WO2023232110A1 - Anti-human cd24 antibody and use thereof - Google Patents

Abstract

Provided in the present invention are an anti-human CD24 antibody or a fragment thereof and the use thereof. The anti-human CD24 antibody of the present invention specifically binds to human CD24, has significant complement-dependent cytotoxic (CDC) activity and antibody-dependent cytotoxic (ADCC) activity on the cell expressing the target, can regulate and control macrophage-mediated anti-tumor immune response, and can be used for tumor immunotherapy.

Description

Anti-human CD24 antibodies and their applications

Cross-references to related applications

This patent application claims the priority rights of the Chinese invention patent application with application number CN202210621670.X submitted on June 1, 2022, the entire content of which is hereby incorporated by reference.

Technical field

The present invention relates to the field of antibody drugs. Specifically, the invention relates to antibodies against human CD24 and their use for preparing drugs.

Background technique

The CD24 molecule is a sialic acid glycoprotein, which consists of a core skeleton structure of 33 amino acid residues and has 16 potential O- or N-glycosylation sites. The surface is highly glycosylated and the molecular weight ranges from 30 to 70kDa. between. The CD24 molecule can be anchored to the surface of the cell membrane through glycosyl-phosphatidyl-inositol (GPI) at the carboxyl terminus. Under physiological conditions, CD24 molecules are only expressed at low levels on immature B cells, mature granulocytes, and a small number of epithelial cells and nerve cells. By analyzing the RNA gene databases of TCGA (The Cancer Genome Atlas) and TARGET (Therapeutically Applicable Research To Generate Effective Treatments), it was found that in more than 20 tumor types, the vast majority of tumor cells highly express CD24, especially ovarian cancer. , the expression level exceeds 9 times. In addition, CD24 expression in triple-negative breast cancer (TNBC) is significantly higher than that in healthy breast cells or estrogen and progesterone receptor-positive (ER+PR+) breast cancer cells.

CD24 inhibits inflammatory responses by interacting with Siglec-10 molecules on immune cells and transmitting immunosuppressive signals. In addition, CD24-Siglec-10, as an innate immune checkpoint, can regulate macrophage-mediated anti-tumor immune responses. Therefore, CD24 may become an ideal target for tumor immunotherapy, especially for breast cancer or ovarian cancer.

Contents of the invention

The technical problem to be solved by the present invention is to obtain mouse antibodies through hybridoma technology, obtain better antibodies through mouse anti-activity analysis and screening, carry out humanized design and further activity analysis of the antibodies, and finally screen to obtain specific binding to human CD24 and Therapeutics with high affinity, selectivity and biological activity Antibody.

In view of the above technical problems, the object of the present invention is to provide an antibody or fragment thereof that specifically binds to human CD24 and provide its use. Among them, the antibody fragments described in the present invention include various functional fragments of antibodies, such as their antigen-binding portions, such as Fab, F(ab') ₂ or scFv fragments.

The technical solution of the present invention is as follows.

In one aspect, the invention provides an antibody or fragment thereof capable of specifically binding to CD24, in particular human CD24. According to a specific embodiment of the present invention, the antibody of the present invention is a murine antibody obtained by using human CD24 as an immunogen, as well as chimeric antibodies and humanized antibodies obtained based on the murine antibody.

Specifically, the present invention provides an antibody or fragment thereof, said antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

The heavy chain variable region (VH) includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3) contained in the following amino acid sequences: SEQ ID NO.1, 3, 5, 7, The amino acid sequence shown in 9, 11, 13 or 15; and

The light chain variable region (VL) includes light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), light chain CDR3 (LCDR3) contained in the following amino acid sequences: SEQ ID NO.2, 4, 6, 8, The amino acid sequence shown in 10, 12, 14 or 16.

The antibody or fragment thereof provided by the present invention is an anti-CD24, especially an antibody or fragment thereof against human CD24, which can bind to the CD24 with high affinity and specificity. The combination of heavy and light chain CDRs comprised by the antibody or fragment thereof is derived from the specific antibodies of the invention (see the Examples section). Based on the heavy and light chain amino acid sequences comprised by these specific antibodies, those skilled in the art can routinely determine where CDRs included. According to the specific embodiment of the present invention, the heavy and light chain CDRs contained in any of the above amino acid sequences can be obtained using Chothia, AbM, Kabat, Contact, IMGT and comprehensive analysis. For details, please refer to the Examples section. Heavy and light chain CDRs divided by other methods known in the art and combinations thereof are also encompassed within the scope of the present invention.

In the context of the present invention, the term "fragment" encompasses various functional fragments of the anti-CD24 antibody that retain the antigen-binding ability of the antibody and the corresponding biological activity. It is well known in the art that the binding ability of antibodies to antigens and the corresponding biological activities can be achieved by fragments of intact antibodies, which can be obtained using conventional techniques known to those skilled in the art and in the same manner as for intact antibodies. The way to filter is based on functionality. For example, antigen-binding fragments of the antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody. For example, the fragment may be a Fab, F(ab')2 or scFv fragment, etc.

According to a specific embodiment of the invention, the antibody or fragment thereof provided by the invention includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) are derived from the following amino acid sequence combinations:

(1) The amino acid sequence shown in SEQ ID NO.1; and the amino acid sequence shown in SEQ ID NO.2;

(2) The amino acid sequence shown in SEQ ID NO.3; and the amino acid sequence shown in SEQ ID NO.4;

(3) The amino acid sequence shown in SEQ ID NO.5; and the amino acid sequence shown in SEQ ID NO.6;

(4) The amino acid sequence shown in SEQ ID NO.7; and the amino acid sequence shown in SEQ ID NO.8;

(5) The amino acid sequence shown in SEQ ID NO.9; and the amino acid sequence shown in SEQ ID NO.10;

(6) The amino acid sequence shown in SEQ ID NO. 11; and the amino acid sequence shown in SEQ ID NO. 12;

(7) The amino acid sequence shown in SEQ ID NO. 13; and the amino acid sequence shown in SEQ ID NO. 14;

(8) The amino acid sequence shown in SEQ ID NO. 15; and the amino acid sequence shown in SEQ ID NO. 16.

Further, the anti-CD24 antibody or fragment thereof provided by the invention includes a combination of heavy chain CDRs and light chain CDRs (HCDR1, HCDR2 and HCDR3; and LCDR1, LCDR2 and LCDR3) selected from the following:

(1) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22; and, the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.24 and SEQ ID NO.25;

(2) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.22; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , ammonia shown in SEQ ID NO.24 and SEQ ID NO.25 amino acid sequence;

(3) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.22; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.24 and SEQ ID NO.25;

(4) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.33 , the amino acid sequence shown in SEQ ID NO.34 and SEQ ID NO.35;

(5) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.39 , the amino acid sequence shown in SEQ ID NO.40 and SEQ ID NO.25;

(6) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.29 and SEQ ID NO.22; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.24 and SEQ ID NO.25;

(7) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.20, SEQ ID NO.41 and SEQ ID NO.42; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.43 , the amino acid sequence shown in SEQ ID NO.44 and SEQ ID NO.45;

(8) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.46 and SEQ ID NO.42; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.43 , the amino acid sequence shown in SEQ ID NO.44 and SEQ ID NO.45;

(9) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.47 and SEQ ID NO.42; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.43 , the amino acid sequence shown in SEQ ID NO.44 and SEQ ID NO.45;

(10) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.30, SEQ ID NO.48 and SEQ ID NO.49; and, the LCDR1, LCDR2 and LCDR3 contains the amino acid sequences shown in SEQ ID NO.50, SEQ ID NO.51 and SEQ ID NO.52 respectively;

(11) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.84 and SEQ ID NO.85; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.86 , the amino acid sequence shown in SEQ ID NO.40 and SEQ ID NO.45;

(12) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.47 and SEQ ID NO.42; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.43 , the amino acid sequence shown in SEQ ID NO.44 and SEQ ID NO.45;

(13) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.53; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.54 and SEQ ID NO.55;

(14) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.56 and SEQ ID NO.53; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.54 and SEQ ID NO.55;

(15) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.57 and SEQ ID NO.53; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.54 and SEQ ID NO.55;

(16) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.30, SEQ ID NO.58 and SEQ ID NO.59; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.33 , the amino acid sequence shown in SEQ ID NO.60 and SEQ ID NO.61;

(17) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.62; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.39 , the amino acid sequence shown in SEQ ID NO.40 and SEQ ID NO.55;

(18) The HCDR1, HCDR2 and HCDR3 respectively contain SEQ ID NO.26, SEQ ID The amino acid sequences shown in NO.57 and SEQ ID NO.53; and, the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.23, SEQ ID NO.54 and SEQ ID NO.55;

(19) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.63, SEQ ID NO.64 and SEQ ID NO.65; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.66 , the amino acid sequence shown in SEQ ID NO.67 and SEQ ID NO.68;

(20) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.69, SEQ ID NO.70 and SEQ ID NO.65; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.66 , the amino acid sequence shown in SEQ ID NO.67 and SEQ ID NO.68;

(21) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.71, SEQ ID NO.72 and SEQ ID NO.65; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.66 , the amino acid sequence shown in SEQ ID NO.67 and SEQ ID NO.68;

(22) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.73, SEQ ID NO.74 and SEQ ID NO.75; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.76 , the amino acid sequence shown in SEQ ID NO.77 and SEQ ID NO.78;

(23) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.79, SEQ ID NO.80 and SEQ ID NO.81; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.82 , the amino acid sequence shown in SEQ ID NO.83 and SEQ ID NO.68;

(24) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.69, SEQ ID NO.72 and SEQ ID NO.65; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.66 , SEQ ID NO.67 and the amino acid sequence shown in SEQ ID NO.68.

Further, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region may comprise the following amino acid sequence or an amino acid sequence having at least 75% identity with the amino acid sequence: SEQ ID NO. 1, 3, 5, 7, 9, 11, 13 or 15; and, the light chain variable region may comprise the following amino acid sequence or an amino acid sequence having at least 75% identity with the amino acid sequence Acid sequence: the amino acid sequence shown in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16.

"At least 75% identity" used in the context of the present invention with respect to a sequence is, for example, at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, Any percentage of identity ≥ 75% such as 96%, 97%, 98% or even 99% identity. In particular, the antibody or fragment thereof of the present invention includes at least a heavy chain variable region and a light chain variable region, both of which include the CDRs described above and an intervening framework region, which includes a structural domain The arrangement is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Therefore, alternatively, the "at least 75% identity" resulting in up to 25% difference in amino acid sequence may be present in any framework region of the heavy chain variable region or the light chain variable region, or may be present in the present invention. Any domain or sequence in the antibody or its antigen-binding fragment other than the heavy chain variable region and the light chain variable region. The differences may result from amino acid deletions, additions or substitutions at any position.

According to a specific embodiment of the present invention, in the antibody or fragment thereof, the heavy chain variable region and the light chain variable region comprise a sequence combination selected from the following:

(1) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.1; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.2 Amino acid sequences with at least 75% identity;

(2) The heavy chain variable region includes an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.3; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.4 Amino acid sequences with at least 75% identity;

(3) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.5; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.6 Amino acid sequences with at least 75% identity;

(4) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.8 Amino acid sequences with at least 75% identity;

(5) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.9; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.10 Amino acid sequences with at least 75% identity;

(6) The heavy chain variable region includes an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO. 12 Amino acid sequences with at least 75% identity;

(7) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.13; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.14 Amino acid sequences with at least 75% identity;

(8) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.15; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.16 Amino acid sequences with at least 75% identity.

The antibody provided by the invention is a mouse anti-, chimeric antibody or a fully or partially humanized antibody that binds to CD24, especially human CD24 (UniProt: P25063); the fragment is a half-antibody or an antigen-binding fragment of the antibody, Includes scFv, dsFv, (dsFv) ₂ , Fab, Fab', F(ab') ₂ or Fv fragment. Preferably, the antibody is a monoclonal antibody or a single chain antibody.

In addition to the variable region, the antibody or fragment thereof also contains a human or murine constant region, preferably a human or murine heavy chain constant region (CH) and/or a light chain constant region (CL); preferably, the The antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region and/or a kappa or lambda light chain selected from the group consisting of IgG, IgA, IgM, IgD or IgE. constant region. According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody. According to a specific embodiment of the invention, the monoclonal antibody is an IgG, in particular an IgG1 (eg human IgG1).

Further preferably, the antibody or fragment thereof provided by the present invention includes a heavy chain constant region, and the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO. 17 or SEQ ID NO. 18 or is at least 75% identical to the amino acid sequence. % identical amino acid sequence; and/or comprising a light chain constant region, the light chain constant region comprising the amino acid sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity with the amino acid sequence. Furthermore, the antibody provided by the present invention can be a monoclonal antibody, including two identical heavy chains and two identical light chains.

On the other hand, the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the present invention or a fragment thereof, or a heavy chain CDR or a light chain CDR encoding the antibody or fragment thereof. , a light chain variable region, a heavy chain variable region, a nucleotide sequence of a heavy chain or a light chain.

The nucleic acid molecules of the present invention can be cloned into vectors and then transformed or transfected into host cells. Therefore, in yet another aspect, the invention provides a vector comprising a nucleic acid molecule of the invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

The vector or nucleic acid molecule of the present invention can be used to transform or transfect host cells or enter host cells in any way for the purpose of preserving or expressing antibodies. Therefore, in yet another aspect, the present invention provides Provide a host cell containing the nucleic acid molecule and/or vector of the present invention, or the host cell being transformed or transfected by the nucleic acid molecule and/or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant or animal cells.

The antibodies or fragments thereof provided by the invention can be obtained using any method known in the art. For example, when the host cell provided by the invention is allowed to express the heavy chain variable region and/or light chain variable region of the antibody or the heavy chain and/or light chain of the antibody to assemble into the antibody, The host cells are cultured. Optionally, the method further includes the step of recovering the produced antibodies.

The antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention can be included in pharmaceutical compositions, more specifically included in pharmaceutical preparations, and thus used for various purposes according to actual needs. Therefore, in yet another aspect, the present invention also provides a composition comprising the antibody of the present invention or a fragment thereof, a nucleic acid molecule, a vector and/or a host cell; preferably, the composition is a drug The composition also optionally includes a pharmaceutically acceptable carrier, auxiliary material or excipient.

In another aspect, the present invention provides the use of the above-mentioned antibodies or fragments thereof, nucleic acid molecules, vectors, host cells and/or compositions in the preparation of medicaments for the treatment of diseases associated with CD24 expression or mediated by CD24. Preferably, the disease is a tumor or cancer; more preferably, the disease is colorectal cancer, ovarian cancer, breast cancer, liver cancer, bladder cancer, prostate cancer, non-small cell lung cancer, rectal cancer, pancreatic cancer or nasopharyngeal cancer cancer.

In yet another aspect, the invention also provides the use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or composition in the preparation of reagents for detecting or diagnosing diseases associated with CD24 expression or mediated by CD24. . Preferably, the disease is a tumor or cancer; more preferably, the disease is colorectal cancer, ovarian cancer, breast cancer, liver cancer, bladder cancer, prostate cancer, non-small cell lung cancer, rectal cancer, pancreatic cancer or nasopharyngeal cancer cancer.

On the other hand, the present invention also provides a method for treating diseases related to CD24 expression or mediated by CD24, which method includes administering the antibody or fragment thereof or nucleic acid molecule of the present invention to a subject in need thereof. , vectors, host cells and/or compositions, and optionally other drugs or means. The optional other drugs or means refer to other drugs or means that can be administered in combination with the antibodies or fragments thereof, nucleic acid molecules, vectors, host cells and/or compositions of the present invention, such as small molecule drugs, targeted drugs , antibodies and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs and radiotherapy. The combined administration of the two can be carried out in any form, such as simultaneously, continuously or at certain intervals.

Preferably, the disease is tumor or cancer; more preferably, the disease is colorectal cancer, egg ovarian cancer, breast cancer, liver cancer, bladder cancer, prostate cancer, non-small cell lung cancer, rectal cancer, pancreatic cancer or nasopharyngeal cancer. Preferably, the subject is a mammal; more preferably, the subject is a human.

Alternatively, the present invention also provides a method for detecting or diagnosing diseases related to CD24 expression or mediated by CD24, which method includes making the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or The composition is contacted with a sample from the subject. Preferably, the disease is a tumor or cancer; more preferably, the disease is colorectal cancer, ovarian cancer, breast cancer, liver cancer, bladder cancer, prostate cancer, non-small cell lung cancer, rectal cancer, pancreatic cancer or nasopharyngeal cancer cancer. Preferably, the subject is a mammal; more preferably, the subject is a human.

Furthermore, the present invention provides kits comprising said antibodies or fragments thereof, nucleic acid molecules, vectors, host cells and/or compositions. The kit is used for treatment or detection or diagnosis as described above. Optionally, the kit may also include instructions for use.

In the present invention, hybridoma technology is used to obtain mouse antibodies. After obtaining candidate mouse antibodies through antibody activity analysis, the mouse antibody light and heavy chain gene variable region sequences are cloned upstream of the human antibody light and heavy chain constant region sequences for expression in mammalian cells. Prepare chimeric antibodies, and then use antibody activity analysis to verify the regulatory effect of the chimeric antibodies on macrophage-mediated tumor cell phagocytosis and the blocking activity of CD24 binding Siglec-10, then select the humanized template according to the Germline database, The antibody sequence was humanized and designed, and the obtained humanized antibody was analyzed again for antibody activity to further verify the regulatory effect of the humanized modified antibody on macrophage-mediated tumor cell phagocytosis and the blocking of CD24 binding to Siglec-10. Activity, combined with in vitro and in vivo physical and chemical properties analysis of humanized antibodies, in vivo efficacy experiments, etc., we finally obtained a new humanized antibody sequence that specifically binds to human CD24 and confirmed the candidate drug molecules.

Description of the drawings

Below, the embodiments of the present invention are described in detail with reference to the accompanying drawings, wherein:

Figure 1 shows the binding of anti-human CD24 chimeric antibodies to human CD24 expressed on the cell surface.

Figure 2 shows the detection results of the regulatory effect of anti-human CD24 chimeric antibodies on macrophage phagocytosis of SKOV-3 cells; among the results of each antibody in the figure, the left side is the 10 μg/mL concentration result, and the right side is the 1 μg/mL concentration result. .

Figure 3 shows the ADCC activity detection results of anti-human CD24 chimeric antibodies.

Figure 4 shows the antigen binding specificity detection results of anti-human CD24 chimeric antibodies, wherein 4A: MCF7 cell line; 4B: MCF7-CD24KO cell line.

Figure 5 shows the detection results of the regulatory effect of anti-human CD24 humanized antibodies on macrophage phagocytosis of SKOV-3 cells.

Figure 6 shows the ADCC activity detection results of anti-human CD24 humanized antibodies.

Figure 7 shows the ADCP activity detection results of anti-human CD24 humanized antibodies.

Figure 8 shows the detection results of the blocking activity of anti-human CD24 humanized antibodies on Siglec-10 binding to huCD24.

Figure 9 shows the test results of the stability of the anti-human CD24 humanized antibody binding to the antigen; wherein 9A, 9C: C55 Hz43; 9B, 9D: B17 Hz10.

Figure 10: 10A shows the concentration versus time curve of the anti-human CD24 humanized antibody; 10B shows the half-life T _1/2β of the anti-human CD24 humanized antibody obtained by compartmental analysis.

Figures 11 to 13 respectively show the detection results of the therapeutic effect of anti-human CD24 humanized antibodies on colorectal cancer.

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The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified. in:

(1) Tool antibodies:

SN3muIgG: anti-human CD24 mouse monoclonal antibody from Abcam, Cat.No.ab134375;

Hu5F9: Anti-CD47 antibody from Forty Seven Company, take the V region sequence and splice it into the huIgG1 constant region sequence to obtain Hu5F9huIgG1;

(2) In the examples, the huIgG1 heavy chain constant region and light chain constant region or the LALA heavy chain constant region (the antibody is labeled as "LALA-huIgG1") are used, and the sequences are as follows:

Heavy chain constant region (SEQ ID NO. 17):

LALA heavy chain constant region (SEQ ID NO. 18):

Light chain constant region (SEQ ID NO. 19):

Example 1 Screening and identification of hybridoma cells

1.1 Mouse immunization

Balb/c mice aged 8-10 weeks were immunized with the antigenic protein human CD24-mFc (Lot: 20190827A, Beijing Kenuo Xincheng Technology Co., Ltd.). Using Freund's adjuvant, the mice were subcutaneously injected with antigen protein at multiple points on days 0, 3, 6, 12, 15 and 18. The first immunization dose was 6 μg. The second and subsequent immunization doses are 3 μg. Mouse serum was taken as a negative control for detection before immunization, and blood was taken from the tail vein on the 21st day after the initial immunization. The serum titer was detected by ELISA using a 96-well enzyme plate coated with recombinant human CD24 protein.

1.2 Cell fusion and hybridoma preparation

P3X63Ag8.653 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., Cat. No. CBP60876) were revived and cultured to expand to prepare myeloma cell suspension.

Aseptically take the spleens and lymph nodes of mice with high serum titers to prepare specific B cells and obtain a B cell suspension.

The above two cell suspensions were mixed at a ratio of 1:2 between B cells and myeloma cells, and fusion was carried out using electrofusion. After fusion, the cells were recovered in complete _medium at 37°C and 8% CO for 30-240 minutes, then added to HAT medium and plated, rehydrated after 5 days, and replaced with HT medium after 7 days. After 8-10 days, take the cell culture supernatant from the well plate, use FACS to analyze the antibodies secreted by the hybridoma cells, and screen out a number of CHOK1 cells that can bind to the cell surface and stably express human CD24 protein but cannot bind to the cell surface and stably express human irrelevant proteins. Cloning of CHOK1 cells. The selected clones were converted into single cells by limiting dilution method, and each hybridoma cell clone obtained secreted only one antibody.

Example 2 Variable region sequence identification of murine monoclonal antibodies

The hybridoma cells secreting anti-human CD24 antibodies were expanded and cultured, and the total RNA of the cells was extracted according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); the total RNA of the hybridoma cells was reacted with 5×PrimeScript RT Master Mix (Takara). Transcribe into cDNA; use degenerate primers (Anke Krebber., 1997) and Extaq PCR reagent (Takara) to amplify the antibody light chain variable region IgVL (κ) and heavy chain variable region VH sequences; use PCR clean-up Gel The PCR amplification product was purified using extraction kit (Macherey-Nagel); according to the instructions of pClone007 Simple Vector Kit (Beijing Qingke Biotechnology Co., Ltd.), the amplified PCR product was connected to the T vector and transformed into E. coli competent cells. The strain was expanded After the plasmid is amplified and extracted, DNA sequencing is performed to obtain the monoclonal antibody variable region sequence.

The mouse monoclonal antibody variable region sequence is shown below.

1. Mouse-derived antibody B17

B17 heavy chain variable region amino acid sequence (SEQ ID NO.1):

B17 light chain variable region amino acid sequence (SEQ ID NO.2):

Among them, the amino acid positions of the heavy chain and light chain CDR sequences in the heavy and light chain variable regions are shown in Table 1.

Table 1. CDR sequence of mouse antibody B17

2. Mouse-derived antibody C01

C01 heavy chain variable region amino acid sequence (SEQ ID NO.3):

C01 light chain variable region amino acid sequence (SEQ ID NO.4):

Among them, the amino acid positions of the heavy chain and light chain CDR sequences in the heavy and light chain variable regions are shown in Table 2.

Table 2. CDR sequence of mouse antibody C01

3. Mouse-derived antibody C55

C55 heavy chain variable region amino acid sequence (SEQ ID NO.5):

C55 light chain variable region amino acid sequence (SEQ ID NO.6):

Among them, the amino acid positions of the heavy chain and light chain CDR sequences in the heavy and light chain variable regions are shown in Table 3.

Table 3. CDR sequence of mouse antibody C55

4. Mouse-derived antibody C60

C60 heavy chain variable region amino acid sequence (SEQ ID NO.7):

C60 light chain variable region amino acid sequence (SEQ ID NO.8):

Among them, the amino acid positions of the heavy chain and light chain CDR sequences in the heavy and light chain variable regions are shown in Table 4.

Table 4. CDR sequence of mouse antibody C60

Example 3 Preparation of anti-human CD24 chimeric antibodies

The heavy chain variable region encoding gene of the murine anti-human CD24 monoclonal antibody and the huIgG1 heavy chain constant region encoding gene were spliced together and constructed into a mammalian cell expression vector; the light chain of the murine anti-human CD24 monoclonal antibody was The gene encoding the variable region and the gene encoding the huIgG1 light chain constant region were spliced together and constructed into a mammalian cell expression vector. The heavy chain vector and light chain vector of the constructed anti-human CD24 chimeric antibody were paired and mixed, and polyethylenimine (PEI) was used to transfect HEK293 cells. After about 7 days, the cell supernatant was collected and purified using Mabselect to obtain the anti-human CD24 chimeric antibody. The antibody was named "mouse-derived antibody xiIgG1".

Example 4 In vitro cell binding experiment of anti-human CD24 chimeric antibodies

The anti-human CD24 chimeric antibody of the present invention, Hu5F9, and unrelated negative antibodies were diluted 2-fold starting from a starting concentration of 10 nM, with a total of 7 concentration points. 10 μl of the antibody at each concentration point was added to a 384-well plate.

Prepare a suspension of OVCAR-3 cells (expressing CD24 on the cell surface, purchased from Nanjing Kebai Biotechnology Co., Ltd., Cat. No. CBP60294) with a density of approximately 2×10 ⁶ cells/mL, and add 10 μl to the 384-well well with the antibody added. in the wells of the plate; and, replace the antibody diluent with an equal volume of PBS in the blank control wells. After incubation at 4°C for 1 hour, fluorescently labeled goat anti-human IgG secondary antibody was added, continued incubation at 4°C for 1 hour, and the average fluorescence reading of the cell population was analyzed by flow cytometry.

The results are shown in Figure 1. The results show that the chimeric antibodies provided by the invention all retain good binding activity to human CD24.

Example 5 In vitro cytological experiments of anti-human CD24 chimeric antibodies

5.1 Detection of the regulation of CD24 antibody on macrophage-mediated phagocytosis of SKOV-3 cells

Use PMA (phorbol 12-myristate 13-acetate, purchased from Sigma, dosage 60ng/mL) and LPS (lipopolysaccharide, purchased from Sigma, dosage 100ng/mL) to treat human peripheral blood mononuclear macrophages Cells were SC (purchased from ATCC, Cat. No. CRL-9855) for 72 hours to differentiate into mature SC macrophages (effector cells), and then stained with Cell-Trace ^TM Far-Red. Anti-CD24 antibodies were diluted and added to the effector cell suspension to final concentrations of 10 μg/mL and 1 μg/mL respectively. Ovarian cancer cell SKOV-3, which stably overexpresses GFP (purchased from Nanjing Kebai Biotechnology Co., Ltd., Cat. No. CBP60291), was used as the target cell. The ratio of effector cells to target cells E:T was 2:1, and equal volumes were added to the above system. , incubated for 6 hours, followed by flow cytometric analysis. The phagocytosis rate was calculated by calculating the ratio of Far-Red and GFP double-stained cells to all Far-Red-stained cells.

The results are shown in Table 5 and Figure 2.

Table 5. Phagocytosis rate (%)

The results show that the chimeric antibodies provided by the invention all improve the phagocytosis of CD24-expressing SKOV-3 cells by macrophages to varying degrees.

5.2 ADCC activity detection

Engineered Jurkat cells were used as effector cells, which stably express the FcγRIIIa-FcεRIaγ hybrid receptor and express firefly luciferase driven by the NFAT response element. The biological activity of the antibody in the ADCC mechanism of action was quantified by luciferase production from activation of the NFAT pathway. Mix effector cells with a density of 6×10 ⁶ cells/mL and anti-CD24 antibody with a concentration of 100 μg/mL to 1.5ng/mL in equal volumes, and then add target cells SKOV with a density of 1×10 ⁶ cells/mL. -3 (E:T ratio of effector cells to target cells 6:1) was added in the same volume and incubated at 37°C for 17 hours; thereafter, detection was performed using Promega's Bio-Glo ^TM Luciferase Assay System. Finally, the LUM value was detected by an MD microplate reader, and a 4-parameter fitting was performed to calculate the EC50 of the sample.

The results are shown in Table 6 and Figure 3.

Table 6.RLU

The results show that the chimeric antibodies provided by the invention all have varying degrees of ADCC activity.

5.3 ADCP activity detection

Engineered Jurkat cells were used as effector cells, which stably express the FcγRIIa-FcεRIaγ hybrid receptor and express firefly luciferase driven by the NFAT response element. The biological activity of the antibody in the ADCP mechanism of action was quantified by luciferase production from activation of the NFAT pathway. Effector cells with a density of 6×10 ⁶ cells/mL were mixed with an equal volume of anti-CD24 antibody with a concentration of 100 μg/mL to 1.5ng/mL, and then SKOV3 cells with a density of 1×10 ⁶ cells/mL ( Effector cells and target cells (E:T ratio 6:1) were added in the same volume and incubated at 37°C for 17 hours; thereafter, detection was carried out through Promega's Bio-Glo ^TM Luciferase Assay System; finally, MD enzyme labeling was used The instrument detects the LUM value, performs 4-parameter fitting, and calculates the EC50 of the sample.

The results are shown in Table 7.

Table 7.RLU

The results show that the chimeric antibodies provided by the invention all have varying degrees of ADCP activity.

Example 6 Humanization of anti-human CD24 murine antibodies

Determine the amino acid sequence regions of the six antigenic complementary determinants (CDRs) of the heavy and light chains of the mouse antibody and the framework region that supports the conservative three-dimensional conformation of the antibody, and then select the human antibody that is closest to the mouse antibody. The heavy chain variable region sequence is used as a template to combine the mouse antibody heavy chain CDR with the framework region in the human antibody heavy chain variable region sequence to generate a humanized antibody heavy chain variable region sequence. The same process is used to generate humanized antibody light chain variable region sequences.

After direct transplantation of CDRs, if the binding activity decreases sharply, individual amino acids in the framework region should be back mutated, and the humanized sequence should also be checked for potential post-translational modification sites.

The humanized antibody heavy chain variable region encoding gene is constructed into a mammalian cell expression vector containing the huIgG1 heavy chain constant region encoding gene or the LALA heavy chain constant region encoding gene; the light chain variable region encoding gene is constructed into a mammalian cell expression vector containing the huIgG1 heavy chain constant region encoding gene. Chain constant region encoding genes in mammalian cell expression vectors. The obtained heavy chain vector and light chain vector were paired and mixed, and HEK293 cells were transfected with polyethylenimine (PEI). After about 7 days, the cell supernatant was collected and purified to obtain humanized anti-human CD24 antibodies.

An exemplary humanized sequence is as follows; the underlines show the heavy chain or light chain CDR1, CDR2 and CDR3 in sequence. The principle of CDR classification is to refer to databases such as Chothia, Kabat and IMGT, and select the longest segment to define the humanized CDR. part:

1.Humanization of B17

Humanized amino acid sequence of B17 heavy chain variable region

>B17_VH_hz1(SEQ ID NO.9)

HCDR1：SEQ ID NO.26

HCDR2：SEQ ID NO.29

HCDR3：SEQ ID NO.22

Humanized amino acid sequence of B17 light chain variable region

>B17_VL_hz0(SEQ ID NO.10)

LCDR1：SEQ ID NO.23

LCDR2：SEQ ID NO.24

LCDR3：SEQ ID NO.25

2. Humanization transformation of C01

Humanized amino acid sequence of C01 heavy chain variable region

>C01_VH_hz1(SEQ ID NO.11)

HCDR1：SEQ ID NO.26

HCDR2：SEQ ID NO.47

HCDR3：SEQ ID NO.42

Humanized amino acid sequence of C01 light chain variable region

>C01_VL_hz0(SEQ ID NO.12)

LCDR1：SEQ ID NO.43

LCDR2：SEQ ID NO.44

LCDR3：SEQ ID NO.45

3.Humanized transformation of C55

Humanized amino acid sequence of C55 heavy chain variable region

>C55_VH_hz4(SEQ ID NO.13)

HCDR1：SEQ ID NO.26

HCDR2: SEQ ID NO.57

HCDR3：SEQ ID NO.53

Humanized amino acid sequence of C55 light chain variable region

>C55_VL_hz3(SEQ ID NO.14)

LCDR1：SEQ ID NO.23

LCDR2：SEQ ID NO.54

LCDR3：SEQ ID NO.55

4.Humanized transformation of C60

Humanized amino acid sequence of C60 heavy chain variable region

>C60_VH_hz4(SEQ ID NO.15)

HCDR1：SEQ ID NO.69

HCDR2：SEQ ID NO.72

HCDR3：SEQ ID NO.65

Humanized amino acid sequence of C60 light chain variable region

>C60_VL_hz8(SEQ ID NO.16)

LCDR1：SEQ ID NO.66

LCDR2：SEQ ID NO.67

LCDR3：SEQ ID NO.68

Name the humanized antibody as "mouse antibody name hzmn" or "mouse antibody name Hzmn", where m and n are the humanized (hz) modified sequences (VH_hz and VL_hz) of VH and VL respectively. Number.

Example 7 Antigen binding specificity verification experiment

Humanized antibodies are as follows:

C01 hz10: VH is C01_VH_hz1 (SEQ ID NO.11); VL is C01_VL_hz0 (SEQ ID NO.12);

C55 hz43: VH is C55_VH_hz4 (SEQ ID NO.13); VL is C55_VL_hz3 (SEQ ID NO.14);

C60 Hz48: VH is C60_VH_hz4 (SEQ ID NO.15); VL is C60_VL_hz8 (SEQ ID NO.16);

B17 Hz10: VH is B17_VH_hz1 (SEQ ID NO.9); VL is B17_VL_hz0 (SEQ ID NO.10).

Human breast cancer cells MCF7 and CD24-knocked-out MCF7 (MCF7-CD24KO) were used as experimental cells respectively. Prepare a cell suspension of 5×10 ⁶ cells/mL and add 80 μL/well into a 96-well plate. The cell wells are grouped as follows: blank control group (without adding antibodies or secondary antibodies); secondary antibody control group (without adding antibodies, Add secondary antibody); experimental group (add antibody and secondary antibody).

Starting with an antibody solution with a concentration of 20 μg/ml, dilute it 5 times to obtain 8 antibody dilutions with test concentrations. Add 20 μL of antibody diluent per well to the cell wells of the experimental group, add an equal volume of FACS Buffer to the blank and secondary antibody control groups, mix and incubate in the dark at 4°C for 40 minutes. Wash the cells three times with FACS Buffer, and finally resuspend the cells with 100 μL FACS Buffer. Add 5 μL of PE-labeled secondary antibody (PE anti-human IgG Fc antibody) to the experimental group and secondary antibody control group, add an equal volume of FACS Buffer to the blank control group, mix and incubate in the dark at 4°C for 40 minutes. Use FACS Buffer to wash the cells three times again, and finally resuspend the cells in 250 μL FACS Buffer.

With an excitation wavelength of 488 nm, the fluorescence intensity (MFI) was detected by flow cytometry, and the FACS data was analyzed using FlowJo software. The results are shown in Table 8 and Figure 4.

Table 8.MFI

The results show that the humanized antibodies and tool antibody SN3 provided by the present invention have binding activity to MCF7 normal cell lines, but have no binding signal to MCF7-CD24KO cells, proving that these antibodies can specifically bind to CD24.

Example 8 In vitro cytological experiments of anti-human CD24 humanized antibodies

8.1 Detection of the regulation of CD24 antibody on macrophage-mediated phagocytosis of SKOV-3 cells

The experimental procedure described in 5.1 in Example 5 was followed, except that the anti-CD24 antibody was diluted to 1, 0.1, and 0.01 μg/mL.

The results are shown in Table 9 and Figure 5.

Table 9. Phagocytosis rate (%)

Experimental results show that the humanized antibodies provided by the invention all have varying degrees of activity in mediating the phagocytosis of SKOV-3 cells by macrophages; and the pro-phagocytic activity of the LALA form antibody is similar to that of the wild-type huIgG1 form antibody.

8.2 ADCC activity detection

The experimental process described in 5.2 in Example 5 was carried out, except that the dilution concentration of the anti-CD24 antibody was 100 μg/mL to 5 ng/mL.

The results are shown in Table 10 and Figure 6.

Table 10.RLU

Experimental results show that the humanized antibodies provided by the present invention all have varying degrees of ADCC activity against ovarian cancer cell SKOV-3.

8.3 ADCP activity detection

The experimental process described in 5.2 in Example 5 was carried out, except that the dilution concentration of the anti-CD24 antibody was 100 μg/mL to 5 ng/mL.

The results are shown in Table 11 and Figure 7.

Table 11.RLU

Experimental results show that the humanized antibodies provided by the invention all have varying degrees of ADCP activity against the ovarian cancer cell SKOV-3.

8.4 Detection of blocking activity of CD24 antibody on Siglec-10 binding to huCD24

A cell suspension of 1×10 ⁵ cells/mL of HEK293-hCD24 cells (Acros, Cat: CHEK-ATP032) was prepared. The antibody to be tested was diluted with 2% BSA, with a starting concentration of 100 μg/mL, and diluted 3 times to obtain 8 concentration dilutions. Add 50 μL of cell suspension to the cell plate, then add 50 μL/well of antibody diluent to the cell suspension, and incubate on ice for 30 min. Then E-Labeled Human Siglec-10 (Acros, Cat: S10-HP2E5) was diluted to 3 μg/mL with 2% BSA, added to the above cell suspension at 50 μL/well, and incubated on ice for 1 hour. After washing the cells with PBS, use a flow cytometer to analyze the MFI data obtained and perform a 4-parameter fitting to calculate the IC50.

The results are shown in Table 12 and Figure 8.

Table 12.MFI

*: Only PE-Labeled Human Siglec-10 alone was added as a control reagent signal

Experimental results show that the humanized antibodies provided by the invention all have varying degrees of blocking activity for PE-human siglec-10 binding to human CD24-positive cells.

Example 9 In vitro monkey serum stability study of anti-human CD24 humanized antibodies

Prepare a 20 μg/ml solution of the antibody to be tested and mix it with fetal bovine serum in equal volumes. The mixture was placed at 37°C, and samples were taken on days 0, 3, 7, 10, 14, 21, and 24 and stored at 4°C for testing. The sample on day 21 was placed at 4°C for at least one day.

The antigenic protein Human CD24-mFc and anti-IgG Fab monoclonal antibody (Sigma, I5260-1ML) were coated with PBS at 0.2μg/ml, 100ul/well in a 96-well enzyme-linked plate, incubated at 4°C overnight, and then Wash three times with PBS at 300 μl/well, then add blocking solution (5% BSA+PBS) at 200 μl/well, block at 37°C for 1 h, and then remove the blocking solution.

The sample to be tested was diluted to 2 μg/ml using diluent (5% BSA+PBS+50% FBS), and diluted 3 times to obtain a total of 8 concentration sample dilutions. Add 100 μl/well of the sample dilution solution to the above two enzyme-linked plates respectively, and incubate at 37°C for 1 hour. Then, wash the plate three times with PBST (0.1% Tween+PBS). Dilute HRP-labeled goat anti-human IgG secondary antibody (Jackson, Cat. No. 109-035-098) at 1:5000, add 100ul/well to the washed enzyme-linked plate, and incubate at 37°C for 40 minutes. Wash the plate three times again with PBST. Add 100ul/well of TMB and develop color in the dark for 10 minutes. Add 50ul 2M HCL to each well to stop the reaction and read the absorbance at 450nm.

Draw a binding curve based on the absorbance at 450nm to evaluate the stability of the antibody-antigen binding. The results are shown in Figure 9 (9A to 9B: coated anti-IgG Fab monoclonal antibody; 9C to 9D: coated antigen). In this example, two humanized antibodies (C55 Hz43 and B17 Hz10) were co-incubated with monkey serum at 37°C to simulate the stability of antibody samples in vivo in monkeys. The experimental results showed that the antibodies Within 24 days of observation, they all had good stability.

Example 10 In vivo drug metabolism analysis of anti-human CD24 humanized antibodies

Female Balb/C mice, 3/group, were administered with 200 μg of the antibody to be tested/animal in the tail vein. Blood was collected from the tail vein at different time points after administration, and the serum was collected by centrifugation and stored at -20°C for testing (the last serum collected was frozen at -20°C for at least 24 hours).

The antigen protein Human CD24-mFc was coated in a 96-well enzyme-linked plate with PBS at 0.2 μg/ml, 100 μl/well, incubated at 4°C overnight, and then washed three times with PBS at 300 μl/well. 200μl/well Add blocking solution (5% BSA+PBS), block at 37°C for 1 hour, and then remove the blocking solution.

Take the mouse serum that has not been administered as a blank control and perform gradient dilution with antibody diluent (5% BSA+PBS+20% blank mouse serum); use blocking solution (5% BSA+PBS) to collect the serum to be tested by centrifugation. ) diluted so that it has the same serum concentration as the gradient dilution of the blank control. The dilution factor of the specific concentration needs to be adjusted according to the preliminary experiment. In principle, the final color value of the serum sample tested should be within the range of the color value of the standard.

Dilute the antibody standard with antibody diluent. The dilution of the standard is still adjusted according to the preliminary experiment so that the standard can fit a linear curve.

Add 100 μl/well of the serum sample to be tested and the standard diluent into the above two enzyme-linked plates respectively, and incubate at 37°C for 1 hour. Then, wash the plate three times with PBST (0.1% Tween+PBS). Dilute HRP-labeled goat anti-human IgG secondary antibody (Jackson, Cat. No. 109-035-098) at 1:5000, add 100ul/well to the washed enzyme-linked plate, and incubate at 37°C for 40 minutes. Wash the plate three times again with PBST. Add 100ul/well of TMB and develop color in the dark for 10 minutes. Add 50ul 2M HCL to each well to stop the reaction and read the absorbance at 450nm.

The results are shown in Table 13 and Figure 10.

Table 13. Changes in antibody concentration (μg/mL) over time and calculated half-life

BQL: lower than the lower detection limit, the lower detection limit is 20ng/mL

The results show that the metabolism levels of the humanized antibodies provided by the invention in mice are within the normal range.

Example 11 In vivo efficacy experiment of anti-human CD24 humanized antibodies

The therapeutic effect of the antibody on subcutaneous tumor formation of MC38-hCD24 colorectal cancer cells in hSiglec-10 humanized mice was tested.

11.1 Drug efficacy experiment one

Female hSiglec-10 humanized mice aged 6-8 weeks (purchased from Southern Model Biology Company) were subcutaneously inoculated with MC38-hCD24 cells at 1×10 ⁶ cells/mouse, and the survival status and subcutaneous tumor formation of the mice were observed the next day. situation, establishing the MC38-hCD24 colorectal cancer model. On the 8th day after inoculation, the average tumor volume was approximately 132mm ³ . The tumor-bearing mice were randomly divided into the following 6 groups, with 6 mice in each group:

Group 1: Isotype control, hIgG1;

Group 2: C01 hz10 huIgG1;

Group 3: C55 hz43 huIgG1;

Group 4: C55 hz43 LALA-huIgG1;

Group 5: C60 Hz48 huIgG1;

Group 6: B17 hz10 huIgG1.

Intraperitoneal administration was started on the day of grouping. The administration volume of each group was 10 mL/kg, and the administration dose was 10 mg/kg. Each group was administered continuously for 3 weeks, twice a week, for a total of 6 administrations.

Measure and record the mouse body weight and tumor size, which are measured during each administration in the first 2 weeks of the administration cycle; observe for 2 weeks after drug withdrawal, measure twice a week, and calculate TGI% (TGI=[1-(TVt -TVinitial)/(CVt-CVinitial)] × 100%, where TVt represents the tumor volume of the treatment group at each measurement; TVinitial represents the tumor volume of the treatment group when administered in groups; CVt represents the tumor volume of the control group at each measurement Volume; CVinitial represents the tumor volume in the control group when administered in groups). Calculate IR% (IR=[(CVt-TVt)/CVt]×100%.

The results are shown in Table 14 and Figure 11.

Table 14. Test results

The results showed that the body weight of mice was monitored throughout the treatment period, and there was no significant difference in the body weight of mice in each group, indicating that the mice had strong resistance to C01 hz10 huIgG1, C55 hz43 huIgG1, C55 hz43 LALA-huIgG1, C60 Hz48 huIgG1 and B17 hz10 huIgG1 antibodies. Well tolerated, these antibodies have minimal toxic side effects.

Compared with the control group (Group 1) and other antibody treatment groups, C55 hz43 huIgG1 (Group 2) and B17 hz10 huIgG1 (Group 6) have good inhibitory activity on tumor growth in mice, with TGIs of 57.40% and 91.42% respectively, while The remaining three groups of drugs had no inhibitory effect on tumors. At the end of the experiment (Day 24), the animals were euthanized, and the tumors were removed and weighed. The specific results are shown in the table above. They are consistent with the tumor volume results. The tumor weights of Group 2 and Group 6 are smaller, and the average tumor weights are 0.9g respectively. and 0.43g.

11.2 Drug efficacy experiment 2

The establishment process of the MC38-hCD24 colorectal cancer model was the same as the drug efficacy experiment one. On the 7th day after inoculation, the average tumor volume was approximately 91mm ³ . The tumor-bearing mice were randomly divided into the following 5 groups, with 6 mice in each group:

Group 1: B17 hz10 huIgG1;

Group 2: B17 hz10 LALA-huIgG1;

Group 3: Isotype control,NC huIgG1;

Group 4: C55 hz43 huIgG1;

Group 5: C55 hz43 LALA-huIgG1

Intraperitoneal administration was started on the day of grouping. The administration volume of each group was 10 mL/kg, and the administration dose was 10 mg/kg. Each group was administered continuously for 2 weeks, twice a week, for a total of 4 times.

Measure and record the mouse body weight and tumor size, which are measured at each dose; observe for 2 weeks after drug withdrawal, and measure twice a week. The results are shown in Table 15 and Figure 12.

Table 15. Test results

The results showed that the body weight of mice was monitored throughout the treatment period, and there was no significant difference in the body weight of mice in each group, indicating that the mice were tolerant to B17 hz10 huIgG1, B17 hz10 LALA-huIgG1, C55 hz43 huIgG1 and C55 hz43 LALA-huIgG1 antibodies. The properties are good and these antibodies have little side effects.

Compared with the control group (Group 3) and other antibody treatment groups, B17 hz10 huIgG1 (Group 1) and C55 hz43 huIgG1 (Group 4) have good inhibitory activity on tumor growth in mice, and the tumor volume has a very significant difference (P<0.01 ), TGI are 102.94% and 113.72% respectively. At the end of the experiment, the animals were euthanized and the tumors were removed and weighed. Among them, the tumors of Group 4 disappeared on the 11th day of administration and no tumors were collected, while the average tumor weight of Group 1 was only 0.16g, less than 1/6 of the control group (Group3). Moreover, the anti-tumor effects of B17 and C55 chimeric with different constant regions are also quite different. The huIgG1 form of antibodies (Group 1 and 4) has a more significant inhibitory effect compared to the LALA-huIgG1 form of antibodies (Group 2 and 5). tumor activity, indicating that the ADCC function mediated by antibody Fc plays a very important role in inhibiting tumor growth.

11.3 Drug efficacy experiment three

An MC38-hCD24 transplanted tumor model was established using 6-8 week old female C57 mice. The modeling process was the same as the drug efficacy experiment 1.

On the 6th day after inoculation, the average tumor volume was approximately 94 mm ³ . The tumor-bearing mice were randomly divided into 7 groups, with 6 rats in each group. Intraperitoneal administration began on the day of grouping. The administration volume of each group was 10 mL/kg, and each group was administered continuously. The drug was administered for 2 weeks, twice a week, for a total of 4 times.

Measure and record the mouse body weight and tumor size, which are measured at each dose; observe 1 week after drug withdrawal, and measure twice. The dosage and results of each group are shown in Table 16 and Figure 13.

Table 16. Test results

The dosages of B17 hz10 huIgG1 and C55 hz43 huIgG1 are 0.5 mg/kg, 2 mg/kg and 10 mg/kg respectively. The results showed that after 18 days of observation, the body weight of the mice was monitored throughout the treatment period. There was little difference in the body weight of the mice in each group, and there was no significant difference, indicating that the mice were tolerant to each dose of B17 hz10 huIgG1 and C55 hz43 huIgG1 antibody drugs. Good, indicating that these antibodies have little toxic side effects.

Compared with the control group (Group 1), the medium and high-dose groups of B17 hz10 huIgG1 and C55 hz43 huIgG1 (Group 4) had good inhibitory activity on tumor growth in mice, and there was a significant difference in tumor volume (P<0.05). Among them, B17 The mid-dose group of hz10 huIgG1 (2mg/kg) and the high-dose group of C55 hz43 huIgG1 (10mg/kg) had better anti-tumor activity, with extremely significant differences (P<0.01), with TGIs of 98.14% and 93.61% respectively. At the end of the experiment (Day 18), the animals were euthanized, and the tumors were removed and weighed. Among them, the average tumor weights of Group 3 and Group 7 were 0.26g and 0.29g respectively, with extremely significant differences (P<0.01).

The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention. As long as they do not deviate from the spirit of the present invention, they should all fall within the scope of the appended claims of the present invention.

Claims (15)

1. An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

   The heavy chain variable region (VH) includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3) contained in the following amino acid sequences: SEQ ID NO.1, 3, 5, 7, The amino acid sequence shown in 9, 11, 13 or 15; and

   The light chain variable region (VL) includes light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), light chain CDR3 (LCDR3) contained in the following amino acid sequences: SEQ ID NO.2, 4, 6, 8, The amino acid sequence shown in 10, 12, 14 or 16.
2. The antibody or fragment thereof according to claim 1, characterized in that the antibody or fragment thereof comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3) and light chain CDR1 (LCDR1 ), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) are derived from the following amino acid sequence combinations:

   (1) The amino acid sequence shown in SEQ ID NO.1; and the amino acid sequence shown in SEQ ID NO.2;

   (2) The amino acid sequence shown in SEQ ID NO.3; and the amino acid sequence shown in SEQ ID NO.4;

   (3) The amino acid sequence shown in SEQ ID NO.5; and the amino acid sequence shown in SEQ ID NO.6;

   (4) The amino acid sequence shown in SEQ ID NO.7; and the amino acid sequence shown in SEQ ID NO.8;

   (5) The amino acid sequence shown in SEQ ID NO.9; and the amino acid sequence shown in SEQ ID NO.10;

   (6) The amino acid sequence shown in SEQ ID NO. 11; and the amino acid sequence shown in SEQ ID NO. 12;

   (7) The amino acid sequence shown in SEQ ID NO. 13; and the amino acid sequence shown in SEQ ID NO. 14;

   (8) The amino acid sequence shown in SEQ ID NO. 15; and the amino acid sequence shown in SEQ ID NO. 16.
3. The antibody or fragment thereof according to claim 1 or 2, characterized in that the antibody or fragment thereof comprises heavy chain CDRs and light chain CDRs selected from the group consisting of (HCDR1, HCDR2 and HCDR3; and, LCDR1, LCDR2 and LCDR3) The combination:

   (1) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22; and, the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.24 and SEQ ID NO.25;

   (2) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.22; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.24 and SEQ ID NO.25;

   (3) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.22; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.24 and SEQ ID NO.25;

   (4) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.33 , the amino acid sequence shown in SEQ ID NO.34 and SEQ ID NO.35;

   (5) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.39 , the amino acid sequence shown in SEQ ID NO.40 and SEQ ID NO.25;

   (6) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.29 and SEQ ID NO.22; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.24 and SEQ ID NO.25;

   (7) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.20, SEQ ID NO.41 and SEQ ID NO.42; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.43 , the amino acid sequence shown in SEQ ID NO.44 and SEQ ID NO.45;

   (8) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.46 and SEQ ID NO.42; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.43 , the amino acid sequence shown in SEQ ID NO.44 and SEQ ID NO.45;

   (9) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.47 and SEQ ID NO.42; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.43 , the amino acid sequence shown in SEQ ID NO.44 and SEQ ID NO.45;

   (10) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.30, SEQ ID NO.48 and SEQ ID NO.49; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.50 , the amino acid sequence shown in SEQ ID NO.51 and SEQ ID NO.52;

   (11) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.84 and SEQ ID NO.85; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.86 , the amino acid sequence shown in SEQ ID NO.40 and SEQ ID NO.45;

   (12) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.47 and SEQ ID NO.42; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.43 , the amino acid sequence shown in SEQ ID NO.44 and SEQ ID NO.45;

   (13) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.53; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.54 and SEQ ID NO.55;

   (14) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.56 and SEQ ID NO.53; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.54 and SEQ ID NO.55;

   (15) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.57 and SEQ ID NO.53; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , ammonia shown in SEQ ID NO.54 and SEQ ID NO.55 amino acid sequence;

   (16) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.30, SEQ ID NO.58 and SEQ ID NO.59; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.33 , the amino acid sequence shown in SEQ ID NO.60 and SEQ ID NO.61;

   (17) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.62; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.39 , the amino acid sequence shown in SEQ ID NO.40 and SEQ ID NO.55;

   (18) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.26, SEQ ID NO.57 and SEQ ID NO.53; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.23 , the amino acid sequence shown in SEQ ID NO.54 and SEQ ID NO.55;

   (19) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.63, SEQ ID NO.64 and SEQ ID NO.65; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.66 , the amino acid sequence shown in SEQ ID NO.67 and SEQ ID NO.68;

   (20) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.69, SEQ ID NO.70 and SEQ ID NO.65; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.66 , the amino acid sequence shown in SEQ ID NO.67 and SEQ ID NO.68;

   (21) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.71, SEQ ID NO.72 and SEQ ID NO.65; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.66 , the amino acid sequence shown in SEQ ID NO.67 and SEQ ID NO.68;

   (22) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.73, SEQ ID NO.74 and SEQ ID NO.75; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.76 , the amino acid sequence shown in SEQ ID NO.77 and SEQ ID NO.78;

   (23) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.79, SEQ ID NO.80 and SEQ ID NO.81; and, the LCDR1, LCDR2 and LCDR3 contains the amino acid sequences shown in SEQ ID NO.82, SEQ ID NO.83 and SEQ ID NO.68 respectively;

   (24) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO.69, SEQ ID NO.72 and SEQ ID NO.65; and the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO.66 , the amino acid sequence shown in SEQ ID NO.67 and SEQ ID NO.68;

   Further, the heavy chain variable region includes the following amino acid sequence or an amino acid sequence having at least 75% identity with the amino acid sequence: SEQ ID NO. 1, 3, 5, 7, 9, 11, 13 or 158 The amino acid sequence is shown; and, the light chain variable region includes the following amino acid sequence or an amino acid sequence having at least 75% identity with the amino acid sequence: SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 Or the amino acid sequence shown in 16.
4. The antibody or fragment thereof according to any one of claims 1 to 3, in the antibody or fragment thereof, the heavy chain variable region and the light chain variable region comprise a sequence combination selected from the following:

   (1) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.1; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.2 Amino acid sequences with at least 75% identity;

   (2) The heavy chain variable region includes an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.3; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.4 Amino acid sequences with at least 75% identity;

   (3) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.5; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.6 Amino acid sequences with at least 75% identity;

   (4) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.8 Amino acid sequences with at least 75% identity;

   (5) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.9; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.10 Amino acid sequences with at least 75% identity;

   (6) The heavy chain variable region includes an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO. 12 Amino acid sequences with at least 75% identity;

   (7) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.13; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.14 Amino acid sequences with at least 75% identity;

   (8) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.15; and the light chain variable region includes an amino acid sequence shown in SEQ ID NO.16 Amino acid sequences with at least 75% identity.
5. The antibody or fragment thereof according to any one of claims 1 to 4, characterized in that the antibody is a mouse antibody, a chimeric antibody or a fully or partially humanized antibody thereof; the fragment is a half-antibody or the scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv fragment of the antibody;

   Preferably, the antibody is a monoclonal antibody or a single chain antibody;

   Preferably, the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine heavy chain constant region (CH) and/or a light chain constant region (CL); preferably, the antibody or its fragment The fragment comprises a heavy chain and a light chain; more preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region;

   Further preferably, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; further preferably, the monoclonal antibody is IgG, especially IgG1.
6. A nucleic acid molecule comprising the heavy chain CDR, light chain CDR, light chain variable region, heavy chain variable region, heavy chain or Nucleotide sequence of the light chain.
7. A vector comprising the nucleic acid molecule of claim 6.
8. A host cell comprising the nucleic acid molecule of claim 6 and/or the vector of claim 7.
9. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7 and/or the host according to claim 8 cell.
10. The composition according to claim 9, characterized in that the composition is a pharmaceutical composition, which further contains optional pharmaceutically acceptable carriers, auxiliary materials or excipients.
11. The antibody or fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8 and/or claim 9 or 10 Use of the composition in the preparation of medicaments for treating diseases related to CD24 expression or mediated by CD24.
12. The antibody or fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8 and/or claim 9 or 10 Use of the composition in the preparation of reagents for detecting or diagnosing diseases associated with CD24 expression or mediated by CD24.
13. A method of treating a disease associated with CD24 expression or mediated by CD24, the method comprising administering to a subject in need thereof the antibody or fragment thereof according to any one of claims 1 to 5, claim 6 The nucleic acid molecule, the vector of claim 7, the host cell of claim 8 and/or the composition of claim 9 or 10, and optional other drugs or means.
14. A method for detecting or diagnosing diseases related to CD24 expression or mediated by CD24, the method comprising making the antibody or fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, The vector of claim 7, the host cell of claim 8 and/or the composition of claim 9 or 10 are contacted with a sample from a subject.
15. An antibody comprising the antibody or fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8 and/or the Kit of the composition described in 9 or 10.