Anti-cd24 antibodies and uses thereof

Abstract

The application discloses an anti-CD24 antibody and application thereof. Specifically disclosed are an anti-CD24 antibody or an antigen binding fragment thereof, including 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-derived monoclonal antibody and three human-derived monoclonal antibodies) is obtained by a hybridoma technology and a CDR grafting method. The anti-CD24 antibody of the application has high affinity, can significantly inhibit the growth of tumors, and the tumor inhibition rate can reach 82.3%, has a significant anti-tumor 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 a prophylactic and therapeutic drug, a diagnostic drug and a detection kit for diseases related to a CD24 target point, and has a very wide clinical application prospect in the fields of tumor treatment and diagnosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an anti-CD24 antibody and application thereof. BACKGROUND

[0002] Phagocytes play an important role in anti-tumor immunity, and macrophages are the main phagocytes that can directly clear tumor cells. Phagocytosis is regulated by a variety of signal molecules, and tumor cells can express anti-phagocytic proteins such as CD47, PD-L1, CD24, etc., to produce a "don't eat me" signal and induce immune escape.

[0003] CD24 (cluster of differentiation antigen 24) is a highly glycosylated glycosylphosphatidylinositol-anchored membrane protein, and is a kind of 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 response. The intracellular region of Siglec-10 contains an immunoreceptor 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, and thus inhibit phagocytosis of macrophages and promote immune escape of tumors. 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 the phagocytosis of macrophages on tumor cells and inhibit immune escape. The high expression of CD24 in ovarian cancer and triple-negative breast cancer is most obvious, and the high expression of CD24 is also closely related to the poor prognosis of ovarian cancer and breast cancer.

[0004] CD24, as an important immune checkpoint molecule, has great potential in the treatment of tumors and other immune system diseases. However, CD24 is a very small and highly glycosylated protein, which brings great challenges to the development of antibody drugs. The 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, developing new CD24 antibodies with high affinity, strong anti-tumor ability, and good tumor selectivity is of great significance for precisely targeting tumor cells, reducing damage to normal cells, improving treatment effect and reducing side effects, and can provide patients with safer and more effective treatment options, and has wide clinical application value in the field of tumor treatment. SUMMARY

[0005] One of the purposes of the present application is to provide an anti-CD24 antibody and application thereof. The technical problems to be solved are not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

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

[0007] The antigen-binding fragment includes, but is not limited to, Fab, Fab', F(ab')2, antibody variable region (Fv), disulfide-stabilized Fv (dsFv), single-chain antibody (ScFv), single-domain antibody (sdAb, nanobody), minibody and minimal recognition unit (MRU), etc.

[0008] The sequence of the complementarity determining region (CDR) can be defined according to the Kabat numbering system.

[0009] The heavy chain variable region and the light chain variable region each comprise a framework region (FR, a region outside the CDR in the variable region). The framework region can be derived from a human or a non-human mammal (such as a mouse, a rat, a guinea pig, a rabbit, a sheep, a camel, etc.).

[0010] Further, the antibody or the 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):

[0011] A1) the amino acid sequence of the heavy chain variable region can be SEQ ID NO: 1, or an amino acid sequence with more than 80% identity to SEQ ID NO: 1 obtained by substitution, deletion and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region can be SEQ ID NO: 3, or an amino acid sequence with more than 80% identity to SEQ ID NO: 3 obtained by substitution, deletion and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 3;

[0012] A2) the amino acid sequence of the heavy chain variable region can be SEQ ID NO: 5, or an amino acid sequence having 80% or more identity to SEQ ID NO: 5 obtained by substitution, deletion, and / or addition of amino acid residues of SEQ ID NO: 5; and the amino acid sequence of the light chain variable region can be SEQ ID NO: 7, or an amino acid sequence having 80% or more identity to SEQ ID NO: 7 obtained by substitution, deletion, and / or addition of amino acid residues of SEQ ID NO: 7;

[0013] A3) the amino acid sequence of the heavy chain variable region can be SEQ ID NO: 9, or an amino acid sequence having 80% or more identity to SEQ ID NO: 9 obtained by substitution, deletion, and / or addition of amino acid residues of SEQ ID NO: 9; and the amino acid sequence of the light chain variable region can be SEQ ID NO: 11, or an amino acid sequence having 80% or more identity to SEQ ID NO: 11 obtained by substitution, deletion, and / or addition of amino acid residues of SEQ ID NO: 11;

[0014] A4) the amino acid sequence of the heavy chain variable region can be SEQ ID NO: 13, or an amino acid sequence having 80% or more identity to SEQ ID NO: 13 obtained by substitution, deletion, and / or addition of amino acid residues of SEQ ID NO: 13; and the amino acid sequence of the light chain variable region can be SEQ ID NO: 15, or an amino acid sequence having 80% or more identity to SEQ ID NO: 15 obtained by substitution, deletion, and / or addition of amino acid residues of SEQ ID NO: 15.

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

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

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

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

[0019] Further, the substitution can be a conservative substitution. The discrepancy in the amino acid sequence can be in a framework region, for example, with one or several amino acid substitutions, deletions, or additions in the framework region. The several can be 2-10 or more.

[0020] Further, the antibody or antigen-binding fragment thereof can further comprise a heavy chain constant region (CH) and a light chain constant region (CL). The heavy chain constant region can be selected from the heavy chain constant region of IgG, IgA, IgM, IgD, or IgE. The heavy chain constant region can further be selected from the CH1, Fc, and CH3 domains. The light chain constant region can be selected from the kappa (K) or lambda (l) type light chain constant region. The heavy chain constant region and the light chain constant region can be derived from human or non-human mammals (e.g., mouse, rat, guinea pig, rabbit, sheep, camel, etc.).

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

[0022] Further, the heavy chain constant region can be the heavy chain constant region of human IgGl, mouse IgGl, or mouse IgG2b.

[0023] Further, the light chain constant region can be the light chain constant region of human kappa or mouse kappa.

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

[0025] The present application also provides a biological material, which can be any one of the following:

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

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

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

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

[0030] B5) a recombinant host cell containing the nucleic acid molecule of B1).

[0031] Among the above-mentioned biological materials, the recombinant vector can be a cloning vector or an expression vector.

[0032] Further, the recombinant vector can be a recombinant expression vector obtained by cloning a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof 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 examples provided herein is a pcMV3 vector, the present application is not limited to this particular vector. Those skilled in the art can use other suitable vectors as long as the vector can express the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

[0033] The prokaryotic expression vector can be selected from the group consisting of E. coli expression vectors (e.g., pET series vectors, etc.). The eukaryotic expression vector can be selected from the group consisting of yeast expression vectors (e.g., pYES2, pPICZaA, etc.), insect cell expression vectors (e.g., pFastBacl, 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 the group consisting of 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 expression vectors.

[0034] Among the above-mentioned biological materials, the nucleic acid molecule of B1) can be any one of the following:

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

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

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

[0038] C4) a DNA molecule having a coding sequence of SEQ ID NO: 14 and SEQ ID NO: 16.

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

[0040] The skilled person can readily employ known methods, such as site-directed mutagenesis (including oligonucleotide primer-directed 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 thereof described herein of the present application. Those artificially engineered nucleotide sequences that are 75% or more identical to the nucleotide sequence encoding any of the antibodies or antigen-binding fragments thereof described herein of the present application, as long as they encode any of the antibodies or antigen-binding fragments thereof described herein, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[0041] In certain embodiments, substitutions, insertions, or deletions can occur within one or more complementarity determining regions or framework regions of any of the antibodies or antigen-binding fragments thereof described herein of the present application, as long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative modifications (e.g., conservative substitutions well known to those skilled in the art that do not change the properties and functions of the protein) can be made to the complementarity determining regions and / or framework regions that do not substantially reduce binding affinity. Such changes can be outside of antigen contacting residues in the complementarity determining regions, for example.

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

[0043] D1) in the manufacture of a product for preventing and / or treating a tumor;

[0044] D2) in the manufacture of a product for preventing and / or treating a CD24 target-related disease;

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

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

[0047] D5) in the manufacture of a product for diagnosing, assisting in the diagnosis or screening for a CD24 target-related disease;

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

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

[0050] In the above uses, 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, cholangiocarcinoma, kidney cancer, thyroid cancer, testicular cancer, melanoma, brain tumor, head and neck tumor, oral tumor, lymphoma, soft tissue tumor, 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) can be a CD24-positive tumor.

[0053] Further, the CD24-positive tumor can be a CD24-positive cancer. The cancer includes early-stage cancer, mid-stage cancer, mid-advanced stage cancer, advanced stage cancer, recurrent cancer, metastatic cancer, and refractory cancer, etc.

[0054] D5) The use can include detecting the expression level of CD24 protein by using the antibody or antigen-binding fragment thereof of the present application based on the principle of antigen-antibody specific reaction, and further used for screening and diagnosis (including prognosis diagnosis, evaluating the malignancy of tumor, 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 thereof described herein can be prepared into an immunohistochemical kit for screening or diagnosing CD24-positive tumor patients, and further guiding drug use and treatment.

[0055] D6) The detection of CD24 protein or CD24-expressing cells 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 detecting whether the sample to be tested contains CD24 protein and / or detecting the content of CD24 protein in the sample to be tested. The CD24-expressing cells can further be CD24-expressing tumor cells.

[0056] D7) The product for binding CD24 protein described in D7) includes CD24 protein inhibitors, products for isolating or purifying CD24 protein, and in vivo imaging products of CD24 protein, but is not limited thereto. For example, the antibody or antigen-binding fragment thereof described in the present application can be prepared into an immunoaffinity chromatography column. Based on the principle that antigens can be captured by antibodies when passing through the chromatography column, environmental conditions such as pH value are changed to make the antigens dissociate from the column, CD24 protein is isolated and screened out. The antibody or antigen-binding fragment thereof described in the present application can also be coupled with molecules having imaging functions (including but not limited to radionuclides, near-infrared dyes, luciferase, magnetic resonance imaging nanoparticles, magnetic resonance imaging quantum dots), and after being injected into the body of a patient, based on the targeting function of the antibody, it can reach the corresponding tissue site by itself, and immunological imaging can be performed, that is, in vivo imaging of CD24 protein can be achieved. In addition, it is well known to those skilled in the art that antibodies are commonly used protein inhibitors, which can inhibit the activity of their ligands, and therefore the antibody or antigen-binding fragment thereof described in the present application can be made into a CD24 inhibitor or a signal pathway inhibitor (such as a CD24-Siglec10 signal pathway inhibitor) involving CD24.

[0057] The products described herein include but are not limited to reagents, kits (such as therapeutic kits or detection kits), preparations, drugs, pharmaceutical compositions, chips, test papers, detection cards, immunosensors, etc.

[0058] The present application also provides an antibody conjugate, which comprises an antibody moiety and a conjugate moiety, wherein the antibody moiety comprises the antibody or antigen-binding fragment thereof.

[0059] Further, the antibody moiety and the conjugate moiety can be directly linked or covalently linked through a linker (such as a hydrazone bond, a disulfide bond, a thioether bond, a peptide bond).

[0060] Further, the conjugate moiety can be selected from a chemical drug, a cytotoxin, and a detectable label.

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

[0062] Further, the cytotoxin can be a substance that inhibits cell proliferation or induces cell apoptosis, including a microtubule inhibitor (such as maytansine, auristatin (MMAE and MMAF), docetaxel, paclitaxel, vincristine, colchicine), a DNA damaging agent (such as ozogamicin, calicheamicin, duocarmycin, camptothecin, duocarmycin, mitomycin C (MMC), cisplatin, carboplatin, nedaplatin, oxaliplatin, iproplatin), an RNA synthesis inhibitor (such as amanitin, teixiston and its analogues, carmustine), or a protein synthesis inhibitor (such as ricin).

[0063] Further, the detectable label includes an enzyme (such as horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase, etc.), a chemiluminescent reagent (such as acridinium ester compounds, acridinium sulfonamide compounds, luminol and its derivatives, ruthenium derivatives, etc.), a fluorescent dye (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 700, BV605, BV480, BV785, BV510, BV711, BV421, etc.), a near-infrared dye (such as cyanine dyes, BODIPY, rhodamine, squarine, porphyrin dyes, etc.), a radionuclide (such as 125I, 18F, 11C, 99mTc, 123I, etc.), biotin, a magnetic resonance imaging nanoparticle, a magnetic resonance imaging quantum dot, a magnetic substance (such as magnetic beads, gadolinium-containing complex nanoparticles, superparamagnetic iron oxide nanoparticles), and colloidal gold, but is not limited thereto.

[0064] The linker (linker) includes a non-cleavable linker and a cleavable linker. The linker can be selected from N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-epsilon-maleimide caproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyl disulfide)-propionate (SPDP), Val-Cit (valine-citrulline) dipeptide, GGFG tetrapeptide, phosphatase and pyrophosphatase linker, beta-galactosidase linker, and sulfatase linker.

[0065] Further, the antibody conjugate can be an antibody drug conjugate (ADC).

[0066] Further, the coupling moiety can also be a tag. In order to facilitate the separation, purification, detection and / or localization of the antibody or antigen-binding fragment thereof of the present application, a tag protein can be linked to the amino-terminal or carboxyl-terminal end of the antibody or antigen-binding fragment thereof. The tag includes, but is not limited to, a GST (glutathione S-transferase) tag protein, a Trx (thioredoxin) tag protein, a NusA tag protein, a His tag protein (His-tag), a MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, a HA (influenza hemagglutinin) tag protein, a Myc tag protein, a LacZ tag protein, a CBD (cellulose binding domain) tag protein, a bacteriophage T7 protein kinase (T7PK) tag protein, a GFP (green fluorescent protein), a CFP (cyan fluorescent protein), a YFP (yellow-green fluorescent protein), a mCherry (monomeric red fluorescent protein) or an AviTag tag protein. Those skilled in the art know how to select a suitable tag protein according to the desired purpose. The use of a tag does not change the function of the antibody, and its purpose is to separate, purify, detect or track, so the tag protein suitable for the present application is not limited to a particular kind. The tag can be separated from the antibody by chemical cleavage or enzymatic cleavage known in the art, such as introducing a protease cleavage site to remove the tag using TEV protease cleavage.

[0067] The present application also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, the biomaterial or the antibody conjugate, and one or more pharmaceutically acceptable carriers.

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

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

[0070] Further, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0071] The pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, co-solvents, diluents (such as water, normal 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 dodecyl sulfate, etc.), fillers (such as starch, dextrin, etc.), adhesives (such as gelatin, pectin, acacia gum, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), penetration enhancers (such as Brij-78), pH regulators, stabilizers (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactants (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium dodecyl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption enhancers (such as chitosan), thickening agents (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidants (such as sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium thiosulfate, vitamin C, etc.), plasticizers (such as glycerol, sorbitol, phthalate esters, etc.), propellants (such as hydrofluoroalkanes, dimethyl ether, etc.), aerosolizing agents, suspending agents, dispersing agents, coloring agents (such as TiO2, pigments, etc.), and flavoring agents. Those skilled in the art know that a carrier usually has multiple functions, for example, starch can be used as a disintegrant and a binder. Those skilled in the art can routinely select the above carriers according to the properties of the drug and the route of administration.

[0072] Excipients are generally used in pharmaceutical products to make the pharmaceutical products into a shape, to change the physical state of the pharmaceutical products, and to act as a support. Excipients include, but are not limited to: (1) excipients for injection solutions: such as solvent water, alcohols, ethers, amides, sulfones, esters, etc.; (2) excipients for injection powders: such as sucrose, lactose, mannitol, etc.; (3) excipients for sprays: such as soybean lecithin, propylene glycol, menthol, 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, ethylenediaminetetraacetic acid disodium salt (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, acacia, etc.; (10) excipients for ointments: such as vaseline, paraffin, liquid paraffin, lanolin, lanolin alcohol, beeswax, lard, vegetable oil, silicone oil, silicone, soap, higher fatty alcohol, fatty alcohol sulfate, polyhydric alcohol, 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, acacia, polyvinyl alcohol compounds, acrylic acid copolymers, etc.

[0073] Biopharmaceutical preparations are susceptible to contamination by adventitious microorganisms during production, transportation, and storage, which can affect the quality of the pharmaceutical products. Therefore, preservatives can be generally added to inhibit the growth and reproduction of microorganisms. The preservatives include, but are not limited to, thiomersal, formaldehyde, phenol, and m-cresol.

[0074] Protectants can generally refer to any substance capable of protecting the activity of a pharmaceutical product, including lyophilization protectants. Lyophilization protectants can be used to change the physicochemical environment of a biological sample during lyophilization, reduce cell damage, maintain original biological activity, and improve the stability of proteins and cells, etc. The lyophilization protectants 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, acetyl tryptophan, etc.), and salts and amines (such as phosphate, acetate, citrate, etc.).

[0075] The dosage form of the pharmaceutical composition includes, but is not limited to, injections (including injection solutions and injection powders for injection), gels, eye drops (including eye drop solutions and intraocular injection solutions), oral solutions, suppositories, effervescent tablets, capsules, ointments, creams, sprays, aerosols, external solutions, tablets, powders, pills, granules, scratches, granules, drops, coatings, patches, and long-acting controlled-release preparations. Those skilled in the art know that various dosage forms can be prepared using active ingredients (such as antibodies or antigen-binding fragments thereof of the present application) with suitable pharmaceutically acceptable carriers according to conventional preparation processes.

[0076] Further, the dosage form of the pharmaceutical composition can be an injection preparation.

[0077] Injections generally refer to solutions, emulsions prepared by extracting and purifying medicinal materials for injection into the body, and sterile powders for preparing solutions before use, which include injection solutions and injection powders for injection. The preparation method of injections is well known to those skilled in the art, for example, injection powders for injection can be prepared by vacuum freeze-drying technology, spray drying technology, and spray freeze-drying technology. Injection solutions can also be prepared by concentrating or diluting the drug with suitable diluents, cosolvents, and / or wetting agents, etc., and then filtering (such as surface filtration and / or depth filtration), filling and sealing, sterilizing, etc.

[0078] In order to prepare the pharmaceutical composition into injection preparations such as solutions, emulsions, lyophilized powders for injection, and suspensions, diluents commonly used 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 solvents to prepare injection solutions. In addition, in order to prepare isotonic injection solutions, an appropriate amount of sodium chloride, glucose, or glycerol, etc. can be added to the injection preparation, and in addition, conventional cosolvents, buffers, pH adjusters, etc. can also be added.

[0079] The administration method of the pharmaceutical composition includes, but is not limited to, injection administration (such as administration by injection), mucosal administration (such as administration by spray, aerosol, tablet, eye drop, suppository, granule, capsule, etc.), and transdermal administration (such as administration by gel, ointment, patch, film, etc.).

[0080] The administration method of the pharmaceutical composition includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumoral injection, intracranial injection, mucosal administration, oral administration, skin smearing, oral and nasal cavity spraying, aerosol inhalation, in vivo implantation, and in vitro carrying device administration.

[0081] The active ingredient of the pharmaceutical composition can be any of the antibodies or antigen-binding fragments thereof described herein.

[0082] The present application also provides a kit containing the antibody or antigen-binding fragment thereof.

[0083] The kit can have at least one of the following uses: (1) diagnosing, aiding diagnosis or screening for a CD24 target-related disease; (2) detecting CD24 protein or CD24-expressing cells; (3) inhibiting or binding CD24 protein (such as isolation, purification and in vivo imaging of CD24 protein).

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

[0085] The detection 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 can be a chemiluminescence immunoassay kit, an enzyme-linked immunoassay 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] Further, the kit can further include reagents required for immunodetection, such as labeled antibodies or antigens, magnetic particles, blocking solution, dilution solution, washing solution, color developing solution, termination solution, etc., but is not limited thereto.

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

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

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

[0091] Further, the preparation method can comprise the following steps: cloning a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof 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 culture of the cultured recombinant host cell.

[0092] Further, the recovering or isolating can be performed by a precipitation method (such as a salting-out method, an organic solvent precipitation method, an octanoic acid-saturated ammonium sulfate precipitation method, and an isoelectric point precipitation method) or a chromatography technique (such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography) from the culture (including all substances in the culture vessel).

[0093] The method of introduction can comprise any one of the following: (1) introducing a target gene or a recombinant vector containing the target gene into a host cell by a chemical transformation method (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation) or a physical transformation method (such as electroporation transformation); (2) introducing the target gene into the host cell by a phage transduction method; (3) directly transforming the target gene into a plant receptor cell by a physical or chemical method, such as a chemical stimulation method, an electric shock method, a liposome-mediated method, a microinjection method, a gene gun method, a laser microbeam method, a pollen tube channel method, an ultrasonic wave method, an air gun method, and a vortex method; (4) introducing the target gene into the plant receptor cell by using a vector as a medium, such as an Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems); (5) introducing the target gene into an isolated animal cell (transfection) by a calcium phosphate coprecipitation method, a cationic polymer method (such as a DEAE-dextran transfection method), a cationic liposome method, an electroporation method (i.e., an electroporation transfection method), a microinjection method, a gene gun method, or a virus-mediated method (such as an adenovirus infection method and a lentivirus infection method).

[0094] The antibody of the present application can 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 on 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. Tumor cells and normal cells express CD24 with different degrees of glycosylation. The present application successfully screened a monoclonal antibody (IMB4H7) with high affinity for non-glycosylated CD24 by hybridoma technology, using KLH-conjugated CD24 antigen polypeptide subcutaneous immunization + cell impact immunization (CHO-hCD24 cells). The present application further utilizes CDR grafting to humanize the obtained murine monoclonal antibody IMB4H7, obtaining three humanized antibodies: IMB4H7h1, IMB4H7h2 and IMB4H7h3.

[0096] The anti-CD24 antibodies (IMB4H7, IMB4H7h1, IMB4H7h2 and IMB4H7h3) of the present application have greater affinity for tumor cells compared to the commercial CD24 antibody SN3, and have strong binding to various tumor cells and tumor tissues, but weak binding to normal cells. The binding of the anti-CD24 antibodies of the present application to deglycosylated CD24 is significantly enhanced, indicating that the binding of the antibodies to CD24 can be blocked by glycosylation in normal cells but not in cancer cells, making the antibodies have good tumor selectivity. The results of in vivo treatment experiments show that the anti-CD24 antibodies of the present application 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 antibodies of the present application can be expressed and produced in prokaryotic cells, eukaryotic cells and various recombinant systems, and can be made into clinical drugs for the prevention and / or treatment of CD24 target-related diseases, diagnostic drugs, detection kits for CD24 protein or in vivo imaging products for CD24 protein, etc., and 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 have very broad clinical application prospects in the fields of tumor treatment and diagnosis, etc.

[0098] Definition of terms

[0099] In the present application, 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 application, the definitions and explanations of related terms are provided as follows.

[0100] The term "identity" generally refers to the extent to which two (nucleotide or amino acid) sequences have the same residues at a given position in the alignment, and is usually expressed as a percentage. Identity as described herein can refer to amino acid sequence or nucleotide sequence. Two copies having exactly the same sequence have 100% identity. One skilled in the art is aware that identity of an amino acid sequence or a nucleotide sequence can be determined using identity search sites on the internet, such as the BLAST page of the NCBI home page website. For example, the value of identity (%) can be obtained by searching in Advanced BLAST 2.1 using blastp as the program, setting Expect value to 10, setting all Filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and then calculating the identity of the amino acid sequence. In addition, it can be determined using sequence analysis software (such as CLC Main Workbench and MegAlign™), for example, computer program BLAST, especially BLASTP or TBLASTN, using default parameters. More than 75% identity as described herein can be at least 75%, 80%, 85%, 90% or 95% or more. More than 80% identity as 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.

[0101] The term "conservative substitution" generally refers to the replacement of one amino acid residue by another possessing a similar physicochemical property. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and He), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gin), 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). Conservative substitutions generally known in the art not to produce a significant change in the conformational structure of a protein, and do not substantially alter the biological activity of the protein. Conservative substitutions in a protein sequence that are expected to have little or no effect on the structure or function of the protein can be readily designed by one of ordinary skill in the art.

[0102] The term "antibody conjugate" herein includes not only an antibody-drug conjugate (ADC) formed by conjugation of an antibody with a drug (a substance for preventing, treating or diagnosing a disease) via a linker, but also a conjugate formed by conjugation of an antibody with a detectable label or tag.

[0103] The term "CD24 positive tumor" generally refers to a tumor expressing CD24 protein. Methods for detecting CD24 protein expression in a tumor 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 reaction (such as immunohistochemistry, FACS, etc.), and the expression of CD24 mRNA can also be determined by real-time fluorescent 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 an antibody analog, which generally includes at least part of the antigen-binding region or variable region (such as one or more CDRs) of the parental antibody. The antigen-binding fragment retains at least some of the binding specificity of the parental antibody. Generally, the antigen-binding fragment retains at least 10% of the binding activity of the parental antibody when the activity is expressed on a molar basis. 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. Those skilled in the art are well aware that the antigen-binding fragment of an antibody can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. BRIEF DESCRIPTION OF DRAWINGS

[0105] Figure 1 SDS-PAGE electrophoretogram of the purified antibody.

[0106] Figure 2 Flow cytometry detection of the subtype of IMB4H7 antibody.

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

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

[0109] Figure 5 Flow cytometry detection of the binding of IMB4H7 and SN3 to CD24 from which N-glycan (SKOV3+PNGase F) and sialic acid (SKOV3+sialic acid) were removed.

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

[0111] Figure 7 Flow cytometry to determine the half maximal effective binding concentration of humanized antibodies IMB4H7h1, IMB4H7h2, IMB4H7h3 and murine antibody IMB4H7.

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

[0113] Figure 9 Flow cytometry to compare the difference in binding of humanized antibodies to human normal cells and cancer cells.

[0114] Figure 10 Tissue microarray to determine the binding of IMB4H7 to different tumor tissues (mean ± SEM).

[0115] Figure 11 Distribution of IMB4H7h2 antibody in a bilateral tumor-bearing mouse.

[0116] Figure 12 Anti-tumor activity of IMB4H7 in vivo.

[0117] Figure 13 Effect of IMB4H7 on the body weight of a mouse. DETAILED DESCRIPTION

[0118] The present application will be further described in conjunction with the specific embodiments thereof, and the examples given are only intended to illustrate the present application and not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

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

[0120] Example 1, Preparation and binding activity detection of CD24 antibody

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

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

[0123] The human CD24 (hCD24, Uniprot: P25063) encoding gene is cloned into a PHBLV lentivirus vector (prepared by Hangzhou Laibo Biotechnology Co., Ltd.) under the promoter of EF1a (EF-1a), to obtain a recombinant vector. The recombinant vector is transfected into CHO-K1 cells (Wuhan Punsai Life Science Co., Ltd., Catalog No. CL-0062), and the limited dilution method is used to obtain a Chinese hamster ovary cancer cell strain with high expression of human CD24, named CHO-hCD24 cells. The above-mentioned cells are cultured using 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 is synthesized by commercial means, and each BALB / c mouse (6-8 weeks old) is subcutaneously immunized with 30 μg of KLH-coupled polypeptide, emulsified with Freund's complete adjuvant before immunization, once every two weeks, for a total of three times; two weeks later, the mice are immunized once again with 1×10 7 CHO-hCD24 cells.

[0126] 3. Cell fusion

[0127] After the above-mentioned immunization, the mouse blood is taken, and the antibody titer is detected using CHO-hCD24 cells. The two mice with the highest positive rates are sacrificed, and the spleen cells are fused with SP2 / 0 cells (Hangzhou Laibo Biotechnology Co., Ltd.). The titer of the fused cells is detected, the positive fused cells are screened, and the single clones are picked up using the limited dilution method.

[0128] 4. Hybridoma cell screening

[0129] The clones with high binding activity to CHO-hCD24 cells are selected by flow cytometry. The flow cytometry detection steps are as follows: 1×10 6After centrifugation at 300g for 5 minutes, the precipitate was collected, and the cells were resuspended with staining solution (pre-cooled PBS containing 1% BSA). Each cell was aliquoted into a flow detection tube at a volume of 100 μL per tube. The antibody to be detected (i.e., hybridoma cell culture supernatant) was added at different dilution ratios, and the mixture was incubated at 4°C for 30 minutes. After washing with staining solution for 3 times, a FITC-labeled anti-mouse IgG secondary antibody was added, and the mixture was incubated at 4°C for 30 minutes. After washing with staining solution for 3 times, the fluorescence intensity was detected using a flow cytometer. The flow-sorted clones were further subcloned and screened, and finally, a positive monoclonal antibody with strong binding activity to CHO-hCD24 cells was obtained. The hybridoma cell strain that secreted the CD24 monoclonal antibody with the strongest binding activity to CHO-hCD24 cells was named 4H7 cells. The monoclonal antibody secreted by the hybridoma cell strain was named IMB4H7.

[0130] The obtained hybridoma cell strain 4H7 was injected into the abdominal cavity of 8-12 week-old BalB / c mice at a concentration of 1×10 6 cells per mouse. After 7-10 days, the ascites containing the monoclonal antibody were collected. The antibody in the ascites was purified using a Protein A affinity column. The purification method was as follows: (1) 1 mL of Protein A affinity column was equilibrated with 20 mM PB, pH 7.0, at a flow rate of 1.0 mL / min; (2) 50 mL of culture solution was loaded at a flow rate of 1 mL / min, and the equilibration solution was used for equilibration; (3) 0.1 M glycine-HCl, pH 2.7 was used for elution at a flow rate of 1 mL / min, and the 280 nm absorption peak was collected; (4) Superdex 200Increase 10 / 300 GL column was equilibrated with 20-50 mM PB, 150 mM NaCl, pH 6.7, at a flow rate of 1 mL / min; and (5) 2 mL of the 280 nm absorption peak was collected after loading. The murine anti-CD24 monoclonal antibody IMB4H7 was obtained. After 10% SDS-PAGE electrophoresis, Coomassie blue staining showed that the antibody had heavy chains and light chains, and no impurities were observed. Figure 1

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

[0132] ​Subtype identification and sequencing analysis of the monoclonal antibody were performed. The steps were as follows: 1) an appropriate amount of hybridoma cells was dispensed into 1.5 mL Ep tubes, centrifuged at 4000 rpm for 5 minutes, and the supernatant was aspirated. 2) 50 μL of Goat anti-mouse IgG-FITC, Goat anti-mouse IgG1-FITC, Goat anti-mouse IgG2a-FITC, Goat anti-mouse IgG2b-FITC (Hangzhou Laibio Biotechnology Co., Ltd.) diluted 1:500 with PBS was added to resuspend the above cells, and the mixture was incubated at 4°C for 15 minutes. 3) The mixture was centrifuged at 4000 rpm for 5 minutes, and the supernatant was aspirated. 6) The cells were resuspended with 400 μL of PBS, and flow cytometry analysis was performed. The results are shown in Figure 2 Figure 1, and the subtype of the IMB4H7 antibody is IgG2b.

[0133] Based on 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 is shown in SEQ ID NO: 1, and the nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region of the monoclonal antibody IMB4H7 is shown in SEQ ID NO: 3, and the nucleotide sequence of the light chain variable region encoding gene is shown in SEQ ID NO: 4.

[0134] wherein:

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

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

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

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

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

[0140] The amino acid sequence of CDR3 of the light chain variable region of the monoclonal antibody IMB4H7 is shown in 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, item number 11-0247-42) to CHO-K1 cells and CHO-hCD24 cells, with mouse IgG as the isotype control (Invitrogen, item number 02-6502). As shown in Table 1, IMB4H7 showed strong binding to CHO-hCD24 cells, and the binding was stronger than that of SN3. Both antibodies showed weak binding to CHO-K1 cells. Figure 3

[0143] 7. Preparation of antibodies by genetic engineering

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

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

[0146] The heavy chain gene and the light chain gene of monoclonal antibody IMB4H7g1 were cloned into pcMV3 vectors (Beijing Yikuo Shenzhou Technology Co., Ltd.) respectively to obtain the heavy chain expression vector and the light chain expression vector.

[0147] The heavy chain expression vector and the light chain expression vector were transfected into HEK293 cells, 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, following the same method as in Step 4 of Example 1. Flow cytometry was used to detect the binding of IMB4H7g1 (4 μg / mL) to CHO-K1 and CHO-hCD24, following the same method as in Step 4 of Example 1, with mouse IgG as the 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 low-glycosylated 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. Ovarian cancer SKOV3 cells (Wuhan Pronosei Biotechnology Co., Ltd., CL-0215) were treated with sialidase (NEW ENGLAND Biolabs, catalog number P0722S) and PNGase F (peptide N-glycosidase F, NEW ENGLAND Biolabs, catalog number P0704S), and the binding of IMB4H7 and SN3 antibodies to the cells was detected by flow cytometry. The experiment was performed according to the manufacturer's instructions, and the specific steps are as follows: SKOV3 cells were collected by trypsin digestion, washed twice with PBS, and divided into 3 EP tubes, each containing 1×10⁻⁶ cells. 6 Cells; Sialidase group: 2 μL sialidase + 2 μL GlycoBuffer 1 (10×, 50 mM CaCl2, 500 mM sodium acetate, pH 5.5) + 18 μL water; PNGase F group: 2 μL PNGase F + 2 μL GlycoBuffer 2 (10×, 500 mM Sodium Phosphate, pH 7.5) + 18 μL water; Control group: 1 μL GlycoBuffer 1 (10×) + 1 μL GlycoBuffer 2 (10×) + 20 μL water; incubate at 37℃ for 24 hours; wash the sample twice with PBS, centrifuge and discard the supernatant, divide each EP tube into two samples, add IMB4H7 and SN3 antibodies (4 μg / 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 500 μL PBS, and perform flow cytometry detection.

[0150] The results are as follows Figure 5 As shown, SKOV3 cells treated with sialidase or PNGase F exhibited decreased binding to SN3, but significantly increased binding to IMB4H7. This indicates that the binding of antibody SN3 to CD24 depends on sialyl polysaccharide and N-glycosylation, while the monoclonal antibody IMB4H7 can bind to low-glycosylated CD24.

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

[0152] The IMB4H7 antibody was humanized using the CDR transplantation method. The mouse CDR was retained, the mouse FR was replaced with the human frame region (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 gene encoding the heavy chain variable region 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 gene encoding the light chain variable region 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 gene encoding the heavy chain variable region 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 gene encoding the light chain variable region 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 gene encoding the heavy chain variable region 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 gene encoding the light chain variable region 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 gene encoding the heavy chain constant region 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 gene encoding the light chain constant region is SEQ ID NO:21.

[0155] Humanized antibodies were prepared using the genetic engineering method described in step 7 of Example 1. The light and heavy chain sequences of the humanized antibodies were cloned into the pcMV3 vector, transiently transfected into HEK293 cells, and cultured at 37°C for 6 days. The culture supernatant was collected, and the antibodies were purified by affinity chromatography. Flow cytometry was used to compare the binding strength of the three humanized antibodies and antibody SN3 to CHO-hCD24 cells, with CHO-K1 cells as a control. The results showed that all three humanized antibodies could bind to CHO-hCD24 cells, and their binding strength was significantly stronger than that of the 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 the three humanized antibodies and the murine IMB4H7 antibody to CHO-hCD24 cells, and the half-maximum effective concentration (EC50) was calculated. 50 Value. Result as follows Figure 7 As shown, ECGs of three humanized antibodies and IMB4H7 antibody... 50The values are 2.039 pg / mL, 1.491 pg / mL, 1.089 pg / mL and 2.608 pg / mL, respectively.

[0156] Example 4, Detection of the binding of CD24 antibodies to various tumor cells

[0157] This example takes the IMB4H7h2 antibody as an example to detect the binding of humanized antibodies to various tumor cells from different tissues by flow cytometry. Human breast cancer MDA-MB-468 cells, human hepatoma HuH-7 cells and human melanoma A375 cells were cultured in DMEM medium, human lung cancer A549 cells were cultured in Ham's F-12K medium, human gastric cancer NUGC-4 cells were cultured in RPMI-640 medium, and human ovarian cancer SKOV3 cells were cultured in McCoy's 5A medium. All cell culture media were supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 pg / mL streptomycin and 100 U / mL penicillin, and cultured in a 37°C incubator with 5% CO2(Thermo Scientific, 3131 carbon dioxide incubator). The above tumor cell lines are common cell lines, which were obtained from the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, or purchased from commercial sources such as ATCC cell bank (Rockville, MD, USA), National Experimental Cell Resource Sharing Platform, etc. The flow detection method is the same as that in Example 1, step 4, and the secondary antibody is anti-human IgG-FITC, and human IgG1 kappa (Beijing Yiqiao God Science and Technology Co., Ltd., Catalog No. HG1K) is used as a homologous control. The experimental results are shown in Figure 8 Figure 5, and the IMB4H7h2 antibody has strong binding to the above tumor cells.

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

[0159] Flow cytometry was used to compare the binding strength of three humanized antibodies to human normal breast epithelial cells MCF 10A and human breast cancer cells MDA-MB-468. MCF 10A cells (Wuhan Punsai Life Science and Technology Co., Ltd., Catalog No. CL-0525) were cultured in DMEM / F12 + 5% horse serum + 20 ng / mL EGF + 0.5 pg / mL hydrocortisone + 10 pg / mL insulin + 1% non-essential amino acids + 100 pg / mL streptomycin and 100 U / mL penicillin medium, and MDA-MB-468 cells were cultured as in Example 4. The flow detection method is the same as that in Example 4. The results show that the three humanized antibodies strongly bind to MDA-MB-468 cells, but do not bind to human normal breast epithelial cells MCF 10A Figure 9 ).

[0160] Example 6, Tissue chip test the binding activity of CD24 antibody to different tumor tissues

[0161] This example takes IMB4H7 antibody as an example to perform immunohistochemical analysis on a tissue chip containing 20 different organs, each organ including 20 cases of cancer tissues (Shanghai Xiebu Kang Biotechnology Co., Ltd.). Including gastric cancer, melanoma (skin, rectum, small intestine), prostate cancer, brain tumor (astrocytoma, glioblastoma), ovarian cancer, breast cancer, testicular cancer, colon cancer, bladder cancer, uterine cancer, thyroid cancer, lung cancer, head and neck / oral tumor, lymphoma, soft tissue tumor, liver cancer, pancreatic cancer, kidney cancer, cervical cancer, esophageal cancer. The specific steps are as follows: the tissue chip is placed in a 59°C incubator for 60 minutes, xylene dewaxing, anhydrous ethanol gradient hydration, 3% H2O2 immersion for 10 minutes for inactivation, EDTA (PH 8.0) antigen retrieval solution for two times, each time high fire for 10-12 minutes, drop 5% BSA blocking solution, room temperature for 35 minutes. Drop IMB4H7 antibody working solution, 4°C overnight, wash 4 times with PBS, drop HRP labeled anti-mouse secondary antibody, 37°C for 35 minutes, wash 4 times with PBS. Color development with DAB color developing agent, after color development, drop hematoxylin staining solution, soak for 2 minutes, rinse for 10 minutes, observe the color. The chip is soaked in 1 cylinder of anhydrous ethanol, 2 cylinders of anhydrous ethanol, and 3 cylinders of xylene for 3 minutes each to dehydrate. Add 1 drop of mounting plastic, cover the chip. The above reagents are all commonly used commercial reagents. After scanning the chip (PANNORAMIC MIDI scanner, 3DHISTECH Ltd), use halo software for quantitative analysis. Figure 10 The results of the histochemical score (H-score) of the binding of IMB4H7 antibody to different tumor tissues are summarized, showing that most tumor tissues show strong binding.

[0162] Example 7, Distribution of CD24 antibody in tumor-bearing mice

[0163] This example first labels IMB4H7h2 antibody with DyLight 680 NHS ester (Thermo scientific, item number 46419). Take 0.37 mL of IMB4H72 (2.73 mg / mL), add 3.17 μL of DyLight 680 NHS ester (10 mg / mL), incubate at room temperature for 1 hour, add PBS to the sample to a total volume of 1 mL, pass through a PD MidiTrap G-25 column (GE Healthcare) to collect the eluate, remove excess unreacted fluorescein, concentrate the sample with an ultrafiltration tube (Amion Ultra-4), quantify with BCA, and obtain DyLight 680-IMB4H7h2. The CHO-hCD24 and CHO-K1 cells are plated at 5x10 6BALB / c nude mice (female, 18-22 g, Beijing Yakang Biotechnology Co., Ltd.) on the left and right sides of the armpit subcutaneously, and when the tumors grew to 200-300 mm 3 When the tumors grew to 200-300 mm 6 , the DyLight 680-IMB4H7h2 antibody was injected into the tail vein, and the live imaging was performed after 0.5 hours and 6 hours. The imaging instrument was IVIS-200 Imaging System (Xenogen, USA), and the excitation light / emission light wavelength was 675 / 760 nm. The results showed that the antibody began to enrich on the side of CHO-hCD24 after 0.5 hours of injection, and was significantly enriched after 6 hours; while on the side of CHO-K1, only a small amount of enrichment was observed after 6 hours of injection. Figure 11 The above results showed that the IMB4H7h2 antibody could significantly enrich on the side of the tumor with high expression of CD24.

[0164] Example 8, animal experimental treatment scheme of IMB4H7 antibody

[0165] The in vivo efficacy of IMB4H7 antibody was evaluated by using the Chinese hamster ovary cancer cell CHO-hCD24 xenograft tumor model with high expression of human CD24. The CHO-hCD24 cells were inoculated subcutaneously in the armpit of BALB / c nude mice (female, 18-22 g, Beijing Yakang Biotechnology Co., Ltd.) at a dose of 5×10 6 / each. When the tumor grew to an average of about 50 mm 3 , the mice were divided into a control group and an IMB4H7 group according to the size of the tumor, and the drug was administered through the tail vein. The dose was 10 mg / kg, 0.2 mL / each, 1 time / 3 days, and a total of 4 times, and the control group was injected with normal saline. During the experiment, the long diameter a and the short diameter b perpendicular to it of the tumor were measured every 2-3 days, and the animal body weight was recorded. The tumor volume was calculated by the formula V=1 / 2ab 2 , and the inhibition rate ((tumor volume of the control group-tumor volume of the experimental group) / tumor volume of the control group x 100%) was calculated. The experimental results showed that the IMB4H7 antibody had significant in vivo efficacy, 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% Figure 12 . Compared with the control group, there was no significant decrease in body weight Figure 13 .

[0166] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. An anti-CD24 antibody or antigen-binding fragment thereof, characterized in that, The antibody or antigen-binding fragment thereof includes a heavy chain variable region including a CDR1 of amino acid sequences of positions 31-35 of SEQ ID NO: 1, a CDR2 of amino acid sequences of positions 50-66 of SEQ ID NO: 1, and a CDR3 of amino acid sequences of positions 99-102 of SEQ ID NO: 1, and a light chain variable region including a CDR1 of amino acid sequences of positions 24-39 of SEQ ID NO: 3, a CDR2 of amino acid sequences of positions 55-61 of SEQ ID NO: 3, and a CDR3 of amino acid sequences of positions 94-102 of SEQ ID NO:

3.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof includes 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 80% or more identity to SEQ ID NO: 1 by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is SEQ ID NO: 3, or an amino acid sequence having 80% or more identity to SEQ ID NO: 3 by substitution, deletion, and / or addition of amino acid residues to 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 having 80% or more identity to SEQ ID NO: 5 by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence of the light chain variable region is SEQ ID NO: 7, or an amino acid sequence having 80% or more identity to SEQ ID NO: 7 by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in 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 having 80% or more identity to SEQ ID NO: 9 by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is SEQ ID NO: 11, or an amino acid sequence having 80% or more identity to SEQ ID NO: 11 by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 11; and A4) the amino acid sequence of the heavy chain variable region is SEQ ID NO: 13, or an amino acid sequence having 80% or more identity to SEQ ID NO: 13 by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO: 13; and the amino acid sequence of the light chain variable region is SEQ ID NO: 15, or an amino acid sequence having 80% or more identity to SEQ ID NO: 15 by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO:

15. A4) the amino acid sequence of the heavy chain variable region is SEQ ID NO: 13, or an amino acid sequence having 80% or more identity to SEQ ID NO: 13 obtained by substitution, deletion, and / or addition of amino acid residues in the amino acid sequence shown in SEQ ID NO: 13; and the amino acid sequence of the light chain variable region is SEQ ID NO: 15, or an amino acid sequence having 80% or more identity to SEQ ID NO: 15 obtained by substitution, deletion, and / or addition of amino acid residues in the amino acid sequence shown in SEQ ID NO:

15.

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

4. The biomaterial of claim 3, wherein, The nucleic acid molecule according to B1) is any one of the following: C1) a DNA molecule whose coding sequences are SEQ ID NO: 2 and SEQ ID NO: 4; C2) a DNA molecule whose coding sequences are SEQ ID NO: 6 and SEQ ID NO: 8; C3) a DNA molecule whose coding sequences are SEQ ID NO: 10 and SEQ ID NO: 12; C4) a DNA molecule whose coding sequences are 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 biological material according to claim 3 or 4, in any one of the following: D1) use in the preparation of a product for preventing and / or treating ovarian cancer or breast cancer; D2) use in the preparation of a product for detecting CD24 protein or CD24-expressing cells; D3) use in the preparation of a product for isolating or purifying CD24 protein or a product for in vivo imaging of CD24 protein.

6. An antibody conjugate comprising an antibody moiety and a conjugate moiety, characterized in that, The antibody moiety comprises the antibody or antigen-binding fragment thereof according to claim 1 or 2, and the conjugation moiety is a detectable label or a tag protein.

7. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to claim 1 or 2, the biological material according to claim 3 or 4, or the antibody conjugate according to claim 6, and one or more pharmaceutically acceptable carriers.

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

9. A method of producing an anti-CD24 antibody or antigen-binding fragment thereof, characterized by, 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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