WO2024007672A1 - 特异性结合cd24的抗体及其用途 - Google Patents

特异性结合cd24的抗体及其用途 Download PDF

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WO2024007672A1
WO2024007672A1 PCT/CN2023/088392 CN2023088392W WO2024007672A1 WO 2024007672 A1 WO2024007672 A1 WO 2024007672A1 CN 2023088392 W CN2023088392 W CN 2023088392W WO 2024007672 A1 WO2024007672 A1 WO 2024007672A1
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antibody
antigen
cancer
binding fragment
tumor
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PCT/CN2023/088392
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English (en)
French (fr)
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杜靓
张红艳
金理娜
陈亚莉
徐溜溜
郭晓月
万婷婷
金夷
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上海吉倍生物技术有限公司
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Publication of WO2024007672A1 publication Critical patent/WO2024007672A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to the fields of disease treatment and immunology. Specifically, the present invention relates to anti-CD24 antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors and host cells encoding the antibodies or antigen-binding fragments thereof, the antibodies or antigen-binding fragments thereof, Derivatives of antigen-binding fragments, and their use in the treatment of disease.
  • Macrophages are an important part of innate immunity. They phagocytose aging and dead cells in the human body and play a role in cleaning the body. In recent years, the role of innate immunity in anti-tumor has received much attention, especially macrophages, which can clear tumor cells through phagocytosis in tumor tissues and present tumor cell-related antigens on their surfaces to further activate the immune system.
  • CD47 is a target related to macrophage immune activation. As research continues to deepen, multiple CD47 monotherapy and combination therapies have entered the clinical trial stage. Among them, FortySeven's Hu5F9 is currently in clinical phase III, and another 5 drugs are in clinical trials. Phase II experiment (Zhongxing Jiang, et al: Targeting CD47 for cancer immunotherapy. J. Hematol. Oncol. 2021; 14:180-202.). Clear single-agent efficacy has been observed for drugs targeting this target in the clinical stage. However, the high expression of this target in red blood cells raises an important safety issue.
  • Drugs targeting CD47 may destroy red blood cells and cause severe hematological toxicity, triggering Side effects such as anemia and thrombocytopenia (Zhongxing Jiang, et al: Targeting CD47 for cancer immunotherapy. J. Hematol. Oncol. 2021; 14:180-202.).
  • CD24 is a highly glycosylated glycolphosphatidylinositol (GPI)-anchored cell surface protein. Highly glycosylated CD24 is anchored on lipid rafts in the cell membrane through glycosyl-phosphatidyl-inositol (GPI). It serves as a mediator of intermolecular interactions at cell junction sites, mediating intercellular, cell-to-cell interactions. and matrix adhesion. CD24 is involved in cell recognition, activation, signal transduction, cell proliferation and differentiation, cell stretching and movement, etc.
  • GPI glycosyl-phosphatidyl-inositol
  • CD24 interacts with siglec10 on macrophages of the innate immune system to inhibit destructive inflammatory responses to infection, sepsis, liver injury, and chronic graft-versus-host disease (Guo-Yun Chen, et al: CD24 and Siglec- 10 Selectively Repress Tissue Damage-Induced Immune Responses.Science 2009;323(5922):1722–1725.).
  • CD24 is a signaling protein similar to CD47 that is overexpressed in a variety of human cancers and its receptor Siglec-10 Expressed on tumor-associated macrophages, CD24 binds to Siglec-10 on the surface of tumor-associated macrophages, activating the SHP-1/SHP-2-mediated inhibitory signaling pathway, thereby blocking Toll-like receptor-mediated inflammation. And the cytoskeletal rearrangement required by macrophage phagocytes causes the suppression of macrophages in the tumor microenvironment and achieves tumor immune escape (Amira A. Barkal, et al: Weissman CD24 signaling through macrophage Siglec-10 is a target for cancer.
  • the inventor of the present application has developed a monoclonal antibody with high affinity for cell surface CD24 after extensive research.
  • the antibody of the present invention can effectively block the binding of CD24 and its ligand (such as Siglec-10), thereby releasing the immunosuppressive signaling pathway mediated by SHP-1/SHP-2 and inducing the phagocytosis of tumor cells by macrophages. . Therefore, the antibody of the present invention has the potential to be used to treat CD24-expressing tumors and has significant clinical value.
  • the invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to CD24, the antibody or an antigen-binding fragment thereof comprising:
  • the variant has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, and VH CDR3 as shown in SEQ ID NO:5; and/or, the following three light chain variable region CDRs: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, VL CDR3 as shown in SEQ ID NO:8 ;or,
  • the three CDRs contained in the heavy chain variable region and the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 1 or a variant thereof; and/or comprising SEQ ID NO: The light chain variable region (VL) of the sequence shown in 2 or a variant thereof;
  • VH heavy chain variable region
  • VL The light chain variable region
  • the variant has one or several amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO: 1 and a VL comprising the sequence set forth in SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof is humanized.
  • the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin, which framework region may optionally comprise one or more (e.g., 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to the corresponding murine residues.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence (i.e., an amino acid sequence encoded by a human heavy chain germline gene), and a heavy chain framework sequence derived from A light chain framework region sequence of a human light chain germline sequence (i.e., an amino acid sequence encoded by a human light chain germline gene), the heavy chain framework region and/or the light chain framework region optionally comprising one or more ( For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from a human residue to the corresponding murine residue.
  • a heavy chain framework region sequence derived from a human heavy chain germline sequence i.e., an amino acid sequence encoded by a human heavy chain germline gene
  • a light chain framework region sequence of a human light chain germline sequence i.e., an amino acid sequence encoded by a human light chain germline gene
  • the heavy chain framework region and/or the light chain framework region optionally comprising one or more ( For
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 9 or a variant thereof; and/or comprising SEQ ID NO: The light chain variable region (VL) of the sequence shown in 10 or a variant thereof; wherein the variant has one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions), or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, Sequences that have at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO: 9 and a VL comprising the sequence set forth in SEQ ID NO: 10.
  • the antibodies of the invention, or antigen-binding fragments thereof may further comprise a constant region derived from a mammalian (eg, murine or human) immunoglobulin.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant derived from a mammalian (e.g., murine or human) immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3, or IgG4).
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a mammalian (eg, murine or human) immunoglobulin (eg, kappa or lambda).
  • the heavy chain of the antibody or antigen-binding fragment thereof of the invention comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof that has a Substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, 1, 2, 3, 4 or substitution, deletion or addition of 5 amino acids); and/or,
  • CH heavy chain constant region
  • the light chain of the antibody or antigen-binding fragment thereof of the invention comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions compared to the sequence from which it is derived. , deletion or addition (for example, substitution, deletion or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, substitution of 1, 2, 3, 4 or 5 amino acids, missing or added).
  • CL light chain constant region
  • the variant of the heavy chain constant region (CH) may have one or more conservative amino acid substitutions compared to the wild-type sequence from which it is derived. In such embodiments, the variant of the heavy chain constant region (CH) may have the same or substantially the same effector function as the wild-type sequence from which it is derived.
  • the variant of the heavy chain constant region may comprise one or more amino acid mutations compared to the wild-type sequence from which it is derived to alter one or more of the following of the antibodies of the invention Characteristics: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function, etc.
  • Functional changes can be produced by replacing at least one amino acid residue with a different residue in the constant region of the antibody, e.g., altering the effector response of the antibody to Affinity for a ligand (e.g. FcR or complement C1q), thereby altering (e.g. reducing) effector function.
  • the Fc region of antibodies mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.
  • the antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4), the antibody or antigen-binding fragment thereof
  • the light chain includes a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) set forth in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) set forth in SEQ ID NO: 12.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabodies, and single domain antibodies (sdAb).
  • the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.
  • the antibodies or antigen-binding fragments thereof of the invention may include variants that differ from the antibodies or antigen-binding fragments thereof from which they are derived by only one or more (e.g., up to 20 conservative substitutions of amino acid residues), or have at least 85%, at least 90%, or at least 95% similarity with the antibody or antigen-binding fragment thereof from which it is derived , at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived (e.g., specific binding to CD24 , blocking activity that blocks the binding of CD24 to its ligand (e.g., Siglec-10)).
  • variants that differ from the antibodies or antigen-binding fragments thereof from which they are derived by only one or more (e.g., up to 20 conservative substitutions of amino acid residues), or have at least 85%, at least 90%, or at least 95% similarity with the antibody
  • the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab′ fragments can be obtained directly from host cells; Fab′ fragments can be chemically coupled to form F(ab′) 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof.
  • the isolated nucleic acid molecule encodes an antibody of the invention, or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof.
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain variable region of an antibody of the invention or an antigen-binding fragment thereof, and/or encoding an antibody of the invention or an antigen-binding fragment thereof The second nucleotide sequence of the light chain variable region of the fragment.
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain of an antibody of the invention or an antigen-binding fragment thereof, and/or a first nucleotide sequence encoding a light chain of an antibody of the invention or an antigen-binding fragment thereof.
  • the second nucleotide sequence of the chain comprises a first nucleotide sequence encoding a heavy chain of an antibody of the invention or an antigen-binding fragment thereof, and/or a first nucleotide sequence encoding a light chain of an antibody of the invention or an antigen-binding fragment thereof.
  • the invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention.
  • vectors of the invention are, for example, plasmids, cosmids, phages, and the like.
  • the vector is capable of expressing an antibody of the invention or an antigen-binding fragment thereof in a subject (eg, a human).
  • the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • a method for preparing an antibody or an antigen-binding fragment thereof of the present invention comprising culturing a host cell as described above under conditions that allow expression of the antibody or an antigen-binding fragment thereof, and from the cultured The antibody or antigen-binding fragment thereof is recovered in host cell culture.
  • Antibodies of the invention or antigen-binding fragments thereof may be derivatized, for example linked to another molecule (e.g. another peptide or protein).
  • another molecule e.g. another peptide or protein.
  • derivatization e.g, labeling
  • the antibodies or antigen-binding fragments thereof of the invention are also intended to include such derivatized forms.
  • an antibody of the invention or an antigen-binding fragment thereof can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming Bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (e.g., avidin or polyhistidine tags).
  • the antibodies of the invention or antigen-binding fragments thereof can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.
  • the antibodies of the invention, or antigen-binding fragments thereof bear a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • a detectable label such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • the detectable label of the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means.
  • Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H , 125I , 35S , 14C , or 32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , Phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridinium ester compounds), Magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., poly
  • Detectable labels as described above can be detected by methods known in the art.
  • radioactive labels can be detected using photographic film or a scintillation calculator
  • fluorescent labels can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and thermometric markers are detected by simply visualizing a colored marker.
  • such labels can be adapted for immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • detectable labels as described above can be linked to the antibodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.
  • the antibodies of the invention, or antigen-binding fragments thereof can be used to form bispecific or multispecific molecules.
  • An antibody or antigen-binding fragment thereof may be part of a bispecific or multispecific molecule that contains a second function having a binding specificity different from that of the antibody or antigen-binding fragment thereof of the invention.
  • a module e.g., a second antibody capable of binding to at least two different binding sites and/or target molecules.
  • an antibody or antigen-binding fragment thereof of the invention may be linked to a second antibody or antigen-binding fragment thereof capable of specifically binding to any protein that may be used as a potential target for combination therapy.
  • the antibodies of the invention or antigen-binding fragments thereof may be linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other Binding molecules (eg, additional antibodies, antibody fragments, peptides, or binding mimetics).
  • Binding molecules e.g, additional antibodies, antibody fragments, peptides, or binding mimetics.
  • the invention provides a bispecific or multispecific molecule comprising an antibody of the invention or an antigen-binding fragment thereof.
  • the bispecific or multispecific molecule specifically binds CD24 and additionally specifically binds one or more other targets.
  • the bispecific or multispecific molecule further comprises at least one molecule (eg, a second antibody) having a second binding specificity for a second target.
  • the antibodies of the invention, or antigen-binding fragments thereof can be linked to therapeutic agents to form immunoconjugates. Because immunoconjugates have the ability to selectively deliver one or more therapeutic agents to target tissues (e.g., CD24-expressing cells), immunoconjugates can improve the performance of the antibodies of the invention or antigen-binding fragments thereof in treating diseases (e.g., CD24-expressing cells). therapeutic efficacy in cancer).
  • target tissues e.g., CD24-expressing cells
  • the invention provides immunoconjugates comprising an antibody of the invention, or an antigen-binding fragment thereof, and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the therapeutic agent is a cytotoxic agent.
  • the cytotoxic agent includes any agent that is harmful to cells (eg, kills cells).
  • the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination.
  • the antibodies of the invention, or antigen-binding fragments thereof are conjugated to the therapeutic agent, optionally via a linker.
  • cytotoxic agents can be coupled to the antibodies of the invention or antigen-binding fragments thereof using linker technologies available in the art.
  • linker types that have been used to couple cytotoxic agents to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers.
  • Linkers may be selected that are susceptible to cleavage at low pH within the lysosomal compartment or to cleavage by proteases such as cathepsins, such as cathepsins B, C, D, which are preferentially expressed in tumor tissue.
  • the antibodies of the invention or antigen-binding fragments thereof can be used to construct chimeric antigen receptors (CARs) that possess the ability to direct T-cell specificity and reactivity toward CD24-expressing cells (e.g., tumor cells) in a non-MHC-restricted manner.
  • CARs chimeric antigen receptors
  • the invention provides a chimeric antigen receptor (CAR) comprising the antigen-binding domain of an antibody of the invention or an antigen-binding fragment thereof.
  • CAR chimeric antigen receptor
  • the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of an antibody of the invention or an antigen-binding fragment thereof.
  • the antigen binding domain is a scFv.
  • the chimeric antigen receptor comprises an antigen-binding fragment (eg, scFv) of an antibody of the invention.
  • an antigen-binding fragment eg, scFv
  • the chimeric antigen receptor is expressed by immune effector cells (eg, T cells).
  • immune effector cells eg, T cells
  • the chimeric antigen receptor further includes a transmembrane domain, one or more intracellular T cell signaling domains (e.g., T cell receptor signaling domain, T cell costimulatory signaling domain, or its combination).
  • T cell receptor signaling domain refers to the portion of the CAR that contains the intracellular domain of the T cell receptor (eg, the intracellular portion of the CD3 ⁇ protein).
  • the costimulatory signaling domain refers to the part of the intracellular domain of the CAR that contains costimulatory molecules, which are cell surface molecules other than antigen receptors or their ligands required for efficient lymphocyte responses to antigens. .
  • a spacer domain comprising a polypeptide sequence may be present between the antigen-binding domain and the transmembrane domain of the CAR.
  • the spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.
  • the spacer domain may comprise an immunoglobulin domain, such as a human immunoglobulin sequence.
  • the immunoglobulin domain comprises immunoglobulin CH2 and CH3 domain sequences. In such embodiments, without being bound by a particular theory, it is believed that the CH2 and CH3 domains extend the antigen-binding domain of the CAR away from the membrane of the CAR-expressing cell and more accurately mimic the size and Domain structure.
  • the transmembrane domain can be derived from natural or synthetic sources.
  • the domain may be derived from any membrane-bound or transmembrane protein.
  • Exemplary transmembrane domains that can be used in the CAR of the invention can include at least the transmembrane region of the alpha, beta or zeta chain of a T cell receptor, which can be selected from the group consisting of CD28, CD3 ⁇ , CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain comprises a transmembrane domain of a T cell receptor, such as a CD8 transmembrane domain.
  • the transmembrane domain comprises the transmembrane domain of a T cell costimulatory molecule (eg, CD137 or CD28).
  • examples of intracellular T cell domains useful for use in the CAR include cytoplasmic sequences and costimulatory molecules of the T cell receptor (TCR)
  • TCR T cell receptor
  • the cytoplasmic sequences and costimulatory molecules cooperate to initiate signal transduction upon antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.
  • the intracellular region of the CAR may comprise a primary cytoplasmic signaling sequence that acts in a stimulatory manner, which may comprise a signal termed an immunoreceptor tyrosine-based activation motif or ITAM Motif.
  • ITAMs containing primary cytoplasmic signal sequences include those from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b, and CD66d proteins.
  • the intracellular region of the CAR may comprise an ITAM containing a primary cytoplasmic signaling domain (eg, CD3 ⁇ ) by itself or in combination with any other desired cytoplasmic domain that may be used in the context of a CAR.
  • a primary cytoplasmic signaling domain eg, CD3 ⁇
  • the cytoplasmic domain of the CAR includes a CD3 ⁇ chain portion and an intracellular costimulatory signaling domain.
  • the costimulatory signaling domain refers to the portion of the CAR that contains the intracellular domain of costimulatory molecules. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand required for efficient lymphocyte response to antigen.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, Lymphocyte Function Associated Antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3.
  • the CAR can comprise a CD3 ⁇ signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain, or any combination thereof.
  • the order of the one or more T cell signaling domains on the CAR can be changed as needed by those skilled in the art.
  • chimeric antigen-binding receptors encoding the invention can be The nucleic acid molecule is included in an expression vector (eg, a lentiviral vector) for expression in a host cell, such as a T cell, to produce the CAR.
  • an expression vector eg, a lentiviral vector
  • methods of using the chimeric antigen receptor include isolating T cells from a subject, transforming the T cells with an expression vector encoding the chimeric antigen receptor (e.g., a lentiviral vector), and converting the expressed
  • the engineered T cells of the chimeric antigen receptor are administered to the subject for treatment, for example, for treating a tumor in the subject.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor of the invention.
  • the isolated nucleic acid molecule encodes a chimeric antigen receptor of the invention.
  • the invention provides a vector (eg a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above.
  • a vector eg a cloning vector or an expression vector
  • vectors of the invention are, for example, plasmids.
  • the invention provides a host cell expressing a chimeric antigen receptor of the invention and/or comprising an isolated nucleic acid molecule or vector as described above.
  • the host cells are immune effector cells (eg, T cells and/or NK cells).
  • the host cell is a chimeric antigen receptor T cell (CAR-T).
  • the present invention provides a pharmaceutical composition, which contains the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the isolated nucleic acid molecule described in the second aspect, the third aspect Vector, the host cell described in the fourth aspect, the bispecific or multispecific molecule described in the fifth aspect, the immunoconjugate described in the sixth aspect, the chimeric antigen receptor described in the seventh aspect, the The isolated nucleic acid molecule described in the eighth aspect, the vector described in the ninth aspect, or the host cell described in the tenth aspect, and pharmaceutically acceptable carriers and/or excipients.
  • compositions of the invention comprise an antibody of the invention or an antigen-binding fragment thereof.
  • compositions of the invention comprise bispecific or multispecific molecules of the invention.
  • compositions of the invention comprise immunoconjugates of the invention.
  • the pharmaceutical composition of the present invention includes the immunoconjugate described in the sixth aspect, the chimeric antigen receptor described in the seventh aspect, the isolated nucleic acid molecule described in the eighth aspect, the ninth aspect The vector described in the aspect or the host cell described in the tenth aspect.
  • the pharmaceutical compositions may also include additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, specific targeting tumor cell antibodies, immune checkpoint inhibitors, etc.
  • a drug with anti-tumor activity such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, specific targeting tumor cell antibodies, immune checkpoint inhibitors, etc.
  • the additional pharmaceutically active agent is an immune checkpoint inhibitor.
  • the additional pharmaceutically active agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the vector or host cell and the additional pharmaceutically active agent may be provided as separate components or as mixed components. Accordingly, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, chimeric antigen receptors, isolated nucleic acid molecules, vectors or host cells of the invention may be combined with said additional pharmaceutically active agent. Administer simultaneously, separately or sequentially.
  • the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, chimeric antigen receptors, isolated nucleic acid molecules, vectors or host cells of the invention or the pharmaceutical compositions of the invention can be formulated into medicines Any dosage form known in the art, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, injections Sterile powders and concentrated solutions for injection), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical compositions of the present invention should be sterile and stable under the conditions of production and storage.
  • One preferred dosage form is an injection.
  • Such injections may be sterile injectable solutions.
  • sterile injectable solutions may be prepared by incorporating in an appropriate solvent the requisite dose of an antibody of the invention, or antigen-binding fragment thereof, and, optionally, other desired ingredients including, but not (limited to, pH adjuster, surfactant, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof), followed by filter sterilization.
  • sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution such as 0.9% (w/v) NaCl
  • Glucose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • compositions of the invention comprise sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
  • sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g., 0.9% (w/v) NaCl
  • dextrose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • Antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and chimeric antigens of the present invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ophthalmic, topical, enteral Externally, rectum, intraleaf sheath, intracytoplasmic reticulum, inguinal, intravesical, topical (eg, powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • parenteral eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection.
  • the route and/or mode of administration will vary depending on the intended purpose.
  • the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, chimeric antigen receptors, isolated nucleic acid molecules, vectors or host cells of the invention or The pharmaceutical composition is administered by intravenous injection or bolus injection.
  • the pharmaceutical composition of the present invention may include a "therapeutic effective amount” or a "preventive effective amount” of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the isolated nucleic acid molecule described in the second aspect, or the third aspect of the invention.
  • “Prophylactically effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of disease.
  • a “therapeutically effective amount” means an amount sufficient to cure or at least partially prevent disease and its complications in a patient who is already suffering from the disease.
  • the therapeutically effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other concurrent treatments administered. wait.
  • the antibody of the present invention specifically binds to human CD24 and can block the binding of CD24 to its ligand (such as Siglec-10), thereby releasing the immunosuppressive signaling pathway mediated by SHP-1/SHP-2 and inducing macrophages to respond to Phagocytosis of tumor cells, thereby providing the following therapeutic applications.
  • ligand such as Siglec-10
  • the invention provides a method for enhancing an immune response or preventing and/or treating tumors in a subject (e.g., a human), the method comprising administering to the subject an effective amount selected from The following reagents: the antibody or antigen-binding fragment thereof described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the vector described in the third aspect, the host cell described in the fourth aspect, the fifth aspect The bispecific or multispecific molecule, the immunoconjugate described in the sixth aspect, the chimeric antigen receptor described in the seventh aspect, the isolated nucleic acid molecule described in the eighth aspect, the ninth aspect
  • the present invention also relates to the use of each of the above reagents in the preparation of medicaments for enhancing immune response or preventing and/or treating tumors in a subject (such as a human). the use of.
  • the immune response includes a macrophage-mediated immune response.
  • the tumor expression involves CD24-positive tumor cells, and/or, involves CD24 ligand (eg, Siglec-10)-positive macrophages.
  • CD24 ligand eg, Siglec-10-positive macrophages.
  • the tumor can be of any tumor type that expresses CD24, including various hematological tumors as well as solid tumors.
  • the tumor is a solid tumor, such as breast cancer (e.g., triple negative breast cancer), ovarian cancer, liver cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, bladder cancer, prostate cancer, renal cell carcinoma Cancer, cervical cancer, endometrial cancer, cholangiocarcinoma, kidney cancer, nasopharyngeal cancer, thyroid cancer, brain cancer, testicular cancer, bone cancer, lymphoma or glioma, etc.
  • the tumor is a hematological neoplasm, eg, leukemia, lymphoma, myeloma.
  • Ovarian Cancer is a common gynecological malignant tumor. It is a highly heterogeneous epithelial tumor with different histological subtypes and genetic and biological characteristics, including serous carcinoma, endometrioid carcinoma, hyaline carcinoma, Cellular and mucinous carcinomas. There are 310,000 new cases of ovarian cancer and more than 200,000 deaths worldwide every year (Hyuna S, et al: Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA CANCER J CLIN 2021; 0:1 –41.). 75% of ovarian cancers are serous cancers, with a five-year survival rate of 35%. They are an extremely malignant type of cancer and are usually diagnosed at an advanced stage.
  • TNBC triple-negative breast cancer
  • CD24 is a signaling protein expressed by ovarian cancer and breast cancer cells that inhibits macrophage immune phagocytosis. Ovarian cancer and triple-negative breast cancer are greatly affected by CD24 signaling blockade (Amira A. Barkal, et al :Weissman CD24 signaling through macrophage Siglec-10 is a target for cancer. Immunotherapy 2019;572:392-399.).
  • CD24 is overexpressed far more than CD47 and PD-L1 in the above two tumor samples; the CD24 antibody has a stronger effect on sensitizing macrophage phagocytosis in the two tumor cell lines than the CD47 antibody; CD24 gene knockout or CD24 antibody in small It can effectively inhibit tumor growth in mouse tumor models.
  • the above relevant data Reflecting the immune activation role of CD24 as a target in ovarian cancer and triple-negative breast cancer, the development of anti-CD24 antibody drugs is expected to provide effective clinical drugs in the treatment of solid tumors including ovarian cancer and triple-negative breast cancer.
  • the tumor is preferably ovarian or breast cancer (eg, triple negative breast cancer).
  • the method further includes administering a second therapeutic agent (e.g., a drug with anti-tumor activity) or a second treatment (e.g., surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy , gene therapy or palliative care).
  • a second therapeutic agent e.g., a drug with anti-tumor activity
  • a second treatment e.g., surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy , gene therapy or palliative care.
  • the second therapeutic agent is an immune checkpoint inhibitor.
  • the second therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the invention provides a method for killing or depleting CD24-positive cells in a subject (e.g., a human), the method comprising administering to the subject an effective amount of an agent selected from: : The antibody or antigen-binding fragment thereof according to the first aspect, the isolated nucleic acid molecule according to the second aspect, the vector according to the third aspect, the host cell according to the fourth aspect, the bispecific protein according to the fifth aspect.
  • an agent selected from: : The antibody or antigen-binding fragment thereof according to the first aspect, the isolated nucleic acid molecule according to the second aspect, the vector according to the third aspect, the host cell according to the fourth aspect, the bispecific protein according to the fifth aspect.
  • the present invention also
  • the CD24-positive cells are tumor cells. Therefore, the method can be used for tumor treatment.
  • an agent provided by the invention selected from the following is administered in combination with a second therapeutic agent or therapy: the antibody or antigen-binding fragment thereof described in the first aspect, the isolated antibody described in the second aspect Nucleic acid molecules, vectors according to the third aspect, host cells according to the fourth aspect, bispecific or multispecific molecules according to the fifth aspect, immunoconjugates according to the sixth aspect, immunoconjugates according to the seventh aspect
  • the second therapeutic agent or treatment may be administered before, simultaneously with, or after the administration of the above-described agent of the invention.
  • the second therapeutic agent is selected from drugs with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • drugs with anti-tumor activity such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • the second therapeutic agent is an immune checkpoint inhibitor.
  • the second therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the second treatment may be any therapy known for tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.
  • the antibody or antigen-binding fragment thereof of the present invention can specifically bind to CD24 and thus can be used to detect the presence or level of CD24 in a sample.
  • the invention provides a kit comprising an antibody of the invention or an antigen-binding fragment thereof.
  • the antibodies of the invention, or antigen-binding fragments thereof are detectably labeled.
  • the kit further includes a second antibody that specifically recognizes an antibody of the invention or an antigen-binding fragment thereof.
  • the second antibody further includes a detectable label.
  • the detectable label may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels are suitable for immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).
  • immunological detection eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.
  • the invention provides a method for detecting the presence or amount of CD24 in a sample, comprising the following steps:
  • the formation of the complex indicates the presence of CD24 or CD24-expressing cells.
  • the antibodies of the invention, or antigen-binding fragments thereof further bear a detectable label.
  • an antibody or antigen-binding fragment thereof of the invention is detected using a detectably labeled reagent.
  • the method may be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).
  • the CD24 is human CD24.
  • the methods are used to diagnose whether a subject has a CD24-expressing tumor or to detect whether a tumor is Can it be treated with anti-tumor therapies targeting CD24?
  • the method may further comprise the step of comparing the amount of CD24 in a sample from the subject to a reference value.
  • the reference value refers to the level of CD24 in a sample from a subject known not to have a tumor that expresses CD24 (also referred to as a "negative reference value”), or to the level of CD24 in a sample from a subject known to have a tumor that expresses CD24.
  • the level in the subject's sample also called the "positive reference value”
  • the amount of CD24 in a sample from the subject is similar to (or not significantly different from) a negative reference value, it indicates that the subject does not have a CD24-expressing tumor, and if the CD24 An increase in the amount in a sample from the subject relative to a negative reference value is an indication that the subject has a CD24-expressing tumor.
  • the amount of CD24 in a sample from the subject is similar to (or not significantly different from) a positive reference value, this indicates that the subject has a CD24-expressing tumor.
  • the sample may be selected from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy or fine needle aspiration tissue), histological preparations, etc.
  • the methods further comprise administering a CD24-targeted immunotherapy to a subject diagnosed with a CD24-expressing tumor.
  • the tumor can be of any tumor type that expresses CD24, including various hematological tumors as well as solid tumors.
  • the tumor is a solid tumor, such as breast cancer (e.g., triple negative breast cancer), ovarian cancer, liver cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, bladder cancer, prostate cancer, renal cell carcinoma Cancer, cervical cancer, endometrial cancer, cholangiocarcinoma, kidney cancer, nasopharyngeal cancer, thyroid cancer, brain cancer, testicular cancer, bone cancer, lymphoma or glioma, etc.
  • the tumor is a hematological neoplasm, eg, leukemia, lymphoma, myeloma.
  • the use of the antibody or antigen-binding fragment or kit thereof of the present invention in preparing a detection reagent is provided, and the detection reagent is used to determine the presence or amount of CD24 in a sample, and to diagnose whether a subject is suffering from the disease.
  • the detection reagent is used to determine the presence or amount of CD24 in a sample, and to diagnose whether a subject is suffering from the disease.
  • an antibody in its broadest sense refers to molecules that specifically bind to an antigenic determinant and may include a variety of antibody structures so long as they exhibit the desired antigen-binding activity.
  • an antibody may be an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 Definition of -917; Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • the variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof are preferably determined by the Kabat, Chothia, or IMGT numbering systems.
  • the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof are preferably determined by the Kabat numbering system.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • antibody is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
  • an antigen-binding moiety which is also called an "antigen-binding moiety.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, di- scFv, (scFv) 2 , and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • the term “Fd” means an antibody fragment consisting of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains;
  • the term “F(ab') 2 fragment” means an antibody fragment consisting of two fragments connected by a disulfide bridge on the hinge region An antibody fragment of a Fab fragment;
  • the term “Fab'fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd of the heavy chain. Fragment (consisting of VH and CH1 domains).
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
  • Fc means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody.
  • scFv can form (scFv) 2 , which refers to two or more individual scFvs joining in parallel to form an antibody.
  • the term "diabody” means one whose VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, This forces the domain to pair with the complementary domain of the other chain and creates two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable An antibody fragment consisting of a region) that retains the ability to specifically bind to the same antigen that the full-length antibody binds.
  • Single domain antibodies are also called nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibodies are screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
  • chimeric antibody refers to an antibody in which a portion of the light chain or/and heavy chain is derived from an antibody (which may originate from a specific species or belong to a specific species). a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but regardless of However, it still retains binding activity to the target antigen.
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and The heavy and light chain constant regions of the antibody are derived from a secondary antibody (eg, a human antibody).
  • a first antibody e.g., a murine antibody
  • a secondary antibody e.g., a human antibody
  • humanized antibody refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology to that of a human antibody.
  • CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) comes from a human source.
  • Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc.
  • the donor antibody can be a mouse, rat, rabbit, or non-human primate (eg, cynomolgus monkey) antibody with desired properties (eg, antigen specificity, affinity, reactivity, etc.).
  • germline antibody gene or “germline antibody gene segment” refers to an immunoglobulin-encoding sequence present in the genome of an organism , which have not undergone the maturation process that leads to genetic rearrangements and mutations that lead to the expression of specific immunoglobulins.
  • expression “heavy chain germline gene” refers to the germline antibody gene or gene fragment encoding the immunoglobulin heavy chain, which includes V gene (variable), D gene (diversity), and J gene (joining).
  • germline gene refers to the germline antibody gene or gene fragment encoding the immunoglobulin light chain, which includes V gene (variable), J gene (joining) and C gene (constant).
  • amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also called a "germline sequence”.
  • Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences Columns are well known to those skilled in the art and can be obtained or queried from professional databases (eg IMGT, UNSWIg, NCBI or VBASE2).
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473).
  • K D , kon and kdis values can be measured by any valid method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • chimeric antigen receptor refers to a recombinant polypeptide construct comprising at least one extracellular antigen-binding domain, spacer domain, transmembrane domain, and intracellular signaling domain , which combines antibody-based specificity for an antigen of interest (eg, CD24) with an immune effector cell-activating intracellular domain to exhibit specific immune activity against cells expressing the antigen of interest (eg, CD24).
  • an antigen of interest eg, CD24
  • an immune effector cell-activating intracellular domain to exhibit specific immune activity against cells expressing the antigen of interest (eg, CD24).
  • CD24 an antigen of interest
  • CD24 an antigen of interest
  • CD24 an immune effector cell-activating intracellular domain
  • immune effector cell refers to any cell of the immune system having one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC) .
  • effector cells are cells that are of hematopoietic origin and play a role in immune responses.
  • effector function refers to a specialized function of an immune effector cell, such as a function or response that enhances or promotes an immune attack on a target cell (eg, kills the target cell, or inhibits its growth or proliferation).
  • the effector function of a T cell may, for example, be cytolytic activity or auxiliary or activity including secretion of cytokines.
  • immune effector cells include T cells (eg, alpha/beta T cells and gamma/delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.
  • cytotoxic agent includes any agent that is harmful to cells (eg, kills cells), such as chemotherapeutic drugs, bacterial toxins, phytotoxins, or radioactive isotopes, and the like.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyoma vacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to,
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes cells, NK cells, monocytes, macrophages or dendritic cells, etc.). Host cells can include single cells or populations of cells.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each polypeptide is occupied by lysine)
  • Percent identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared ⁇ 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions).
  • comparisons are made when two sequences are aligned to yield maximum identity.
  • Such alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17) integrated into the ALIGN program (version 2.0) (1988)), uses the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine percent identity between two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm can be used using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • dextrose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom (eg, tumor) in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no further worsening) of the disease state, delaying or slowing the progression of the disease, ameliorating or alleviating the symptoms of the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject eg, human
  • the present invention provides monoclonal antibodies with high affinity for cell surface CD24, which can effectively block CD24 Binding to its ligand (e.g., Siglec-10) relieves the immunosuppressive signaling pathway mediated by SHP-1/SHP-2 and induces macrophage phagocytosis of tumor cells. Therefore, the antibody of the present invention has the potential to be used to treat CD24-expressing tumors and has significant clinical value.
  • its ligand e.g., Siglec-10
  • Figure 1A shows the flow cytometric evaluation results of measuring the binding activity of mouse antibody 20G10 to MCF7.
  • Figure 1B shows the flow cytometric evaluation results of measuring the binding activity of mouse antibody 20G10 to Huh.
  • Figure 2A shows the flow cytometric evaluation results of measuring the binding activity of chimeric antibody ch20G10 to MCF7.
  • Figure 2B shows the results of ELISA evaluation to determine the binding activity of chimeric antibody ch20G10 to recombinantly expressed CD24.
  • Figure 2C is an ELISA evaluation result for measuring the binding activity of chimeric antibody ch20G10 to chemically synthesized CD24 polypeptide.
  • Figure 3A is a flow cytometric evaluation result of measuring the binding activity of humanized antibody GB7011-ORG1 to HepG2 cells that naturally express human CD24.
  • Figure 3B is a flow cytometric evaluation result of measuring the binding activity of humanized antibody GB7011-ORG1 to Huh7 cells that naturally express human CD24.
  • Figure 3C is a flow cytometric evaluation result of measuring the binding activity of humanized antibody GB7011-ORG1 to MCF7 cells that naturally express human CD24.
  • Figure 4 shows the blocking activity of humanized antibody GB7011-ORG1 in blocking the binding of CD24 on the cell membrane surface to its receptor protein Siglec10.
  • Figure 5A shows the phagocytosis of NALM6 tumor cells by M1 macrophages induced by humanized antibody GB7011-ORG1.
  • Figure 5B shows the phagocytosis of NALM6 tumor cells by M2 macrophages induced by humanized antibody GB7011-ORG1.
  • Figure 6 shows the weight of mouse tumors in each group of animals in Example 10 after 7 days of administration and sacrifice.
  • Figure 7 shows the proportion of PD1-positive T cells (Tc cells, Th cells) after anti-CD24 antibody treatment in Example 11.
  • Figure 8 shows the volume changes of mouse tumors 21 days after administration to each group of animals in Example 11.
  • Table 1 Information on the sequences covered by this application is described in the table below.
  • the lentivirus was provided by Shanghai Jikai Gene Medical Technology Co., Ltd. After the cells were infected for 72 hours, corresponding antibiotics were added and cultured for 2-4 weeks, amplified and cryopreserved to obtain HEK293-CD24 and CHOS-CD24 cells for subsequent experiments. .
  • the constructed CHOS-CD24 cells overexpressing human CD24 were used to immunize Balb/c mice (Beijing Vital River Experimental Animal Technology Co., Ltd., strain code 216); the primary immunization adjuvant was completely free. His adjuvant CFA (InvivoGen Company, product number vac-cfa-60), and subsequent immune adjuvants use IFA (InvivoGen Company, product number vac-ifa-60); the immune route is subcutaneous multiple points.
  • the spleen cells of the immunized mice were fused with mouse myeloma cells SP2/0 using the polyethylene glycol method to obtain B cell fusions that can express antibodies and proliferate indefinitely in vitro, and were selectively cultured in HAT Cultured in the substrate.
  • the fused hybridoma cells were plated in a 96-well cell culture plate, and the antibodies in the supernatant were used to bind CD24 at the cellular level.
  • Naturally expressing human breast cancer tumor cells MCF7, CD24-overexpressing HEK293-CD24, and CD24-negative cells HEK293
  • the target positive clones antibodies that bind MCF7 and HEK293-CD24, but do not bind HEK293
  • the target positive clones are screened out and subjected to 2-3 rounds of subcloning to obtain monoclonal cell lines.
  • High-throughput screening of mouse anti-binding cell levels Human CD24-expressing cells and negative control cells (MCF7/HEK293-CD24; HEK293) were plated separately in the screening. Dilute 10,000 cells into 100 ⁇ L of complete culture medium, use a flat-bottomed 96-well plate, allow the cells to adhere or sink to the bottom of the well overnight, and remove the supernatant the next day. Add 100 ⁇ L of hybridoma supernatant to be screened to the cell plate and incubate at room temperature for 1 hour.
  • the imaging obtained by the fluorescence channel counts the antibody-bound cells according to the fluorescently labeled cell morphology and fluorescence intensity setting parameters.
  • the imaging obtained by the brightfield channel counts adherent cells according to the cell morphology setting parameters, and then the two sets of data are compared.
  • the percentage of cells showing fluorescence that binds to the antibody to the total number of cells is obtained. Based on this ratio, the binding effect of the antibody in the fusion tumor supernatant to CD24-expressing cells was determined.
  • the data of murine antibody 20G10-derived fusion tumor supernatant binding to several cells in high-throughput screening are shown in Table 2.
  • VH and VL sequences of the mouse-derived antibodies are shown in Table 3. Further, the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647-669 page), the CDR sequence of the mouse monoclonal antibody was determined.
  • variable region of the mouse antibody was connected to the constant region of the human antibody (human IgG4, whose heavy chain constant region and light chain constant region amino acid sequences are shown in SEQ ID NO: 11 and 12 respectively) , construct chimeric antibody ch20G10, and transfect the expression vector containing the antibody gene into mammalian cells.
  • the supernatant of mammalian cells containing antibody clones grown in culture flasks was harvested, purified using a protein A column, and the antibody protein was eluted using 100mM acetic acid pH 3.0. The purified antibody protein is then loaded onto a size exclusion chromatography column for further separation and purification.
  • Antibody proteins corresponding to the monomers were formulated in PBS buffer supplemented with 20% glycerol.
  • Plates were washed 3 times in wash buffer 10, and HRP-conjugated goat anti-human IgG Fc fragment secondary antibody was added for 1 hr at room temperature to detect bound anti-CD24 antibodies.
  • the plate was washed 3 times in wash buffer and bound secondary antibodies were revealed by adding TMB (HRP substrate) and incubating the plate in the dark at room temperature for 5 to 10 minutes.
  • the enzymatic reaction was stopped by adding 1M sulfuric acid solution and the light absorption was measured at 450 nm. Draw a curve with the light absorption value as the ordinate and the logarithm of antibody concentration as the abscissa, and use GraphPad Prism software to calculate EC50.
  • chimeric antibody ch20G10 has good binding activity to both recombinantly expressed CD24 (Figure 2B) and chemically synthesized CD24 polypeptide ( Figure 2C).
  • the mouse antibody provided in the above embodiments can be humanized designed and prepared using methods known in the art.
  • Mouse CDR regions were inserted into human framework sequences (see Winter, U.S. Patent Nos. 5,225,539; Queen et al., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols , volume 248, Humana Press, New Jersey, 2004).
  • the heavy chain and light chain CDR regions of the mouse antibody 20G10 were transplanted onto the FR framework of the corresponding humanized template, and a series of back mutations were performed on the amino acid residues in the FR region of the humanized template. , so that the humanized antibody retains the antigen-binding ability of the mouse antibody as much as possible.
  • the inventors prepared a humanized antibody of murine antibody 20G10, named GB7011-ORG1 (the amino acids of its heavy chain variable region and light chain variable region The sequences are shown in SEQ ID NO:9 and 10 respectively).
  • the heavy chain constant region of the antibody is SEQ ID NO: 11, and the light chain constant region is SEQ ID NO: 12.
  • Antibodies block the binding of Siglec10 to CD24 using fluorescently labeled Siglec-10 protein (ACRO biosystems catalog number SI0-HP2E5) as the receptor and HEK293T-CD24 cells (high-expressing human CD24; human embryonic kidney cell line) as the ligand , titrations were performed using flow cytometry measurements.
  • the specific detection steps are to place 100,000 HEK293T-CD24 cells in 50 ⁇ L FACS buffer (PBS+2% FBS)/well for use.
  • the experiment uses a round-bottom low-adsorption 96-well plate; the antibody sample is diluted in FACS buffer (use ISO as Negative control antibody), add 50 ⁇ L of diluted antibody to each well of the cell plate, add 50 ⁇ L of FACS buffer to the corresponding negative control well, and incubate at 4°C for 1 hour; after the first stage of incubation is completed, add 50 ⁇ L of fluorescently labeled Siglec to each well.
  • FACS buffer use ISO as Negative control antibody
  • the ratio of the antibody blocking binding 100 ⁇ [(max – PE value) / max].
  • the higher the ratio the higher the ratio of the measured anti-CD24 antibody resistance.
  • the lower the value the better the ability of the measured anti-CD24 antibody to block the binding of Siglec10 protein to CD24 high-expressing cell lines.
  • GB7011-ORG1 can effectively block the binding of CD24 on the cell membrane surface to its receptor protein Siglec10. The results are shown in Figure 4.
  • Example 8 Anti-CD24 antibody induces phagocytosis of tumor cells by macrophages
  • PBMC cells derived from healthy people, purchased from Miaoshun Biotechnology
  • IFN ⁇ nearshore protein, C014
  • M-CSF nearshore protein, C417) was added to stimulate for 7 days, and IL4 (nearshore protein, GMP-CD03) + IL10 (R&D, 217-IL-025) was used to stimulate for two days to obtain M2 macrophages.
  • Collect the cells by centrifugation at 1000 rpm for 5 minutes, remove the supernatant, add 5ug/ml APC-anti CD11b antibody (Biolegend, 101212) and incubate at 4°C for 30 minutes. Collect the cells by centrifugation at 1000 rpm for 5 minutes, remove the supernatant, wash twice with DPBS, and use a flow cytometer (BD Company, model CantoII) to detect the cells in the experimental plate.
  • APC-anti CD11b antibody Biolegend, 101212
  • the cell positions were first circled based on FCS and SSC, and then the red fluorescent channel (APC) corresponding to CD11b-labeled macrophages and the green fluorescent channel (FITC) corresponding to CFSE-labeled NALM6 cells were selected to analyze the cells.
  • APC red fluorescent channel
  • FITC green fluorescent channel
  • the proportion of cells is determined It means the percentage of NALM6 tumor cells that do not undergo phagocytosis. The higher the proportion of double-positive cells, the better the ability of the anti-CD24 antibody to induce phagocytosis, and vice versa.
  • the phagocytosis of NALM6 tumor cells by M1 macrophages and M2 macrophages induced by anti-CD24 antibody GB7011-ORG1 is shown in Figure 5A (M1) and Figure 5B (M2), in which Anti-CD47 5F9 is recombinantly expressed.
  • the antibody sequence is derived from Magrolimab (Forty Seven).
  • Example 9 Anti-CD24 antibody and anti-PD1 antibody synergistically induce cytokine secretion
  • GB7011-ORG1 and anti-PD1 antibody Pieriszumab
  • the specific detection steps are: culture monocytes (TPCS, A19K207021(#21)/A19K349043(#43)) with 50ng/ml recombinant M-CSF (nearshore protein, C417) for 6 days to produce monocyte-derived macrophages.
  • MCF7 cells 200,000/well starved for 24 hours were added for stimulation, and then allogeneic CD4+ T cells (200,000/well) were added to macrophages together with antibodies and continued to be cultured for 6 days. The supernatant was collected for use.
  • cytokine IFN ⁇ As shown in Table 4, the release of cytokine IFN ⁇ under the combination of anti-CD24 antibody GB7011-ORG1 and anti-PD1 antibody increased compared with the use of PD1 antibody alone. These data indicate that the anti-CD24 antibody GB7011-ORG1 combined with the anti-PD1 antibody induces T cell activation and increased cytokine secretion.
  • Example 10 Anti-tumor efficacy of anti-CD24 antibody alone in mouse tumor model
  • B6-Siglec10 humanized mice (Southern model) were subcutaneously transplanted with MC38-hCD24 colorectal cancer cells to establish the MC38-hCD24 colorectal cancer model.
  • the average tumor volume was about 130-132mm 3 .
  • the tumor-bearing mice were divided into 4 groups using random block method, including group 1ISO CTRL 3mg/kg, group 2GB7011-ORG1 3mg/kg, and group 3GB7011-ORG1. 1mg/kg and group 4GB7011-ORG1 0.3mg/kg, groups 1/2/3/4, 6 animals in each group.
  • Each group was administered drugs through the tail vein. The drugs were administered once after grouping and observed for one week.
  • the tumor volume of group 1 was 368.44 ⁇ 51.64mm 3 and that of group 2 was 101.02 ⁇ 17.61mm 3 .
  • the tumor volume inhibition rate (TGI) was 112.30%. Compared with the tumor volume of control group 1, there was a significant difference.
  • Statistical difference P ⁇ 0.01; the tumor volumes of groups 3 and 4 were 276.29 ⁇ 67.19mm 3 and 272.42 ⁇ 77.33mm 3 respectively, and the tumor volume inhibition rates (TGI) were 38.60% and 40.69%, which were significantly lower than those of the control group 1.
  • the animals were After euthanasia, the tumor mass was removed and weighed.
  • the average tumor volume in group 1 was 0.2523 ⁇ 0.0667g, and the average tumor weight in group 2 was 0.0573 ⁇ 0.0090g.
  • the tumor weight inhibition rate was 77.31%.
  • the average tumor weights of groups 3 and 4 were 0.2755 ⁇ 0.0580g and 0.2090 ⁇ 0.0576g, and the tumor weight inhibition rates were -9.19% and 17.17% respectively.
  • P>0.05 Figure 6 shows the comparison of the weight of mouse tumors in the four groups of animals after 7 days of administration. It shows that a single administration of GB7011-ORG1 3mg/kg can effectively inhibit the growth of mouse tumors.
  • GB7011-ORG1 1mg/kg and GB7011-ORG1 Although no statistical TGI was observed with a single dose of 0.3 mg/kg, individual mice responded to treatment.
  • Example 11 Anti-tumor efficacy of anti-CD24 antibody combined with anti-PD1 antibody in mouse tumor model
  • TILs tumor tissue infiltrating lymphocytes
  • B6-Siglec10 humanized mice (Southern model) were subcutaneously transplanted with MC38-hCD24 colorectal cancer cells to establish the MC38-hCD24 colorectal cancer model.
  • the average tumor volume was about 88-92mm 3 .
  • the tumor-bearing mice were divided into 2 groups using random block method, including group 1 ISO CTRL 5mg/kg and group 2GB7011-ORG1 5mg/kg, with 6 rats in each group. .
  • Each group was administered via tail vein, and the drugs were administered once a week after grouping.
  • the tumor volume and tumor growth inhibition percentage on days 0/3/7/10/14/17/21 after the start of administration are shown in Table 5.
  • GB7011-ORG1 showed better tumor growth inhibition in the early stage.
  • the animals were sacrificed on the 21st day after administration.
  • the mouse tumors were removed, weighed, cut into small pieces, ground, and processed through a filter to obtain a single-cell suspension.
  • the red blood cell lysate was added and washed twice with FACS buffer. 500,000 Place the cells in FACS buffer for later use. Use a 2 ml experimental tube.
  • Fc receptor blocker (biolegend, catalog number 422301) is diluted in FACS buffer. Add the diluted blocker to the tube and incubate at room temperature for 5 minutes.
  • a flow cytometer (BD Company, model CantoII) was used to measure and read the cells in the experimental tube. During the measurement, the cell positions were first circled based on FCS and SSC, then viable cells were circled based on L/D, CD45-positive cells were circled based on CD45, and T cells were circled based on CD3. The results are shown in Figure 7. TIL analysis showed anti-CD24 treatment After the T cell population increased.
  • B6-Siglec10 humanized mice (Southern model) were subcutaneously transplanted with MC38-hCD24 colorectal cancer cells to establish the MC38-hCD24 colorectal cancer model. On the 5th day after vaccination, the average tumor volume was approximately 88-90mm 3 .
  • the tumor-bearing mice were divided into 3 groups using random block method, including Group 1 ISO CTRL 3+3mg/kg, Group 2 anti-PD1 antibody 3mg/kg and Group 2. 3 Anti-PD1 antibody 3mg/kg was used in combination with GB7011-ORG1 3mg/kg, groups 1/2/3 had 6 animals in each group.
  • GB7011-ORG1 is administered through the tail vein, once a week after grouping, and the anti-PD1 antibody is administered intraperitoneally, twice a week.
  • the tumor volume of group 1 was 303.67 ⁇ 31.11mm 3
  • that of group 2 was 253.46 ⁇ 92.36mm 3
  • that of group 3 was 62.06 ⁇ 7.93mm 3
  • the tumor volume inhibition rates (TGI) were 23.07% and 112.22 respectively. %.
  • Example 12 Evaluation of antibody GB7011-ORG1 specificity using cell microarray technology (Retrogenix)
  • ZsGreen1 green fluorescent protein
  • the protein to be tested including 6052 full-length human plasma membrane proteins (secreted or cell surface-tethered human secreted proteins) and 397 human heterodimers. Please see Appendix 1-2 for specific targets.
  • vector was placed on a microarray slide and reverse transfected/expressed with human HEK293 cells, and then added to the slide containing 0.5 ⁇ g/mL GB7011-ORG1, 1 ⁇ g/mL anti-CD20 rituximab biosimilar ( Positive control) or no test molecule (negative control), detect AF647 fluorescence signal after incubation with secondary antibody (AF647 anti-hIgG Fc).
  • the binding strength of the antibody to the antigen to be tested on the cell surface was evaluated by fluorescence analysis and quantification (ImageQuant software, GE healthcare, Version 8.2).
  • ImageQuant software GE healthcare, Version 8.2
  • a total of 30 proteins were selected from the library that may bind to GB7011-ORG1, with binding strengths ranging from very weak to very strong (Table 6).
  • a secondary verification was conducted for these 30 targets, using the same method as the primary screening. In the secondary verification, after cells expressing 30 proteins were fixed, 23 proteins (marked "*" in Table 6) were consistent with GB7011-ORG1 and the control antibody (anti-CD20 rituximab biosimilar). Binding is classified as non-specific binding.
  • the binding may be mediated by the Fc domain, such as Fc ⁇ receptors (FCGR1A, FCGR2A(isoform2), FCGR2A(isoform1), FCGR2A+FCER1G and FCGR2A+CD247); or the protein to be tested may be directly combined with a fluorescent secondary antibody, such as IGHG( IGHG1, IGHG2, IGHG3 and IGHG4).
  • Fc ⁇ receptors FCGR1A, FCGR2A(isoform2), FCGR2A(isoform1), FCGR2A+FCER1G and FCGR2A+CD247
  • a fluorescent secondary antibody such as IGHG( IGHG1, IGHG2, IGHG3 and IGHG4
  • 6 proteins (XG, PRR7, SLC38A4, MYMK, ST6GAL1 and EDN1) have very weak binding to the test antibodies, and the signal level is extremely low and can be ignored.
  • CD24 was the only protein that showed specific binding to the test antibody and

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Abstract

提供了抗CD24的抗体或其抗原结合片段，编码所述抗体或其抗原结合片段的核酸分子、载体及宿主细胞，所述抗体或其抗原结合片段的衍生物，以及它们用于疾病治疗的用途。

Description

特异性结合CD24的抗体及其用途 技术领域
本发明涉及疾病治疗及免疫学领域，具体而言，本发明涉及抗CD24的抗体或其抗原结合片段，编码所述抗体或其抗原结合片段的核酸分子、载体及宿主细胞，所述抗体或其抗原结合片段的衍生物，以及它们用于疾病治疗的用途。
背景技术
巨噬细胞是先天性免疫的重要组成部分，在人体内吞噬衰老和死亡细胞起到清洁人体的作用。近年来，先天性免疫在抗肿瘤的作用受到重视，尤其是巨噬细胞，其在肿瘤组织中可以通过吞噬作用清除肿瘤细胞，并在其表面呈递肿瘤细胞相关抗原从而进一步激活免疫系统。
CD47是巨噬细胞免疫激活相关靶点，随着研究的不断深入，多个CD47的单一疗法和联合疗法进入临床试验阶段，其中FortySeven的Hu5F9目前处于临床三期，另有5个药品在进行临床二期实验(Zhongxing Jiang,et al:Targeting CD47 for cancer immunotherapy.J.Hematol.Oncol.2021；14:180-202.)。针对这个靶点的药物在临床阶段观察到了清晰的单药疗效，但是该靶点在红细胞中的高表达引起一个重要的安全性问题，靶向CD47的药物可能破坏红细胞导致严重的血液毒性，引发贫血和血小板减少等副作用(Zhongxing Jiang,et al:Targeting CD47 for cancer immunotherapy.J.Hematol.Oncol.2021；14:180-202.)。
CD24是一种高度糖基化糖磷脂酰肌醇(glycolphosphatidylinositol，GPI)锚定的细胞表面蛋白。高度糖基化的CD24通过磷脂酰肌醇(glycosyl-phoshphatidyl-inositol，GPI)锚定在细胞膜内的脂质筏上，作为细胞连接位点上分子间相互作用的媒介，介导细胞间、细胞和基质间的黏附。CD24参与细胞的识别、活化、信号转导、细胞增殖与分化、细胞的伸展与运动等。CD24与先天免疫系统的巨噬细胞上的siglec10相互作用，抑制对感染、脓毒症、肝损伤和慢性移植物抗宿主病的破坏性炎症反应(Guo-Yun Chen,et al:CD24 and Siglec-10 Selectively Repress Tissue Damage-Induced Immune Responses.Science 2009；323(5922):1722–1725.)。
CD24是一种与CD47相似的信号蛋白，在多种人类癌症中过表达，其受体Siglec-10 在肿瘤相关巨噬细胞上表达，CD24与肿瘤相关巨噬细胞表面的Siglec-10结合，激活SHP-1/SHP-2介导的抑制性信号通路，从而阻断Toll样受体介导的炎症和巨噬细胞吞噬细胞所需要的细胞骨架重排，引起肿瘤微环境中巨噬细胞的抑制，实现了肿瘤的免疫逃逸(Amira A.Barkal,et al:Weissman CD24 signalling through macrophage Siglec-10 is a target for cancer.Immunotherapy 2019；572:392-399.；Shan-Shan Yin,et al:Molecular Mechanism of Tumor Cell Immune Escape Mediated by CD24/Siglec-10.Front.Immunol.2020；11:1324.)。敲除CD24或Siglec-10，以及使用CD24单克隆抗体来阻断CD24与Siglec-10的相互作用，都可以强有力地增强实验中巨噬细胞对所有表达CD24的人类肿瘤细胞的吞噬能力。在移植了人类癌细胞的小鼠中，阻断CD24可恢复巨噬细胞对癌症的攻击(Amira A.Barkal,et al:Weissman CD24 signalling through macrophage Siglec-10 is a target for cancer.Immunotherapy 2019；572:392-399.)。这些证据表明，CD24是一个非常有前景的免疫疗法新靶点，CD24抗体有望成为巨噬细胞免疫检查点抑制剂家族的新力量。
发明内容
本申请的发明人经过大量的研究，开发了对细胞表面CD24具备高亲和力的单克隆抗体。本发明的抗体能够有效阻断CD24与其配体(如，Siglec-10)的结合，从而解除SHP-1/SHP-2介导的免疫抑制性信号通路，诱导巨噬细胞对肿瘤细胞的吞噬作用。因此，本发明的抗体具有用于治疗CD24表达肿瘤的潜力，具有重大的临床价值。
本发明的抗体
在第一方面，本发明提供了能够特异性结合CD24的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
包含如SEQ ID NO:3或其变体所示的VH CDR1、如SEQ ID NO:4或其变体所示的VH CDR2以及如SEQ ID NO:5或其变体所示的VH CDR3的重链可变区(VH)；和/或，包含如SEQ ID NO:6或其变体所示的VL CDR1、如SEQ ID NO:7或其变体所示的VL CDR2以及如SEQ ID NO:8或其变体所示的VL CDR3的轻链可变区(VL)；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。
在某些实施方案中，所述的置换为保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)如下的三个重链可变区CDRs：如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:4所示的VH CDR2、如SEQ ID NO:5所示的VH CDR3；和/或，如下的三个轻链可变区CDRs：如SEQ ID NO:6所示的VL CDR1、如SEQ ID NO:7所示的VL CDR2、如SEQ ID NO:8所示的VL CDR3；或，
(b)如下的三个重链可变区CDRs：SEQ ID NO:1或9所示的重链可变区中含有的VH CDR1、VH CDR2和VH CDR3；和/或，如下的三个轻链可变区CDRs：SEQ ID NO:2或10所示的轻链可变区中含有的VL CDR1、VL CDR2和VL CDR3。
在某些实施方案中，所述重链可变区中含有的3个CDR和所述轻链可变区中含有的3个CDR由Kabat、Chothia或IMGT编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含SEQ ID NO:1所示的序列或其变体的重链可变区(VH)；和/或，包含SEQ ID NO:2所示的序列或其变体的轻链可变区(VL)；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:1所示的序列的VH和包含如SEQ ID NO:2所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段是人源化的。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的框架区序列，所述框架区可以任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至相应的鼠源残基的回复突变。
在某些实施方案中，所述抗体或其抗原结合片段包含：来源于人重链胚系序列(即，人重链胚系基因所编码的氨基酸序列)的重链框架区序列，以及来源于人轻链胚系序列(即，人轻链胚系基因所编码的氨基酸序列)的轻链框架区序列,所述重链框架区和/或轻链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至相应的鼠源残基的回复突变。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含SEQ ID NO:9所示的序列或其变体的重链可变区(VH)；和/或，包含SEQ ID NO:10所示的序列或其变体的轻链可变区(VL)；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:9所示的序列的VH和包含如SEQ ID NO:10所示的序列的VL。
在某些实施方案中，本发明的抗体或其抗原结合片段可以进一步包含来源于哺乳动物(例如，鼠或人)免疫球蛋白的恒定区。在某些实施方案中，所述抗体或其抗原结合片段的重链包含来源于哺乳动物(例如，鼠或人)免疫球蛋白(例如IgG，如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗体或其抗原结合片段的轻链包含来源于哺乳动物(例如，鼠或人)免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，本发明的抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，
本发明的抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。
在一些实施方案中，所述重链恒定区(CH)的变体与其所源自的野生型序列相比可以具有一个或多个氨基酸的保守置换。在此类实施方案中，所述重链恒定区(CH)的变体与其所源自的野生型序列相比可以具有相同或基本相同的效应子功能。
在另一些实施方案中，所述重链恒定区(CH)的变体与其所源自的野生型序列相比可以包含一个或多个氨基酸突变以改变本发明抗体的下列中的一个或更多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能等。可以通过将抗体恒定区中的至少一个氨基酸残基替换为不同残基，产生功能改变，例如，改变抗体对效应子 配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低)。抗体的Fc区介导几种重要的效应子功能，例如ADCC、吞噬作用、CDC等。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的恒定区。在某些实施方案中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:11所示的重链恒定区(CH)。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:12所示的轻链恒定区(CL)。
在某些实施方案中，所述抗原结合片段选自Fab、Fab’、(Fab’)2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)。在某些实施方案中，所述抗体为鼠源抗体、嵌合抗体、人源化抗体、双特异性抗体或多特异性抗体。
在本发明中，本发明的抗体或其抗原结合片段可以包括这样的变体，所述变体与其所源自的抗体或其抗原结合片段相比差异仅在于一个或多个(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的保守置换)氨基酸残基的保守置换，或者与其所源自的抗体或其抗原结合片段具有至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，且基本保留了其所源自的抗体或其抗原结合片段的生物学功能(例如特异性结合CD24，阻断CD24与其配体(如，Siglec-10)结合的阻断活性)。
抗体的制备
本发明的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。 另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’)2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’)2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
第二方面，本发明提供了一种分离的核酸分子，其包含编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。在某些实施方案中，所述分离的核酸分子编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
在某些实施方案中，所述分离的核酸分子包含编码本发明的抗体或其抗原结合片段的重链可变区的第一核苷酸序列，和/或编码本发明的抗体或其抗原结合片段的轻链可变区的第二核苷酸序列。
在某些实施方案中，所述分离的核酸分子包含编码本发明的抗体或其抗原结合片段的重链的第一核苷酸序列，和/或编码本发明的抗体或其抗原结合片段的轻链的第二核苷酸序列。
第三方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含本发明的分离的核酸分子。在某些实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等。在某些实施方案中，所述载体能够在受试者(例如人)体内表达本发明的抗体或其抗原结合片段。
第四方面，本发明提供了一种宿主细胞，其包含如上所述的分离的核酸分子或载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
在另一个方面，提供了制备本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
衍生的抗体
本发明的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一 个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会不利影响其对CD24的结合。因此，本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本发明的抗体或其抗原结合片段功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。此外，本发明的抗体或其抗原结合片段还可以用化学基团进行衍生，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。这些基团可用于改善抗体的生物学特性，例如增加血清半衰期。
经标记的抗体
在某些实施方案中，本发明的抗体或其抗原结合片段带有可检测的标记，例如酶、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物)、磁珠(例如，)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。如上所述的可检测的标记可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
双特异性或多特异性分子
本发明的抗体或其抗原结合片段可用于形成双特异性或多特异性分子。本发明的 抗体或其抗原结合片段可以是双特异性或多特异性分子的一部分，所述双特异性或多特异性分子包含具有不同于本发明的抗体或其抗原结合片段的结合特异性的第二功能模块(例如第二抗体)，从而能够结合至少两个不同的结合位点和/或靶分子。例如，本发明的抗体或其抗原结合片段可连接至能够特异性结合可用作联合治疗的潜在靶标的任何蛋白质的第二抗体或其抗原结合片段。为产生所述双特异性或多特异性分子，可以将本发明的抗体或其抗原结合片段连接(例如通过化学偶联、基因融合、非共价缔合或其他方式)至一个或多个其他结合分子(例如另外的抗体、抗体片段、肽或结合模拟物)。
因此，第五方面，本发明提供了一种双特异性或多特异性分子，其包含本发明的抗体或其抗原结合片段。
在某些实施方案中，所述双特异性或多特异性分子特异性结合CD24，并且额外地特异性结合一个或多个其他靶标。
在某些实施方案中，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
免疫缀合物
本发明的抗体或其抗原结合片段可以与治疗剂连接以形成免疫缀合物。由于免疫缀合物具有选择性递送一种或多种治疗剂至靶组织(例如表达CD24的细胞)的能力，因此，免疫缀合物可以提高本发明的抗体或其抗原结合片段在治疗疾病(例如癌症)中的治疗效力。
在第六方面，本发明提供了免疫缀合物，其包含本发明的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂。
在某些实施方案中，所述免疫缀合物是抗体-药物偶联物(ADC)。
在某些实施方案中，所述治疗剂是细胞毒剂。在本发明中，所述细胞毒剂包括对细胞有害(例如杀伤细胞)的任何试剂。
在某些实施方案中，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合。
在某些实施方案中，本发明的抗体或其抗原结合片段任选地通过接头与所述治疗剂缀合。
在本发明中，使用本领域现有的接头技术可以将细胞毒剂偶联至本发明的抗体或其抗原结合片段。已经用于将细胞毒剂偶联至抗体的接头类型的实例包括但不限于腙、硫醚、酯、二硫化物和含肽的接头。可以选择例如易于在溶酶体隔室内被低pH切割或易于被蛋白酶(例如优先在肿瘤组织中表达的蛋白酶，如组织蛋白酶，诸如组织蛋白酶B、C、D)切割的接头。
嵌合抗原受体
本发明的抗体或其抗原结合片段可用于构建嵌合抗原受体(CAR)，其具备以非MHC限制的方式将T-细胞特异性和反应性指向表达CD24的细胞(例如肿瘤细胞)的能力。
在第七方面，本发明提供了一种嵌合抗原受体(CAR)，其包含本发明的抗体或其抗原结合片段的抗原结合结构域。
在某些实施方案中，所述抗原结合结构域包含本发明的抗体或其抗原结合片段的重链可变区和轻链可变区。
在某些实施方案中，所述抗原结合结构域是scFv。
在某些实施方案中，所述嵌合抗原受体包含本发明抗体的抗原结合片段(例如scFv)。
在某些实施方案中，所述嵌合抗原受体由免疫效应细胞(例如T细胞)所表达。
在某些实施方案中，所述嵌合抗原受体还包括跨膜结构域、一个或多个细胞内T细胞信号结构域(例如T细胞受体信号结构域、T细胞共刺激信号结构域或其组合)。T细胞受体信号结构域是指CAR中包含T细胞受体的细胞内结构域(例如CD3ζ蛋白的细胞内部分)的部分。共刺激信号结构域是指CAR中包含共刺激分子的细胞内结构域的部分，所述共刺激分子是除了淋巴细胞对抗原的高效应答所需的抗原受体或其配体以外的细胞表面分子。
在某些实施方案中，在所述CAR的抗原结合结构域与跨膜结构域之间，可存在包含多肽序列的间隔结构域。所述间隔结构域可包含多达300个氨基酸，优选10至100个氨基酸，并且最优选25至50个氨基酸。在一些实施方案中，所述间隔结构域可包含免疫球蛋白结构域，例如人免疫球蛋白序列。在某些示例性实施方案中，所述免疫球蛋白结构域包含免疫球蛋白CH2和CH3结构域序列。在此类实施方案中，不受特定理论的约束，认为CH2和CH3结构域使所述CAR的抗原结合结构域从表达CAR的细胞的膜延伸出去，并且可更精确地模拟天然TCR的大小和结构域结构。
在某些实施方案中，所述跨膜结构域可衍生自天然来源或合成来源。在此类实施方案中，所述结构域可衍生自任何膜结合的或跨膜的蛋白质。可用于本发明的CAR中的示例性跨膜结构域可包含至少T细胞受体的α、β或ζ链的跨膜区，所述T细胞受体可以选自CD28、CD3ε、CD45、CD4、CD5、CDS、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154。或者，所述跨膜结构域可为合成的，在这种情况下，其将主要包含疏水残基例如亮氨酸和缬氨酸。在某些示例性实施方案中，所述跨膜结构域包含T细胞受体的跨膜结构域，例如CD8跨膜结构域。在某些示例性实施方案中，所述跨膜结构域包含T细胞共刺激分子(例如CD137或CD28)的跨膜结构域。
在某些实施方案中，可用于在所述CAR中使用的细胞内T细胞结构域的实例包括T细胞受体(TCR)的胞质序列和共刺激分子，所述T细胞受体(TCR)的胞质序列和共刺激分子在抗原受体接合后协力启动信号转导，以及这些序列和任何具有相同功能性能力的合成序列的任何衍生物或变体。
在某些实施方案中，所述CAR的细胞内区可包含以刺激的方式起作用的初级胞质信号序列，其可包含被称为基于免疫受体酪氨酸的活化基序或ITAM的信号基序。可包含于所述CAR中的包含初级胞质信号序列的ITAM的实例包括来自CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CDS、CD22、CD79a、CD79b、和CD66d蛋白的那些。
在某些实施方案中，所述CAR的细胞内区可包含含有初级胞质信号结构域(例如CD3ζ)本身或与可用于CAR的环境中的任何其他期望的胞质结构域组合的ITAM。例如，所述CAR的胞质结构域包含CD3ζ链部分和细胞内共刺激信号结构域。所述共刺激信号结构域是指所述CAR的包含共刺激分子的细胞内结构域的部分。共刺激分子是除了淋巴细胞对抗原的高效应答所需的抗原受体或其配体以外的细胞表面分子。这样的分子的实例包括CD27、CD28、4-1BB(CD137)、OX40(CD134)、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原1(LFA-1)、CD2、CD7、LIGHT、NKG2C和B7-H3。
在某些实施方案中，所述CAR可包含CD3ζ信号结构域、CD8信号结构域、CD28信号结构域、CD137信号结构域或其任意组合。所述一个或多个T细胞信号结构域在CAR上的顺序可由本领域技术人员根据需要而改变。
生成嵌合抗原受体、包含这样的受体的T细胞的方法以及它们的用途(例如，用于治疗癌症)是本领域已知的，例如，可将编码本发明的嵌合抗原结合受体的核酸分子包含于用于在宿主细胞例如T细胞中表达的表达载体(例如，慢病毒载体)中，以制造所述CAR。在某 些实例性实施方案中，使用所述嵌合抗原受体的方法包括从受试者分离T细胞，采用编码嵌合抗原受体的表达载体(例如慢病毒载体)转化T细胞，以及将表达所述嵌合抗原受体的工程化的T细胞施用至所述受试者以进行治疗，例如用于治疗所述受试者中的肿瘤。
在第八方面，本发明提供了一种分离的核酸分子，其包含编码本发明的嵌合抗原受体的核苷酸序列。在某些实施方案中，所述分离的核酸分子编码本发明的嵌合抗原受体。
在第九方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含如上所述的分离的核酸分子。在某些实施方案中，本发明的载体例如是质粒。
在第十方面，本发明提供了一种宿主细胞，其表达本发明的嵌合抗原受体和/或包含如上所述的分离的核酸分子或载体。在某些实施方案中，所述宿主细胞是免疫效应细胞(例如，T细胞和/或NK细胞)。在某些实施方案中，所述宿主细胞是嵌合抗原受体T细胞(CAR-T)。
药物组合物
在第十一方面，本发明提供了一种药物组合物，其含有本发明第一方面所述的抗体或其抗原结合片段、第二方面所述的分离的核酸分子、第三方面所述的载体、第四方面所述的宿主细胞、第五方面所述的双特异性或多特异性分子、第六方面所述的免疫缀合物、第七方面所述的嵌合抗原受体、第八方面所述的分离的核酸分子、第九方面所述的载体、或第十方面所述的宿主细胞，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，本发明的药物组合物包含本发明的抗体或其抗原结合片段。
在某些实施方案中，本发明的药物组合物包含本发明的双特异性或多特异性分子。
在某些实施方案中，本发明的药物组合物包含本发明的免疫缀合物。
在某些实施方案中，本发明的药物组合物包含第六方面所述的免疫缀合物、第七方面所述的嵌合抗原受体、第八方面所述的分离的核酸分子、第九方面所述的载体或第十方面所述的宿主细胞。
在某些实施方案中，所述药物组合物还可以包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、特异性靶向肿瘤细胞抗体、免疫检查点抑制剂等。
在某些实施方案中，所述另外的药学活性剂是免疫检查点抑制剂。
在某些实施方案中，所述另外的药学活性剂是抗PD-1抗体或抗PD-L1抗体。
在某些实施方案中，在所述药物组合物中，本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、嵌合抗原受体、分离的核酸分子、载体或宿主细胞与所述另外的药学活性剂可以作为分离的组分或作为混合的组分提供。因此，本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、嵌合抗原受体、分离的核酸分子、载体或宿主细胞与所述另外的药学活性剂可以同时、分开或相继施用。
本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、嵌合抗原受体、分离的核酸分子、载体或宿主细胞或本发明的药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的本发明的抗体或其抗原结合片段，以及任选地，同时掺入其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
因此，在某些示例性实施方案中，本发明的药物组合物包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、嵌合抗原 受体、分离的核酸分子、载体或宿主细胞或本发明的药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、嵌合抗原受体、分离的核酸分子、载体或宿主细胞或本发明的药物组合物通过静脉注射或推注给予。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明第一方面所述的抗体或其抗原结合片段、第二方面所述的分离的核酸分子、第三方面所述的载体、第四方面所述的宿主细胞、第五方面所述的双特异性或多特异性分子、第六方面所述的免疫缀合物、第七方面所述的嵌合抗原受体、第八方面所述的分离的核酸分子、第九方面所述的载体、或第十方面所述的宿主细胞。“预防有效量”是指，足以预防，阻止，或延迟疾病的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
治疗应用
本发明的抗体特异性结合人CD24，并能阻断CD24与其配体(如Siglec-10)的结合，从而解除SHP-1/SHP-2介导的免疫抑制性信号通路，诱导巨噬细胞对肿瘤细胞的吞噬作用，由此提供了以下治疗应用。
在一个方面，本发明提供了一种用于在受试者(例如人)中增强免疫应答或预防和/或治疗肿瘤的方法，所述方法包括向所述受试者施用有效量的选自下列的试剂：第一方面所述的抗体或其抗原结合片段、第二方面所述的分离的核酸分子、第三方面所述的载体、第四方面所述的宿主细胞、第五方面所述的双特异性或多特异性分子、第六方面所述的免疫缀合物、第七方面所述的嵌合抗原受体、第八方面所述的分离的核酸分子、第九方面所述的载体、或第十方面所述的宿主细胞、或第十一方面所述的药物组合物。本发明还涉及上述各试剂在制备用于在受试者(例如人)中增强免疫应答或预防和/或治疗肿瘤的药物中 的用途。
在某些实施方案中，所述免疫应答包括巨噬细胞介导的免疫应答。
在某些实施方案中，所述肿瘤表达涉及CD24阳性的肿瘤细胞，和/或，涉及CD24配体(例如Siglec-10)阳性的巨噬细胞。
在某些实施方案中，所述肿瘤可以是表达CD24的任意肿瘤类型，包括各种血液瘤以及实体瘤。在某些实施方案中，所述肿瘤为实体瘤，例如乳腺癌(如，三阴性乳腺癌)、卵巢癌、肝癌、肺癌、结直肠癌、胃癌、胰腺癌、膀胱癌、前列腺癌、肾细胞癌、宫颈癌、子宫内膜癌、胆管癌、肾癌、鼻咽癌、甲状腺癌、脑癌、睾丸癌、骨癌、淋巴癌或胶质瘤等。在某些实施方案中，所述肿瘤为血液瘤，例如，白血病、淋巴瘤、骨髓瘤。
卵巢癌(Ovarian Cancer,OC)是常见的妇科恶性肿瘤，属于高度异质性的上皮肿瘤，具有不同的组织学亚型以及遗传和生物学特征，包括浆液性癌，子宫内膜样癌，透明细胞癌和粘液性癌。卵巢癌全球每年新发病例31万，死亡超过20万例(Hyuna S,et al:Global cancer statistics 2020:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA CANCER J CLIN 2021；0:1–41.)。75％的卵巢癌为浆液性癌，五年生存率35％，是恶质性极强的一类癌症，通常被诊断时已经是晚期。
乳腺癌是女性年新发比率最高的癌症，死亡率的比率也很高(约50％)。目前，乳腺癌分为4个分子亚型，ER/PR和/或HER2的阳性表达决定了ER阳性和/或HER2阳性亚型，而ER、PR和HER2表达的缺失定义了三阴性乳腺癌(TNBC)。相比之下，TNBC缺乏靶向治疗，仍在使用全身化疗药物进行治疗。此外，TNBC往往表现出更具侵袭性的临床特征并且往往更早且更频繁地复发，这使其成为最具侵袭性的乳腺癌亚型(Plasilova ML,et al:Features of triple-negative breast cancer:Analysis of 38,813 cases from the National Cancer Database.Medicine(Baltimore)2016；95:e4614.)。
Weissman博士团队发现，CD24是卵巢癌和乳腺癌细胞表达的抑制巨噬细胞免疫吞噬的信号蛋白，卵巢癌和三阴乳腺癌受CD24信号传导阻断的影响很大(Amira A.Barkal,et al:Weissman CD24 signalling through macrophage Siglec-10 is a target for cancer.Immunotherapy 2019；572:392-399.)。CD24在上述两种肿瘤样本中的过表达远超过CD47和PD-L1；CD24抗体在两种肿瘤细胞系中致敏巨噬细胞吞噬的作用强于CD47抗体；CD24基因敲除或CD24抗体在小鼠肿瘤模型中能有效抑制肿瘤生长。上述相关数据 反映了CD24这一靶点在卵巢癌和三阴性乳腺癌中的免疫激活作用，抗CD24抗体药物的研发有望在包括卵巢癌和三阴性乳腺癌的固体瘤治疗中提供有效的临床用药。
因此，在某些实施方案中，所述肿瘤优选为卵巢癌或乳腺癌(例如三阴性乳腺癌)。
在某些实施方案中，所述方法还包括施用第二治疗剂(如具有抗肿瘤活性的药物)或第二治疗术(例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗)。
在某些实施方案中，所述第二治疗剂是免疫检查点抑制剂。
在某些实施方案中，所述第二治疗剂是抗PD-1抗体或抗PD-L1抗体。
在一个方面，本发明提供了一种用于在受试者(例如人)中对CD24阳性细胞杀伤或耗竭的方法，所述方法包括向所述受试者施用有效量的选自下列的试剂：第一方面所述的抗体或其抗原结合片段、第二方面所述的分离的核酸分子、第三方面所述的载体、第四方面所述的宿主细胞、第五方面所述的双特异性或多特异性分子、第六方面所述的免疫缀合物、第七方面所述的嵌合抗原受体、第八方面所述的分离的核酸分子、第九方面所述的载体、或第十方面所述的宿主细胞、或第十一方面所述的药物组合物。本发明还涉及上述各试剂在制备用于在受试者(例如人)中对CD24阳性细胞杀伤或耗竭的药物中的用途。
在某些实施方案中，所述CD24阳性细胞是肿瘤细胞。因此，所述方法可用于肿瘤治疗。
在某些实施方案中，将本发明所提供的选自下列的试剂与第二治疗剂或治疗术组合施用：第一方面所述的抗体或其抗原结合片段、第二方面所述的分离的核酸分子、第三方面所述的载体、第四方面所述的宿主细胞、第五方面所述的双特异性或多特异性分子、第六方面所述的免疫缀合物、第七方面所述的嵌合抗原受体、第八方面所述的分离的核酸分子、第九方面所述的载体、或第十方面所述的宿主细胞、或第十一方面所述的药物组合物。所述第二治疗剂或治疗术可以在施用本发明的上述试剂之前、同时或之后施用。
在某些实施方案中，所述第二治疗剂选自具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，所述第二治疗剂是免疫检查点抑制剂。
在某些实施方案中，所述第二治疗剂是抗PD-1抗体或抗PD-L1抗体。
在某些实施方案中，所述第二治疗术可以是已知用于肿瘤的任何疗法，例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗。
检测应用
本发明的抗体或其抗原结合片段能够特异性结合CD24，从而可用于检测CD24在样品中的存在或其水平。
因此，在另一个方面，本发明提供了一种试剂盒，其包括本发明的抗体或其抗原结合片段。
在某些实施方案中，本发明的抗体或其抗原结合片段带有可检测的标记。
在某些实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的抗体或其抗原结合片段。优选地，所述第二抗体还包括可检测的标记。
在本发明中，所述可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。
在另一个方面，本发明提供了检测CD24在样品中的存在或其量的方法，其包括以下步骤：
(1)将所述样品与本发明的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与CD24之间复合物的形成或检测所述复合物的量。
所述复合物的形成表明存在CD24或表达CD24的细胞。
在某些实施方案中，本发明的抗体或其抗原结合片段还带有可检测的标记。在某些实施方案中，在步骤(2)中，使用带有可检测的标记的试剂来检测本发明的抗体或其抗原结合片段。
所述方法可以用于诊断目的，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。在某些实施方案中，所述CD24是人CD24。
在某些实施方案中，所述方法用于诊断受试者是否患有表达CD24的肿瘤或检测肿瘤是 否能够通过靶向CD24的抗肿瘤疗法来治疗。在此类实施方案中，所述方法还可以包括：将所述CD24在来自所述受试者的样品中的量与参考值进行比较的步骤。
所述参考值是指CD24在来自已知不具有表达CD24的肿瘤的受试者的样品中的水平(也称作“阴性参考值”)，或者是指CD24在已知患有表达CD24的肿瘤的受试者的样品中的水平(也称作“阳性参考值”)。例如，如果所述CD24在来自所述受试者的样品中的量与阴性参考值相似(或者无显著差异)，则指示所述受试者未患有表达CD24的肿瘤，以及如果所述CD24在来自所述受试者的样品中的量相对于阴性参考值升高时，则指示所述受试者患有表达CD24的肿瘤。此外，如果所述CD24在来自所述受试者的样品中的量与阳性参考值相似(或者无显著差异)，则指示所述受试者患有表达CD24的肿瘤。
在某些实施方案中，所述样品可以选自尿液、血液、血清、血浆、唾液、腹水、循环细胞、循环肿瘤细胞、非组织缔合的细胞(即游离细胞)、组织(例如手术切除的肿瘤组织、活体组织切片或细针抽吸组织)、组织学制备物等。
在某些实施方案中，所述方法还包括给被诊断为患有表达CD24的肿瘤的受试者施用靶向CD24的免疫疗法。
在某些实施方案中，所述肿瘤可以是表达CD24的任意肿瘤类型，包括各种血液瘤以及实体瘤。在某些实施方案中，所述肿瘤为实体瘤，例如乳腺癌(如，三阴性乳腺癌)、卵巢癌、肝癌、肺癌、结直肠癌、胃癌、胰腺癌、膀胱癌、前列腺癌、肾细胞癌、宫颈癌、子宫内膜癌、胆管癌、肾癌、鼻咽癌、甲状腺癌、脑癌、睾丸癌、骨癌、淋巴癌或胶质瘤等。在某些实施方案中，所述肿瘤为血液瘤，例如，白血病、淋巴瘤、骨髓瘤。
在另一个方面，提供了本发明的抗体或其抗原结合片段或试剂盒在制备检测试剂中的用途，所述检测试剂用于测定CD24在样品中的存在或其量、诊断受试者是否患有表达CD24的肿瘤、和/或检测肿瘤是否能够通过靶向CD24的抗肿瘤疗法来治疗。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子生物学、生物化学、核酸化学、免疫学等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”在其最广泛意义上是指特异性结合抗原决定簇的分子，可以包括各种抗体结构，只要它们表现出所需的抗原结合活性。典型地，抗体可以是由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat编号系统确定。
如本文中所使用的，术语“构架区(framework region)”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、(Fab’)2、Fv、二硫键连接的Fv、scFv、di-scFv、(scFv)2和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv)2，其指的是两个或两个以上单个scFv并联而形成抗体。
如本文中所使用的，术语“双抗体”意指，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure2:1121-1123(1994))。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力。单域抗体也称为纳米抗体(nanobody)。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性。在某些实施方案中，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性等)的小鼠、大鼠、兔或非人灵长类动物(例如，食蟹猴)抗体。
如本文中所使用的，术语“胚系抗体基因(germline antibody gene)”或“胚系抗体基因片段(germline antibody gene segment)”是指，存在于生物体的基因组中的编码免疫球蛋白的序列，其没有经历过能够导致表达特异性免疫球蛋白的遗传学重排及突变的成熟过程。在本发明中，表述“重链胚系基因”是指，编码免疫球蛋白重链的胚系抗体基因或基因片段，其包括V基因(variable)、D基因(diversity)、J基因(joining)和C基因(constant)；类似地，表述“轻链胚系基因”是指，编码免疫球蛋白轻链的胚系抗体基因或基因片段，其包括V基因(variable)、J基因(joining)和C基因(constant)。在本发明中，由所述胚系抗体基因或胚系抗体基因片段所编码的氨基酸序列也称为“胚系序列(germline sequence)”。胚系抗体基因或胚系抗体基因片段及其相应的胚系序 列是本领域技术人员熟知的，并且可从专业数据库(例如，IMGT、UNSWIg、NCBI或VBASE2)获得或查询。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，术语“嵌合抗原受体(CAR)”是指包含至少一个细胞外抗原结合结构域、间隔结构域、跨膜结构域和胞内信号传导结构域的重组多肽构建体，其将针对目的抗原(例如CD24)的基于抗体的特异性与免疫效应细胞活化胞内结构域组合以展现针对表达该目的抗原(例如CD24)细胞的特异性免疫活性。在本发明中，表述“表达CAR的细胞”是指表达CAR并且具有由该CAR的靶向结构域决定的抗原特异性的细胞。
如本文中使用的，术语“免疫效应细胞”是指具有一种或多种效应功能(例如，细胞毒性细胞杀伤活性、细胞因子的分泌、ADCC和/或CDC的诱导)的免疫系统的任何细胞。典型地，免疫效应细胞是具有造血的起源并在免疫应答中起作用的细胞。术语“效应功能”指免疫效应细胞的特化功能，例如增强或促进对靶细胞的免疫攻击(例如对靶细胞的杀伤，或者抑制其生长或增殖)的功能或反应。T细胞的效应功能，例如，可以是细胞溶解活性或者辅助或者包括细胞因子的分泌在内的活性。免疫效应细胞的实例包括T细胞(例如α/βT细胞和γ/δT细胞)、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞和骨髓来源巨噬细胞。
如本文中所使用的，术语“细胞毒剂”包括对细胞有害(例如杀死细胞)的任何试剂，例如化疗药物、细菌毒素、植物毒素或放射性同位素等。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞，免疫细胞(如T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞等)。宿主细胞可以包括单个细胞或细胞群体。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17 (1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania: Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤。
有益效果
本发明提供了对细胞表面CD24具备高亲和力的单克隆抗体，其能够有效阻断CD24 与其配体(如，Siglec-10)的结合，从而解除SHP-1/SHP-2介导的免疫抑制性信号通路，诱导巨噬细胞对肿瘤细胞的吞噬作用。因此，本发明的抗体具有用于治疗CD24表达肿瘤的潜力，具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1A为测定鼠源抗体20G10与MCF7的结合活性的流式评价结果。
图1B为测定鼠源抗体20G10与Huh的结合活性的流式评价结果。
图2A为测定嵌合抗体ch20G10对MCF7的结合活性的流式评价结果。
图2B为测定嵌合抗体ch20G10对重组表达CD24的结合活性的ELISA评价结果。
图2C为测定嵌合抗体ch20G10对化学合成CD24多肽的结合活性的ELISA评价结果。
图3A为测定人源化抗体GB7011-ORG1对天然表达人CD24的HepG2细胞的结合活性的流式评价结果。
图3B为测定人源化抗体GB7011-ORG1对天然表达人CD24的Huh7细胞的结合活性的流式评价结果。
图3C为测定人源化抗体GB7011-ORG1对天然表达人CD24的MCF7细胞的结合活性的流式评价结果。
图4显示了人源化抗体GB7011-ORG1阻断细胞膜表面CD24与其受体蛋白Siglec10结合的阻断活性。
图5A显示了人源化抗体GB7011-ORG1所诱导的M1型巨噬细胞对于NALM6肿瘤细胞的吞噬作用。
图5B显示了人源化抗体GB7011-ORG1所诱导的M2型巨噬细胞对于NALM6肿瘤细胞的吞噬作用。
图6显示了实施例10中各组动物给药7天处死后小鼠肿瘤的重量。
图7显示了实施例11中抗CD24抗体治疗后PD1阳性T细胞(Tc细胞、Th细胞)的比例。
图8显示了实施例11中各组动物给药后21天鼠肿瘤的体积变化。
序列信息
表1：本申请涉及的序列的信息描述于下面的表中。

具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本申请所要求保护的范围。实施例中的实验方法，如无特殊说明，均为常规方法。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：鼠抗人CD24抗体的产生
通过慢病毒感染的方法(MOI＝3-10，5μg/ml polybrene)在HEK293细胞(ATCC)、CHOS细胞(Invitrogen)上分别过表达人源CD24(氨基酸序列如SEQ ID NO:13)。慢病毒由上海吉凯基因医学科技股份有限公司提供，细胞感染72小时后加相应抗生素继续培养2-4周，扩增并冻存，得到HEK293-CD24和CHOS-CD24细胞，以用于后续实验。
为了获得抗人CD24抗体，使用构建的过表达人源CD24的CHOS-CD24细胞免疫Balb/c小鼠(北京维通利华实验动物技术有限公司，品系代码216)；初免佐剂使用完全弗氏佐剂CFA(InvivoGen公司，货号vac-cfa-60)，之后免疫佐剂都使用IFA(InvivoGen公司，货号vac-ifa-60)；免疫途径为皮下多点。多次免疫后将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0使用聚乙二醇法进行融合，得到既能表达抗体又能在体外无限增殖的B细胞融合，并且在HAT选择培养基中培养。将融合后的杂交瘤细胞铺在96孔细胞培养板中，并且通过对上清中抗体在细胞水平结合CD24天然表达人源乳腺癌肿瘤细胞MCF7，CD24过表达HEK293-CD24，CD24阴性细胞HEK293的情况，筛选出目的阳性克隆(结合MCF7和HEK293-CD24，不结合HEK293的抗体)进行2-3轮亚克隆，得到单克隆细胞株。
鼠抗结合细胞水平高通量筛选：在筛选中通过使用人源CD24表达的细胞和阴性对照细胞(MCF7/HEK293-CD24；HEK293)分别进行铺板。将10000个细胞稀释于100μL完全培养基中，使用平底96孔板，过夜使细胞贴壁或沉于孔底，第二天去掉上清。将100μL待筛选杂交瘤上清加入细胞板中，室温孵育1小时。去掉上清后，每孔加入100μL第二抗体(DyLight488山羊抗鼠IgG(Abcam目录编号ab97015))，浓度为5μg/mL，室温孵 育0.5小时。染色完成后去掉上清后每孔加入100μL DPBS，上机器进行读数。使用全视野细胞扫描分析仪(Nexcelom，型号Image Cytometer)对实验板进行测定读数。测定时选择第二抗体对应荧光通道和明场通道同时对孔内细胞进行高速扫描成像。荧光通道得到的成像根据有荧光标记的细胞形态和荧光强度设定参数对抗体结合的细胞进行计数，明场通道得到的成像根据细胞形态设定参数对贴壁细胞进行计数，然后两组数据相除得到和抗体结合的显示荧光的细胞占细胞总数的百分比。根据该比例判定融合瘤上清中抗体与表达CD24的细胞的结合效果。鼠源抗体20G10来源融合瘤上清在高通量筛选中结合几种细胞的数据如表2所示。
表2：鼠源抗体在细胞水平高通量筛选中的结合表现
鼠抗与CD24结合的流式评价：将500,000个CD24表达细胞(人源乳腺癌肿瘤细胞MCF7,人源肝肿瘤细胞Huh7)置于FACS buffer(PBS+2％FBS)/孔中，加入待测鼠源抗体后4℃孵育1小时(使用ISO作为阴性对照抗体，其特异性结合鸡卵清蛋白)。离心去掉上清，使用FACS buffer清洗两遍，加入第二抗体(DyLight488山羊抗鼠IgG，Abcam目录编号ab97015)4℃孵育0.5小时。染色完成后离心去掉上清，用FACS buffer清洗两次后使用FACS buffer重悬细胞，然后上机器进行读数。使用流式细胞分析仪(BD公司，型号CantoII)对实验细胞进行测定读数。测定时先根据FCS和SSC圈定细胞位置，然后选择第二抗体对应荧光通道和SSC对细胞进行分析。鼠源抗体20G10与MCF7,Huh7结合情况如图1A(MCF7)和1B(Huh7)所示。
实施例2：鼠抗人源CD24抗体的可变区序列测定
离心收集杂交瘤细胞，每5-10×106细胞加入1ml TRIzol和0.2ml氯仿，剧烈振荡15秒，室温放置3分钟，离心取水相加入0.5ml异丙醇，室温放置10分钟后收集沉淀，乙醇洗涤后干燥得到RNA。在冰浴离心管里面加入模板RNA和引物，使引物和模板正确配对后进行反转录过程，在进行PCR扩增。4个微量离心管中各加入dNTP/ddNTP混合物2.5μl，混合物37℃温浴5min，备用。在一个空的微量离心管中加入1pmol的PCR扩增双链DNA，10pmol测序引物，2μl 5×测序缓冲液，加双蒸水至总体积10μl，96℃加热8min，冰浴泠却1min，4℃10000g离心10s。加入2μl预冷的标记混合物(dCTP、dGTP、dTTP各0.75μmol/L)，α-32P-dATP 5μCi，1μl 0.1mol/L DDT，测序酶2U，加水至15μl， 混匀后置冰上2min，标记新合成的DNA链。3.5μl标记反应混合物加入到准备好的4个微量离心管中，37℃温浴5min，每管各加入4μl终止液。样品在80℃的水浴中热变性5min，每一泳道加2μl加到测序胶上，电泳分离这些片段，收集序列信息。
鼠源抗体的VH和VL序列如表3所示。进一步，还使用Kabat等人描述的方法(Kabat等，Sequences of Proteins of Immunological Interest,第五版，Public Health Service，美国国立卫生研究院，贝塞斯达、马里兰州(1991)，第647-669页)，确定了鼠源单抗的CDR序列。
表3：鼠源抗体的序列信息
实施例3：嵌合抗CD24抗体的重组表达
在抗体基因序列验证后，鼠源抗体的可变区连接人源抗体的恒定区(人源IgG4，其重链恒定区和轻链恒定区氨基酸序列分别如SEQ ID NO:11和12所示)，构建嵌合抗体ch20G10，将含有抗体基因的表达载体转染至哺乳动物细胞。收获培养瓶生长的含有抗体克隆的哺乳动物细胞上清液，使用protein A柱纯化，并且使用100mM醋酸pH3.0洗脱抗体蛋白。然后将纯化的抗体蛋白上样到分子排阻层析柱上进一步分离纯化。将对应于单体的抗体蛋白配制于PBS缓冲液中，配制品缓冲液补充有20％甘油。
实施例4：嵌合抗CD24抗体的抗原结合活性评价
嵌合抗体与CD24结合的流式评价：
将500,000个CD24天然表达MCF7细胞置于FACS buffer中待用，使用圆底低吸附96孔板。抗体样本使用FACS buffer进行梯度稀释。细胞板中加入经稀释的抗体(使用ISO作为阴性对照抗体)，相应阴性对照孔加入FACS buffer，4℃孵育1小时。离心去掉上清后，使用FACS buffer清洗两遍，每孔加入第二抗体(DyLight488山羊抗人IgG，Abcam目录编号ab97003)，4℃再孵育0.5小时。染色完成后离心去掉上清，用FACS buffer清洗两次后每孔加入FACS buffer重悬细胞，然后上机器进行读数。使用流式细胞分析仪(BD公司，型号CantoII)对实验板中细胞进行测定读数。测定时先根据FCS和SSC圈 定细胞位置，然后选择第二抗体对应荧光通道和SSC对细胞进行分析，数据分析使用GraphPad，横坐标使用抗体浓度的对数，纵坐标使用平均荧光强度数值，根据曲线拟合出抗CD24抗体的EC50。
嵌合抗体ch20G10对MCF7的结合情况如图2A所示。结果表明，嵌合抗体ch20G10对膜表面CD24具备良好的结合活性。
嵌合抗体与CD24结合的ELISA评价:
分别将1μg/ml重组人CD24蛋白-重组CD24(恺佧生物，重组人源CD24蛋白)或化学合成CD24-多肽CD24(吉尔生化)包被在PBS中的ELISA板上，在4℃下过夜。将板在洗涤缓冲液(PBS，0.05％吐温20)中洗涤3次，并且通过添加200μl/w PBS+2％BSA阻断使非特异性位点饱和。将剂量范围的抗CD24抗体梯度稀释100μL加入包被好抗原的ELISA板中在37℃下孵育1h。将板在洗涤缓冲液中10洗涤3次，并且在室温下添加HRP偶联的山羊抗人IgG Fc片段第二抗体持续1hr以检测结合的抗CD24抗体。将板在洗涤缓冲液中洗涤3次，通过添加TMB(HRP底物)并且在室温下在黑暗中孵育板5至10分钟来揭示结合的第二抗体。通过添加硫酸溶液1M终止酶反应，并且在450nm处测量光吸收。以光吸收值为纵坐标，以抗体浓度对数值为横坐标绘制曲线图，并且使用图板棱柱(GraphPad Prism)软件计算EC50。
如图2B和2C所示，嵌合抗体ch20G10对重组表达CD24(图2B)和化学合成CD24多肽(图2C)均具备良好的结合活性。
实施例5：鼠抗人CD24抗体的人源化
为提高候选抗体与人源抗体的序列的同源性，减少抗体对人的免疫原性，可以对以上实施例提供的鼠源抗体进行人源化设计和制备，使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539；Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370；以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。
具体而言，将鼠源抗体20G10的重链和轻链CDR区分别移植到对应的人源化模板的FR框架上，并对人源化模板的FR区氨基酸残基进行了一系列的回复突变，以使人源化抗体尽可能保留鼠源抗体的抗原结合能力。根据以上方法，本发明人制备获得了鼠源抗体20G10的人源化抗体，命名为GB7011-ORG1(其重链可变区及轻链可变区氨基酸 序列分别如SEQ ID NO:9和10所示)。抗体的重链恒定区为SEQ ID NO:11，轻链恒定区为SEQ ID NO:12。
实施例6：人源化抗CD24抗体的抗原结合活性评价
人源化抗体与CD24结合的流式评价：
将500,000个CD24天然表达肿瘤细胞(人源肝肿瘤细胞HepG2,Huh7；人源化乳腺肿瘤细胞MCF7)置于FACS buffer中待用，使用圆底低吸附96孔板。抗体样本使用FACS buffer进行梯度稀释(使用ISO作为阴性对照抗体)。细胞板中加入经稀释的抗体，相应阴性对照孔加入FACS buffer，4℃孵育1小时。离心去掉上清后，使用FACS buffer清洗两遍，每孔加入第二抗体(DyLight488山羊抗人IgG，Abcam目录编号ab97003)4℃再孵育0.5小时。染色完成后离心去掉上清，用FACS buffer清洗两次后每孔加入FACS buffer重悬细胞，然后上机器进行读数。使用流式细胞分析仪(BD公司，型号CantoII)对实验板中细胞进行测定读数。测定时先根据FCS和SSC圈定细胞位置，然后选择第二抗体对应荧光通道和SSC对细胞进行分析，数据分析使用GraphPad，横坐标使用抗体浓度的对数，纵坐标使用平均荧光强度数值，根据曲线拟合出抗CD24抗体的EC50。
人源化抗体GB7011-ORG1对天然表达人CD24的三种肿瘤细胞(HepG2/Huh7/MCF7)的结合情况分别如图3A(HepG2)，图3B(Huh7)，图3C(MCF7)所示。结果表明，人源化抗体GB7011-ORG1对三种细胞的膜表面CD24均具备良好的结合活性。
实施例7：抗CD24抗体阻断Siglec10与CD24的结合
抗体阻断Siglec10与CD24的结合，使用荧光标记的Siglec-10蛋白(ACRO biosystems目录编号SI0-HP2E5)作为受体，使用HEK293T-CD24细胞(高表达人CD24；人胚肾细胞系)作为配体，利用流式细胞仪测量方法进行滴定。具体检测步骤为将100,000个HEK293T-CD24细胞置于50μL FACS buffer(PBS+2％FBS)/孔中待用，实验使用圆底低吸附96孔板；抗体样本稀释于FACS buffer中(使用ISO作为阴性对照抗体)，细胞板的每一孔中加入50μL经稀释的抗体，相应阴性对照孔加入50μL FACS buffer，4℃孵育1小时；第一阶段孵育完成后，每孔加入50μL标记荧光标记的Siglec-10蛋白(终浓度1/40，稀释于FACS buffer中)，4℃继续孵育1小时；孵育完成后离心去掉上清，用 FACS buffer清洗两次后每孔加入100μL FACS buffer重悬细胞，然后使用流式细胞分析仪(BD公司，型号CantoII)对实验板中细胞进行测定读数。测定时先根据FCS和SSC圈定细胞位置，然后选择荧光标记蛋白对应红色荧光通道(PE)和SSC对细胞进行分析，根据PE数值计算嵌合抗体阻断Siglec10-CD24结合效果。以阴性对照孔数值为max值计算抗体阻断Siglec10与CD24的结合的比例，抗体阻断结合的比例＝100×[(max–PE数值)/max]，比例越高代表被测定抗CD24抗体阻断Siglec10蛋白和CD24高表达细胞株结合的能力越强，反之，数值越低代表被测定抗CD24抗体阻断Siglec10蛋白和CD24高表达细胞株结合的能力越好。GB7011-ORG1能够有效阻断细胞膜表面CD24与其受体蛋白Siglec10的结合，结果如图4所示。
实施例8：抗CD24抗体诱导巨噬细胞对肿瘤细胞的吞噬作用
以NALM6细胞为靶细胞，以M1型或M2型巨噬细胞为效应细胞，使用流式细胞仪检测吞噬作用。具体检测步骤为：复苏PBMC细胞(来源于健康人，购自妙顺生物)，培养72h后，弃去上清悬浮细胞。加入GM-CSF(近岸蛋白，GMP-CC79)刺激8天，改用IFNγ(近岸蛋白，C014)刺激一天得到M1型巨噬细胞。加入M-CSF(近岸蛋白，C417)刺激7天，改用IL4(近岸蛋白，GMP-CD03)+IL10(R&D，217-IL-025)刺激两天得到M2型巨噬细胞。消化收集处理好的巨噬细胞，重悬于1640培养基，调整密度至5×105/ml。取NALM6细胞(富衡生物，CRL-3273)，以DPBS清洗两次后，调整细胞密度为1×106/ml,加入终浓度为1.6uM的CFSE染料(Thermo Scientific，C34554)，37℃避光染色20分钟。加入5倍体积2％FBS-PBS避光终止5分钟后，以DPBS离心清洗2次，重悬于1640培养基，调整密度至1×106/ml。抗体样本按终浓度的5倍稀释于1640培养基中(使用ISO作为阴性对照抗体)。使用圆底低吸附96孔板(Corning，7007)，在对应孔中加入50ul CFSE染色的NALM6细胞悬液和25ul经稀释的抗体，37℃静置孵育20分钟。孵育完成后加入50ul巨噬细胞悬液，于37℃，5％CO2细胞培养箱中静置3小时。1000rpm'离心5分钟收集细胞，去上清，加入5ug/ml APC-anti CD11b抗体(Biolegend,101212)于4℃孵育30分钟。1000rpm离心5分钟收集细胞，去上清，以DPBS清洗2次后使用流式细胞仪(BD公司，型号CantoII)对实验板中细胞进行检测。测定时先根据FCS和SSC圈定细胞位置，然后选择CD11b标记巨噬细胞对应红色荧光通道(APC)和CFSE标记NALM6细胞对应绿色荧光通道(FITC)对细胞进行分析，根据APC和FITC双阳性门内的细胞比例定 义未发生吞噬的肿瘤细胞NALM6的百分比，双阳性细胞比例越高代表被测定抗CD24抗体诱导吞噬作用能力越好，反之则反。抗CD24抗体GB7011-ORG1所诱导的M1型巨噬细胞和M2型巨噬细胞对于NALM6肿瘤细胞的吞噬作用如图5A(M1)和图5B(M2)所示，其中Anti-CD47 5F9为重组表达靶向CD47诱导巨噬细胞吞噬的抗体，该抗体序列来源于Magrolimab(Forty Seven)。
实施例9：抗CD24抗体与抗PD1抗体协同诱导细胞因子分泌
混合白细胞反应测定以评估GB7011-ORG1与抗PD1抗体(Pembrolizumab)组合潜在协同或相加效应。具体检测步骤为：50ng/ml重组M-CSF(近岸蛋白，C417)培养单核细胞(TPCS，A19K207021(#21)/A19K349043(#43))6天生产单核细胞来源巨噬细胞。第6天，加入饥饿24小时的MCF7细胞(200000/孔)刺激，然后将异基因CD4+T细胞(200000/孔)与抗体一起加入巨噬细胞然后继续培养6天，收集上清液用于分泌的细胞因子的检测分析(TH1-CBA试剂盒)。如表4所示，抗CD24抗体GB7011-ORG1与抗PD1抗体联用条件下细胞因子IFNγ的释放相比PD1抗体单用增加。这些数据表明，抗CD24抗体GB7011-ORG1与抗PD1抗体联用诱导T细胞活化和细胞因子分泌增加。
表4细胞因子释放
实施例10：抗CD24抗体单用在小鼠肿瘤模型中的抗肿瘤药效
B6-Siglec10人源化小鼠(南方模式)皮下移植MC38-hCD24结直肠癌细胞，建立MC38-hCD24结直肠癌模型。接种后第11天，肿瘤平均体积约为130-132mm3，采用随机区组法将荷瘤鼠分为4组，包括组1ISO CTRL 3mg/kg、组2GB7011-ORG1 3mg/kg、组3GB7011-ORG1 1mg/kg和组4GB7011-ORG1 0.3mg/kg,组1/2/3/4每组6只。各组均采用尾静脉给药，分组后给药一次，进行一周观察。给药开始后第7天，组1肿瘤体积为368.44±51.64mm3，组2为101.02±17.61mm3，肿瘤体积抑制率(TGI)为112.30％，与对照组1的肿瘤体积比较，存在显著统计学差异(P<0.01)；组3与组4的肿瘤体积分别为276.29±67.19mm3和272.42±77.33mm3，肿瘤体积抑制率(TGI)为38.60％和40.69％，与对照组组1的肿瘤体积比较，没有统计学差异(P>0.05)。试验结束时，对动物实施安 乐死，剥取瘤块称重，组1平均肿瘤体积为0.2523±0.0667g，组2平均肿瘤重为0.0573±0.0090g，瘤重抑制率为77.31％，与组1瘤重比较，有显著统计学差异(P<0.01)。组3和组4平均肿瘤重为0.2755±0.0580g和0.2090±0.0576g，瘤重抑制率分别为-9.19％和17.17％，与组1瘤重比较，无统计学差异(P>0.05)。图6为四组动物给药7天处死动物后小鼠肿瘤的重量比较，显示GB7011-ORG1 3mg/kg单次给药能够有效抑制小鼠肿瘤的生长，GB7011-ORG1 1mg/kg和GB7011-ORG1 0.3mg/kg单次给药尽管未观察到统计TGI，但单个小鼠对治疗有反应。
实施例11：抗CD24抗体与抗PD1抗体联用在小鼠肿瘤模型中的抗肿瘤药效
1.抗CD24抗体在小鼠肿瘤模型中的抗肿瘤药效及肿瘤组织浸润淋巴细胞(TIL)分析
B6-Siglec10人源化小鼠(南方模式)皮下移植MC38-hCD24结直肠癌细胞，建立MC38-hCD24结直肠癌模型。接种后第6天，肿瘤平均体积约为88-92mm3，采用随机区组法将荷瘤鼠分为2组，包括组1ISO CTRL 5mg/kg和组2GB7011-ORG1 5mg/kg,每组6只。各组均采用尾静脉给药，分组后每周给药一次。给药开始后第0/3/7/10/14/17/21天肿瘤体积和肿瘤生长抑制百分比如表5所示，GB7011-ORG1在早期表现出更好的肿瘤生长抑制。
表5：肿瘤大小
动物给药后第21天处死动物，将小鼠肿瘤取出称重后切成小块磨碎后经滤网处理得到单细胞悬液，加入红细胞裂解液处理后使用FACS buffer清洗两遍，将500,000个细胞置于FACS buffer中待用，使用2毫升实验管，Fc受体阻断剂(biolegend，目录编号422301)使用FACS buffer进行稀释，管中加入经稀释的阻断剂，室温孵育5分钟，完成后离心去掉上清，用FACS buffer清洗两次后每管加入FACS buffer重悬细胞，检测抗体(L/D  FVS700(BD，目录编号564997)，CD45APC-Cy7(Biolegend，目录编号103116)，CD3FITC(Biolegend，目录编号100204)，CD4 BV605(BD，目录编号563151)，CD8 Percp-Cy5.5(BD，目录编号551162)，)使用FACS buffer进行稀释，管中加入经稀释的抗体，室温孵育15分钟，染色完成后离心去掉上清，用FACS buffer清洗两次后每管加入FACS buffer重悬细胞，然后上机器进行读数。使用流式细胞分析仪(BD公司，型号CantoII)对实验管中细胞进行测定读数。测定时先根据FCS和SSC圈定细胞位置，然后根据L/D圈出活细胞，根据CD45圈出CD45阳性细胞，最后根据CD3圈出T细胞，结果如图7所示，TIL分析显示抗CD24治疗后T细胞群增高。
基于图7显示的CD24治疗后T细胞群增高，以及表4显示的抗CD24抗体和抗PD-1抗体联用T细胞活化和细胞因子分泌的协同增加这两项实验结果，本申请发明人将本申请抗体与抗PD1抗体联合施用，以尝试进一步提高本申请抗CD24抗体的抗肿瘤效果。
2.抗CD24抗体与抗PD1抗体联用在小鼠肿瘤模型中的抗肿瘤药效
B6-Siglec10人源化小鼠(南方模式)皮下移植MC38-hCD24结直肠癌细胞，建立MC38-hCD24结直肠癌模型。接种后第5天，肿瘤平均体积约为88-90mm3，采用随机区组法将荷瘤鼠分为3组，包括组1ISO CTRL 3+3mg/kg、组2抗PD1抗体3mg/kg和组3抗PD1抗体3mg/kg与GB7011-ORG1 3mg/kg联用，组1/2/3每组6只。GB7011-ORG1采用尾静脉给药，分组后每周给药一次，抗PD1抗体采用腹腔给药，每周给药两次。给药开始后第7天，组1肿瘤体积为303.67±31.11mm3，组2为253.46±92.36mm3，组3为62.06±7.93mm3，肿瘤体积抑制率(TGI)分别为23.07％和112.22％。
组3与对照组1的肿瘤体积比较，存在显著统计学差异(P<0.01)；给药开始后第16天，组3肿瘤全部清除。试验结束时，对动物实施安乐死，剥取瘤块称重，组1平均肿瘤体积为1.7369±0.2017g，组2平均肿瘤重为0.4071±0.1857g，瘤重抑制率为82.1％，与组1瘤重比较，有显著统计学差异(P<0.01)。组3肿瘤全部消除，瘤重抑制率为104.81％，与组1瘤重比较，有显著统计学差异(P<0.01)。图8为三组动物给药后21天鼠肿瘤的体积变化，显示抗PD1抗体3mg/kg与GB7011-ORG1 3mg/kg联合使用能够显著抑制小鼠肿瘤的生长。
实施例12：用细胞微阵列技术(Retrogenix)评估抗体GB7011-ORG1特异性
用细胞微阵列技术(Retrogenix)评估抗体GB7011-ORG1特异性，确认测试抗体的 主要靶标。
将编码绿色荧光蛋白(ZsGreen1)和待测蛋白(包含6052个全长人质膜蛋白(分泌或细胞表面束缚的人类分泌蛋白)以及397个人类异源二聚体，具体靶标详见附录1-2)的载体置于微阵列载玻片上并用人HEK293细胞反向转染/表达，然后向载玻片上添加含有0.5μg/mL GB7011-ORG1、1μg/mL抗CD20利妥昔单抗生物仿制药(阳性对照)或无测试分子(阴性对照)，与二抗(AF647抗hIgG Fc)孵育后检测AF647荧光信号。通过荧光分析和定量(ImageQuant软件，GE healthcare,Version 8.2)评估抗体与细胞表面待测抗原的结合强度。第一轮初筛共在文库中选出30个可能与GB7011-ORG1结合的蛋白，结合强度从非常弱到很强不等(表6)。之后针对这30个靶标外进行二次验证，方法与初筛时相同。二次验证中，分别表达30个蛋白的细胞经固定处理后，有23个蛋白(表6中标记“*”)与GB7011-ORG1和对照抗体(抗CD20利妥昔单抗生物类似药)均结合，故被归为非特异性结合。其结合可能是Fc结构域介导，例如Fcγ受体(FCGR1A，FCGR2A(isoform2)，FCGR2A(isoform1)，FCGR2A+FCER1G和FCGR2A+CD247)；或待测蛋白与荧光二抗直接结合，如IGHG(IGHG1，IGHG2，IGHG3和IGHG4)。此外，有6个蛋白(XG、PRR7、SLC38A4、MYMK、ST6GAL1和EDN1)与测试抗体有非常弱的结合，信号水平极低，可忽略。排除不可重复的，非特异性的和非显著的结合后，CD24是唯一1个对测试抗体呈现特异性结合蛋白，是其主要靶标，该结果在活细胞结合实验上得到进一步的确认。
表6：抗体与靶标结合强度

尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。
附录1单个蛋白(6052)







附录2异二聚体(397对)

Claims (26)

  1. 能够特异性结合CD24的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    包含如SEQ ID NO:3或其变体所示的VH CDR1、如SEQ ID NO:4或其变体所示的VH CDR2以及如SEQ ID NO:5或其变体所示的VH CDR3的重链可变区(VH)；和/或，包含如SEQ ID NO:6或其变体所示的VL CDR1、如SEQ ID NO:7或其变体所示的VL CDR2以及如SEQ ID NO:8或其变体所示的VL CDR3的轻链可变区(VL)；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；
    优选地，所述的置换是保守置换。
  2. 权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)如下的三个重链可变区CDRs：如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:4所示的VH CDR2、如SEQ ID NO:5所示的VH CDR3；和/或，如下的三个轻链可变区CDRs：如SEQ ID NO:6所示的VL CDR1、如SEQ ID NO:7所示的VL CDR2、如SEQ ID NO:8所示的VL CDR3；或，
    (b)如下的三个重链可变区CDRs：SEQ ID NO:1或9所示的重链可变区中含有的VH CDR1、VH CDR2和VH CDR3；和/或，如下的三个轻链可变区CDRs：SEQ ID NO:2或10所示的轻链可变区中含有的VL CDR1、VL CDR2和VL CDR3；
    优选地，所述重链可变区中含有的3个CDR和所述轻链可变区中含有的3个CDR由Kabat、Chothia或IMGT编号系统定义。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    包含SEQ ID NO:1所示的序列或其变体的重链可变区(VH)；和/或，包含SEQ ID NO:2所示的序列或其变体的轻链可变区(VL)；或，
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、 至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：
    包含如SEQ ID NO:1所示的序列的VH和包含如SEQ ID NO:2所示的序列的VL。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的框架区序列；
    优选地，所述抗体或其抗原结合片段包含：来源于人重链胚系序列的重链框架区序列，以及来源于人轻链胚系序列的轻链框架区序列。
  5. 权利要求4所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    包含SEQ ID NO:9所示的序列或其变体的重链可变区(VH)；和/或，包含SEQ ID NO:10所示的序列或其变体的轻链可变区(VL)；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：
    包含如SEQ ID NO:9所示的序列的VH和包含如SEQ ID NO:10所示的序列的VL。
  6. 权利要求1-5任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含来源于人免疫球蛋白的恒定区；
    优选地，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:11所示的重链恒定区(CH)；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:12所示的轻链恒定区(CL)。
  7. 权利要求1-6任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’)2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)；和/或，所述抗体为鼠源抗体、嵌合抗体、人源化抗体、双特异性抗体或多特异性抗体。
  8. 权利要求1-7任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  9. 分离的核酸分子，其编码权利要求1-8任一项所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
  10. 载体，其包含权利要求9所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
  11. 宿主细胞，其包含权利要求9所述的分离的核酸分子或权利要求10所述的载体。
  12. 制备权利要求1-8任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求11所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  13. 双特异性或多特异性分子，其包含权利要求1-8任一项所述的抗体或其抗原结合片段；
    优选地，所述双特异性或多特异性分子特异性结合CD24，并且额外地特异性结合一个或多个其他靶标；
    优选地，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
  14. 免疫缀合物，其包含权利要求1-8任一项所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂；
    优选地，所述治疗剂选自细胞毒剂；
    优选地，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合；
    优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
  15. 嵌合抗原受体，其包含权利要求1-8任一项所述的抗体或其抗原结合片段的抗原结合结构域；
    优选地，所述抗原结合结构域包含权利要求1-8任一项所述的抗体或其抗原结合片段的重链可变区和轻链可变区；
    优选地，所述抗原结合结构域是scFv；
    优选地，所述嵌合抗原受体包含权利要求1-8任一项所述的抗体的抗原结合片段；
    优选地，所述嵌合抗原受体由免疫效应细胞(例如T细胞)所表达。
  16. 分离的核酸分子，其编码权利要求15所述的嵌合抗原受体。
  17. 载体，其包含权利要求16所述的分离的核酸分子；优选地，其用于制备嵌合抗原受体T细胞。
  18. 宿主细胞，其表达权利要求15所述的嵌合抗原受体，和/或包含权利要求16所述的分离的核酸分子或权利要求17所述的载体；
    优选地，所述宿主细胞是免疫效应细胞(例如，T细胞和/或NK细胞)；
    优选地，所述宿主细胞是嵌合抗原受体T细胞(CAR-T)。
  19. 药物组合物，其含有权利要求1-8任一项所述的抗体或其抗原结合片段、权利要求9所述的分离的核酸分子、权利要求10所述的载体、权利要求11所述的宿主细胞、权 利要求13所述的双特异性或多特异性分子、权利要求14所述的免疫缀合物、权利要求15所述的嵌合抗原受体、权利要求16所述的分离的核酸分子、权利要求17所述的载体或权利要求18所述的宿主细胞，以及药学上可接受的载体和/或赋形剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、特异性靶向肿瘤细胞抗体、免疫检查点抑制剂或免疫调节剂；
    优选地，所述另外的药学活性剂是免疫检查点抑制剂；
    优选地，所述另外的药学活性剂是抗PD-1抗体或抗PD-L1抗体。
  20. 一种用于在受试者(例如人)中增强免疫应答或预防和/或治疗肿瘤的方法，所述方法包括向所述受试者施用有效量的权利要求1-8任一项所述的抗体或其抗原结合片段、权利要求9所述的分离的核酸分子、权利要求10所述的载体、权利要求11所述的宿主细胞、权利要求13所述的双特异性或多特异性分子、权利要求14所述的免疫缀合物、权利要求15所述的嵌合抗原受体、权利要求16所述的分离的核酸分子、权利要求17所述的载体、权利要求18所述的宿主细胞、或权利要求19所述的药物组合物；
    优选地，所述肿瘤涉及CD24阳性的肿瘤细胞，和/或，涉及CD24配体(例如Siglec-10)阳性的巨噬细胞；
    优选地，所述肿瘤为实体瘤，例如乳腺癌(如，三阴性乳腺癌)、卵巢癌、肝癌、肺癌、结直肠癌、胃癌、胰腺癌、膀胱癌、前列腺癌、肾细胞癌、宫颈癌、子宫内膜癌、胆管癌、肾癌、鼻咽癌、甲状腺癌、脑癌、睾丸癌、骨癌、淋巴癌或胶质瘤；
    优选地，所述肿瘤为血液瘤，例如，白血病、淋巴瘤、骨髓瘤；
    优选地，所述方法还包括施用第二治疗剂(如具有抗肿瘤活性的药物)或第二治疗术(例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗)；
    优选地，所述第二治疗剂是免疫检查点抑制剂；
    优选地，所述第二治疗剂是抗PD-1抗体或抗PD-L1抗体。
  21. 一种在受试者(例如人)中对CD24阳性细胞杀伤或耗竭的方法，所述方法包括向所述受试者施用有效量的权利要求1-8任一项所述的抗体或其抗原结合片段、权利要求9所述的分离的核酸分子、权利要求10所述的载体、权利要求11所述的宿主细胞、权利要求13所述的双特异性或多特异性分子、权利要求14所述的免疫缀合物、权利要求15所述的嵌合抗原受体、权利要求16所述的分离的核酸分子、权利要求17所述的载体、权利要求18所述的宿主细胞、或权利要求19所述的药物组合物。
  22. 权利要求1-8任一项所述的抗体或其抗原结合片段、权利要求9所述的分离的核酸分子、权利要求10所述的载体、权利要求11所述的宿主细胞、权利要求13所述的双特异性或多特异性分子、权利要求14所述的免疫缀合物、权利要求15所述的嵌合抗原受体、权利要求16所述的分离的核酸分子、权利要求17所述的载体、权利要求18所述的宿主细胞、或权利要求19所述的药物组合物，在制备用于在受试者(例如人)中增强免疫应答或预防和/或治疗肿瘤的药物中的用途；
    优选地，所述肿瘤涉及CD24阳性的肿瘤细胞，和/或，涉及CD24配体(例如Siglec-10)阳性的巨噬细胞；
    优选地，所述肿瘤为实体瘤，例如乳腺癌(如，三阴性乳腺癌)、卵巢癌、肝癌、肺癌、结直肠癌、胃癌、胰腺癌、膀胱癌、前列腺癌、肾细胞癌、宫颈癌、子宫内膜癌、胆管癌、肾癌、鼻咽癌、甲状腺癌、脑癌、睾丸癌、骨癌、淋巴癌或胶质瘤；
    优选地，所述肿瘤为血液瘤，例如，白血病、淋巴瘤、骨髓瘤；
    优选地，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、双特异性或多特异性分子、免疫缀合物、嵌合抗原受体、分离的核酸分子、载体、宿主细胞、或药物组合物与另外的药学活性剂(例如具有抗肿瘤活性的药物)的联合施用；优选地，所述另外的药学活性剂是免疫检查点抑制剂；
    优选地，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、双特异性或多特异性分子、免疫缀合物、嵌合抗原受体、分离的核酸分子、载体、宿主细胞、或药物组合物与抗PD-1抗体或抗PD-L1抗体联合施用。
  23. 权利要求1-8任一项所述的抗体或其抗原结合片段、权利要求9所述的分离的核酸分子、权利要求10所述的载体、权利要求11所述的宿主细胞、权利要求13所述的双特异性或多特异性分子、权利要求14所述的免疫缀合物、权利要求15所述的嵌合抗原受体、权利要求16所述的分离的核酸分子、权利要求17所述的载体、权利要求18所述的宿主细胞、或权利要求19所述的药物组合物，在制备用于在受试者(例如人)中对CD24阳性细胞杀伤或耗竭的药物中的用途。
  24. 试剂盒，其含有权利要求1-8任一项所述的抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
    优选地，所述试剂盒还包括第二抗体，其特异性识别权利要求1-8任一项所述的抗体或其抗原结合片段；优选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  25. 一种检测CD24在样品中的存在或其量的方法，其包括以下步骤：
    (1)将所述样品与权利要求1-8任一项所述的抗体或其抗原结合片段接触；
    (2)检测所述抗体或其抗原结合片段与CD24之间复合物的形成或检测所述复合物的量；
    优选地，所述方法用于诊断受试者是否患有表达CD24的肿瘤；
    优选地，所述方法用于检测肿瘤是否能够通过靶向CD24的抗肿瘤疗法来治疗；
    优选地，所述肿瘤为实体瘤，例如乳腺癌(如，三阴性乳腺癌)、卵巢癌、肝癌、肺癌、结直肠癌、胃癌、胰腺癌、膀胱癌、前列腺癌、肾细胞癌、宫颈癌、子宫内膜癌、胆管癌、肾癌、鼻咽癌、甲状腺癌、脑癌、睾丸癌、骨癌、淋巴癌或胶质瘤；
    优选地，所述肿瘤为血液瘤，例如，白血病、淋巴瘤、骨髓瘤。
  26. 权利要求1-8任一项所述的抗体或其抗原结合片段或权利要求24所述的试剂盒在制备检测试剂中的用途，所述检测试剂用于测定CD24在样品中的存在或其量、诊断受试者是否患有表达CD24的肿瘤、和/或检测肿瘤是否能够通过靶向CD24的抗肿瘤疗法来治 疗；
    优选地，所述肿瘤为实体瘤，例如乳腺癌(如，三阴性乳腺癌)、卵巢癌、肝癌、肺癌、结直肠癌、胃癌、胰腺癌、膀胱癌、前列腺癌、肾细胞癌、宫颈癌、子宫内膜癌、胆管癌、肾癌、鼻咽癌、甲状腺癌、脑癌、睾丸癌、骨癌、淋巴癌或胶质瘤；
    优选地，所述肿瘤为血液瘤，例如，白血病、淋巴瘤、骨髓瘤。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009063461A1 (en) * 2007-11-14 2009-05-22 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Methods of treating cancer using anti cd24 antibodies
CN103819561A (zh) * 2014-01-22 2014-05-28 中国药科大学 抗cd24单克隆抗体、其可变区序列及其应用
CN108373504A (zh) * 2017-01-30 2018-08-07 亘喜生物科技（上海）有限公司 Cd24特异性抗体和抗cd24-car-t细胞
WO2020261280A1 (en) * 2019-06-25 2020-12-30 Ichilov Tech Ltd. Anti-cd24 antibody and uses thereof
CN113831412A (zh) * 2021-10-13 2021-12-24 宜明昂科生物医药技术（上海）有限公司 靶向cd24的抗体及其制备和用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113956363B (zh) * 2021-10-13 2023-03-31 宜明昂科生物医药技术（上海）股份有限公司 靶向cd47和cd24的重组融合蛋白及其制备和用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009063461A1 (en) * 2007-11-14 2009-05-22 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Methods of treating cancer using anti cd24 antibodies
CN103819561A (zh) * 2014-01-22 2014-05-28 中国药科大学 抗cd24单克隆抗体、其可变区序列及其应用
CN108373504A (zh) * 2017-01-30 2018-08-07 亘喜生物科技（上海）有限公司 Cd24特异性抗体和抗cd24-car-t细胞
WO2020261280A1 (en) * 2019-06-25 2020-12-30 Ichilov Tech Ltd. Anti-cd24 antibody and uses thereof
CN113831412A (zh) * 2021-10-13 2021-12-24 宜明昂科生物医药技术（上海）有限公司 靶向cd24的抗体及其制备和用途

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