sialidase group: add 2μL sialidase + 2μL GlycoBuffer 1 (10×, 50mM CaCl2, 500mMsodium acetate, pH 5.5) + 18μL water; PNGase F group: add 2μL PNGase F + 2μL GlycoBuffer 2 (10×, 500mM Sodium Phosphate, pH 7.5) + 18μL water; blank group: add 1μL GlycoBuffer 1 (10×) + 1μL GlycoBuffer 2(10×)+20μL water; incubate at 37℃ for 24 hours; wash the samples twice with PBS, centrifuge and discard the supernatant, divide each EP tube into two samples, add IMB4H7 and SN3 antibodies (4μg / mL) respectively, incubate at 4℃ for 45 minutes, wash twice with PBS, centrifuge and discard the supernatant, add FITC-labeled secondary antibody (1:100), incubate at 4℃ for 45 minutes, wash twice with PBS, add 500μL PBS, and detect by flow cytometry.

[0150] The results are as follows Figure 5 As shown, after SKOV3 cells were treated with sialidase or PNGase F, the binding to SN3 was weakened, but the binding to IMB4H7 was significantly enhanced. This indicates that the binding of antibody SN3 to CD24 depends on sialic acid glycans and N-glycosylation, while monoclonal antibody IMB4H7 can bind to low-glycosylated CD24.

[0151] Example 3 Humanization of monoclonal antibody IMB4H7 and detection of its binding activity

[0152] The IMB4H7 antibody was humanized using the CDR transplantation method. The mouse CDR was retained, the mouse framework region (FR) was replaced by the human FR, and the human constant region (C region) was selected to obtain three humanized antibodies: IMB4H7h1, IMB4H7h2 and IMB4H7h3.

[0153] The amino acid sequence of the heavy chain variable region of the humanized antibody IMB4H7h1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID NO: 6; the amino acid sequence of the light chain variable region of the humanized antibody IMB4H7h1 is shown in SEQ ID NO: 7, and the nucleotide sequence of the light chain variable region encoding gene is shown in SEQ ID NO: 8. The amino acid sequence of the heavy chain variable region of the humanized antibody IMB4H7h2 is shown in SEQ ID NO: 9, and the nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID NO: 10; the amino acid sequence of the light chain variable region of the humanized antibody IMB4H7h2 is shown in SEQ ID NO: 11, and the nucleotide sequence of the light chain variable region encoding gene is shown in SEQ ID NO: 12. The amino acid sequence of the heavy chain variable region of the humanized antibody IMB4H7h3 is shown in SEQ ID NO:13, and the nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID NO:14; the amino acid sequence of the light chain variable region of the humanized antibody IMB4H7h3 is shown in SEQ ID NO:15, and the nucleotide sequence of the light chain variable region encoding gene is shown in SEQ ID NO:16.

[0154] The heavy chain constant regions of humanized antibodies IMB4H7h1, IMB4H7h2 and IMB4H7h3 are all human IgG1, and the nucleotide sequence of the heavy chain constant region encoding gene is SEQ ID NO: 20. The light chain constant regions of humanized antibodies IMB4H7h1, IMB4H7h2 and IMB4H7h3 are all human kappa chains, and the nucleotide sequence of the light chain constant region encoding gene is SEQ ID NO: 21.

[0155] The humanized antibody was prepared by the genetic engineering method of step 7 in Example 1, and the light and heavy chain sequences of the humanized antibody were cloned into the pcMV3 vector, transiently transfected into HEK293 cells, cultured at 37°C for 6 days, and the culture supernatant was collected and the antibody was affinity purified. Flow cytometry was used to compare the binding ability of the three humanized antibodies and antibody SN3 to CHO-hCD24 cells, while CHO-K1 cells were used as a control. The results showed that all three humanized antibodies could bind to CHO-hCD24 cells, and the binding ability was significantly stronger than that of SN3 antibody ( Figure 6 ), none of the four antibodies could bind to CHO-K1 cells. Flow cytometry was used to detect the binding ability of different concentrations of three humanized antibodies and mouse IMB4H7 antibody to CHO-hCD24 cells, and the half effective concentration EC was calculated. 50 The result is Figure 7 As shown, the EC values ​​of the three humanized antibodies and IMB4H7 antibody are 50The values ​​were 2.039 μg / mL, 1.491 μg / mL, 1.089 μg / mL and 2.608 μg / mL respectively.

[0156] Example 4. Detection of binding of CD24 antibody to various tumor cells

[0157] In this embodiment, IMB4H7h2 antibody is used as an example, and flow cytometry is used to detect the binding of humanized antibodies to tumor cells from various tissues. Human breast cancer MDA-MB-468 cells, human liver cancer HuH-7 cells and human melanoma cells A375 were cultured in DMEM culture medium, human lung cancer A549 cells were cultured in Ham's F-12K culture medium, human gastric cancer NUGC-4 cells were cultured in RPMI-640 culture medium, and human ovarian cancer cells SKOV3 were cultured in McCoy's 5A culture medium. 10% fetal bovine serum, 2mM glutamine, 100μg / mL streptomycin and 100U / mL penicillin were added to all cell culture media, and cultured in an incubator (Thermo Scientific, 3131 carbon dioxide incubator) containing 5% CO2 at 37°C. The above tumor cell lines are all common cell lines, which are preserved from the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, or purchased from commercial channels such as ATCC Cell Bank (Rockville, MD, USA), National Experimental Cell Resource Sharing Platform, etc. The flow cytometry detection method was the same as step 4 of Example 1. The secondary antibody was anti-human IgG-FITC, and human IgG1 kappa (Beijing Sino Biological Technology Co., Ltd., catalog number: HG1K) was used as an isotype control. Figure 8 As shown, IMB4H7h2 antibody has strong binding to the above tumor cells.

[0158] Example 5. Detection of the binding of CD24 antibody to tumor cells and normal tissue cells

[0159] Flow cytometry was used to compare the binding strength of three humanized antibodies to normal human mammary epithelial cells MCF 10A and human breast cancer cells MDA-MB-468. MCF 10A cells (Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CL-0525) were cultured in DMEM / F12 + 5% horse serum + 20ng / mL EGF + 0.5μg / mL hydrocortisone + 10μg / mL insulin + 1% non-essential amino acids + 100μg / mL streptomycin and 100U / mL penicillin culture medium. The MDA-MB-468 cell culture method was the same as in Example 4, and the flow cytometry detection method was the same as in Example 4. The results showed that the three humanized antibodies strongly bound to MDA-MB-468 cells, but did not bind to normal human mammary epithelial cells MCF 10A ( Fig. 9 ).

[0160] Example 6: Tissue chip detection of CD24 antibody binding activity to different tumor tissues

[0161] In this embodiment, IMB4H7 antibody is used as an example to perform immunohistochemical analysis on a tissue chip containing 20 different organs, each of which includes 20 cancer tissues (Shanghai Xiabukang Biotechnology Co., Ltd.). Including gastric cancer, melanoma (skin, rectum, small intestine), prostate cancer, brain tumors (astrocytoma, glioblastoma), ovarian cancer, breast cancer, testicular cancer, colon cancer, bladder cancer, uterine cancer, thyroid cancer, lung cancer, head and neck / oral tumors, lymphoma, soft tissue tumors, liver cancer, pancreatic cancer, kidney cancer, cervical cancer, esophageal cancer. The specific steps are as follows: the tissue chip is baked in a 59°C constant temperature box for 60 minutes, dewaxed with xylene, hydrated with anhydrous ethanol gradient, inactivated by immersion in 3% H2O2 for 10 minutes, and the antigen is repaired twice with EDTA (PH8.0) repair solution, each time at high temperature for 10-12 minutes, and 5% BSA blocking solution is added dropwise at room temperature for 35 minutes. Add IMB4H7 antibody working solution, incubate at 4°C overnight, wash 4 times with PBS, add HRP-labeled anti-mouse secondary antibody, incubate at 37°C for 35 minutes, and wash 4 times with PBS. DAB colorimetric agent was used for color development. After color development, hematoxylin staining solution was added, immersion for 2 minutes, rinsed for 10 minutes, and the color was observed. The chip was rinsed in 1 cylinder of anhydrous ethanol, and dehydrated in 2 cylinders of anhydrous ethanol and 3 cylinders of xylene for 3 minutes each. Add 1 drop of sealing plastic and cover the slide. The above reagents are all commonly used commercial reagents. After scanning the slide (PANNORAMIC MIDI scanner, 3DHISTECH Ltd), the halo software was used for quantitative analysis. Fig.10 The histochemical scoring (H-score) results of the binding of IMB4H7 antibody to different tumor tissues were summarized, showing that most tumor tissues exhibited strong binding.

[0162] Example 7: Distribution of CD24 Antibody in Tumor-Bearing Mice

[0163] In this example, DyLight 680NHS ester (Thermo scientific, catalog number 46419) was first used to label the IMB4H7h2 antibody. 0.37 mL of IMB4H72 (2.73 mg / mL) was taken, 3.17 μL of DyLight 680NHS ester (10 mg / mL) was added and incubated at room temperature for 1 hour, PBS was added to the sample to a total volume of 1 mL, and the eluate was collected by passing through a PD MidiTrap G-25 column (GE Healthcare), and the excess unreacted fluorescein was removed. The sample was concentrated by ultrafiltration tube (Amion Ultra-4), and BCA quantification was performed to obtain DyLight 680-IMB4H7h2. CHO-hCD24 and CHO-K1 cells were cultured at 5×10 6Each mouse was inoculated into the subcutaneous tissue of the left and right axillae of BALB / c nude mice (female, 18-22 g, Beijing Yaokang Biotechnology Co., Ltd.) and the tumors were grown to 200-300 mm. 3 The DyLight 680-IMB4H7h2 antibody was injected into the tail vein at 1:00 pm. In vivo imaging was performed 0.5 and 6 hours later using IVIS-200 Imaging System (Xenogen, USA) with excitation / emission wavelengths of 675 / 760 nm. The results showed that the antibody began to accumulate on the CHO-hCD24 side 0.5 hours after injection and was significantly enriched 6 hours later. However, only a small amount of antibody was enriched on the CHO-K1 side 6 hours after injection ( Fig.11 The above results indicate that IMB4H7h2 antibody can be significantly enriched on the side of the tumor where CD24 is highly expressed.

[0164] Example 8: Animal Experimental Treatment Scheme of IMB4H7 Antibody

[0165] The in vivo efficacy of IMB4H7 antibody was evaluated using a Chinese hamster ovarian cancer cell CHO-hCD24 xenograft model that highly expresses human CD24. CHO-hCD24 cells were expressed at 5×10 6 / BALB / c nude mice (female, 18-22 g, Beijing Yaokang Biotechnology Co., Ltd.) were inoculated subcutaneously in the axilla. 3 At 14:00, the animals were divided into a control group and an IMB4H7 group according to the size of the tumor. The animals were given the drug through the tail vein at a dose of 10 mg / kg, 0.2 mL / animal, once every 3 days, for a total of 4 times. The control group was injected with normal saline. The long diameter a and the short diameter b perpendicular to the tumor were measured every 2-3 days during the experiment, and the animal weight was recorded. The formula V = 1 / 2ab 2 The tumor volume was calculated, and the inhibition rate was calculated at the same time ((control group tumor volume - test group tumor volume) / control group tumor volume × 100%). The experimental results showed that the IMB4H7 antibody had significant therapeutic effects in vivo and could significantly inhibit the growth of CHO-hCD24 subcutaneous tumors. On the 13th day of the experiment, the tumor inhibition rate reached 82.3% ( Fig.12 Compared with the control group, there was no significant decrease in body weight ( Fig.13 ).

[0166] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An anti-CD24 antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions whose amino acid sequences are respectively positions 31-35 of SEQ ID NO:1, positions 50-66 of SEQ ID NO:1 and positions 99-102 of SEQ ID NO:1; and the light chain variable region comprises three complementary determining regions whose amino acid sequences are respectively positions 24-39 of SEQ ID NO:3, positions 51-61 of SEQ ID NO:3 and positions 94-102 of SEQ ID NO:

3.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from any one of the following A1) to A4): A1) the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, or an amino acid sequence having more than 80% identity with SEQ ID NO: 1 obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region is SEQ ID NO: 3, or an amino acid sequence having more than 80% identity with SEQ ID NO: 3 obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 3; A2) the amino acid sequence of the heavy chain variable region is SEQ ID NO:5, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:5 and having an amino acid sequence with more than 80% identity with SEQ ID NO:5; the amino acid sequence of the light chain variable region is SEQ ID NO:7, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:7 and having an amino acid sequence with more than 80% identity with SEQ ID NO:7; A3) the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 9 and having an amino acid sequence with more than 80% identity with SEQ ID NO: 9; the amino acid sequence of the light chain variable region is SEQ ID NO: 11, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 11 and having an amino acid sequence with more than 80% identity with SEQ ID NO: 11; A4) the amino acid sequence of the heavy chain variable region is SEQ ID NO: 13, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 13 and having an amino acid sequence with more than 80% identity with SEQ ID NO: 13; the amino acid sequence of the light chain variable region is SEQ ID NO: 15, or an amino acid sequence obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 15 and having an amino acid sequence with more than 80% identity with SEQ ID NO:

15.

3. Biomaterial, characterized in that The biological material is any of the following: B1) a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof according to claim 1 or 2; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1); B4) a recombinant microorganism containing the nucleic acid molecule described in B1); B5) A recombinant host cell containing the nucleic acid molecule described in B1).

4. The biomaterial according to claim 3, characterized in that B1) The nucleic acid molecule is any of the following: C1) a DNA molecule whose coding sequence is SEQ ID NO: 2 and SEQ ID NO: 4; C2) a DNA molecule whose coding sequence is SEQ ID NO: 6 and SEQ ID NO: 8; C3) a DNA molecule whose coding sequence is SEQ ID NO: 10 and SEQ ID NO: 12; C4) The coding sequence of the DNA molecule is SEQ ID NO:14 and SEQ ID NO:

16.

5. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 2, or the biomaterial according to claim 3 or 4 in any of the following: D1) Use in the preparation of products for preventing and / or treating tumors; D2) Use in the preparation of products for preventing and / or treating CD24 target related diseases; D3) Use in the preparation of a product for inhibiting the proliferation of CD24-positive tumor cells; D4) Use in the preparation of a product for inhibiting the growth of CD24-positive tumors; D5) Use in the preparation of products for diagnosis, auxiliary diagnosis or screening of CD24 target related diseases; D6) Use in the preparation of a product for detecting CD24 protein or cells expressing CD24; D7) Use in the preparation of a product for binding to CD24 protein.

6. The use according to claim 5, characterized in that: The CD24 target-related diseases include breast cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, lung cancer, rectal cancer, colon cancer, pancreatic cancer, nasopharyngeal cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, head and neck cancer, bile duct cancer, kidney cancer, thyroid cancer, testicular cancer, melanoma, brain tumors, head and neck tumors, oral tumors, lymphomas, soft tissue tumors and leukemia.

7. An antibody conjugate, comprising an antibody portion and a conjugate portion, characterized in that: The antibody portion comprises the antibody or antigen-binding fragment thereof according to claim 1 or 2.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to claim 1 or 2, the biomaterial according to claim 3 or 4 or the antibody conjugate according to claim 7, and one or more pharmaceutically acceptable carriers.

9. A kit, characterized in that The kit contains the antibody or antigen-binding fragment thereof according to claim 1 or 2.

10. A method for preparing an anti-CD24 antibody or an antigen-binding fragment thereof, characterized in that: The preparation method comprises expressing the antibody or antigen-binding fragment thereof according to claim 1 or 2 in a host cell, and recovering or isolating the antibody or antigen-binding fragment thereof.

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