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WO2023104066A1 - 抗cd24抗体及其用途 - Google Patents

抗cd24抗体及其用途 Download PDF

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Publication number
WO2023104066A1
WO2023104066A1 PCT/CN2022/137123 CN2022137123W WO2023104066A1 WO 2023104066 A1 WO2023104066 A1 WO 2023104066A1 CN 2022137123 W CN2022137123 W CN 2022137123W WO 2023104066 A1 WO2023104066 A1 WO 2023104066A1
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seq
amino acid
acid sequence
variable region
chain variable
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PCT/CN2022/137123
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English (en)
French (fr)
Inventor
冯晓
柯潇
李剑红
任鹏飞
雷刚
Original Assignee
成都康弘生物科技有限公司
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Priority to IL313389A priority Critical patent/IL313389A/en
Priority to KR1020247019827A priority patent/KR20240112870A/ko
Priority to EP22903488.9A priority patent/EP4446344A1/en
Priority to JP2024534548A priority patent/JP2024545174A/ja
Priority to CA3240338A priority patent/CA3240338A1/en
Priority to AU2022404358A priority patent/AU2022404358A1/en
Publication of WO2023104066A1 publication Critical patent/WO2023104066A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/54—F(ab')2
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • This application relates to the technical field of biotechnology, especially antibody engineering, and in particular to anti-CD24 antibody and its application.
  • the immune system can identify and eliminate tumor cells in the tumor microenvironment.
  • tumor cells can adopt different strategies, such as relying on their own high mutation characteristics, evading immune surveillance, and gradually establishing immune suppression microenvironment. environment, to resist and suppress the body's anti-tumor immune response, so that it can break through the limit and continue to expand, and eventually develop into a clinically visible tumor.
  • Tumor immunotherapy is a treatment method to control and eliminate tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response, including monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer Vaccines, cell therapy and small molecule inhibitors, etc. At present, it has been widely used in various tumors such as melanoma, non-small cell lung cancer, kidney cancer and prostate cancer. Tumor-targeting antibodies are the most widely used immunotherapy in the treatment of hematologic cancers and solid tumors due to their high specificity and excellent pharmacokinetic characteristics, among others.
  • Anti-tumor antibodies can exert anti-tumor properties through several mechanisms, including: (1) enhancing/activating Fc-mediated effector functions (ADCC, ADCP, CDC); (2) blocking tumor growth signals; (3) inhibiting tumor Angiogenesis; (4) induce tumor cell apoptosis signal; (5) activate immune cells.
  • ADCC Fc-mediated effector functions
  • ADCP ADCP
  • CDC blocking tumor growth signals
  • Angiogenesis inhibiting tumor Angiogenesis
  • (4) induce tumor cell apoptosis signal (5) activate immune cells.
  • CD24 is a highly glycosylated protein with an apparent molecular weight between 30 and 70 kDa, which is anchored to the membrane by glycosylphosphatidylinositol (GPI). In humans, CD24 is a short peptide of only 31 amino acids with 16 potential O- and N-glycosylation sites.
  • CD24 molecules expressed at a significantly high level can be detected on the surface of most malignant tumors such as adenocarcinoma, ovarian cancer, bladder cancer, prostate cancer, non-small cell lung cancer, nasopharyngeal carcinoma and other tumor tissue cells. Its expression level is closely related to the occurrence and development of tumors, so CD24 has been identified as a cancer stem cell marker in various cancer types.
  • CD24 was identified as the ligand of P-selectin, which is mainly expressed on the surface of activated vascular endothelium, and as an important adhesion molecule, it can promote cell migration.
  • CD24 on the surface of tumor cells acts as a ligand for P-selectin, and P-selectin mediates the combination of tumor cells with activated platelets and endothelial cells, and promotes the extension, invasion and metastasis of tumor cells.
  • CD24 can mediate the proliferation, adhesion, metastasis and invasion of tumor cells through a variety of mechanisms. Therefore, CD24 is expected to become a new target for tumor immunotherapy.
  • the application provides an anti-CD24 antibody or an antigen-binding fragment thereof, wherein the anti-CD24 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), the light chain variable region includes light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), where:
  • the amino acid sequence of HCDR1 comprises X 1 GYX 2 WH (SEQ ID NO: 152), wherein X 1 is R, N or S, and X 2 is S or T; or the amino acid sequence of HCDR1 is TYGVS (SEQ ID NO: 5);
  • the amino acid sequence of HCDR2 comprises X 1 X 2 X 3 X 4 X 5 GX 6 TX 7 YX 8 X 9 X 10 LX 11 X 12 (SEQ ID NO: 153), wherein X 1 is Y, V or F, and X 2 is I or T, X 3 is H, W or Q; X 4 is H, G, Y or F; X 5 is R, S or D; X 6 is S, T, N or A; X 7 is N or K ; X 8 is N or H; X 9 is P or S; X 10 is S or A; X 11 is K or I; X 12 is S or N;
  • the amino acid sequence of HCDR3 comprises GX 1 X 2 X 3 X 4 X 5 DX 6 (SEQ ID NO: 154), wherein X 1 is S or T; X 2 is N, D or T; X 3 is S, R, W , G or T; X4 is A, S or G; X5 is F or L; X6 is Y or H; or the amino acid sequence of HCDR3 is YYGYDFFAY (SEQ ID NO:25) or YYGYDFFPY (SEQ ID NO:26 );
  • the amino acid sequence of LCDR1 comprises X 1 SSQSLLYSX 2 X 3 QKNYX 4 X 5 (SEQ ID NO: 155), wherein X 1 is K or Q, X 2 is N, S or K, X 3 is N or D; X 4 is L or S; X 5 is A, T or V; or the amino acid sequence of LCDR1 is RASQDISNYLN (SEQ ID NO: 35), SASSSISYMH (SEQ ID NO: 36),
  • SASSSLSYMH (SEQ ID NO:37), SASSSVSSSYLY (SEQ ID NO:38) or SASSSVSYMH (SEQ ID NO:39);
  • the amino acid sequence of LCDR2 comprises X 1 X 2 SX 3 X 4 X 5 S (SEQ ID NO: 156), wherein X 1 is W, L, D, S or Y; X 2 is A or T; X 3 is T, K, N or R; X4 is R, K or L; X5 is E, A, Y or H;
  • the amino acid sequence of LCDR3 comprises X 1 QX 2 X 3 X 4 X 5 PX 6 X 7 (SEQ ID NO: 157), wherein X 1 is Q, H or F; X 2 is Y, G, R or W; X 3 is Y, S, N or F; X 4 is I, G, S, T or L; X 5 is Y or L; X 6 is L, F, C, P, H or M; X 7 is T or S ;
  • HCDR1-3 and LCDR1-3 are defined according to Kabat.
  • the present application provides an anti-CD24 antibody or an antigen-binding fragment thereof, wherein the anti-CD24 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region includes a heavy chain CDR1 (HCDR1), a heavy chain chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), wherein:
  • HCDR1 heavy chain CDR1
  • HCDR2 heavy chain chain CDR2
  • HCDR3 heavy chain CDR3
  • the amino acid sequence of HCDR1 comprises X 1 GYX 2 WH (SEQ ID NO: 152), wherein X 1 is R, N or S, and X 2 is S or T; or the amino acid sequence of HCDR1 is TYGVS (SEQ ID NO: 5);
  • the amino acid sequence of HCDR2 comprises X 1 X 2 X 3 X 4 X 5 GX 6 TX 7 YX 8 X 9 X 10 LX 11 X 12 (SEQ ID NO: 153), wherein X 1 is Y, V or F, and X 2 is I or T, X 3 is H, W or Q; X 4 is H, G, Y or F; X 5 is R, S or D; X 6 is S, T, N or A; X 7 is N or K ; X 8 is N or H; X 9 is P or S; X 10 is S or A; X 11 is K or I; X 12 is S or N;
  • the amino acid sequence of HCDR3 comprises GX 1 X 2 X 3 X 4 X 5 DX 6 (SEQ ID NO: 154), wherein X 1 is S or T; X 2 is N, D or T; X 3 is S, R, W , G or T; X4 is A, S or G; X5 is F or L; X6 is Y or H; or the amino acid sequence of HCDR3 is YYGYDFFAY (SEQ ID NO:25) or YYGYDFFPY (SEQ ID NO:26 );
  • HCDR1-3 is defined according to Kabat.
  • the present application provides an isolated nucleic acid molecule (polynucleotide), which encodes the anti-CD24 antibody or antigen-binding fragment described in the first to second aspects.
  • the present application provides a vector comprising a nucleic acid molecule (polynucleotide) encoding the anti-CD24 antibody or antigen-binding fragment described in the first to second aspects.
  • the present application provides an isolated host cell comprising the nucleic acid molecule described in the third aspect or the vector described in the fourth aspect.
  • the present application provides an immunoconjugate, which comprises the anti-CD24 antibody or antigen-binding fragment thereof described in the first to second aspects coupled to a therapeutic agent.
  • the present application provides a pharmaceutical composition, which comprises the anti-CD24 antibody or antigen-binding fragment thereof described in the first to second aspects or the immunoconjugate described in the sixth aspect, and a pharmaceutically acceptable excipient excipients, diluents or carriers.
  • the present application provides the anti-CD24 antibody or its antigen-binding fragment described in the first to second aspects, or the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect, or the host described in the fifth aspect Use of the cell or the immunoconjugate described in the sixth aspect in the preparation of a medicament for preventing or treating tumors (such as malignant tumors) or immune diseases.
  • the present application provides a method for treating a tumor (such as a malignant tumor) or an immune disease in an individual, comprising administering a therapeutically effective amount of the anti-CD24 antibody or its antigen described in the first to second aspects to the individual in need.
  • a tumor such as a malignant tumor
  • an immune disease in an individual, comprising administering a therapeutically effective amount of the anti-CD24 antibody or its antigen described in the first to second aspects to the individual in need
  • the binding fragment, the immunoconjugate of the sixth aspect or the pharmaceutical composition of the seventh aspect comprising administering a therapeutically effective amount of the anti-CD24 antibody or its antigen described in the first to second aspects.
  • the present application provides a method for preparing the anti-CD24 antibody or antigen-binding fragment thereof described in the first to second aspects, which includes the step of culturing the host cell described in the fifth aspect.
  • Figures 1A-1G show the time-dependent changes in tumor volume in tumor-bearing mice treated with different exemplary anti-CD24 antibodies of the present application.
  • Figures 2A-2F show tumor weights at the end of the experiment in tumor-bearing mice treated with different exemplary anti-CD24 antibodies of the present application.
  • an “antibody” may refer to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, thus encompassing whole antibodies, antibody single chains, or antibody fragments. Any antigen-binding fragment (also referred to as an "antigen-binding portion").
  • antigen-binding portion also referred to as an "antigen-binding portion”.
  • Full-length antibody refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated VL) and a light chain constant region.
  • the light chain constant region comprises one domain, CL.
  • the VH and VL regions can be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • These variable regions of the heavy and light chains contain the binding domains that interact with the antigen.
  • the constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system.
  • a full-length antibody may be of any class of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses of the above), but the antibody need not belong to any particular class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional structures of the different classes of immunoglobulins are well known.
  • Chimeric or humanized antibodies are also encompassed by antibodies according to the present application.
  • CDRs complementarity determining regions
  • CDRs are regions in the variable region that have the greatest influence on the affinity and specificity of antibodies.
  • CDR amino acid sequence of VH or VL There are two common ways of defining the CDR amino acid sequence of VH or VL, i.e. Chothia definition and kabat definition.
  • the CDR amino acid sequences in the VH and VL amino acid sequences can generally be determined according to the Chothia definition or the Kabat definition. In an embodiment of the present application, Kabat is used to define the CDR amino acid sequences.
  • the amino acid sequence of the CDR in the amino acid sequence of the variable region can be analyzed in various ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragment refers in particular to antibody fragments such as Fv, Fab, F(ab') 2 or Fab', or any fragment whose half-life should be increased by chemical modification or by incorporation into liposomes.
  • Fragments preferably, said functional fragments will consist of or comprise partial sequences of the heavy or light variable chains of the antibody from which they are derived, said partial sequences being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which they are derived, which Such a functional fragment will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which it is derived.
  • antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having a VL-CL chain and a VH-CH1 chain; ( 2 ) F(ab') fragments, which may be monovalent fragments having two A bivalent fragment of a Fab' fragment, the two Fab' fragments are connected by a disulfide bridge in the hinge region (ie a dimer of Fab'); (3) an Fv fragment with the VL and VH domains of a single arm of an antibody.
  • single-chain antibody refers to a single polypeptide chain that is connected by a VH domain and a VL domain via a peptide linker.
  • (scFv) 2 comprises two VH domains and two VL domains connected by a peptide linker, the two The VL domain is combined with the two VH domains via a disulfide bridge.
  • Fc fragment refers to a portion of the constant region of an antibody heavy chain, including the hinge, CH2 and CH3 fragments of the constant region.
  • binding refers to a non-random binding reaction between two molecules, eg binding of an antibody to an antigenic epitope.
  • humanized antibody means an antibody obtained by grafting CDR sequences derived from the germline of another mammalian species, such as a mouse, onto human framework sequences. Additional framework region modifications can be made in the human framework sequences. Humanized antibodies or fragments thereof according to the present application can be prepared by techniques known to those skilled in the art.
  • chimeric antibody refers to an antibody in which the variable region sequences are from one species and the constant region sequences are from another species, eg, an antibody in which the variable region sequences are from a mouse antibody and the constant region sequences are from a human antibody.
  • the chimeric antibody or fragment thereof according to the present application can be produced by using gene recombination technology.
  • said chimeric antibodies can be produced by cloning recombinant DNA comprising a promoter and sequences encoding the variable regions of non-human, especially murine monoclonal antibodies according to the present application, and sequences encoding the constant regions of human antibodies .
  • the chimeric antibody of the present application encoded by such a recombinant gene will be, for example, a mouse-human chimera whose specificity is determined by a variable region derived from mouse DNA and whose isotype is determined by a variable region derived from human DNA. to determine the constant region.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations in a small number of individuals.
  • the monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding amino acid sequence in an antibody derived from a particular species or belonging to a particular class or subclass of antibodies, and The remainder of the heavy and/or light chain is identical or homologous to the corresponding amino acid sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, so long as they express expected biological activity.
  • bispecific antibody refers to an antibody that has the ability to bind two epitopes at the same time.
  • the two epitopes can be on different antigens or on the same antigen.
  • Bispecific antibodies can have a variety of structural configurations.
  • a bispecific antibody can be composed of two Fc fragments and two binding parts fused to them respectively (similar to natural antibodies, the difference is that the two arms bind different antigenic targets or epitopes), and the antigen-binding part can be a single chain Antibody (scfv) format or Fab fragment format.
  • pharmaceutical composition refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or adjuvant combined together to achieve a specific purpose.
  • the pharmaceutical composition includes combinations that are separated in time and/or space, as long as they can work together to achieve the purpose of the present application.
  • the components contained in the pharmaceutical composition such as antibodies, nucleic acid molecules, nucleic acid molecule combinations and/or conjugates according to the present application
  • the ingredients contained in the pharmaceutical composition are administered to the individual separately, the ingredients may be administered to the individual simultaneously or sequentially.
  • the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline solution such as, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol or polyalkylene glycol Such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the present application is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the compositions according to the present application may comprise wetting agents, emulsifiers or buffer substances as additives.
  • the pharmaceutical composition or pharmaceutical formulation according to the present application may be administered by any suitable route, for example may be administered orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
  • terapéuticaally effective amount refers to a dose sufficient to demonstrate benefit to the subject to which it is administered.
  • the actual amount administered, as well as the rate and time course of administration, will depend upon the individual condition and severity of the individual being treated.
  • the prescribing of treatment e.g. decisions on dosage etc.
  • the prescribing of treatment is ultimately the responsibility of and relies on the general practitioner and other medical practitioners to make decisions, usually taking into account the disease being treated, the individual patient's condition, the site of delivery, the method of administration and what is known to the physician. other known factors.
  • mammals such as humans, but can also be other animals, such as wild animals (such as herons, storks, cranes, etc.), domestic animals (such as ducks, geese, etc.) or experimental animals (such as orangutans, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).
  • wild animals such as herons, storks, cranes, etc.
  • domestic animals such as ducks, geese, etc.
  • experimental animals such as orangutans, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.
  • homology/identity/identity with respect to amino acid or nucleic acid sequences is defined as the percentage of residues that are identical in amino acid or nucleotide sequence variants after alignment of the sequences and introduction of gaps, if desired, to Maximum percent homology. Methods and computer programs for alignment are well known in the art.
  • the application provides an anti-CD24 antibody or an antigen-binding fragment thereof, wherein the anti-CD24 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), the light chain variable region includes light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), where:
  • the amino acid sequence of HCDR1 comprises X 1 GYX 2 WH (SEQ ID NO: 152), wherein X 1 is R, N or S, and X 2 is S or T; or the amino acid sequence of HCDR1 is TYGVS (SEQ ID NO: 5);
  • the amino acid sequence of HCDR2 comprises X 1 X 2 X 3 X 4 X 5 GX 6 TX 7 YX 8 X 9 X 10 LX 11 X 12 (SEQ ID NO: 153), wherein X 1 is Y, V or F, and X 2 is I or T, X 3 is H, W or Q; X 4 is H, G, Y or F; X 5 is R, S or D; X 6 is S, T, N or A; X 7 is N or K ; X 8 is N or H; X 9 is P or S; X 10 is S or A; X 11 is K or I; X 12 is S or N;
  • the amino acid sequence of HCDR3 comprises GX 1 X 2 X 3 X 4 X 5 DX 6 (SEQ ID NO: 154), wherein X 1 is S or T; X 2 is N, D or T; X 3 is S, R, W , G or T; X4 is A, S or G; X5 is F or L; X6 is Y or H; or the amino acid sequence of HCDR3 is YYGYDFFAY (SEQ ID NO:25) or YYGYDFFPY (SEQ ID NO:26 );
  • the amino acid sequence of LCDR1 comprises X 1 SSQSLLYSX 2 X 3 QKNYX 4 X 5 (SEQ ID NO: 155), wherein X 1 is K or Q, X 2 is N, S or K, X 3 is N or D; X 4 is L or S; X5 is A, T or V; or the amino acid sequence of LCDR1 is RASQDISNYLN (SEQ ID NO:35), SASSSISYMH (SEQ ID NO:36), SASSSLSYMH (SEQ ID NO:37), SASSSVSSSYLY (SEQ ID NO:38) or SASSSVSYMH (SEQ ID NO:39);
  • the amino acid sequence of LCDR2 comprises X 1 X 2 SX 3 X 4 X 5 S (SEQ ID NO: 156), wherein X 1 is W, L, D, S or Y; X 2 is A or T; X 3 is T, K, N or R; X4 is R, K or L; X5 is E, A, Y or H;
  • the amino acid sequence of LCDR3 comprises X 1 QX 2 X 3 X 4 X 5 PX 6 X 7 (SEQ ID NO: 157), wherein X 1 is Q, H or F; X 2 is Y, G, R or W; X 3 is Y, S, N or F; X 4 is I, G, S, T or L; X 5 is Y or L; X 6 is L, F, C, P, H or M; X 7 is T or S .
  • HCDR1 has any amino acid sequence in SEQ ID NO: 1-5
  • HCDR2 has any amino acid sequence in SEQ ID NO: 6-18
  • HCDR3 has any amino acid sequence in SEQ ID NO: 19-26
  • LCDR1 has any amino acid sequence in SEQ ID NO:27-39
  • LCDR2 has any amino acid sequence in SEQ ID NO:40-47
  • LCDR3 has any amino acid sequence in SEQ ID NO:48-56.
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 19, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 35, SEQ ID NO: 47. SEQ ID NO: 53;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 17, and SEQ ID NO: 21, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 27, SEQ ID NO: 43, and SEQ ID NO: 55;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 28, SEQ ID NO: 45, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO19: respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 28, SEQ ID NO: 41, SEQ ID NO: 48 ;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 20, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 27, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 20, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 27, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 16, and SEQ ID NO: 24, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 34, SEQ ID NO: 40, and SEQ ID NO: 49;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 25, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 37, SEQ ID NO: 42, and SEQ ID NO: 50;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 26, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 39, SEQ ID NO: 42, and SEQ ID NO: 50;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 19, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 28, SEQ ID NO: 41, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 21, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 31, SEQ ID NO: 46, and SEQ ID NO: 49;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 21, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 27, SEQ ID NO: 43, and SEQ ID NO: 56;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 20, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 30, SEQ ID NO: 40, and SEQ ID NO: 49;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 20, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 30, SEQ ID NO: 40, and SEQ ID NO: 49;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 22, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 28, SEQ ID NO: 41, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 3, SEQ ID NO: 18, and SEQ ID NO: 22, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 28, SEQ ID NO: 41, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 28, SEQ ID NO: 41, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 20, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 31, SEQ ID NO: 40, and SEQ ID NO: 54;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 20, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 27, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 28, SEQ ID NO: 41, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 3, SEQ ID NO: 15, and SEQ ID NO: 20, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 27, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 27, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 20, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 28, SEQ ID NO: 41, SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 27, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 19, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 33, SEQ ID NO: 40, and SEQ ID NO: 49;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 4, SEQ ID NO: 11, and SEQ ID NO: 23, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 33, SEQ ID NO: 40, and SEQ ID NO: 49;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 4, SEQ ID NO: 11, and SEQ ID NO: 23, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 14, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 29, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 19, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 29, SEQ ID NO: 40, and SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 22, respectively, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 48;
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 38, SEQ ID NO: 44, and SEQ ID NO: 52; or
  • amino acid sequences of HCDR1-HCDR3 are SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 19, and the amino acid sequences of LCDR1-LCDR3 are SEQ ID NO: 36, SEQ ID NO: 42, and SEQ ID NO: 51.
  • the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NO:57-71 and 98-120 or with any one of SEQ ID NO:57-71 and 98-120
  • the amino acid sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98% or 99% homologous amino acid sequences.
  • the amino acid sequence of the heavy chain variable region differs from the amino acid sequence set forth in any one of SEQ ID NOs: 57-71 and 98-120 by about 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 amino acid substitutions, deletions and/or additions.
  • the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NOS: 57-71 and 98-120 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the heavy chain variable region of the antibody.
  • 1, 2, 3, 4, 5, 6, 7, 8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids the resulting amino acid sequence still maintains a function similar to the heavy chain variable region of the antibody.
  • 1, 2, 3, 4, 5, 1, 2, 3, 4, 5, 1, 2, 3, 4, 5, or 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids as long as the altered amino acid sequence remains substantially similar to that of the heavy chain variable region of the antibody Function.
  • the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 72-94 and 121-147 or the same as shown in any one of SEQ ID NOs: 72-94 and 121-147
  • the amino acid sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98% or 99% homologous amino acid sequences.
  • the amino acid sequence of the light chain variable region differs by about 1, 2, 3, 4, 5, 6, 7 from the amino acid sequence set forth in any one of SEQ ID NOs: 72-94 and 121-147 , 8, 9 or 10 amino acid substitutions, deletions and/or additions.
  • the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 72-94 and 121-147 can also be truncated by about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining a similar light chain variable region function of the antibody.
  • 1, 2, 3, 4, 5, 6, 7 can also be added to the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 72-94 and 121-147 , 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, the resulting amino acid sequence still maintains a similar light chain variable region function of the antibody.
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:57, and the amino acid sequence of the light chain variable region is SEQ ID NO:72;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:58, and the amino acid sequence of the light chain variable region is SEQ ID NO:73;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:59, and the amino acid sequence of the light chain variable region is SEQ ID NO:74;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:60, and the amino acid sequence of the light chain variable region is SEQ ID NO:75;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:61, and the amino acid sequence of the light chain variable region is SEQ ID NO:76;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:62, and the amino acid sequence of the light chain variable region is SEQ ID NO:76;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:63, and the amino acid sequence of the light chain variable region is SEQ ID NO:77;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:64, and the amino acid sequence of the light chain variable region is SEQ ID NO:78;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:65, and the amino acid sequence of the light chain variable region is SEQ ID NO:79;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:59, and the amino acid sequence of the light chain variable region is SEQ ID NO:80;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:59, and the amino acid sequence of the light chain variable region is SEQ ID NO:81;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:66, and the amino acid sequence of the light chain variable region is SEQ ID NO:82;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:66, and the amino acid sequence of the light chain variable region is SEQ ID NO:83;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:61, and the amino acid sequence of the light chain variable region is SEQ ID NO:84;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:61, and the amino acid sequence of the light chain variable region is SEQ ID NO:85;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:67, and the amino acid sequence of the light chain variable region is SEQ ID NO:86;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:68, and the amino acid sequence of the light chain variable region is SEQ ID NO:87;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:57, and the amino acid sequence of the light chain variable region is SEQ ID NO:88;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:61, and the amino acid sequence of the light chain variable region is SEQ ID NO:89;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:61, and the amino acid sequence of the light chain variable region is SEQ ID NO:76;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:57, and the amino acid sequence of the light chain variable region is SEQ ID NO:90;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:69, and the amino acid sequence of the light chain variable region is SEQ ID NO:76;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:57, and the amino acid sequence of the light chain variable region is SEQ ID NO:76;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:69, and the amino acid sequence of the light chain variable region is SEQ ID NO:90;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:59, and the amino acid sequence of the light chain variable region is SEQ ID NO:76;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:59, and the amino acid sequence of the light chain variable region is SEQ ID NO:91;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:70, and the amino acid sequence of the light chain variable region is SEQ ID NO:91;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:70, and the amino acid sequence of the light chain variable region is SEQ ID NO:80;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:71, and the amino acid sequence of the light chain variable region is SEQ ID NO:92;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:57, and the amino acid sequence of the light chain variable region is SEQ ID NO:92;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:67, and the amino acid sequence of the light chain variable region is SEQ ID NO:92;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:57, and the amino acid sequence of the light chain variable region is SEQ ID NO:93;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:57, and the amino acid sequence of the light chain variable region is SEQ ID NO:94;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:101, and the amino acid sequence of the light chain variable region is SEQ ID NO:137;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and the amino acid sequence of the light chain variable region is SEQ ID NO:144;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:99, and the amino acid sequence of the light chain variable region is SEQ ID NO:122;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:114, and the amino acid sequence of the light chain variable region is SEQ ID NO:143;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and the amino acid sequence of the light chain variable region is SEQ ID NO:142;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:101, and the amino acid sequence of the light chain variable region is SEQ ID NO:124;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and the amino acid sequence of the light chain variable region is SEQ ID NO:122;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:99, and the amino acid sequence of the light chain variable region is SEQ ID NO:121;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and the amino acid sequence of the light chain variable region is SEQ ID NO:124;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and the amino acid sequence of the light chain variable region is SEQ ID NO:137;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and the amino acid sequence of the light chain variable region is SEQ ID NO:121;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:101, and the amino acid sequence of the light chain variable region is SEQ ID NO:121;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:101, and the amino acid sequence of the light chain variable region is SEQ ID NO:133;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:99, and the amino acid sequence of the light chain variable region is SEQ ID NO:124;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:99, and the amino acid sequence of the light chain variable region is SEQ ID NO:134;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:111, and the amino acid sequence of the light chain variable region is SEQ ID NO:136;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and the amino acid sequence of the light chain variable region is SEQ ID NO:134;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and the amino acid sequence of the light chain variable region is SEQ ID NO:133;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:99, and the amino acid sequence of the light chain variable region is SEQ ID NO:133;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:101, and the amino acid sequence of the light chain variable region is SEQ ID NO:122;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:118, and the amino acid sequence of the light chain variable region is SEQ ID NO:146;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:99, and the amino acid sequence of the light chain variable region is SEQ ID NO:137;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:115, and the amino acid sequence of the light chain variable region is SEQ ID NO:122;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:117, and the amino acid sequence of the light chain variable region is SEQ ID NO:121;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:116, and the amino acid sequence of the light chain variable region is SEQ ID NO:145;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:104, and the amino acid sequence of the light chain variable region is SEQ ID NO:125;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:105, and the amino acid sequence of the light chain variable region is SEQ ID NO:123;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:110, and the amino acid sequence of the light chain variable region is SEQ ID NO:126;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:109, and the amino acid sequence of the light chain variable region is SEQ ID NO:123;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:110, and the amino acid sequence of the light chain variable region is SEQ ID NO:123;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:104, and the amino acid sequence of the light chain variable region is SEQ ID NO:130;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:109, and the amino acid sequence of the light chain variable region is SEQ ID NO:126;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:108, and the amino acid sequence of the light chain variable region is SEQ ID NO:130;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:105, and the amino acid sequence of the light chain variable region is SEQ ID NO:126;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:113, and the amino acid sequence of the light chain variable region is SEQ ID NO:141;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO: 108, and the amino acid sequence of the light chain variable region is SEQ ID NO: 125;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:103, and the amino acid sequence of the light chain variable region is SEQ ID NO:139;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:107, and the amino acid sequence of the light chain variable region is SEQ ID NO:140;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:106, and the amino acid sequence of the light chain variable region is SEQ ID NO:140;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:106, and the amino acid sequence of the light chain variable region is SEQ ID NO:129;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:107, and the amino acid sequence of the light chain variable region is SEQ ID NO:129;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:107, and the amino acid sequence of the light chain variable region is SEQ ID NO:128;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:103, and the amino acid sequence of the light chain variable region is SEQ ID NO:128;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO: 103, and the amino acid sequence of the light chain variable region is SEQ ID NO: 140;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:103, and the amino acid sequence of the light chain variable region is SEQ ID NO:129;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:120, and the amino acid sequence of the light chain variable region is SEQ ID NO:139;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:106, and the amino acid sequence of the light chain variable region is SEQ ID NO:128;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:112, and the amino acid sequence of the light chain variable region is SEQ ID NO:147;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:112, and the amino acid sequence of the light chain variable region is SEQ ID NO:138;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:119, and the amino acid sequence of the light chain variable region is SEQ ID NO:138;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:100, and the amino acid sequence of the light chain variable region is SEQ ID NO:127;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:102, and the amino acid sequence of the light chain variable region is SEQ ID NO:127;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:100, and the amino acid sequence of the light chain variable region is SEQ ID NO:135;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:102, and the amino acid sequence of the light chain variable region is SEQ ID NO:131;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:100, and the amino acid sequence of the light chain variable region is SEQ ID NO:132;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:102, and the amino acid sequence of the light chain variable region is SEQ ID NO:135;
  • amino acid sequence of the heavy chain variable region is SEQ ID NO: 102, and the amino acid sequence of the light chain variable region is SEQ ID NO: 132; or
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO:100, and the amino acid sequence of the light chain variable region is SEQ ID NO:131.
  • the present application provides an anti-CD24 antibody or an antigen-binding fragment thereof, wherein the anti-CD24 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region includes a heavy chain CDR1 (HCDR1), a heavy chain chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), wherein:
  • HCDR1 heavy chain CDR1
  • HCDR2 heavy chain chain CDR2
  • HCDR3 heavy chain CDR3
  • the amino acid sequence of HCDR1 comprises X 1 GYX 2 WH (SEQ ID NO: 152), wherein X 1 is R, N or S, and X 2 is S or T; or the amino acid sequence of HCDR1 is TYGVS (SEQ ID NO: 5);
  • the amino acid sequence of HCDR2 comprises X 1 X 2 X 3 X 4 X 5 GX 6 TX 7 YX 8 X 9 X 10 LX 11 X 12 (SEQ ID NO: 153), wherein X 1 is Y, V or F, and X 2 is I or T, X 3 is H, W or Q; X 4 is H, G, Y or F; X 5 is R, S or D; X 6 is S, T, N or A; X 7 is N or K ; X 8 is N or H; X 9 is P or S; X 10 is S or A; X 11 is K or I; X 12 is S or N;
  • the amino acid sequence of HCDR3 comprises GX 1 X 2 X 3 X 4 X 5 DX 6 (SEQ ID NO: 154), wherein X 1 is S or T; X 2 is N, D or T; X 3 is S, R, W , G or T; X4 is A, S or G; X5 is F or L; X6 is Y or H; or the amino acid sequence of HCDR3 is YYGYDFFAY (SEQ ID NO:25) or YYGYDFFPY (SEQ ID NO:26 ).
  • VHH antibody is a single-domain antibody, also known as a heavy chain variable region gene antibody (Variable domain of heavy chain of heavy-chain antibody, VHH), which consists of only one heavy chain variable region, Also known as nanobodies.
  • VHH antibody is a natural light chain-deficient antibody originally found in the peripheral blood of alpaca, which contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.
  • the cloned and expressed VHH structure has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind the target antigen.
  • the anti-CD24 antibody is a whole antibody, a single chain antibody (scFv) or a bispecific antibody.
  • the antigen-binding fragment of the anti-CD24 antibody is Fab, Fab', Fv or F(ab') 2 .
  • the anti-CD24 antibody is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the anti-CD24 antibody is a monoclonal antibody.
  • the anti-CD24 antibody is a bispecific antibody.
  • the light chain constant region of the anti-CD24 antibody or antigen-binding fragment thereof is a light chain constant region of a human antibody.
  • the light chain constant region of the anti-CD24 antibody or antigen-binding fragment thereof is selected from human kappa-type or lambda-type light chain constant regions.
  • the light chain constant region of the anti-CD24 antibody or antigen-binding fragment thereof is a human kappa-type light chain constant region.
  • the human ⁇ -type light chain constant region has an amino acid sequence as set forth in SEQ ID NO: 151 or a mutant thereof.
  • the heavy chain constant region of the anti-CD24 antibody or antigen-binding fragment thereof is the heavy chain constant region of a human antibody.
  • the heavy chain constant region of the anti-CD24 antibody or antigen-binding fragment thereof is selected from any of human IgD, IgE, IgM, IgA, IgG1, IgG2a, IgG2b, IgG3, and IgG4 types. A sort of.
  • the heavy chain constant region of the antibody or antigen-binding fragment thereof is selected from IgG1, IgG2 or IgG4 type.
  • the IgG1 type heavy chain constant region has the amino acid sequence as described in SEQ ID NO: 148 and mutants thereof; the IgG2 type heavy chain constant region has the amino acid sequence as described in SEQ ID NO: 149 and Its mutant; the IgG4 type heavy chain constant region has the amino acid sequence described in SEQ ID NO: 150 and its mutants.
  • the present application provides an isolated nucleic acid molecule (polynucleotide), which encodes the anti-CD24 antibody or antigen-binding fragment described in the first to second aspects.
  • the polynucleotide is operably linked to a regulatory sequence that is recognized by a host cell transformed with the vector.
  • the present application provides a vector (such as an expression vector), which comprises a polynucleotide encoding the anti-CD24 antibody or antigen-binding fragment described in the first to second aspects.
  • a vector such as an expression vector
  • the choice of expression vector depends on the choice of the host cell and can be selected so as to have the desired expression and regulatory characteristics in the host cell of choice.
  • An "expression vector” is a vector that includes one or more expression control sequences, which are DNA sequences that control and regulate the transcription and/or translation of another DNA sequence.
  • the nucleic acid in the vector may be operably linked to one or more expression control sequences.
  • "operably linked” means incorporated into a genetic construct such that expression control sequences effectively control the expression of a coding sequence of interest.
  • expression control sequences include promoters, enhancers, and transcription termination regions.
  • a promoter is an expression control sequence consisting of a region of a DNA molecule usually within 100 nucleotides upstream of the start of transcription (usually near the initiation site for RNA polymerase II).
  • the translation initiation site of the translational reading frame of the polypeptide must be positioned between 1 and about 50 nucleotides downstream of the promoter.
  • Enhancers provide expression specificity in terms of time, location and level. Unlike promoters, enhancers can function when located at different distances from the transcription site. Enhancers can also be located downstream of the transcription initiation site.
  • a coding sequence is "operably linked to” and “under the control of” an expression control sequence in a cell when RNA polymerase is capable of transcribing the coding sequence into mRNA, which can then be translated into the protein encoded by the coding sequence ".
  • Suitable expression vectors include, but are not limited to, plasmid and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus.
  • Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).
  • Expression vectors can include tag sequences.
  • Tag sequences are usually expressed as fusions to the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including the carboxyl or amino termini. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, FlagTM tags (Kodak, New Haven, CT), maltose E-binding protein and protein A.
  • a nucleic acid molecule encoding a Siglec-15 fusion polypeptide is present in a vector comprising nucleic acid encoding one or more domains of an Ig heavy chain constant region, e.g., corresponding to human immunoglobulin C ⁇ 1
  • an Ig heavy chain constant region e.g., corresponding to human immunoglobulin C ⁇ 1
  • the amino acid sequence of the hinge region, CH2 region and CH3 region of the chain (Fc fragment).
  • the present application provides an isolated host cell comprising the nucleic acid molecule described in the third aspect or the vector described in the fourth aspect.
  • the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a phage.
  • the prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.
  • Eukaryotic host cells can be mammalian cells, such as 293 cells, CHO cells, BHK cells, COS cells, myeloma cells, etc.; yeast cells, such as Pichia pastoris, Saccharomyces cerevisiae, etc.; insect cells, Such as lawn armyworm, etc.; plant cells, such as tobacco, etc.
  • the application provides an immunoconjugate (also known as an antibody-drug conjugate (ADC)), which comprises the anti-CD24 antibody or its antigen described in the first to second aspects coupled to a therapeutic agent Combine fragments.
  • ADC antibody-drug conjugate
  • the therapeutic agent is an antineoplastic drug, eg, a cytotoxic drug, an immunopotentiator, or a radioisotope.
  • the type of cytotoxic drug includes tubulin inhibitors (eg, alkaloids), DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics.
  • tubulin inhibitors eg, alkaloids
  • DNA topoisomerase inhibitors e.g., DNA damaging agents, antimetabolites, or antitumor antibiotics.
  • tubulin inhibitors include, but are not limited to, auristatin derivatives (such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (such as DM1, DM4, Ansamitocin, Mertansine or dolastatin and its derivatives).
  • auristatin derivatives such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)
  • maytansine alkaloid derivatives such as DM1, DM4, Ansamitocin, Mertansine or dolastatin and its derivatives.
  • the DNA topoisomerase inhibitor is a camptothecin analog or a DNA topoisomerase I inhibitor and derivatives thereof, e.g., DXD, SN38, irinotecan, irinotecan hydrochloride, Camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxycamptothecin, topotecan, le Totecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)benzene Base]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2 -Cyano-3-(3,4-dihydroxyphenyl)
  • DNA damaging agents include, but are not limited to, calicheamicins, duocarmycins, antramycin derivatives PBD (pyrrolobenzodiazepine, pyrrolobenzodiazepine ).
  • antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine, or 5-fluorouracil.
  • antitumor antibiotics include, but are not limited to, polypeptide antibiotics (such as actinomycin D or bleomycin) or anthraquinones (such as doxorubicin or mitoxantrone hydrochloride).
  • radioactive isotopes include, but are not limited to, 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 60Co, or 177Lu.
  • immune potentiating agents include, but are not limited to, levamisole, pidotimod, imiquimod, isoprinosine, polyinosine, or polyinosine glycosides.
  • the anti-CD24 antibody of the present application is covalently linked to the drug moiety via a linker.
  • the linker is a cleavable linker.
  • linkers are cleavable under intracellular conditions.
  • the linker is hydrolyzable at a pH of less than 5.5.
  • linkers are cleavable by intracellular proteases.
  • the linker is a cathepsin cleavable linker.
  • the linker comprises a dipeptide.
  • the dipeptide is valine (Val)-citrulline (Cit).
  • the antibody is attached to the linker through a cysteine sulfhydryl group of the antibody.
  • the present application provides a pharmaceutical composition, which comprises the anti-CD24 antibody or antigen-binding fragment thereof described in the first to second aspects or the immunoconjugate described in the sixth aspect, and a pharmaceutically acceptable excipient excipients, diluents or carriers.
  • the pharmaceutical composition is an intravitreal injection, a subretinal injection, an intrachoroidal injection, an intravenous injection, an intratumoral injection, or an intramuscular injection.
  • the pharmaceutical composition may also contain one or more of: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preservatives, Such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.
  • lubricants such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying agents such as benzoic acid, sorbic acid and calcium propionate
  • preservatives such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and/or flavoring agents etc.
  • the pharmaceutical compositions of the present application can be formulated as tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules and the like.
  • the pharmaceutical composition of the present application can be delivered by any physiologically acceptable mode of administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration Drugs, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.
  • pharmaceutical compositions for therapeutic use may be formulated in the form of lyophilized formulations or aqueous solutions by mixing reagents of desired purity with, as appropriate, pharmaceutically acceptable carriers, excipients, etc. storage.
  • the pharmaceutical composition is used to prevent or treat tumors (eg, malignant tumors) or immune diseases.
  • tumors are tumors with high expression of CD24 on the surface of tumor cells, including but not limited to, lung cancer, breast cancer, ovarian cancer, lymphatic cancer, colon/rectal cancer, ovarian cancer, liver cancer, cervical cancer , kidney, prostate, esophagus, bladder, or nasopharyngeal cancer.
  • the immune disease is rheumatoid arthritis or systemic lupus erythematosus.
  • the present application provides the anti-CD24 antibody or its antigen-binding fragment described in the first to second aspects, or the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect, or the host described in the fifth aspect Use of the cell or the immunoconjugate described in the sixth aspect in the preparation of a medicament for preventing or treating tumors (such as malignant tumors) or immune diseases.
  • tumors are tumors with high expression of CD24 on the surface of tumor cells, including but not limited to, lung cancer, breast cancer, ovarian cancer, lymphatic cancer, colon/rectal cancer, ovarian cancer, liver cancer, cervical cancer , kidney, prostate, esophagus, bladder, or nasopharyngeal cancer.
  • the immune disease is rheumatoid arthritis or systemic lupus erythematosus.
  • the present application provides a method for treating a tumor (such as a malignant tumor) or an immune disease in an individual, comprising administering a therapeutically effective amount of the anti-CD24 antibody or its antigen described in the first to second aspects to the individual in need
  • a tumor such as a malignant tumor
  • tumors are tumors with high expression of CD24 on the surface of tumor cells, including but not limited to, lung cancer, breast cancer, ovarian cancer, lymphatic cancer, colon/rectal cancer, ovarian cancer, liver cancer, cervical cancer , kidney, prostate, esophagus, bladder, or nasopharyngeal cancer.
  • the immune disease is rheumatoid arthritis or systemic lupus erythematosus.
  • the present application provides a method for preparing the anti-CD24 antibody or antigen-binding fragment thereof described in the first to second aspects, which includes the step of culturing the host cell described in the fifth aspect. In some embodiments, the method further comprises the step of isolating said anti-CD24 antibody or antigen-binding fragment thereof.
  • Embodiment 1 hybridoma preparation and screening
  • Immune BALB/C female mice aged 6-8 weeks, purchased from Dashuo Experimental Animal Co., Ltd.
  • two methods of standard peritoneal immunization or plantar immunization to immunize mice, 10 ⁇ g/mouse, each Inject once every 2 to 3 weeks.
  • mice were first immunized with an equal volume of CFA (SIGMA, product number: SLBW7430) mixed with the antigen and emulsified, and the booster immunization (about the second immunization to the fourth immunization) before the sprint immunization was performed with IFA ( SIGMA, product number: F5506) is mixed with the antigen and emulsified to form a water-in-oil state, and the emulsification is completed when it is dropped into water and shaken gently without dispersion.
  • CFA CFA
  • IFA product number: F5506
  • the antigen used is a commercially available recombinant human CD24Fc chimeric protein (product number: 5247-CD-050, https://www.rndsystems.com/cn/products/recombinant-human-cd24-fc-chimera-protein-cf_5247-cd) .
  • the antigen used in the mouse spur immunization before hybridoma fusion was prepared in PBS (20 ⁇ g/mouse, 100 ⁇ l/mouse).
  • mice immunized with the antigen were immunized by sprint three days before the fusion of the hybridomas, the mice were sacrificed by twisting their necks, and the mice were sterilized by immersing them in 70% ethanol for 5 minutes.
  • the cells were resuspended in 5ml DMEM (GIBCO, catalog number: 12491023/10x 500mL) and set aside; after adding 12ml red blood cell lysate to the spleen B cells to blow off the cells, they were placed at room temperature for 8 minutes, and then centrifuged at 2000rpm for 5 minutes at room temperature. Discard the supernatant, add DMEM medium to resuspend, count the cells separately, and set aside.
  • Collect P3X63Ag8.653 myeloma cells (Cat. No.: CRL-1580, purchased from ATCC) or FO myeloma cells (product number: TCM31, purchased from the Cell Bank of Chinese Academy of Sciences), cell counting; myeloma cells and B cells (1:1) were mixed, ECF solution resuspended, cell counting; Add ECF solution to adjust the cell density to 2x 10 6 to 1x 10 7 cells/ml, add the cell suspension to the fusion tank, run the ECM2001 electric fusion instrument, and transfer the cell suspension from the fusion tank to a sterile In the hybridoma fusion medium containing HAT (GIBCO, catalog number: 20160017), mix thoroughly and add to a 96-well plate; culture in an 8% carbon dioxide incubator at 37°C.
  • Trizol method to extract the total RNA of hybridoma cells, take 5 ⁇ g of total RNA for reverse transcription, and obtain cDNA; then use the cDNA as a template, use 5'RACE technology for PCR amplification, and connect the PCR product into the enzyme-cut vector pcDNA3.4
  • the ligation product was transformed into competent DH5 ⁇ cells by heat shock, colony PCR identification was performed, a small amount of plasmid was extracted from the positive clones and sent for sequencing, and the light and heavy chain variable regions of the hybridoma antibody gene were obtained, as shown in Table 1.
  • Antibody number heavy chain variable region light chain variable region KH01 (SEQ ID NO:57) (SEQ ID NO:72) KH02 (SEQ ID NO:58) (SEQ ID NO:73) KH03 (SEQ ID NO:59) (SEQ ID NO:74) KH04 (SEQ ID NO:60) (SEQ ID NO:75) KH05 (SEQ ID NO:61) (SEQ ID NO:76) KH06 (SEQ ID NO:62) (SEQ ID NO:76) KH07 (SEQ ID NO:63) (SEQ ID NO:77)
  • KH08 (SEQ ID NO:64) (SEQ ID NO:78) KH09 (SEQ ID NO:65) (SEQ ID NO:79) KH10 (SEQ ID NO:59) (SEQ ID NO:80) KH11 (SEQ ID NO:59) (SEQ ID NO:81) KH12 (SEQ ID NO:66) (SEQ ID NO:82) KH13 (SEQ ID NO:66) (SEQ ID NO:83) KH14 (SEQ ID NO:61) (SEQ ID NO:84) KH15 (SEQ ID NO:61) (SEQ ID NO:85) KH16 (SEQ ID NO:67) (SEQ ID NO:86) KH17 (SEQ ID NO:68) (SEQ ID NO:87) KH18 (SEQ ID NO:57) (SEQ ID NO:88) KH19 (SEQ ID NO:61) (SEQ ID NO:89) KH20 (SEQ ID NO:61) (SEQ ID NO:76) KH21 (SEQ ID
  • KH01-04, KH08-13, KH16-18, KH21, KH23, KH32-33 murine antibody heavy chain constant region is mouse IgG2a, and the amino acid sequence is shown in SEQ ID NO:95.
  • KH05, KH07, KH14-15, KH19-20, KH22, KH24-31 murine antibody heavy chain constant region is mouse IgG2b, the amino acid sequence is shown in SEQ ID NO:96.
  • the light chain constant region of the KH01-33 mouse antibody is a mouse ⁇ light chain constant region, and its amino acid sequence is shown in SEQ ID NO:97.
  • Antibodies and engineered cells with high CD24 expression such as CD24 highly expressed CHO engineered cells/293 cells, and tumor cells, such as human lung cancer cells (A549 cells), human breast cancer cells (MCF-7, Ovcar- 3 cells), human B-cell lymphoma cells, and human colorectal cancer cells.
  • tumor cells such as human lung cancer cells (A549 cells), human breast cancer cells (MCF-7, Ovcar- 3 cells), human B-cell lymphoma cells, and human colorectal cancer cells.
  • This embodiment evaluates the phagocytic activity mediated by macrophages caused by anti-CD24 antibodies.
  • the test principle is: CD24 on tumor cells (for example, A549 in this embodiment and MCF-7 in Example 5) and Siglec10 on macrophages Combined to form a "don't eat me” signal, thereby inhibiting the phagocytosis of tumor cells by macrophages, the CD24 antibody can block the combination of CD24 and Siglec10, block the "don't eat me” signal, and stimulate the phagocytosis of macrophages.
  • M2 primary macrophages induced by human peripheral blood cells for 7 days adjust the cell density of the macrophage suspension to 0.7x10 6 cells/ml, and set aside.
  • A549-GFP-2 (self-made) tumor cells were prepared, and the cell density of the tumor cell suspension was adjusted to 1.4x10 6 cells/ml.
  • macrophages and tumor cells were co-incubated: 50 ⁇ l of macrophage suspension, 50 ⁇ l of tumor cell suspension, 50 ⁇ l of negative control antibody (mouse IgG) and the mouse-derived antibody prepared in the above examples were added to each well of a 96-well plate.
  • Antibody the antibody was prepared with PBS to a final concentration of 5 ⁇ g/ml), and mixed evenly.
  • This example evaluates macrophage-mediated phagocytic activity induced by anti-CD24 antibodies.
  • MCF-7 luciferase-GFP (self-made) tumor cells were prepared, and the cell density of the tumor cell suspension was adjusted to 1.4x10 6 cells/ml.
  • macrophages and tumor cells were co-incubated: 50 ⁇ l of macrophage cell suspension, 50 ⁇ l of tumor cell suspension and 50 ⁇ l of negative control antibody (mouse IgG) and mouse IgG prepared in the above-mentioned examples were added to each well of a 96-well plate.
  • Source antibody antibody was prepared with PBS to a final concentration of 5 ⁇ g/ml), and mixed well. Place the plate in a 7°C 5% CO 2 cell incubator and incubate for 2h.
  • Primers were designed according to the mouse hybridoma antibody variable region gene sequence and human light and heavy chain constant regions, and the mouse hybridoma antibody light and heavy chain variable region gene sequences were PCR amplified to obtain the chimeric antibody Q fragment;
  • the human light chain constant region (hIgkCL) and human heavy chain constant region (hIgG1CH) were amplified by PCR to obtain the H fragment of the chimeric antibody; the Q fragment and the H fragment were simultaneously connected into the enzyme-cut vector pcDNA3.4 by homologous recombination for expression in the carrier.
  • murine antibodies are likely to cause human HAMA reactions, in order to reduce the immunogenicity of murine antibodies in humans, it is necessary to transform murine antibodies into humanized antibodies.
  • multiple candidate molecules were humanized by means of CDR transplantation, frame shuffling, key amino acid reverse mutation design and DS (Discovery Studio) software.
  • CDR transplantation, framework shuffling method Firstly, through NCBI database comparison, the mouse antibody light and heavy chain CDR regions are respectively transplanted into the human germline gene FR region with a highly similar sequence, and the humanized version is obtained by gene synthesis. Antibody variable regions.
  • the antibody humanization method assisted by DS software is to input the amino acid sequence of the mouse antibody variable region obtained by hybridoma antibody sequencing into DS software to analyze the CDR region and FR region, and use the method of homology modeling to model the antibody and complete the construction. Add a force field after the modeling, and use the humanization module to humanize the antibody to obtain four humanized sequences, namely: Germline, Best_Single_Mutations, Germline_Substitutions, and Frequent_Residue_Substitutions.
  • the constant regions of the light and heavy chains (hIgkCL and hIgG1CH) were spliced with the corresponding antibody variable regions by homologous recombination and ligated into pcDNA3.4
  • the correct antibody light and heavy chain expression plasmids were obtained through colony PCR screening and sequencing identification. Then, the light and heavy chain plasmids with correct sequencing were transfected into expiCHO or HEK293 cells for expression; 7-10 days later, the cell supernatant was affinity purified using an AKTA protein purifier to obtain the full-length humanized antibody protein, as shown in Table 4.
  • Antibody number heavy chain variable region light chain variable region KH34 SEQ ID NO:101 SEQ ID NO:137 KH35 SEQ ID NO:98 SEQ ID NO:144 KH36 SEQ ID NO:99 SEQ ID NO:122 KH37 SEQ ID NO:114 SEQ ID NO:143 KH38 SEQ ID NO:98 SEQ ID NO:142 KH39 SEQ ID NO:101 SEQ ID NO:124 KH40 SEQ ID NO:98 SEQ ID NO:122 KH41 SEQ ID NO:99 SEQ ID NO:121 KH42 SEQ ID NO:98 SEQ ID NO:124 KH43 SEQ ID NO:98 SEQ ID NO:137
  • the antibody heavy chain constant region of KH34-KH91 is human IgG1
  • the amino acid sequence is shown in SEQ ID NO:148
  • the antibody light chain constant region is a kappa light chain constant region
  • the amino acid sequence is shown in SEQ ID NO:151.
  • Embodiment eight animal efficacy research
  • mice BALB/c nude mice, 5 weeks old, female, purchased from Jizui Yaokang.
  • mice were given 100 ⁇ l/animal via the tail vein, 2 times/week, 6 times in total, 10 animals/group.
  • the murine antibody in Example 2 and the humanized antibody in Example 7 were tested.
  • Monitoring index 1 2 times/week after the first administration, measure the long side (L) and short side (W) of the tumor with a vernier caliper, and calculate according to the following formula: L ⁇ W 2 /2 to calculate the tumor volume.
  • Monitoring index 2 3 weeks after the first administration, the animals were sacrificed, and the subcutaneous tumor was dissected, and the tumor weight was weighed.
  • Monitoring index 1 Tumor volume As shown in Figures 1A-1G, it can be seen from the continuous monitoring statistics of subcutaneous tumor volume that the tumor volume of each administration group was significantly reduced compared with the control group (PBS).

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Abstract

提供了抗CD24 抗体或其抗原结合片段，包含所述抗体或抗原结合片段的药物组合物和免疫偶联物，编码所述抗体或其抗原结合片段的核酸分子、包含所述核酸分子的载体、包含所述载体的宿主细胞、所述抗体或抗原结合片段的制备方法及所述抗体或抗原结合片段的生物医药用途，包括预防或治疗肿瘤(例如恶性肿瘤)或免疫性疾病。

Description

抗CD24抗体及其用途
相关申请的引用
本申请要求2021年12月7日提交的发明名称为“一种抗CD24的抗体及其用途”的202111487167.1号中国专利申请的优先权，在此通过引用的方式将上述专利申请的全部内容并入本文，用于所有目的。
技术领域
本申请涉及生物技术、特别是抗体工程技术领域，具体涉及抗CD24的抗体及其用途。
背景技术
正常情况下，免疫系统可以识别并清除肿瘤微环境中的肿瘤细胞，在机体免疫选择压力下，肿瘤细胞能够采用不同策略，例如依靠自身的高突变特性，逃避免疫监视，逐步建立起免疫抑制微环境，以抵抗和抑制机体抗肿瘤免疫反应，从而能够突破限制而持续扩增，最终发展成为临床可见的肿瘤。
肿瘤免疫治疗是通过重新启动并维持肿瘤-免疫循环，恢复机体正常的抗肿瘤免疫反应，从而控制与清除肿瘤的一种治疗方法，包括单克隆抗体类免疫检查点抑制剂、治疗性抗体、癌症疫苗、细胞治疗和小分子抑制剂等。目前已在多种肿瘤如黑色素瘤，非小细胞肺癌、肾癌和前列腺癌等实体瘤中得到了广泛应用。靶向肿瘤的抗体由于其高度特异性和卓越的药代动力学特征等，在血液癌症和实体肿瘤治疗中使用最广泛的免疫疗法。抗肿瘤抗体可以通过以下几种机制发挥抗肿瘤特性，包括：(1)增强/激活Fc介导的效应功能(ADCC、ADCP、CDC)；(2)阻断肿瘤生长信号；(3)抑制肿瘤血管生成；(4)诱发肿瘤细胞凋亡信号；(5)激活免疫细胞。
CD24是一种表观分子量介于30～70kDa之间的高度糖基化的蛋白质，它通过糖基磷脂酰肌醇(GPI)锚定连接到膜上。在人类中，CD24是一个仅含31个氨基酸的短肽，具有16个潜在的O-和N-糖基化位点。它主要在造血谱系的细胞上表达，例如B淋巴细胞、单核细胞、粒细胞和活化的T淋巴细胞，可作为判断多种细胞(如T细胞、B细胞等)成熟与否的标志，参与调节机体的免疫应答，与系统性红斑狼疮、风湿性关节炎等自身免疫性疾病密切相关。此外，在多数恶性肿瘤如腺癌、卵巢癌、膀胱癌、前列腺癌、非小细胞肺癌、鼻咽癌等肿瘤组织细胞的表面均能检测到显著高水平表达的CD24分子。其表达水平的高低与肿瘤的发生和发展密切相关，因此CD24被鉴定为多种癌症类型的癌症干细胞标志物。
CD24被鉴定为P-选择素的配体，P-选择素主要表达于活化的血管内皮表面，作为一种重要的黏附分子，具有促进细胞迁移的作用。肿瘤细胞表面上的CD24作为P-选择素的配体，P-选择素介导肿瘤细胞与活化的血小板、内皮细胞结合，促进肿瘤细胞的伸展、侵袭及转移。其次，作为细胞膜表面信号转导分子，CD24能够通过多种作用机制介导肿瘤细胞的的增殖、粘附、转移和侵袭。因此CD24有望成为肿瘤免疫治疗方案的新靶标。
目前关于以抗CD24抗体作为治疗性抗体的报道很少，本领域亟需对于以CD24作为治疗靶点的药物研发。
发明概述
第一方面，本申请提供了抗CD24抗体或其抗原结合片段，其中所述抗CD24抗体或其抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包括重链CDR1(HCDR1)、重链CDR2(HCDR2)和重链CDR3(HCDR3)，轻链可变区包括轻链CDR1(LCDR1)、轻链CDR2(LCDR2)和轻链CDR3(LCDR3)，其中：
HCDR1的氨基酸序列包含X 1GYX 2WH(SEQ ID NO:152)，其中X 1为R、N或S，X 2为S或T；或者HCDR1的氨基酸序列为TYGVS(SEQ ID NO:5)；
HCDR2的氨基酸序列包含X 1X 2X 3X 4X 5GX 6TX 7YX 8X 9X 10LX 11X 12(SEQ ID NO:153)，其中X 1为Y、V或F，X 2为I或T，X 3为H、W或Q；X 4为H、G、Y或F；X 5为R、S或D；X 6为S、T、N或A；X 7为N或K；X 8为N或H；X 9为P或S；X 10为S或A；X 11为K或I；X 12为S或N；
HCDR3的氨基酸序列包含GX 1X 2X 3X 4X 5DX 6(SEQ ID NO:154)，其中X 1为S或T；X 2为N、D或T；X 3为S、R、W、G或T；X 4为A、S或G；X 5为F或L；X 6为Y或H；或者HCDR3的氨基酸序列为YYGYDFFAY(SEQ ID NO:25)或YYGYDFFPY(SEQ ID NO:26)；
LCDR1的氨基酸序列包含X 1SSQSLLYSX 2X 3QKNYX 4X 5(SEQ ID NO:155)，其中X 1为K或Q，X 2为N、S或K，X 3为N或D；X 4为L或S；X 5为A、T或V；或者LCDR1的氨基酸序列为RASQDISNYLN(SEQ ID NO:35)、SASSSISYMH(SEQ ID NO:36)、
SASSSLSYMH(SEQ ID NO:37)、SASSSVSSSYLY(SEQ ID NO:38)或SASSSVSYMH(SEQ ID NO:39)；
LCDR2的氨基酸序列包含X 1X 2SX 3X 4X 5S(SEQ ID NO:156)，其中X 1为W、L、D、S或Y；X 2为A或T；X 3为T、K、N或R；X 4为R、K或L；X 5为E、A、Y或H；
LCDR3的氨基酸序列包含X 1QX 2X 3X 4X 5PX 6X 7(SEQ ID NO:157)，其中X 1为Q、H或F；X 2为Y、G、R或W；X 3为Y、S、N或F；X 4为I、G、S、T或L；X 5为Y或L；X 6为L、F、C、P、H或M；X 7为T或S；
其中HCDR1-3和LCDR1-3的序列根据Kabat定义。
第二方面，本申请提供了抗CD24抗体或其抗原结合片段，其中所述抗CD24抗体或其抗原结合片段包含重链可变区，其中重链可变区包括重链CDR1(HCDR1)、重链CDR2(HCDR2)和重链CDR3(HCDR3)，其中：
HCDR1的氨基酸序列包含X 1GYX 2WH(SEQ ID NO:152)，其中X 1为R、N或S，X 2为S或T；或者HCDR1的氨基酸序列为TYGVS(SEQ ID NO:5)；
HCDR2的氨基酸序列包含X 1X 2X 3X 4X 5GX 6TX 7YX 8X 9X 10LX 11X 12(SEQ ID NO:153)，其中X 1为Y、V或F，X 2为I或T，X 3为H、W或Q；X 4为H、G、Y或F；X 5为R、S或D；X 6为S、T、N或A；X 7为N或K；X 8为N或H；X 9为P或S；X 10为S或A；X 11为K或I；X 12为S或N；
HCDR3的氨基酸序列包含GX 1X 2X 3X 4X 5DX 6(SEQ ID NO:154)，其中X 1为S或T；X 2为N、D或T；X 3为S、R、W、G或T；X 4为A、S或G；X 5为F或L；X 6为Y或H；或者HCDR3的氨基酸序列为YYGYDFFAY(SEQ ID NO:25)或YYGYDFFPY(SEQ ID NO:26)；
其中HCDR1-3的序列根据Kabat定义。
第三方面，本申请提供了分离的核酸分子(多核苷酸)，其编码第一至第二方面所述的抗CD24抗体或抗原结合片段。
第四方面，本申请提供了载体，其包含编码第一至第二方面所述的抗CD24抗体或抗原结合片段的核酸分子(多核苷酸)。
第五方面，本申请提供了分离的宿主细胞，其包含第三方面所述的核酸分子或第四方面所述的载体。
第六方面，本申请提供了免疫偶联物，其包含与治疗剂偶联的第一至第二方面所述的抗CD24抗体或其抗原结合片段。
第七方面，本申请提供了药物组合物，其包含第一至第二方面所述的抗CD24抗体或其抗原结合片段或第六方面所述的免疫偶联物，以及药学上可接受的赋形剂、稀释剂或载体。
第八方面，本申请提供了第一至第二方面所述的抗CD24抗体或其抗原结合片段或第三方面所述的核酸分子或第四方面所述的载体或第五方面所述的宿主细胞或第六方面所述的免疫偶联物在制备用于预防或治疗肿瘤(例如恶性肿瘤)或免疫性疾病的药物中的用途。
第九方面，本申请提供了治疗个体中肿瘤(例如恶性肿瘤)或免疫性疾病的方法，包括 向有需要的个体给予治疗有效量的第一至第二方面所述的抗CD24抗体或其抗原结合片段、第六方面所述的免疫偶联物或第七方面所述的药物组合物。
第十方面，本申请提供了制备第一至第二方面所述的抗CD24抗体或其抗原结合片段的方法，其包括培养第五方面所述的宿主细胞的步骤。
附图简要描述
图1A-图1G显示了荷瘤小鼠在接受本申请的不同示例性抗CD24抗体治疗后肿瘤体积的随时间变化。
图2A-图2F显示了荷瘤小鼠在接受本申请的不同示例性抗CD24抗体治疗后实验结束时的肿瘤重量。
发明详细描述
定义
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
尽管本申请的广义范围所示的数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
广义而言，“抗体”可以指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到靶标的免疫球蛋白分子，因此涵盖完整抗体、抗体单链或抗体的任何抗原结合片段(也称为“抗原结合部分”)。当“抗体”和“抗原结合片段/抗原结合部分”出现在同一语境下时，“抗体”可以理解为相对于“抗原结合片段/抗原结合部分”的完整体，二者共同对应于广义的抗体概念。
“全长抗体”系指包含通过二硫键而互连的至少两条重(H)链和两条轻(L)链的蛋白。每条重链包含重链可变区(缩写为VH)和重链恒定区。重链恒定区包含三个域(domain)，CH1、CH2和CH3。每条轻链包含轻链可变区(缩写为VL)和轻链恒定区。该轻链恒定区包含一个域，CL。VH和VL区域还可再细分为具有高可变性的多个区，被称为互补决定区(CDR)，其间散布有更为保守的被称为框架区(FR)的多个区域。每个VH和VL均由三个CDR和四个FR构成，按照以下顺序从氨基端至羧基端排布：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的这些可变区包含与抗原相互作用的结合域。抗体的恒定区可介导免疫球蛋白与宿主的组织或因子结合，包括免疫系统的各种细胞(如效应细胞)和经典补体系统的第一成分(Clq)。全长抗体可以是任何种类的抗体，例如IgD、IgE、IgG、IgA或IgM(或上述的子类)，但抗体不需要属于任何特定的类别。根据重链的恒定域的抗体氨基酸序列，可以将免疫球蛋白指定为不同的类别。通常，免疫球蛋白有五种主要的类别：IgA、IgD、IgE、IgG及IgM，而且这些类别中有几个可以再被进一步区分成子类(同型)，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同免疫球蛋白类别的重链恒定域分别称为α、δ、ε、γ、以及μ。不同类别的免疫球蛋白的子单元结构和三维结构是公知的。嵌合或人源化抗体也涵盖在根据本申请的抗体中。本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR氨基酸序列有两种常见 的定义方式，即Chothia定义和kabat定义。对于给定抗体的可变区氨基酸序列，通常可以根据Chothia定义或者Kabat定义来确定VH和VL氨基酸序列中的CDR氨基酸序列。在本申请的实施方案中，利用Kabat定义CDR氨基酸序列。对于给定抗体的可变区氨基酸序列，可以通过多种方式分析可变区氨基酸序列的中CDR氨基酸序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
本文所使用的术语“抗原结合片段”尤其是指抗体片段如Fv、Fab、F(ab’) 2或Fab’，或者通过化学修饰或通过掺入脂质体中应能够增加半寿期的任何片段，优选地，所述功能片段将由其来源抗体的重链或轻链可变链的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，这种功能片段将包含最少5个氨基酸，优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。抗原结合片段的实例包括但不限于：(1)Fab片段，其可以是具有VL-CL链和VH-CH1链的单价片段；(2)F(ab') 2片段，其可以是具有两个Fab'片段的二价片段，该两个Fab'片段由铰链区的二硫桥(即Fab'的二聚物)连接；(3)具有抗体的单臂的VL和VH域的Fv片段。
术语“单链抗体(scFv)指是由VH域和VL域经由肽连接子连接的单一多肽链。(scFv) 2包含两个由肽连接子连接的VH域和两个VL域，该两个VL域是经由二硫桥与该两个VH域组合。
术语“Fc片段”、“Fc结构域”、“Fc部分”或类似的术语是指抗体重链恒定区的一部分，包括铰链区(hinge)、恒定区的CH2片段和CH3片段。
如本文所用术语“特异性结合”是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
术语“人源化抗体”意指将源自另一哺乳动物物种，诸如小鼠种系的CDR序列嫁接到人框架序列上获得的抗体。可以在人框架序列中进行别的框架区修饰。通过本领域技术人员已知的技术可以制备根据本申请的人源化抗体或其片段。
术语“嵌合抗体”系指以下抗体，其中的可变区序列来自一物种而恒定区序列来自另一物种，例如可变区序列来自小鼠抗体而恒定区序列来自人抗体的抗体。通过使用基因重组技术可以制备根据本申请的嵌合抗体或其片段。例如，所述嵌合抗体可以通过克隆重组DNA来生产，所述重组DNA包含启动子和编码根据本申请的非人尤其是鼠单克隆抗体可变区的序列、以及编码人抗体恒定区的序列。由这种重组基因编码的本申请嵌合抗体将是，例如，鼠-人嵌合体，该抗体的特异性由来源于鼠DNA的可变区确定，并且其同种型由来源于人DNA的恒定区来确定。
如本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚类的抗体中的对应氨基酸序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应氨基酸序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性。
术语“双特异性抗体”是指同时具有两种抗原表位结合能力的抗体。两种抗原表位可以在不同抗原上，也可是在同一抗原上。双特异性抗体可以具有多种结构构型。例如，双特异性抗体可以由两个Fc片段以及分别与其融合的两个结合部分组成(与天然抗体相似，区别在于两个臂结合不同的抗原靶标或表位)，抗原结合部可以为单链抗体(scfv)的形式或者Fab片段的形式。
本文使用的术语“药物组合物”其表示组合在一起以实现某种特定目的的至少一种药物以及任选地可药用载体或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本申请的目的。例如，所述药物组合物中所含的成分(例如根据本申请的抗体、核酸分子、核酸分子组合和/或缀合物)可以以整体施用于 个体，或者分开施用于个体。当所述药物组合物中所含的成分分开地施用于个体时，所述成分可以同时或依次施用于个体。优选地，所述可药用载体是水、缓冲水溶液、等渗盐溶液如、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本申请的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本申请的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。根据本申请的药物组合物或者药物制剂可通过任何适宜的途径施用，例如可口服、鼻、皮内、皮下、肌内或静脉内施用。
本文使用的“治疗有效量”或“有效量”是指足以显示其对于所施用个体益处的剂量。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。
本文所使用的术语“个体”是指哺乳动物，如人类，但也可以是其它动物，如野生动物(如苍鹭、鹳、鹤等)，家畜(如鸭、鹅等)或实验动物(如猩猩、猴子、大鼠、小鼠、兔子、豚鼠、土拨鼠、地松鼠等)。
关于氨基酸或核酸序列的术语“同源性/同一性/一致性”被定义为经过序列比对和引入空位后，氨基酸或核苷酸序列变体中相同的残基的百分比，如果需要，达到最大百分比的同源性。用于比对的方法和计算机程序在本领域内是公知的。
通常，为了制备单克隆抗体或其功能片段，尤其是鼠源的单克隆抗体或其功能片段，可以参考尤其描述在手册“Antibodies”中的技术(Harlow and Lane，Antibodies：A Laboratory Manual，Cold Spring Harbor Laboratory，Cold Spring Harbor NY，pp.726，1988)或者参考Kohler和Milstein描述的从杂交瘤细胞制备的技术(Nature，256：495-497，1975)。
第一方面，本申请提供了抗CD24抗体或其抗原结合片段，其中所述抗CD24抗体或其抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包括重链CDR1(HCDR1)、重链CDR2(HCDR2)和重链CDR3(HCDR3)，轻链可变区包括轻链CDR1(LCDR1)、轻链CDR2(LCDR2)和轻链CDR3(LCDR3)，其中：
HCDR1的氨基酸序列包含X 1GYX 2WH(SEQ ID NO:152)，其中X 1为R、N或S，X 2为S或T；或者HCDR1的氨基酸序列为TYGVS(SEQ ID NO:5)；
HCDR2的氨基酸序列包含X 1X 2X 3X 4X 5GX 6TX 7YX 8X 9X 10LX 11X 12(SEQ ID NO:153)，其中X 1为Y、V或F，X 2为I或T，X 3为H、W或Q；X 4为H、G、Y或F；X 5为R、S或D；X 6为S、T、N或A；X 7为N或K；X 8为N或H；X 9为P或S；X 10为S或A；X 11为K或I；X 12为S或N；
HCDR3的氨基酸序列包含GX 1X 2X 3X 4X 5DX 6(SEQ ID NO:154)，其中X 1为S或T；X 2为N、D或T；X 3为S、R、W、G或T；X 4为A、S或G；X 5为F或L；X 6为Y或H；或者HCDR3的氨基酸序列为YYGYDFFAY(SEQ ID NO:25)或YYGYDFFPY(SEQ ID NO:26)；
LCDR1的氨基酸序列包含X 1SSQSLLYSX 2X 3QKNYX 4X 5(SEQ ID NO:155)，其中X 1为K或Q，X 2为N、S或K，X 3为N或D；X 4为L或S；X 5为A、T或V；或者LCDR1的氨基酸序列为RASQDISNYLN(SEQ ID NO:35)、SASSSISYMH(SEQ ID NO:36)、SASSSLSYMH(SEQ ID NO:37)、SASSSVSSSYLY(SEQ ID NO:38)或SASSSVSYMH(SEQ ID NO:39)；
LCDR2的氨基酸序列包含X 1X 2SX 3X 4X 5S(SEQ ID NO:156)，其中X 1为W、L、D、S或Y；X 2为A或T；X 3为T、K、N或R；X 4为R、K或L；X 5为E、A、Y或H；
LCDR3的氨基酸序列包含X 1QX 2X 3X 4X 5PX 6X 7(SEQ ID NO:157)，其中X 1为Q、H或F；X 2为Y、G、R或W；X 3为Y、S、N或F；X 4为I、G、S、T或L；X 5为Y或L；X 6为L、F、C、P、H或M；X 7为T或S。
在一些实施方案中，HCDR1具有SEQ ID NO:1-5中任一个氨基酸序列，HCDR2具有SEQ ID NO:6-18中任一个氨基酸序列，HCDR3具有SEQ ID NO:19-26中任一个氨基酸序列；LCDR1具有SEQ ID NO:27-39中任一个氨基酸序列，LCDR2具有SEQ ID NO:40-47中任一个氨基酸序列，LCDR3具有SEQ ID NO:48-56中任一个氨基酸序列。
在一些实施方案中，HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：35、SEQ ID NO：47、SEQ ID NO：53；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：17、SEQ ID NO：21，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：43、SEQ ID NO：55；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：45、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO19：，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：13、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：16、SEQ ID NO：24，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：34、SEQ ID NO：40、SEQ ID NO：49；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：5、SEQ ID NO：9、SEQ ID NO：25，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：37、SEQ ID NO：42、SEQ ID NO：50；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：5、SEQ ID NO：9、SEQ ID NO：26，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：39、SEQ ID NO：42、SEQ ID NO：50；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：32、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：10、SEQ ID NO：21，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：31、SEQ ID NO：46、SEQ ID NO：49；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：10、SEQ ID NO：21，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：43、SEQ ID NO：56；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：30、SEQ ID NO：40、SEQ ID NO：49；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：30、SEQ ID NO：40、SEQ ID NO：49；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：12、SEQ ID NO：22，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：18、SEQ ID NO：22，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：31、SEQ ID NO：40、SEQ ID NO：54；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：15、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：15、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：33、SEQ ID NO：40、SEQ ID NO：49；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：4、SEQ ID NO：11、SEQ ID NO：23，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：33、SEQ ID NO：40、SEQ ID NO：49；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：4、SEQ ID NO：11、SEQ ID NO：23，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：32、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：14、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：29、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：29、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：12、SEQ ID NO：22，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：29、SEQ ID NO：40、SEQ ID NO：48；
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：38、SEQ ID NO：44、SEQ ID NO：52；或
HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：36、SEQ ID NO：42、SEQ ID NO：51。
在一些实施方案中，重链可变区具有SEQ ID NO：57-71和98-120中任一项所示氨基酸序列或与SEQ ID NO：57-71和98-120中任一项所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的氨基酸序列。
在一些实施方案中，重链可变区的氨基酸序列与SEQ ID NO：57-71和98-120中任一项所示的氨基酸序列相差约1、2、3、4、5、6、7、8、9或10个氨基酸的取代、缺失和/或添加。在一些实施方案中，SEQ ID NO：57-71和98-120中任一项所示的氨基酸序列的C端或N端区域可以被截短约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，而仍然保持类似的所述抗体的重链可变区的功能。在一些实施方案中，SEQ ID NO：57-71和98-120中任一项所示的氨基酸序列的C端或N端区域添加1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，得到的氨基酸序列仍然保持类似的所述抗体的重链可变区的功能。在一些实施方案中，可以在SEQ ID NO：57-71和98-120中任一项所示的氨基酸序列的C端或N端以外的区域添加或缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，只要改变后的氨基酸序列基本上保持类似的所述抗体的重链可变区的功能。
在一些实施方案中，轻链可变区具有SEQ ID NO：72-94和121-147中任一项所示氨基酸序列或与SEQ ID NO：72-94和121-147中任一项所示氨基酸序列具有至少80％、81％、82％、 83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的氨基酸序列。
在一些实施方案中，轻链可变区的氨基酸序列与SEQ ID NO：72-94和121-147中任一项所示的氨基酸序列相差约1、2、3、4、5、6、7、8、9或10个氨基酸的取代、缺失和/或添加。在一些实施方案中，SEQ ID NO：72-94和121-147中任一项所示的氨基酸序列的C端或N端区域还可以被截短约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，而仍然保持类似的所述抗体的轻链可变区的功能。在一些实施方案中，还可以在SEQ ID NO：72-94和121-147中任一项所示的氨基酸序列的C端或N端区域添加1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，得到的氨基酸序列仍然保持类似的所述抗体的轻链可变区的功能。在一些实施方案中，还可以在SEQ ID NO：72-94和121-147中任一项所示的氨基酸序列的C端或N端以外的区域添加或缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，只要改变后的氨基酸序列基本上保持类似的所述抗体的轻链可变区的功能。
在一些实施方案中，重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:72；
重链可变区的氨基酸序列为SEQ ID NO:58，轻链可变区的氨基酸序列为SEQ ID NO:73；
重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:74；
重链可变区的氨基酸序列为SEQ ID NO:60，轻链可变区的氨基酸序列为SEQ ID NO:75；
重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:76；
重链可变区的氨基酸序列为SEQ ID NO:62，轻链可变区的氨基酸序列为SEQ ID NO:76；
重链可变区的氨基酸序列为SEQ ID NO:63，轻链可变区的氨基酸序列为SEQ ID NO:77；
重链可变区的氨基酸序列为SEQ ID NO:64，轻链可变区的氨基酸序列为SEQ ID NO:78；
重链可变区的氨基酸序列为SEQ ID NO:65，轻链可变区的氨基酸序列为SEQ ID NO:79；
重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:80；
重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:81；
重链可变区的氨基酸序列为SEQ ID NO:66，轻链可变区的氨基酸序列为SEQ ID NO:82；
重链可变区的氨基酸序列为SEQ ID NO:66，轻链可变区的氨基酸序列为SEQ ID NO:83；
重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:84；
重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:85；
重链可变区的氨基酸序列为SEQ ID NO:67，轻链可变区的氨基酸序列为SEQ ID NO:86；
重链可变区的氨基酸序列为SEQ ID NO:68，轻链可变区的氨基酸序列为SEQ ID NO:87；
重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:88；
重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:89；
重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:76；
重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:90；
重链可变区的氨基酸序列为SEQ ID NO:69，轻链可变区的氨基酸序列为SEQ ID NO:76；
重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:76；
重链可变区的氨基酸序列为SEQ ID NO:69，轻链可变区的氨基酸序列为SEQ ID NO:90；
重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:76；
重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:91；
重链可变区的氨基酸序列为SEQ ID NO:70，轻链可变区的氨基酸序列为SEQ ID NO:91；
重链可变区的氨基酸序列为SEQ ID NO:70，轻链可变区的氨基酸序列为SEQ ID NO:80；
重链可变区的氨基酸序列为SEQ ID NO:71，轻链可变区的氨基酸序列为SEQ ID NO:92；
重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:92；
重链可变区的氨基酸序列为SEQ ID NO:67，轻链可变区的氨基酸序列为SEQ ID NO:92；
重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:93；
重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:94；
重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:137；
重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:144；
重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:122；
重链可变区的氨基酸序列为SEQ ID NO:114，轻链可变区的氨基酸序列为SEQ ID NO:143；
重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:142；
重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:124；
重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:122；
重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:121；
重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:124；
重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:137；
重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:121；
重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:121；
重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:133；
重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:124；
重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:134；
重链可变区的氨基酸序列为SEQ ID NO:111，轻链可变区的氨基酸序列为SEQ ID NO:136；
重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:134；
重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:133；
重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:133；
重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:122；
重链可变区的氨基酸序列为SEQ ID NO:118，轻链可变区的氨基酸序列为SEQ ID NO:146；
重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:137；
重链可变区的氨基酸序列为SEQ ID NO:115，轻链可变区的氨基酸序列为SEQ ID NO:122；
重链可变区的氨基酸序列为SEQ ID NO:117，轻链可变区的氨基酸序列为SEQ ID NO:121；
重链可变区的氨基酸序列为SEQ ID NO:116，轻链可变区的氨基酸序列为SEQ ID NO:145；
重链可变区的氨基酸序列为SEQ ID NO:104，轻链可变区的氨基酸序列为SEQ ID NO:125；
重链可变区的氨基酸序列为SEQ ID NO:105，轻链可变区的氨基酸序列为SEQ ID NO:123；
重链可变区的氨基酸序列为SEQ ID NO:110，轻链可变区的氨基酸序列为SEQ ID NO:126；
重链可变区的氨基酸序列为SEQ ID NO:109，轻链可变区的氨基酸序列为SEQ ID NO:123；
重链可变区的氨基酸序列为SEQ ID NO:110，轻链可变区的氨基酸序列为SEQ ID NO:123；
重链可变区的氨基酸序列为SEQ ID NO:104，轻链可变区的氨基酸序列为SEQ ID NO:130；
重链可变区的氨基酸序列为SEQ ID NO:109，轻链可变区的氨基酸序列为SEQ ID NO:126；
重链可变区的氨基酸序列为SEQ ID NO:108，轻链可变区的氨基酸序列为SEQ ID NO:130；
重链可变区的氨基酸序列为SEQ ID NO:105，轻链可变区的氨基酸序列为SEQ ID NO:126；
重链可变区的氨基酸序列为SEQ ID NO:113，轻链可变区的氨基酸序列为SEQ ID NO:141；
重链可变区的氨基酸序列为SEQ ID NO:108，轻链可变区的氨基酸序列为SEQ ID NO:125；
重链可变区的氨基酸序列为SEQ ID NO:103，轻链可变区的氨基酸序列为SEQ ID NO:139；
重链可变区的氨基酸序列为SEQ ID NO:107，轻链可变区的氨基酸序列为SEQ ID NO:140；
重链可变区的氨基酸序列为SEQ ID NO:106，轻链可变区的氨基酸序列为SEQ ID NO:140；
重链可变区的氨基酸序列为SEQ ID NO:106，轻链可变区的氨基酸序列为SEQ ID NO:129；
重链可变区的氨基酸序列为SEQ ID NO:107，轻链可变区的氨基酸序列为SEQ ID NO:129；
重链可变区的氨基酸序列为SEQ ID NO:107，轻链可变区的氨基酸序列为SEQ ID NO:128；
重链可变区的氨基酸序列为SEQ ID NO:103，轻链可变区的氨基酸序列为SEQ ID NO:128；
重链可变区的氨基酸序列为SEQ ID NO:103，轻链可变区的氨基酸序列为SEQ ID NO:140；
重链可变区的氨基酸序列为SEQ ID NO:103，轻链可变区的氨基酸序列为SEQ ID NO:129；
重链可变区的氨基酸序列为SEQ ID NO:120，轻链可变区的氨基酸序列为SEQ ID NO:139；
重链可变区的氨基酸序列为SEQ ID NO:106，轻链可变区的氨基酸序列为SEQ ID NO:128；
重链可变区的氨基酸序列为SEQ ID NO:112，轻链可变区的氨基酸序列为SEQ ID NO:147；
重链可变区的氨基酸序列为SEQ ID NO:112，轻链可变区的氨基酸序列为SEQ ID NO:138；
重链可变区的氨基酸序列为SEQ ID NO:119，轻链可变区的氨基酸序列为SEQ ID NO:138；
重链可变区的氨基酸序列为SEQ ID NO:100，轻链可变区的氨基酸序列为SEQ ID NO:127；
重链可变区的氨基酸序列为SEQ ID NO:102，轻链可变区的氨基酸序列为SEQ ID NO:127；
重链可变区的氨基酸序列为SEQ ID NO:100，轻链可变区的氨基酸序列为SEQ ID NO:135；
重链可变区的氨基酸序列为SEQ ID NO:102，轻链可变区的氨基酸序列为SEQ ID NO:131；
重链可变区的氨基酸序列为SEQ ID NO:100，轻链可变区的氨基酸序列为SEQ ID NO:132；
重链可变区的氨基酸序列为SEQ ID NO:102，轻链可变区的氨基酸序列为SEQ ID NO:135；
重链可变区的氨基酸序列为SEQ ID NO:102，轻链可变区的氨基酸序列为SEQ ID NO:132；或
重链可变区的氨基酸序列为SEQ ID NO:100，轻链可变区的氨基酸序列为SEQ ID NO:131。
第二方面，本申请提供了抗CD24抗体或其抗原结合片段，其中所述抗CD24抗体或其抗原结合片段包含重链可变区，其中重链可变区包括重链CDR1(HCDR1)、重链CDR2(HCDR2)和重链CDR3(HCDR3)，其中：
HCDR1的氨基酸序列包含X 1GYX 2WH(SEQ ID NO:152)，其中X 1为R、N或S，X 2为S或T；或者HCDR1的氨基酸序列为TYGVS(SEQ ID NO:5)；
HCDR2的氨基酸序列包含X 1X 2X 3X 4X 5GX 6TX 7YX 8X 9X 10LX 11X 12(SEQ ID NO:153)，其中X 1为Y、V或F，X 2为I或T，X 3为H、W或Q；X 4为H、G、Y或F；X 5为R、S或D；X 6为S、T、N或A；X 7为N或K；X 8为N或H；X 9为P或S；X 10为S或A；X 11为K或I；X 12为S或N；
HCDR3的氨基酸序列包含GX 1X 2X 3X 4X 5DX 6(SEQ ID NO:154)，其中X 1为S或T；X 2为N、D或T；X 3为S、R、W、G或T；X 4为A、S或G；X 5为F或L；X 6为Y或H；或者HCDR3的氨基酸序列为YYGYDFFAY(SEQ ID NO:25)或YYGYDFFPY(SEQ ID NO:26)。
第二方面所述抗体的一个实例是单域抗体，也称为重链可变区基因抗体(Variable domain of heavy chain of heavy-chain antibody，VHH)，其仅由一个重链可变区组成，又被称为纳米抗体。VHH抗体是最初在羊驼外周血液中发现的一种天然缺失轻链的抗体，该抗体只包含一个 重链可变区(VHH)和两个常规的CH2与CH3区。克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是已知的可结合目标抗原的最小单位。
在不存在矛盾的情况下，第一方面所述的技术方案和技术特征也适用于第二方面。
在第一至第二方面的一些实施方案中，抗CD24抗体为全抗体、单链抗体(scFv)或双特异性抗体。在第一至第二方面的一些实施方案中，抗CD24抗体的抗原结合片段为Fab、Fab’、Fv或F(ab’) 2。在第一至第二方面的一些实施方案中，抗CD24抗体为鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。在第一至第二方面的一些实施方案中，抗CD24抗体为单克隆抗体。在第一至第二方面的一些实施方案中，抗CD24抗体为双特异性抗体。
在第一至第二方面的一些实施方案中，抗CD24抗体或其抗原结合片段的轻链恒定区为人类抗体的轻链恒定区。在一些具体实施方案中，抗CD24抗体或其抗原结合片段的轻链恒定区选自人κ型或λ型轻链恒定区。在一些具体实施方案中，抗CD24抗体或其抗原结合片段的轻链恒定区为人κ型轻链恒定区。在一些具体的实施方案中，人κ型轻链恒定区具有如SEQ ID NO:151所述的氨基酸序列或其突变体。
在第一至第二方面的一些实施方案中，抗CD24抗体或其抗原结合片段的重链恒定区为人类抗体的重链恒定区。在第一至第二方面的一些实施方案中，抗CD24抗体或其抗原结合片段的重链恒定区选自人IgD、IgE、IgM、IgA、IgG1、IgG2a、IgG2b、IgG3和IgG4型中的任一种。在第一至第二方面的一些实施方案中，抗体或其抗原结合片段的重链恒定区选自IgG1、IgG2或IgG4型。在一些具体的实施方案中，IgG1型重链恒定区具有如SEQ ID NO：148所述的氨基酸序列及其突变体；IgG2型重链恒定区具有如SEQ ID NO：149所述的氨基酸序列及其突变体；IgG4型重链恒定区具有选SEQ ID NO：150所述的氨基酸序列及其突变体。
第三方面，本申请提供了分离的核酸分子(多核苷酸)，其编码第一至第二方面所述的抗CD24抗体或抗原结合片段。在一些实施方案中，所述多核苷酸可操作地连接到调控序列，调控序列可以被用所述载体转化过的宿主细胞识别。
第四方面，本申请提供了载体(例如表达载体)，其包含编码第一至第二方面所述的抗CD24抗体或抗原结合片段的多核苷酸。表达载体的选择取决于宿主细胞的选择，并且可以选择以便在所选宿主细胞中具有所需的表达和调控特征。
“表达载体”是包括一个或多个表达控制序列的载体，“表达控制序列”是控制和调节另一DNA序列的转录和/或翻译的DNA序列。
载体中的核酸可以与一个或多个表达控制序列可操作地连接。如本文所用，“可操作地连接”意指并入遗传构建体中，使得表达控制序列有效地控制目标编码序列的表达。表达控制序列的示例包括启动子、增强子和转录终止区。启动子是由DNA分子的通常在转录起始点上游100个核苷酸内(通常在RNA聚合酶II的起始位点附近)的区域组成的表达控制序列。为了使编码序列处于启动子的控制下，必须将多肽翻译阅读框的翻译起始位点定位在启动子下游的1至约50个核苷酸之间。增强子在时间、位置和水平方面提供表达特异性。与启动子不同，增强子可以在位于距转录位点的不同距离时起作用。增强子也可位于转录起始位点的下游。当RNA聚合酶能够将编码序列转录成mRNA，然后mRNA可以翻译成由编码序列所编码的蛋白质时，编码序列与细胞中的表达控制序列“可操作地连接”且处于表达控制序列的“控制下”。
合适的表达载体包括但不限于源自例如噬菌体、杆状病毒、烟草花叶病毒、疱疹病毒、巨细胞病毒、逆转录病毒、痘苗病毒、腺病毒和腺相关病毒的质粒和病毒载体。许多载体和表达系统可从诸如Novagen(Madison,WI)、Clontech(Palo Alto,CA)、Stratagene(LaJolla,CA)和Invitrogen Life Technologies(Carlsbad,CA)等公司商购获得。
表达载体可包括标签序列。标签序列通常表达为与编码的多肽的融合体。此类标签可以插入多肽内的任何位置，包括羧基或氨基末端。有用标签的示例包括但不限于Fc片段、聚组 氨酸、绿色荧光蛋白(GFP)、谷胱甘肽S-转移酶(GST)、c-myc、血凝素、FlagTM标签(Kodak,NewHaven,CT)、麦芽糖E结合蛋白和蛋白A。在一些实施方案中，编码Siglec-15融合多肽的核酸分子存在于含有编码Ig重链恒定区的一个或多个结构域的核酸的载体中，所述结构域例如，对应与人免疫球蛋白Cγ1链的铰链区、CH 2区和CH3区的氨基酸序列(Fc片段)。
第五方面，本申请提供了分离的宿主细胞，其包含第三方面所述的核酸分子或第四方面所述的载体。
在一些实施方案中，宿主细胞可以为原核宿主细胞、真核宿主细胞或噬菌体。原核宿主细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。真核宿主细胞可以为哺乳动物细胞，如293细胞、CHO细胞、BHK细胞、COS细胞、骨髓瘤细胞等；酵母细胞，如巴斯德毕赤酵母、酿酒酵母、裂殖酵母等；昆虫细胞，如草地粘虫等；植物细胞，如烟草等。
第六方面，本申请提供了免疫偶联物(也称为抗体-药物缀合物(ADC))，其包含与治疗剂偶联的第一至第二方面所述的抗CD24抗体或其抗原结合片段。
在一些实施方案中，治疗剂为抗肿瘤药物，例如，细胞毒性药物、免疫增强剂或放射性同位素。
在一些实施方案中，细胞毒性药物的类型包括微管蛋白抑制剂(例如生物碱)、DNA拓扑异构酶抑制剂、DNA损伤剂、抗代谢药或抗肿瘤抗生素。
在一些实施方案中，微管蛋白抑制剂包括但不限于，奥瑞斯他汀衍生物(例如MMAE(Monomethyl auristatin E)、MMAF(Monomethyl auristatin F))或美登素生物碱衍生物(例如DM1、DM4、安丝菌素(Ansamitocin)、美登素(Mertansine)或海兔毒素肽(dolastatin)及其衍生物)。
在一些实施方案中，DNA拓扑异构酶抑制剂为喜树碱类似物或DNA拓扑异构酶I抑制剂及其衍生物，例如，DXD、SN38、伊立替康、伊立替康盐酸盐、喜树碱、9-氨基喜树碱、9-硝基喜树碱、10-羟基喜树碱、9-氯-10-羟基喜树碱、22-羟基旱莲木碱、拓扑替康、勒托替康、贝洛替康、依喜替康、硅基高喜树碱(homosilatecan)、6,8-二溴-2-甲基-3-[2-(D-吡喃木糖基氨基)苯基]-4(3H)-喹唑啉酮、2-氰基-3-(3,4-二羟基苯基)-N-(苯基甲基)-(2E)-2-丙烯酰胺、2-氰基-3-(3,4-二羟基苯基)-N-(3-羟基苯基丙基)-(E)-2-丙烯酰胺、12-β-D-吡喃萄萄糖基-12,13-二氢-2,10-二羟基-6-[[2-羟基-1-(羟基甲基)乙基]氨基]-5H-吲哚并[2,3-a]吡咯并[3,4-c]咔唑-5,7(6H)-二酮、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺二盐酸盐、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺。
在一些实施方案中，DNA损伤剂包括但不限于，卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD(pyrrolobenzodiazepine，吡咯并苯并二氮杂)。
在一些实施方案中，抗代谢药包括但不限于，甲氨蝶呤、6-巯嘌呤或5-氟尿嘧啶。
在一些实施方案中，抗肿瘤抗生素包括但不限于，多肽类抗生素(例如放线菌素D或博来霉素)或蒽醌类药物(例如阿霉素或盐酸米托蒽醌)。在一些实施方案中，放射性同位素包括但不限于，211At、131I、125I、90Y、186Re、188Re、153Sm、212Bi、32P、60Co或177Lu。
在一些实施方案中，免疫增强剂包括但不限于，左旋咪唑(levamisole)、匹多莫德(pidotimod)、咪喹莫特(imiquimod)、异丙肌苷、聚肌胞苷酸或聚肌尿苷酸。
在一些实施方案中，本申请的抗CD24抗体抗体通过接头与药物部分共价连接。在一些实施方案中，接头是可切割接头。在一些实施方案中，接头在细胞内条件下可被切割。在一个实施方案中，接头在小于5.5的pH下可水解。在一些实施方案中，接头可被细胞内蛋白酶切割。在一些实施方案中，接头是组织蛋白酶可切割接头。在一些实施方案中，接头包含二肽。在一些实施方案中，二肽是缬氨酸(Val)-瓜氨酸(Cit)。在一些实施方案中，抗体通过抗体的半胱氨酸巯基与接头连接。
第七方面，本申请提供了药物组合物，其包含第一至第二方面所述的抗CD24抗体或其 抗原结合片段或第六方面所述的免疫偶联物，以及药学上可接受的赋形剂、稀释剂或载体。在一些实施方案中，药物组合物为玻璃体内注射剂、视网膜下注射制剂、脉络膜内注射制剂、静脉注射制剂、肿瘤内注射制剂或肌肉注射制剂。在一些实施方案中，药物组合物还可包含下述中的一种或多种：润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。在一些实施方案中，可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
在一些实施方案中，药物组合物用于预防或治疗肿瘤(例如恶性肿瘤)或免疫性疾病。在一些实施方案中，肿瘤(例如恶性肿瘤)为肿瘤细胞表面CD24高表达的肿瘤，包括但不限于，肺癌、乳腺癌、卵巢癌、淋巴癌、结/直肠癌、卵巢癌、肝癌、宫颈癌、肾癌、前列腺癌、食管癌、膀胱癌或鼻咽癌。在一些实施方案中，免疫性疾病为类风湿性关节炎或系统性红斑狼疮。
第八方面，本申请提供了第一至第二方面所述的抗CD24抗体或其抗原结合片段或第三方面所述的核酸分子或第四方面所述的载体或第五方面所述的宿主细胞或第六方面所述的免疫偶联物在制备用于预防或治疗肿瘤(例如恶性肿瘤)或免疫性疾病的药物中的用途。在一些实施方案中，肿瘤(例如恶性肿瘤)为肿瘤细胞表面CD24高表达的肿瘤，包括但不限于，肺癌、乳腺癌、卵巢癌、淋巴癌、结/直肠癌、卵巢癌、肝癌、宫颈癌、肾癌、前列腺癌、食管癌、膀胱癌或鼻咽癌。在一些实施方案中，免疫性疾病为类风湿性关节炎或系统性红斑狼疮。
第九方面，本申请提供了治疗个体中肿瘤(例如恶性肿瘤)或免疫性疾病的方法，包括向有需要的个体给予治疗有效量的第一至第二方面所述的抗CD24抗体或其抗原结合片段、第六方面所述的免疫偶联物或第七方面所述的药物组合物。在一些实施方案中，肿瘤(例如恶性肿瘤)为肿瘤细胞表面CD24高表达的肿瘤，包括但不限于，肺癌、乳腺癌、卵巢癌、淋巴癌、结/直肠癌、卵巢癌、肝癌、宫颈癌、肾癌、前列腺癌、食管癌、膀胱癌或鼻咽癌。在一些实施方案中，免疫性疾病为类风湿性关节炎或系统性红斑狼疮。
第十方面，本申请提供了制备第一至第二方面所述的抗CD24抗体或其抗原结合片段的方法，其包括培养第五方面所述的宿主细胞的步骤。在一些实施方案中，所述方法进一步包括分离所述抗CD24抗体或其抗原结合片段的步骤。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
实施例
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。
本申请描述的抗体重轻链的CDR移植，PCR引入位点突变的筛选是通过基因重组技术及基于抗原抗体相互作用的免疫学技术所完成，具体实验方法步骤参考如<<分子克隆>>第三版(Joseph Sambrook，科学出版社，2002年8月1日)及类似实验手册所记载。
实施例一、杂交瘤制备及筛选
1、小鼠免疫
免疫BALB/C雌性小鼠(鼠龄6-8周，购于达硕实验动物有限公司)，采用了标准腹腔免疫或足底免疫两种方式对小鼠进行免疫，10μg/只小鼠，每2至3周进行一次注射，所有小 鼠初次免疫使用等体积CFA(SIGMA，货号：SLBW7430)与抗原混合乳化，冲刺免疫前的加强免疫(约第二次免疫-第四次免疫)使用IFA(SIGMA，货号：F5506)与抗原混合乳化，形成油包水的状态，滴入水中轻晃不分散即乳化完成。所用抗原为商购重组人CD24Fc嵌合蛋白(货号：5247-CD-050，https://www.rndsystems.com/cn/products/recombinant-human-cd24-fc-chimera-protein-cf_5247-cd)。杂交瘤融合前的小鼠冲刺免疫所用抗原为PBS配制(20μg/只小鼠，100μl/只小鼠)。
2、杂交瘤融合和培养
对于免疫抗原的小鼠，在杂交瘤融合前三天对小鼠进行冲刺免疫，拧脖处死小鼠，用70％乙醇浸泡小鼠5min进行消毒。摘取脾脏及淋巴结，适当研磨脾脏和淋巴结，分别研磨后细胞经70μm细胞滤网过滤，将细胞悬液分别转入无菌离心管中2000rpm室温离心5分钟，离心后弃上清，将淋巴B细胞用5ml DMEM(GIBCO，货号：12491023/10x 500mL)重悬，备用；在脾脏B细胞中加入12ml红细胞裂解液吹散细胞后，室温放置8min后，2000rpm室温离心5分钟。弃上清，加入DMEM培养基重悬，分别细胞计数，备用。
收集P3X63Ag8.653骨髓瘤细胞(货号：
Figure PCTCN2022137123-appb-000001
CRL-1580，购于ATCC)或FO骨髓瘤细胞(货号：TCM31，购于中科院细胞库)，细胞计数；骨髓瘤细胞和B细胞(1:1)混匀，ECF溶液重悬，细胞计数；加ECF溶液将细胞密度调节至2x 10 6至1x 10 7个细胞/ml，将细胞悬液加入融合槽中，运行ECM2001电融合仪，运行结束后将细胞悬液从融合槽转入一个无菌的含HAT(GIBCO，货号：20160017)的杂交瘤融合培养基中，充分混匀加入96孔板；置于37℃，8％二氧化碳培养箱中培养。
融合后第7-12天，根据杂交瘤生长状况，取一半培养上清丢弃，补加新鲜杂交瘤培养基，在2-3次换液后，在细胞最佳状态时，进行ELISA或流式检测。根据检测结果，挑选阳性杂交瘤细胞(OD值为阴性对照OD值的3倍或logshift值为阴性对照logshift值的3倍)，扩大培养。2-3天后进行第二次检测，将有阳性杂交瘤细胞株扩大培养。
3、候选鼠源抗体IgG亚型鉴定
采用Mouse Monoclonal Antibody Isotyping Kit(Roche,cat no.:11493027001)进行候选鼠源抗体的IgG亚型鉴定。具体步骤如下：
使用含1％BSA的PBS将候选抗体稀释至0.1μg/ml-1μg/ml，将150μl稀释好的样品加入显影管，15℃-25℃孵育30s；将试纸条黑色端放入显影管，至少保持1min，取出试纸条；放置5-10min内，蓝色条纹的指示位置，即为小鼠IgG亚型。
实施例二、杂交瘤单克隆抗体基因测序
使用Trizol法提取杂交瘤细胞的总RNA，取5μg总RNA进行反转录，得到cDNA；然后以cDNA为模板，使用5’RACE技术进行PCR扩增，将PCR产物连接进酶切载体pcDNA3.4中，连接产物通过热激转化进感受态DH5α细胞中，进行菌落PCR鉴定，将阳性克隆提取少量质粒送测序，获得杂交瘤抗体基因轻、重链可变区，如表1。
表1
抗体编号 重链可变区 轻链可变区
KH01 (SEQ ID NO:57) (SEQ ID NO:72)
KH02 (SEQ ID NO:58) (SEQ ID NO:73)
KH03 (SEQ ID NO:59) (SEQ ID NO:74)
KH04 (SEQ ID NO:60) (SEQ ID NO:75)
KH05 (SEQ ID NO:61) (SEQ ID NO:76)
KH06 (SEQ ID NO:62) (SEQ ID NO:76)
KH07 (SEQ ID NO:63) (SEQ ID NO:77)
KH08 (SEQ ID NO:64) (SEQ ID NO:78)
KH09 (SEQ ID NO:65) (SEQ ID NO:79)
KH10 (SEQ ID NO:59) (SEQ ID NO:80)
KH11 (SEQ ID NO:59) (SEQ ID NO:81)
KH12 (SEQ ID NO:66) (SEQ ID NO:82)
KH13 (SEQ ID NO:66) (SEQ ID NO:83)
KH14 (SEQ ID NO:61) (SEQ ID NO:84)
KH15 (SEQ ID NO:61) (SEQ ID NO:85)
KH16 (SEQ ID NO:67) (SEQ ID NO:86)
KH17 (SEQ ID NO:68) (SEQ ID NO:87)
KH18 (SEQ ID NO:57) (SEQ ID NO:88)
KH19 (SEQ ID NO:61) (SEQ ID NO:89)
KH20 (SEQ ID NO:61) (SEQ ID NO:76)
KH21 (SEQ ID NO:57) (SEQ ID NO:90)
KH22 (SEQ ID NO:69) (SEQ ID NO:76)
KH23 (SEQ ID NO:57) (SEQ ID NO:76)
KH24 (SEQ ID NO:69) (SEQ ID NO:90)
KH25 (SEQ ID NO:59) (SEQ ID NO:76)
KH26 (SEQ ID NO:59) (SEQ ID NO:91)
KH27 (SEQ ID NO:70) (SEQ ID NO:91)
KH28 (SEQ ID NO:70) (SEQ ID NO:80)
KH29 (SEQ ID NO:71) (SEQ ID NO:92)
KH30 (SEQ ID NO:57) (SEQ ID NO:92)
KH31 (SEQ ID NO:67) (SEQ ID NO:92)
KH32 (SEQ ID NO:57) (SEQ ID NO:93)
KH33 (SEQ ID NO:57) (SEQ ID NO:94)
其中，KH01-04、KH08-13、KH16-18、KH21、KH23、KH32-33鼠源抗体重链恒定区为鼠IgG2a，氨基酸序列如SEQ ID NO:95所示。KH05、KH07、KH14-15、KH19-20、KH22、KH24-31鼠源抗体重链恒定区为鼠IgG2b，氨基酸序列如SEQ ID NO:96所示。KH01-33鼠源抗体轻链恒定区为鼠κ型轻链恒定区，氨基酸序列如SEQ ID NO:97所示。
实施例三、流式细胞术抗体结合实验
通过流式细胞术检测抗体与高表达CD24的工程细胞，例如CD24高表达CHO工程细胞/293细胞，以及肿瘤细胞，例如人肺癌细胞(A549细胞)、人乳腺癌细胞(MCF-7、Ovcar-3细胞)、人B细胞淋巴瘤细胞、人结直肠癌细胞的结合能力。将5×10 6/ml的细胞与5μg/ml的上述鼠源抗体按20μl:20μl加入96孔板中，每种细胞设有阴/阳性孔，室温孵育1h后，用FACS缓冲液洗一次，弃上清，加入20μl/孔的5μg/ml PE山羊抗小鼠IgG，室温孵育20min后，用FACS缓冲液洗一次，弃上清，用FACS缓冲液重悬后上流式细胞分析仪(BD FACS Celesta)检测。结果显示本申请的抗体和高表达CD24的工程细胞或肿瘤细胞，例如CD24高表达的CHO工程细胞、293工程细胞，人肺癌细胞(A549细胞)、人乳腺癌细胞(MCF-7、Ovcar-3细胞)、人B细胞淋巴瘤细胞、人结直肠癌细胞等均具有良好的结合能力。
实施例四、人PBMC诱导的M2型原代巨噬细胞吞噬A549肿瘤细胞实验
本实施例评价抗CD24抗体引起巨噬细胞介导的吞噬活性，测试原理为：肿瘤细胞(例 如，本实施例的A549以及实施例五的MCF-7)上的CD24与巨噬细胞上的Siglec10结合，形成“别吃我”信号，从而抑制巨噬细胞吞噬肿瘤细胞，CD24抗体能够阻断CD24与Siglec10的结合，封闭“别吃我”信号，激发巨噬细胞的吞噬作用。
准备人外周血细胞诱导7天的M2型原代巨噬细胞，将巨噬细胞悬液细胞密度调整到0.7x10 6细胞/ml，备用。同时准备A549-GFP-2(自制)肿瘤细胞，将肿瘤细胞悬液细胞密度调整到1.4x10 6细胞/ml。然后将巨噬细胞及肿瘤细胞共孵育：在96孔板每孔中分别加入50μl巨噬细胞悬液、50μl肿瘤细胞悬液及50μl阴性对照抗体(小鼠IgG)和上述实施例制备的鼠源抗体(抗体用PBS配制成终浓度5μg/ml)，混匀。将平板放入37℃，5％CO 2细胞培养箱孵育2h。孵育结束后，离心去上清；每孔加入50μl 2μg/ml抗人BV421-抗人CD11b抗体，重悬细胞，室温孵育30min；洗涤：每孔加入200μl FACS缓冲液重悬细胞，离心去上清；加入100μl 2μg/ml 7-AAD染液重悬细胞。通过BD FACS Celestra TM(JC345)进行上机检测。结果如表2所示。
表2
抗体编号 吞噬率％
KH01 11.80
KH02 10.30
KH03 11.95
KH04 12.75
KH05 10.45
KH06 10.90
KH07 10.05
KH10 12.05
KH12 11.05
KH14 11.25
KH16 12.35
KH19 10.35
KH21 10.15
KH25 11.00
KH27 10.60
KH29 8.95
KH33 18.30
IgG 2.7
实施例五、人PBMC诱导的M2型原代巨噬细胞吞噬MCF-7肿瘤细胞实验
本实施例评价抗CD24抗体引起巨噬细胞介导的吞噬活性。准备人外周血细胞诱导7天的M2型原代巨噬细胞，将巨噬细胞悬液细胞密度调整到0.7x10 6细胞/ml，备用。同时准备MCF-7荧光素酶-GFP(自制)肿瘤细胞，将肿瘤细胞悬液细胞密度调整到1.4x10 6细胞/ml。然后将巨噬细胞及肿瘤细胞共孵育：在96孔板每孔中分别加入50μl巨噬细胞细胞悬液、50μl肿瘤细胞细胞悬液及50μl阴性对照抗体(mouse IgG)和上述实施例制备的鼠源抗体(抗体用PBS配制成终浓度5μg/ml)，混匀。将平板放入7℃5％CO 2细胞培养箱孵育2h。孵育结束后，离心去上清；每孔加入50μl 2μg/ml BV421-抗人CD11b抗体，重悬细胞，室温孵育30min；洗涤：每孔加入200μl FACS缓冲液重悬细胞，离心去上清；加入100μl 2μg/ml 7-AAD染液重悬细胞。通过BD FACS Celestra  TM(JC345)进行上机检测。结果如表3所示。
表3
编号 吞噬率(％)
KH01 6.2
KH02 3.0
KH03 3.6
KH05 3.2
KH06 4.3
KH14 3.4
KH16 6.8
KH25 6.5
IgG 1.3
实施例六、嵌合抗体构建
根据小鼠杂交瘤抗体可变区基因序列与人轻、重链恒定区设计引物，对小鼠杂交瘤抗体轻、重链可变区基因序列进行PCR扩增，得到嵌合抗体Q片段；对人轻链恒定区(hIgkCL)和人重链恒定区(hIgG1CH)PCR扩增，得到嵌合抗体H片段；利用同源重组的方法将Q片段和H片段同时连接进酶切载体pcDNA3.4表达载体中。将测序正确的质粒转染进expiCHO细胞或者HEK293细胞进行蛋白表达，细胞培养约7-10天左右，离心收集细胞上清；细胞上清利用AKTA蛋白纯化仪进行亲和纯化，得到嵌合抗体蛋白。
实施例七、抗体人源化
由于鼠源抗体容易引起人的HAMA反应，为了减少鼠源抗体在人体内的的免疫原性，需要对鼠源抗体进行人源化抗体改造。本实施例采用CDR移植，框架改组和关键氨基酸回复突变设计和结合DS(Discovery Studio)软件等方法对多个候选分子进行人源化。CDR移植，框架改组方法首先通过NCBI数据库比对，将鼠源抗体轻、重链CDR区分别移植进与之序列高度相似的人胚系基因FR区中，通过基因合成方法得到人源化后的抗体可变区。DS软件辅助下的抗体人源化方法为将杂交瘤抗体测序得到的鼠源抗体可变区氨基酸序列输入DS软件分析CDR区和FR区，利用同源模建的方法对抗体建模，完成建模后添加力场，用人源化模块进行抗体人源化，得到四条人源化序列，分别为：Germline、Best_Single_Mutations、Germline_Substitutions、Frequent_Residue_Substitutions。在得到的Germline序列基础上，进行回复突变氨基酸位点选择：利用DS软件选择抗体CDR区
Figure PCTCN2022137123-appb-000002
范围内序列和抗体轻、重链接触面序列，将该区域人源氨基酸回复成鼠源氨基酸，通过基因合成方法得到人源化后的抗体可变区。
通过以上方法，获得人源化的抗体可变区后，再利用同源重组方法分别将轻、重链的恒定区(hIgkCL和hIgG1CH)同相应的抗体可变区进行拼接并连接进pcDNA3.4表达载体中，通过菌落PCR筛选，测序鉴定获得正确的抗体轻、重链表达质粒。然后将测序正确的轻、重链质粒转染expiCHO或HEK293细胞中进行表达；7-10天后，细胞上清利用AKTA蛋白纯化仪进行亲和纯化，得到全长的人源化抗体蛋白，如表4。
表4
抗体编号 重链可变区 轻链可变区
KH34 SEQ ID NO:101 SEQ ID NO:137
KH35 SEQ ID NO:98 SEQ ID NO:144
KH36 SEQ ID NO:99 SEQ ID NO:122
KH37 SEQ ID NO:114 SEQ ID NO:143
KH38 SEQ ID NO:98 SEQ ID NO:142
KH39 SEQ ID NO:101 SEQ ID NO:124
KH40 SEQ ID NO:98 SEQ ID NO:122
KH41 SEQ ID NO:99 SEQ ID NO:121
KH42 SEQ ID NO:98 SEQ ID NO:124
KH43 SEQ ID NO:98 SEQ ID NO:137
KH44 SEQ ID NO:98 SEQ ID NO:121
KH45 SEQ ID NO:101 SEQ ID NO:121
KH46 SEQ ID NO:101 SEQ ID NO:133
KH47 SEQ ID NO:99 SEQ ID NO:124
KH48 SEQ ID NO:99 SEQ ID NO:134
KH49 SEQ ID NO:111 SEQ ID NO:136
KH50 SEQ ID NO:98 SEQ ID NO:134
KH51 SEQ ID NO:98 SEQ ID NO:133
KH52 SEQ ID NO:99 SEQ ID NO:133
KH53 SEQ ID NO:101 SEQ ID NO:122
KH54 SEQ ID NO:118 SEQ ID NO:146
KH55 SEQ ID NO:99 SEQ ID NO:137
KH56 SEQ ID NO:115 SEQ ID NO:122
KH57 SEQ ID NO:117 SEQ ID NO:121
KH58 SEQ ID NO:116 SEQ ID NO:145
KH59 SEQ ID NO:104 SEQ ID NO:125
KH60 SEQ ID NO:105 SEQ ID NO:123
KH61 SEQ ID NO:110 SEQ ID NO:126
KH62 SEQ ID NO:109 SEQ ID NO:123
KH63 SEQ ID NO:110 SEQ ID NO:123
KH64 SEQ ID NO:104 SEQ ID NO:130
KH65 SEQ ID NO:109 SEQ ID NO:126
KH66 SEQ ID NO:108 SEQ ID NO:130
KH67 SEQ ID NO:105 SEQ ID NO:126
KH68 SEQ ID NO:113 SEQ ID NO:141
KH69 SEQ ID NO:108 SEQ ID NO:125
KH70 SEQ ID NO:103 SEQ ID NO:139
KH71 SEQ ID NO:107 SEQ ID NO:140
KH72 SEQ ID NO:106 SEQ ID NO:140
KH73 SEQ ID NO:106 SEQ ID NO:129
KH74 SEQ ID NO:107 SEQ ID NO:129
KH75 SEQ ID NO:107 SEQ ID NO:128
KH76 SEQ ID NO:103 SEQ ID NO:128
KH77 SEQ ID NO:103 SEQ ID NO:140
KH78 SEQ ID NO:103 SEQ ID NO:129
KH79 SEQ ID NO:120 SEQ ID NO:139
KH80 SEQ ID NO:106 SEQ ID NO:128
KH81 SEQ ID NO:112 SEQ ID NO:147
KH82 SEQ ID NO:112 SEQ ID NO:138
KH83 SEQ ID NO:119 SEQ ID NO:138
KH84 SEQ ID NO:100 SEQ ID NO:127
KH85 SEQ ID NO:102 SEQ ID NO:127
KH86 SEQ ID NO:100 SEQ ID NO:135
KH87 SEQ ID NO:102 SEQ ID NO:131
KH88 SEQ ID NO:100 SEQ ID NO:132
KH89 SEQ ID NO:102 SEQ ID NO:135
KH90 SEQ ID NO:102 SEQ ID NO:132
KH91 SEQ ID NO:100 SEQ ID NO:131
其中，KH34-KH91的抗体重链恒定区为人IgG1，氨基酸序列如SEQ ID NO:148所示，抗体轻链恒定区为κ型轻链恒定区，氨基酸序列如SEQ ID NO:151所示。
实施例八、动物药效研究
1.1动物药效建模
动物：BALB/c裸鼠，5周龄，雌性，购自集萃药康。
建模：对裸鼠皮下(左侧背部)接种A549细胞悬液，5×10 7个细胞/ml，200μl/只。
1.2样品准备
将待测抗体样品用PBS配制后，1.6ml/支分装。
1.3给药
接种次日给药，尾静脉，100μl/只，2次/周，共给药6次，10只动物/组。本实施例测试了实施例二中的鼠源抗体以及实施例七中的人源化抗体。
1.4实验监测
监测指标一：首次给药后2次/周，游标卡尺测出肿瘤的长边(L)和短边(W)，按如下公式计算：L×W 2/2计算肿瘤体积。
监测指标二：首次给药后3周，处死动物，皮下瘤解剖后，称取瘤重。
1.5实验结果
监测指标一：肿瘤体积如图1A-1G所示，从皮下瘤体积持续监测统计图可以看出，各给药组相对于对照组(PBS)的肿瘤体积显著减小。
监测指标二：皮下瘤解剖后肿瘤称量结果如图2A-2F所示，从图中可以看出，与对照组(PBS)相比，各给药组中肿瘤重量显著下降。

Claims (26)

  1. 抗CD24抗体或其抗原结合片段，其中所述抗CD24抗体或其抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包括重链CDR1(HCDR1)、重链CDR2(HCDR2)和重链CDR3(HCDR3)，轻链可变区包括轻链CDR1(LCDR1)、轻链CDR2(LCDR2)和轻链CDR3(LCDR3)，其中：
    HCDR1的氨基酸序列包含X 1GYX 2WH(SEQ ID NO:152)，其中X 1为R、N或S，X 2为S或T；或者HCDR1的氨基酸序列为TYGVS(SEQ ID NO:5)；
    HCDR2的氨基酸序列包含X 1X 2X 3X 4X 5GX 6TX 7YX 8X 9X 10LX 11X 12(SEQ ID NO:153)，其中X 1为Y、V或F，X 2为I或T，X 3为H、W或Q；X 4为H、G、Y或F；X 5为R、S或D；X 6为S、T、N或A；X 7为N或K；X 8为N或H；X 9为P或S；X 10为S或A；X 11为K或I；X 12为S或N；
    HCDR3的氨基酸序列包含GX 1X 2X 3X 4X 5DX 6(SEQ ID NO:154)，其中X 1为S或T；X 2为N、D或T；X 3为S、R、W、G或T；X 4为A、S或G；X 5为F或L；X 6为Y或H；或者HCDR3的氨基酸序列为YYGYDFFAY(SEQ ID NO:25)或YYGYDFFPY(SEQ ID NO:26)；
    LCDR1的氨基酸序列包含X 1SSQSLLYSX 2X 3QKNYX 4X 5(SEQ ID NO:155)，其中X 1为K或Q，X 2为N、S或K，X 3为N或D；X 4为L或S；X 5为A、T或V；或者LCDR1的氨基酸序列为RASQDISNYLN(SEQ ID NO:35)、SASSSISYMH(SEQ ID NO:36)、SASSSLSYMH(SEQ ID NO:37)、SASSSVSSSYLY(SEQ ID NO:38)或SASSSVSYMH(SEQ ID NO:39)；
    LCDR2的氨基酸序列包含X 1X 2SX 3X 4X 5S(SEQ ID NO:156)，其中X 1为W、L、D、S或Y；X 2为A或T；X 3为T、K、N或R；X 4为R、K或L；X 5为E、A、Y或H；
    LCDR3的氨基酸序列包含X 1QX 2X 3X 4X 5PX 6X 7(SEQ ID NO:157)，其中X 1为Q、H或F；X 2为Y、G、R或W；X 3为Y、S、N或F；X 4为I、G、S、T或L；X 5为Y或L；X 6为L、F、C、P、H或M；X 7为T或S；
    其中HCDR1-3和LCDR1-3的序列根据Kabat定义。
  2. 根据权利要求1所述的抗CD24抗体或其抗原结合片段，其中HCDR1具有SEQ ID NO:1-5中任一个氨基酸序列，HCDR2具有SEQ ID NO:6-18中任一个氨基酸序列，HCDR3具有SEQ ID NO:19-26中任一个氨基酸序列；LCDR1具有SEQ ID NO:27-39中任一个氨基酸序列，LCDR2具有SEQ ID NO:40-47中任一个氨基酸序列，LCDR3具有SEQ ID NO:48-56中任一个氨基酸序列。
  3. 根据权利要求1所述的抗CD24抗体或其抗原结合片段，其中：
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：35、SEQ ID NO：47、SEQ ID NO：53；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：17、SEQ ID NO：21，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：43、SEQ ID NO：55；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：45、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO19：，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：13、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：16、SEQ ID NO：24，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：34、SEQ ID NO：40、SEQ ID NO：49；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：5、SEQ ID NO：9、SEQ ID NO：25， LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：37、SEQ ID NO：42、SEQ ID NO：50；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：5、SEQ ID NO：9、SEQ ID NO：26，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：39、SEQ ID NO：42、SEQ ID NO：50；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：32、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：10、SEQ ID NO：21，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：31、SEQ ID NO：46、SEQ ID NO：49；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：10、SEQ ID NO：21，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：43、SEQ ID NO：56；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：30、SEQ ID NO：40、SEQ ID NO：49；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：30、SEQ ID NO：40、SEQ ID NO：49；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：12、SEQ ID NO：22，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：18、SEQ ID NO：22，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：31、SEQ ID NO：40、SEQ ID NO：54；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：15、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：15、SEQ ID NO：20，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：28、SEQ ID NO：41、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：27、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：33、SEQ ID NO：40、SEQ ID NO：49；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：4、SEQ ID NO：11、SEQ ID NO：23，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：33、SEQ ID NO：40、SEQ ID NO：49；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：4、SEQ ID NO：11、SEQ ID NO：23，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：32、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：14、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：29、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：29、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：3、SEQ ID NO：12、SEQ ID NO：22，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：29、SEQ ID NO：40、SEQ ID NO：48；
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：38、SEQ ID NO：44、SEQ ID NO：52；或
    HCDR1-HCDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：7、SEQ ID NO：19，LCDR1-LCDR3的氨基酸序列分别为SEQ ID NO：36、SEQ ID NO：42、SEQ ID NO：51。
  4. 根据前述任一项权利要求所述的抗CD24抗体或其抗原结合片段，其中所述重链可变区具有SEQ ID NO：57-71和98-120中任一项所示氨基酸序列或与SEQ ID NO：57-71和98-120中任一项所示氨基酸序列具有至少80％、至少90％、至少95％或至少99％同源性的氨基酸序列。
  5. 根据前述任一项权利要求所述的抗CD24抗体或其抗原结合片段，其中轻链可变区具有SEQ ID NO：72-94和121-147中任一项所示氨基酸序列或与SEQ ID NO：72-94和121-147中任一项所示氨基酸序列具有至少80％、至少90％、至少95％或至少99％同源性的氨基酸序列。
  6. 根据前述任一项权利要求所述的抗CD24抗体或其抗原结合片段，其中：
    所述重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:72；
    所述重链可变区的氨基酸序列为SEQ ID NO:58，轻链可变区的氨基酸序列为SEQ ID NO:73；
    所述重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:74；
    所述重链可变区的氨基酸序列为SEQ ID NO:60，轻链可变区的氨基酸序列为SEQ ID NO:75；
    所述重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:76；
    所述重链可变区的氨基酸序列为SEQ ID NO:62，轻链可变区的氨基酸序列为SEQ ID NO:76；
    所述重链可变区的氨基酸序列为SEQ ID NO:63，轻链可变区的氨基酸序列为SEQ ID NO:77；
    所述重链可变区的氨基酸序列为SEQ ID NO:64，轻链可变区的氨基酸序列为SEQ ID NO:78；
    所述重链可变区的氨基酸序列为SEQ ID NO:65，轻链可变区的氨基酸序列为SEQ ID NO:79；
    所述重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:80；
    所述重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:81；
    所述重链可变区的氨基酸序列为SEQ ID NO:66，轻链可变区的氨基酸序列为SEQ ID NO:82；
    所述重链可变区的氨基酸序列为SEQ ID NO:66，轻链可变区的氨基酸序列为SEQ ID NO:83；
    所述重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:84；
    所述重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:85；
    所述重链可变区的氨基酸序列为SEQ ID NO:67，轻链可变区的氨基酸序列为SEQ ID NO:86；
    所述重链可变区的氨基酸序列为SEQ ID NO:68，轻链可变区的氨基酸序列为SEQ ID NO: 87；
    所述重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:88；
    所述重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:89；
    所述重链可变区的氨基酸序列为SEQ ID NO:61，轻链可变区的氨基酸序列为SEQ ID NO:76；
    所述重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:90；
    所述重链可变区的氨基酸序列为SEQ ID NO:69，轻链可变区的氨基酸序列为SEQ ID NO:76；
    所述重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:76；
    所述重链可变区的氨基酸序列为SEQ ID NO:69，轻链可变区的氨基酸序列为SEQ ID NO:90；
    所述重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:76；
    所述重链可变区的氨基酸序列为SEQ ID NO:59，轻链可变区的氨基酸序列为SEQ ID NO:91；
    所述重链可变区的氨基酸序列为SEQ ID NO:70，轻链可变区的氨基酸序列为SEQ ID NO:91；
    所述重链可变区的氨基酸序列为SEQ ID NO:70，轻链可变区的氨基酸序列为SEQ ID NO:80；
    所述重链可变区的氨基酸序列为SEQ ID NO:71，轻链可变区的氨基酸序列为SEQ ID NO:92；
    所述重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:92；
    所述重链可变区的氨基酸序列为SEQ ID NO:67，轻链可变区的氨基酸序列为SEQ ID NO:92；
    所述重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:93；
    所述重链可变区的氨基酸序列为SEQ ID NO:57，轻链可变区的氨基酸序列为SEQ ID NO:94；
    所述重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:137；
    所述重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:144；
    所述重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:122；
    所述重链可变区的氨基酸序列为SEQ ID NO:114，轻链可变区的氨基酸序列为SEQ ID NO:143；
    所述重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:142；
    所述重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:124；
    所述重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:122；
    所述重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:121；
    所述重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:124；
    所述重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:137；
    所述重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:121；
    所述重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:121；
    所述重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:133；
    所述重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:124；
    所述重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:134；
    所述重链可变区的氨基酸序列为SEQ ID NO:111，轻链可变区的氨基酸序列为SEQ ID NO:136；
    所述重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:134；
    所述重链可变区的氨基酸序列为SEQ ID NO:98，轻链可变区的氨基酸序列为SEQ ID NO:133；
    所述重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:133；
    所述重链可变区的氨基酸序列为SEQ ID NO:101，轻链可变区的氨基酸序列为SEQ ID NO:122；
    所述重链可变区的氨基酸序列为SEQ ID NO:118，轻链可变区的氨基酸序列为SEQ ID NO:146；
    所述重链可变区的氨基酸序列为SEQ ID NO:99，轻链可变区的氨基酸序列为SEQ ID NO:137；
    所述重链可变区的氨基酸序列为SEQ ID NO:115，轻链可变区的氨基酸序列为SEQ ID NO:122；
    所述重链可变区的氨基酸序列为SEQ ID NO:117，轻链可变区的氨基酸序列为SEQ ID NO:121；
    所述重链可变区的氨基酸序列为SEQ ID NO:116，轻链可变区的氨基酸序列为SEQ ID NO:145；
    所述重链可变区的氨基酸序列为SEQ ID NO:104，轻链可变区的氨基酸序列为SEQ ID NO:125；
    所述重链可变区的氨基酸序列为SEQ ID NO:105，轻链可变区的氨基酸序列为SEQ ID NO:123；
    所述重链可变区的氨基酸序列为SEQ ID NO:110，轻链可变区的氨基酸序列为SEQ ID NO:126；
    所述重链可变区的氨基酸序列为SEQ ID NO:109，轻链可变区的氨基酸序列为SEQ ID NO:123；
    所述重链可变区的氨基酸序列为SEQ ID NO:110，轻链可变区的氨基酸序列为SEQ ID NO:123；
    所述重链可变区的氨基酸序列为SEQ ID NO:104，轻链可变区的氨基酸序列为SEQ ID  NO:130；
    所述重链可变区的氨基酸序列为SEQ ID NO:109，轻链可变区的氨基酸序列为SEQ ID NO:126；
    所述重链可变区的氨基酸序列为SEQ ID NO:108，轻链可变区的氨基酸序列为SEQ ID NO:130；
    所述重链可变区的氨基酸序列为SEQ ID NO:105，轻链可变区的氨基酸序列为SEQ ID NO:126；
    所述重链可变区的氨基酸序列为SEQ ID NO:113，轻链可变区的氨基酸序列为SEQ ID NO:141；
    所述重链可变区的氨基酸序列为SEQ ID NO:108，轻链可变区的氨基酸序列为SEQ ID NO:125；
    所述重链可变区的氨基酸序列为SEQ ID NO:103，轻链可变区的氨基酸序列为SEQ ID NO:139；
    所述重链可变区的氨基酸序列为SEQ ID NO:107，轻链可变区的氨基酸序列为SEQ ID NO:140；
    所述重链可变区的氨基酸序列为SEQ ID NO:106，轻链可变区的氨基酸序列为SEQ ID NO:140；
    所述重链可变区的氨基酸序列为SEQ ID NO:106，轻链可变区的氨基酸序列为SEQ ID NO:129；
    所述重链可变区的氨基酸序列为SEQ ID NO:107，轻链可变区的氨基酸序列为SEQ ID NO:129；
    所述重链可变区的氨基酸序列为SEQ ID NO:107，轻链可变区的氨基酸序列为SEQ ID NO:128；
    所述重链可变区的氨基酸序列为SEQ ID NO:103，轻链可变区的氨基酸序列为SEQ ID NO:128；
    所述重链可变区的氨基酸序列为SEQ ID NO:103，轻链可变区的氨基酸序列为SEQ ID NO:140；
    所述重链可变区的氨基酸序列为SEQ ID NO:103，轻链可变区的氨基酸序列为SEQ ID NO:129；
    所述重链可变区的氨基酸序列为SEQ ID NO:120，轻链可变区的氨基酸序列为SEQ ID NO:139；
    所述重链可变区的氨基酸序列为SEQ ID NO:106，轻链可变区的氨基酸序列为SEQ ID NO:128；
    所述重链可变区的氨基酸序列为SEQ ID NO:112，轻链可变区的氨基酸序列为SEQ ID NO:147；
    所述重链可变区的氨基酸序列为SEQ ID NO:112，轻链可变区的氨基酸序列为SEQ ID NO:138；
    所述重链可变区的氨基酸序列为SEQ ID NO:119，轻链可变区的氨基酸序列为SEQ ID NO:138；
    所述重链可变区的氨基酸序列为SEQ ID NO:100，轻链可变区的氨基酸序列为SEQ ID NO:127；
    所述重链可变区的氨基酸序列为SEQ ID NO:102，轻链可变区的氨基酸序列为SEQ ID NO:127；
    所述重链可变区的氨基酸序列为SEQ ID NO:100，轻链可变区的氨基酸序列为SEQ ID NO:135；
    所述重链可变区的氨基酸序列为SEQ ID NO:102，轻链可变区的氨基酸序列为SEQ ID NO:131；
    所述重链可变区的氨基酸序列为SEQ ID NO:100，轻链可变区的氨基酸序列为SEQ ID NO:132；
    所述重链可变区的氨基酸序列为SEQ ID NO:102，轻链可变区的氨基酸序列为SEQ ID NO:135；
    所述重链可变区的氨基酸序列为SEQ ID NO:102，轻链可变区的氨基酸序列为SEQ ID NO:132；或
    所述重链可变区的氨基酸序列为SEQ ID NO:100，轻链可变区的氨基酸序列为SEQ ID NO:131。
  7. 抗CD24抗体或其抗原结合片段，其中所述抗CD24抗体或其抗原结合片段包含重链可变区，其中重链可变区包括重链CDR1(HCDR1)、重链CDR2(HCDR2)和重链CDR3(HCDR3)，其中：
    HCDR1的氨基酸序列包含X 1GYX 2WH(SEQ ID NO:152)，其中X 1为R、N或S，X 2为S或T；或者HCDR1的氨基酸序列为TYGVS(SEQ ID NO:5)；
    HCDR2的氨基酸序列包含X 1X 2X 3X 4X 5GX 6TX 7YX 8X 9X 10LX 11X 12(SEQ ID NO:153)，其中X 1为Y、V或F，X 2为I或T，X 3为H、W或Q；X 4为H、G、Y或F；X 5为R、S或D；X 6为S、T、N或A；X 7为N或K；X 8为N或H；X 9为P或S；X 10为S或A；X 11为K或I；X 12为S或N；
    HCDR3的氨基酸序列包含GX 1X 2X 3X 4X 5DX 6(SEQ ID NO:154)，其中X 1为S或T；X 2为N、D或T；X 3为S、R、W、G或T；X 4为A、S或G；X 5为F或L；X 6为Y或H；或者HCDR3的氨基酸序列为YYGYDFFAY(SEQ ID NO:25)或YYGYDFFPY(SEQ ID NO:26)；
    其中HCDR1-3的序列根据Kabat定义。
  8. 根据前述任一项所述的抗CD24抗体或其抗原结合片段，其中所述抗CD24抗体或其抗原结合片段的轻链恒定区为人类抗体的轻链恒定区；优选地，所述抗CD24抗体或其抗原结合片段的轻链恒定区选自人κ型或λ型轻链恒定区；更优选地，所述抗CD24抗体或其抗原结合片段的轻链恒定区为κ型轻链恒定区。
  9. 根据前述任一项所述的抗CD24抗体或其抗原结合片段，其中所述抗CD24抗体或其抗原结合片段的重链恒定区为人类抗体的重链恒定区；优选地，所述抗CD24抗体或其抗原结合片段的重链恒定区选自人IgD、IgE、IgM、IgA、IgG1、IgG2a、IgG2b、IgG3和IgG4型中的任一种；更优选地，所述抗体或其抗原结合片段的重链恒定区选自IgG1、IgG2或IgG4型。
  10. 根据前述任一项所述的抗CD24抗体或其抗原结合片段，其中：
    所述抗CD24抗体为全抗体、单链抗体(scFv)或双特异性抗体；和/或
    所述抗CD24抗体的抗原结合片段为Fab、Fab’、Fv或F(ab’) 2；和/或
    所述抗CD24抗体为鼠源抗体、嵌合抗体、人源化抗体或全人源抗体；和/或
    所述抗CD24抗体为单克隆抗体。
  11. 分离的核酸分子，其编码权利要求1-10中任一项所述的抗CD24抗体或其抗原结合片段。
  12. 包含权利要求11所述的核酸分子的载体，优选地，所述载体是表达载体。
  13. 分离的宿主细胞，其包含权利要求11所述的核酸分子或权利要求12所述的载体。
  14. 免疫偶联物，其包含与治疗剂偶联的权利要求1-10中任一项所述的抗CD24抗体或其抗原结合片段。
  15. 根据权利要求14所述的免疫偶联物，其中所述治疗剂为抗肿瘤药物，例如，细胞毒性药物、免疫增强剂或放射性同位素。
  16. 药物组合物，其包含权利要求1-10中任一项所述的抗CD24抗体或其抗原结合片段或权利要求14或15所述的免疫偶联物，以及药学上可接受的赋形剂、稀释剂或载体。
  17. 根据权利要求16所述的药物组合物，其用于预防或治疗肿瘤(例如恶性肿瘤)或免疫性疾病。
  18. 根据权利要求17所述的药物组合物，其中所述肿瘤(例如恶性肿瘤)为肿瘤细胞表面CD24高表达的肿瘤，例如，肺癌、乳腺癌、卵巢癌、淋巴癌、结/直肠癌、卵巢癌、肝癌、宫颈癌、肾癌、前列腺癌、食管癌、膀胱癌或鼻咽癌。
  19. 根据权利要求17所述的药物组合物，其中所述免疫性疾病为类风湿性关节炎或系统性红斑狼疮。
  20. 权利要求1-10中任一项所述的抗CD24抗体或其抗原结合片段或权利要求11所述的分离的核酸分子或权利要求12所述的载体或权利要求13所述的分离的宿主细胞或权利要求14或15所述的免疫偶联物在制备用于预防或治疗肿瘤(例如恶性肿瘤)或免疫性疾病的药物中的用途。
  21. 根据权利要求20所述的用途，其中所述肿瘤(例如恶性肿瘤)为肿瘤细胞表面CD24高表达的肿瘤，例如，肺癌、乳腺癌、卵巢癌、淋巴癌、结/直肠癌、卵巢癌、肝癌、宫颈癌、肾癌、前列腺癌、食管癌、膀胱癌或鼻咽癌。
  22. 根据权利要求20所述的用途，其中所述免疫性疾病为类风湿性关节炎或系统性红斑狼疮。
  23. 治疗个体中肿瘤(例如恶性肿瘤)或免疫性疾病的方法，包括向有需要的个体给予治疗有效量的权利要求1-10中任一项所述的抗CD24抗体或其抗原结合片段或权利要求14或15所述的免疫偶联物或权利要求16所述的药物组合物。
  24. 根据权利要求23所述的方法，其中所述肿瘤(例如恶性肿瘤)为肿瘤细胞表面CD24高表达的肿瘤，例如，肺癌、乳腺癌、卵巢癌、淋巴癌、结/直肠癌、卵巢癌、肝癌、宫颈癌、肾癌、前列腺癌、食管癌、膀胱癌或鼻咽癌。
  25. 根据权利要求23所述的方法，其中所述免疫性疾病为类风湿性关节炎或系统性红斑狼疮。
  26. 制备权利要求1-10任一项所述的抗CD24抗体或其抗原结合片段的方法，其包括培养权利要求13所述的分离的宿主细胞的步骤；任选地，所述方法进一步包括分离所述抗CD24抗体或其抗原结合片段的步骤。
PCT/CN2022/137123 2021-12-07 2022-12-07 抗cd24抗体及其用途 WO2023104066A1 (zh)

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