CD24 antibody, antigen-binding fragment thereof and use thereof

Abstract

Disclosed are a CD24 antibody or an antigen-binding fragment thereof and the medical use thereof. Further, disclosed are a chimeric antibody comprising CDRs of the CD24 antibody, a humanized antibody, a pharmaceutical composition comprising the CD24 antibody or the antigen-binding fragment thereof, and the use thereof as an anticancer drug.

Description

CD24 antibody, its antigen-binding fragment and its application Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to a CD24 antibody or its antigen-binding fragment, a chimeric antibody thereof, a humanized antibody thereof, and a pharmaceutical composition comprising the CD24 antibody or its antigen-binding fragment thereof, and its use as an anticancer drug. Background Technology

[0002] CD24 (27-59aa) is a glycosylphosphatidylinositol (GPI)-modified protein composed of a small protein core containing 33 amino acids. It was first discovered as a B-cell differentiation antigen in 1978. It is expressed in various tissues and cell types, including hematopoietic stem cells, B and T lymphocytes, epithelial cells, and nerve cells. It possesses distinct domains: an intracellular domain, a transmembrane domain, and a highly glycosylated extracellular domain. The extracellular domain of CD24 has a varying number of N-linked and O-linked glycosylation sites, which play a role in regulating CD24-mediated cell adhesion and signal transduction. It has one N-glycosylation site and multiple O-glycosylation sites, which function in its glycosylation. This protein attaches to the cell membrane via its terminal (C-terminus) glycosylated phosphatidylinositol (GPI) anchor, interacts with various cell surface receptors such as P-selectin, Siglec-10, and β1 integrin, and participates in regulating cell adhesion, migration, differentiation, and apoptosis through binding to the Notch signaling pathway.

[0003] CD24 is an important biomarker for various cancers. It is highly expressed in a variety of malignant tumors, especially solid tumors, including esophageal squamous cell carcinoma, ovarian cancer, prostate cancer, small cell and non-small cell lung cancer, breast cancer, and B-cell lymphoma. Furthermore, CD24 overexpression is significantly associated with survival time in patients with various cancers. Increased CD24 expression has been reported to promote tumor growth and metastasis (Zhuo J, Wang X. The combined effects of CD24 inhibition and autophagy inhibition can suppress colorectal ischemia and promote its inhibition. Mol Med Rep. 2019; 20: 539–48), etc. Barkal et al. found that CD24 overexpression is closely associated with ovarian cancer and triple-negative breast cancer; ovarian cancer and triple-negative breast cancer cells highly express CD24 on their surface, and tumor-associated macrophages express high levels of Siglec-10. Studies have found that CD24 on the tumor surface promotes immune escape of tumor cells by interacting with the inhibitory receptor sialic acid-binding immunoglobulin-like lectin 10 (Siglec-10). The "don't eat me" signal generated after CD24 binds to Siglec-10 inhibits the anti-tumor activity of macrophages. These data indicate that CD24 is a highly expressed anti-phagocytic signal in various cancers and demonstrate the therapeutic potential of CD24 blockade in cancer immunotherapy (CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy[J].Nature,2019,572:392-396). Currently, there are no CD24-related antibody drugs on the market; therefore, further research is necessary to meet clinical needs. Summary of the Invention

[0004] This invention provides a CD24 antibody or its antigen-binding fragment, which can specifically bind to CD24, exhibiting strong phagocytic activity (ADCP) and ADCC effects, as well as good antitumor efficacy. At the same time, the antibody described in this invention has no toxic side effects on normal cells or organisms and has good safety.

[0005] This invention provides a CD24 antibody or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0006] 1) The heavy chain variable region (VH) comprises HCDR1, HCDR2, and HCDR3; HCDR1 comprises any sequence of SEQ ID NO.1–5, SEQ ID NO.24–28, or SEQ ID NO.42–46, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequence; HCDR2 comprises any sequence of SEQ ID NO.6–9, SEQ ID NO.29–32, or SEQ ID NO.47–50, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequence; and HCDR3 comprises any sequence of SEQ ID NO.10–12 or SEQ ID NO.33–35, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequence; and

[0007] 2) The light chain variable region (VL) comprises LCDR1, LCDR2, and LCDR3; LCDR1 comprises any sequence of SEQ ID NO.13-15, SEQ ID NO.36-38, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequence; LCDR2 comprises any sequence of SEQ ID NO.16-20, SEQ ID NO.39-41, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequence; and LCDR3 comprises any sequence of SEQ ID NO.21-23, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequence; each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is encoded according to a common analytical method of KABAT, Chothia, or IMGT.

[0008] In some embodiments, this application provides a CD24 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein:

[0009] (1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 has any of the HCDR1 sequences shown in Table 1 below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence, HCDR2 has any of the HCDR2 sequences shown in Table 1 below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence, and HCDR3 has any of the HCDR3 sequences shown in Table 1 below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence.

[0010] Table 1

[0011] and,

[0012] (2) The light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 has any of the LCDR1 sequences shown in Table 2 below, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown in Table 2 below; LCDR2 has any of the LCDR2 sequences shown in Table 2 below, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown in Table 2 below; and LCDR3 has any of the LCDR3 sequences shown in Table 2 below, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown in Table 2 below.

[0013] Table 2

[0014] Specifically, for example, the CD24 antibody or antigen-binding fragment of the present invention, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), encoded according to the KABAT analytical method, wherein,

[0015] The heavy chain variable region (VH) contains HCDR1, HCDR2, and HCDR3 selected from any of the following sequences (1)-(6) or sequences having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequences:

[0016] (1) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.1, 6 and 10 respectively;

[0017] (2) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.2, 6 and 11 respectively;

[0018] (3) The HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO.3, 7 and 12;

[0019] (4) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.4, 8 and 12 respectively;

[0020] (5) The HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO.5, 9, and 12, respectively; or

[0021] (6) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.5, 8 and 12 respectively;

[0022] Furthermore, according to the KABAT analysis method, the LCDR1, LCDR2, and LCDR3 contained in the light chain variable region (VL) are selected from any of the following sequences (7)-(11) or sequences having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequences:

[0023] (7) The LCDR1, LCDR2 and LCDR3 are respectively as shown in the sequences SEQ ID NO.13, 16 and 21;

[0024] (8) The LCDR1, LCDR2 and LCDR3 are respectively as shown in the sequences SEQ ID NO.14, 17 and 22;

[0025] (9) The LCDR1, LCDR2 and LCDR3 are respectively as shown in the sequences SEQ ID NO.15, 18 and 23;

[0026] (10) The LCDR1, LCDR2, and LCDR3 are respectively in the sequences shown in SEQ ID NO.15, 19, and 23; or

[0027] (11) The LCDR1, LCDR2 and LCDR3 are respectively as shown in the sequences SEQ ID NO.15, 20 and 23.

[0028] Specifically, for example, the CD24 antibody or antigen-binding fragment of the present invention, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), encoded according to the IMGT analysis method, wherein,

[0029] The heavy chain variable region (VH) contains HCDR1, HCDR2, and HCDR3 selected from any of the following sequences (12)-(17) or sequences having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequences:

[0030] (12) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.24, 29 and 33 respectively;

[0031] (13) The HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO.24, 30 and 34;

[0032] (14) The HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO.25, 31 and 35;

[0033] (15) The HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO.26, 31 and 35;

[0034] (16) The HCDR1, HCDR2, and HCDR3 are respectively as shown in the sequences SEQ ID NO.27, 32, and 35; or

[0035] (17) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.28, 31 and 35 respectively;

[0036] Furthermore, according to the IMGT analysis method, the LCDR1, LCDR2, and LCDR3 contained in the light chain variable region (VL) are selected from any of the following sequences (18)-(20) or sequences having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequences:

[0037] (18) The LCDR1, LCDR2 and LCDR3 are respectively in the sequences shown in SEQ ID NO.36, 39 and 21;

[0038] (19) The LCDR1, LCDR2, and LCDR3 are respectively in the sequences shown in SEQ ID NO.37, 40, and 22; or

[0039] (20) The LCDR1, LCDR2 and LCDR3 are respectively shown in the sequences of SEQ ID NO.38, 41 and 23;

[0040] Specifically, for example, the CD24 antibody or antigen-binding fragment of the present invention, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), encoded according to the Chothia analytical method, wherein,

[0041] The heavy chain variable region (VH) contains HCDR1, HCDR2, and HCDR3 selected from any of the following sequences (21)-(26) or sequences having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequences:

[0042] (21) The HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO.42, 47 and 10;

[0043] (22) The HCDR1, HCDR2 and HCDR3 are respectively shown as sequences SEQ ID NO.42, 48 and 11;

[0044] (23) The HCDR1, HCDR2 and HCDR3 are respectively shown as sequences in SEQ ID NO.43, 49 and 12;

[0045] (24) The HCDR1, HCDR2 and HCDR3 are respectively shown as sequences in SEQ ID NO.44, 49 and 12;

[0046] (25) The HCDR1, HCDR2, and HCDR3 are respectively as shown in the sequences SEQ ID NO.45, 50, and 12; or

[0047] (26) The HCDR1, HCDR2 and HCDR3 are respectively shown as sequences in SEQ ID NO.46, 49 and 12;

[0048] Furthermore, according to Chothia's analytical method, the LCDR1, LCDR2 and LCDR3 contained in the light chain variable region (VL) are selected from any of the sequences in (7)-(11) above or sequences with 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequences.

[0049] The present invention provides a CD24 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or fragments thereof, and / or humanized antibodies or fragments thereof.

[0050] In some embodiments, the present invention provides a CD24 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises a murine heavy chain FR region or a mutant sequence thereof; and the light chain variable region of the antibody comprises a murine light chain FR region or a mutant sequence thereof.

[0051] In one specific embodiment, the present invention provides a CD24 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0052] 1) The heavy chain variable region (VH) is selected from any of the sequences in SEQ ID NO.51 to SEQ ID NO.56 or the heavy chain variable region having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it, as shown in Table 3;

[0053] Table 3. Amino acid sequence of the variable region of the heavy chain of murine antibody

[0054] 2) The light chain variable region (VL) is selected from any of the sequences in SEQ ID NO.57 to SEQ ID NO.62 or the light chain variable region having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence, as shown in Table 4.

[0055] Table 4. Amino acid sequence of the variable region of the light chain of murine antibodies.

[0056] Furthermore, the CD24 antibody or its antigen-binding fragment provided in this application, wherein the antibody comprises: a heavy chain variable region comprising any one of the nucleotide sequences SEQ ID NO: 63-68 or a nucleotide sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it, and a light chain variable region comprising any one of the nucleotide sequences SEQ ID NO: 69-74 or a light chain variable region having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it.

[0057] In some embodiments, the present invention provides that the CD24 antibody or its antigen-binding fragment is a chimeric antibody or its antigen-binding fragment.

[0058] In some embodiments, the CD24 antibody or its antigen-binding fragment provided by the present invention is a humanized antibody or its antigen-binding fragment, wherein the FR region of the heavy chain variable region is subjected to a combination of 0 to multiple amino acid combinations of site-directed mutations based on a mouse sequence, preferably comprising any sequence of mouse sequence SEQ ID NO. 51 and / or SEQ ID NO. 55 or its mutant sequence; and / or the FR region of the light chain variable region of the humanized antibody or its antigen-binding fragment is subjected to a combination of 0 to multiple amino acid combinations of site-directed mutations based on a mouse sequence, preferably comprising any sequence of SEQ ID NO. 57 and / or SEQ ID NO. 61 or its mutant sequence.

[0059] In some specific embodiments, the FR region on the variable region of the humanized antibody or its antigen-binding fragment provided by the present invention contains a combination of site-directed mutations of multiple amino acids.

[0060] Furthermore, the humanized antibody or its antigen-binding fragment provided by the present invention is mutated based on the FR region of the murine heavy chain variable region SEQ ID NO. 55 to obtain mutated sequences SEQ ID NO. 75-76, which contain one or more of the following site mutations: K3Q, Q5L, Q5V, R13K, R13Q, M18L, K19R, V23A, V23T, S40A, E42G, I71T, S79N, S80T, H82Y, N87S, R89T, A90S, G94A, I95V, P107R, T110M, L111V; and / or mutated based on the FR region of the murine light chain variable region SEQ ID NO. 61 to obtain mutated sequences SEQ ID NO. 75-76. NO.80~82 contains one or more of the following site mutations: T10S, S12P, I15L, I15P, L42Q, K50Q, K66R, T68S, L88F, L88V, G105P, I90V, and G105Q, as shown in Table 5.

[0061] Furthermore, the humanized antibody or its antigen-binding fragment provided by the present invention, based on the FR region of the murine heavy chain variable region SEQ ID NO. 51, yields mutated sequences SEQ ID NO. 77–79, which contain mutations at the following sites: a combination of one or more of the following sites: E1Q, K3Q, V5L, A13K, Q16E, Q16G, S17T, I20L, V37I, L48V, L48I, L67F, L67V, S68T, S76N, F79Y, F79S, M82L, N83R, L85V, Q86R, Q86T, T87S, T87A, D88E, D88A, M92L, M92V, P109R, P109Q, S112L, and L113V; and / or mutations are performed based on the FR region of the murine light chain variable region SEQ ID NO. 57 to obtain the mutated sequence SEQ ID NO. 77–79. NO.83~85 contains combinations of one or more mutation sites from the following sites: V3Q, T9S, I10S, M11L, P15L, E17Q, E17D, K18R, M21I, S39P, S40G, T41K, S42P, S42A, G59S, S69E, S71T, A79S, A79P, E78Q, V82F, A99G, A99Q, L105I, and L103V, as shown in Table 5.

[0062] Table 5. List of mutant amino acid sequences in the variable region of humanized antibodies.

[0063] In some embodiments, a CD24 antibody or its antigen-binding fragment, the humanized antibody or its antigen-binding fragment, wherein the heavy chain variable region is selected from any sequence of SEQ ID NO. 75 to SEQ ID NO. 79 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it; the light chain variable region is selected from any sequence of SEQ ID NO. 80 to SEQ ID NO. 85 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it.

[0064] In a further embodiment, a CD24 antibody or its antigen-binding fragment thereof, wherein the humanized CD24 antibody or its antigen-binding fragment comprises: a heavy chain amino acid sequence as shown in SEQ ID NO:75 and a light chain amino acid sequence as shown in SEQ ID NO:80;

[0065] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:75 and a light chain amino acid sequence as shown in SEQ ID NO:81;

[0066] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:75 and a light chain amino acid sequence as shown in SEQ ID NO:82;

[0067] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:76 and a light chain amino acid sequence as shown in SEQ ID NO:80;

[0068] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:76 and a light chain amino acid sequence as shown in SEQ ID NO:81;

[0069] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:76 and a light chain amino acid sequence as shown in SEQ ID NO:82;

[0070] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:77 and a light chain amino acid sequence as shown in SEQ ID NO:83;

[0071] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:77 and a light chain amino acid sequence as shown in SEQ ID NO:84;

[0072] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:77 and a light chain amino acid sequence as shown in SEQ ID NO:85;

[0073] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:78 and a light chain amino acid sequence as shown in SEQ ID NO:83;

[0074] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:78 and a light chain amino acid sequence as shown in SEQ ID NO:84;

[0075] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:78 and a light chain amino acid sequence as shown in SEQ ID NO:85;

[0076] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:79 and a light chain amino acid sequence as shown in SEQ ID NO:83;

[0077] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:79 and a light chain amino acid sequence as shown in SEQ ID NO:84;

[0078] Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:79 and a light chain amino acid sequence as shown in SEQ ID NO:85.

[0079] In some embodiments, the CD24 chimeric antibody or antigen fragment thereof provided in this application, and / or the CD24 humanized antibody or antigen fragment thereof, comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or variants thereof, and a light chain constant region of human Kappa(k), lambda(λ) chain or a mutant sequence thereof.

[0080] Preferably, it comprises the heavy chain constant region of human IgG1 or a mutant sequence thereof and the light chain constant region of human Kappa chain or a mutant sequence thereof.

[0081] In some embodiments, the CD24 antibody or antigen-binding fragment provided in this application is chimeric or humanized; preferably, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies.

[0082] Furthermore, the present invention provides a multispecific antigen-binding molecule; preferably, the multispecific antigen-binding molecule comprises a CD24 antibody or an antigen-binding fragment thereof disclosed in the present invention.

[0083] Furthermore, the present invention provides an isolated nucleic acid molecule that encodes a CD24 antibody or an antigen-binding fragment thereof disclosed in the present invention.

[0084] Furthermore, the present invention provides an expression vector encoding a CD24 antibody or its antigen-binding fragment disclosed in the present invention.

[0085] Furthermore, the present invention provides an expression vector comprising the isolated nucleic acid disclosed in the present invention.

[0086] Furthermore, the present invention provides a host cell transformed with the expression vector disclosed according to the present invention. Even further, the host cell is selected from prokaryotic cells and eukaryotic cells. In a specific embodiment, the eukaryotic cell is a mammalian cell; preferably, the host cell is selected from HEK293.

[0087] In some embodiments, the present invention provides a method for preparing a CD24 antibody or an antigen fragment thereof, comprising culturing the host cells described above under conditions that appropriately express the CD24 antibody or antigen fragment disclosed herein, and isolating and purifying the CD24 antibody or antigen fragment disclosed herein from the host cells.

[0088] In some embodiments, the present invention provides a pharmaceutical composition comprising the CD24 antibody or antigen-binding fragment disclosed herein, the multispecific antigen-binding molecule disclosed herein, the isolated nucleic acid molecule disclosed herein, the expression vector disclosed herein, the cell disclosed herein, or a product prepared by the method disclosed herein (e.g., antibody and antigen-binding fragment), and a pharmaceutically acceptable carrier.

[0089] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient; more preferably, the pharmaceutical composition further comprises an additional antitumor agent.

[0090] In some embodiments, the present invention provides the use of the CD24 antibody or antigen-binding fragment disclosed in the present invention, the multispecific antigen-binding molecule disclosed in the present invention, the isolated nucleic acid molecule disclosed in the present invention, the expression vector disclosed in the present invention, the cell disclosed in the present invention, or the product prepared by the method disclosed in the present invention (e.g., antibody and antigen-binding fragment), or the pharmaceutical composition disclosed in the present invention in the preparation of a medicament for the prevention and / or treatment of CD24-mediated diseases. Preferably, CD24-mediated diseases are B-cell lymphoma, leukemia, ovarian cancer, breast cancer, glioma, small cell lung cancer, esophageal squamous cell carcinoma, liver cancer, pancreatic cancer, or prostate cancer.

[0091] In some embodiments, the present invention provides a method for preventing and / or treating CD24-mediated diseases, comprising administering to a patient in need a CD24 antibody or antigen-binding fragment disclosed herein, a multispecific antigen-binding molecule disclosed herein, an isolated nucleic acid molecule disclosed herein, an expression vector disclosed herein, a cell disclosed herein, or a product prepared by the method disclosed herein (e.g., antibody and antigen-binding fragment), or a pharmaceutical composition disclosed herein; wherein the CD24-mediated disease is B-cell lymphoma, leukemia, ovarian cancer, breast cancer, glioma, small cell lung cancer, esophageal squamous cell carcinoma, liver cancer, pancreatic cancer, or prostate cancer.

[0092] In some embodiments, the present invention provides a kit comprising the CD24 antibody or antigen-binding fragment disclosed in the present invention, the multispecific antigen-binding molecule disclosed in the present invention, the isolated nucleic acid molecule disclosed in the present invention, the expression vector disclosed in the present invention, the cells disclosed in the present invention, or a product prepared by the method disclosed in the present invention (e.g., antibody and antigen-binding fragment), or the pharmaceutical composition disclosed in the present invention, and instructions for use. Attached Figure Description

[0093] Figure 1 shows the reduced SDS-PAGE protein electrophoresis images of the antigen and control antibody (where M: Marker; 1: AF01; 2: AF02; 3: AF03; 4: AF04; 5: PC; 6: NC).

[0094] Figure 2 shows the concentration-absorbance curve of the positive control antibody binding to human CD24-His antigen.

[0095] Figure 3 shows the reduced SDS-PAGE protein electrophoresis image of the chimeric antibody (where M: Marker; 1-6: CMAB01-CMAB06).

[0096] Figure 4 shows the concentration-absorbance curve of the chimeric antibody binding to the human CD24-His antigen.

[0097] Figure 5 shows the concentration-absorbance curve of the chimeric antibody binding to the human CD24-Fc(AF01) antigen.

[0098] Figure 6 shows the mean fluorescence intensity (MFI) value of the chimeric antibody binding to hCD24-MC38 cells.

[0099] Figure 7 shows the mean fluorescence intensity (MFI) value of the chimeric antibody binding to MDA-MB-468 cells.

[0100] Figure 8 shows the mean fluorescence intensity (MFI) value of the chimeric antibody binding to OVCAR-3 cells.

[0101] Figure 9 shows the mean fluorescence intensity (MFI) value of the chimeric antibody binding to HeLa cells.

[0102] Figure 10 shows the mean fluorescence intensity (MFI) values ​​of the chimeric antibody binding to HT-29 cells.

[0103] Figure 11 shows the mean fluorescence intensity (MFI) values ​​of the chimeric antibody binding to cynoCD24-293 cells.

[0104] Figure 12 shows the mean fluorescence intensity (MFI) values ​​of the chimeric antibody binding to HEK 293 cells.

[0105] Figure 13 shows the reduced SDS-PAGE protein electrophoresis image of the humanized antibody (where M: Marker; 1–15: ...).

[0106] HAB01~HAB15).

[0107] Figure 14 shows the non-reduced SDS-PAGE protein electrophoresis image of the humanized antibody (where M: Marker; 1-15: HAB01-HAB15).

[0108] Figure 15 shows the concentration-absorbance curve of the binding of humanized antibody to human CD24-His antigen.

[0109] Figure 16 shows the concentration-mean fluorescence intensity (MFI) curves of the humanized antibody binding to MDA-MB-468 cells.

[0110] Figure 17 shows the concentration-mean fluorescence intensity (MFI) curves of humanized antibody binding to OVCAR-3 cells.

[0111] Figure 18 shows the concentration-mean fluorescence intensity (MFI) curves of the humanized antibody binding to HT-29 cells.

[0112] Figure 19 shows the concentration-light intensity curve of the effect of humanized antibody on ADCC in MDA-MB-468 cells.

[0113] Figure 20 shows the concentration-light intensity curve of the effect of humanized antibody on ADCP in MDA-MB-468 cells.

[0114] Figure 21 Time-mouse tumor volume (mm) of humanized antibody. 3 )curve.

[0115] Figure 22 shows the time-mouse body weight (g) curve of humanized antibody. Detailed Implementation

[0116] the term

[0117] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0118] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. Chem, 243, p3558 (1968).

[0119] CD24 is a highly glycosylated glycosylphosphatidylinositol (GPI)-anchored surface protein. Siglec is a member of the immunoglobulin superfamily expressed on the cell surface, including sialic acid-binding immunoglobulin-like lectin 10 (Siglec-10) on tumor-associated macrophages (TAMs), which regulates cell migration and phagocytic activity through ligand contact on the cell surface. Overexpression of CD24 on the surface of cancer cells reduces phagocytic activity by binding to Siglec-10 on the macrophage surface and transmitting an inhibitory signal, thereby causing immune escape of cancer cells. This signal is figuratively described as a "don't eat me" signal.

[0120] In this article, the term "antibody" refers to immunoglobulin. Immunoglobulin (Ig) refers to a globulin with antibody (Ab) activity or chemical structure, similar to an antibody molecule. Immunoglobulins are tetrapeptide chains composed of two identical light chains and two identical heavy chains linked by interchain disulfide bonds. Immunoglobulins are classified into five classes: immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE), with corresponding heavy chains of μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ and λ chains based on differences in their constant regions. Each of the five classes of Ig can have both κ and λ chains.

[0121] The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably, forming the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or its antigen-binding fragment described in this invention conform to the known KABAT, IMGT, and Chothia numbering rules in terms of both number and position.

[0122] The present invention discloses that the antibody heavy chain may further include a heavy chain constant region, wherein the heavy chain constant region includes the constant regions of mouse or human IgG1, IgG2, IgG3, IgG4 or their mutant sequences.

[0123] The present invention discloses that the antibody light chain may further include a light chain constant region, wherein the light chain constant region includes the constant region of the κ chain or λ chain of mouse or human origin or a mutant sequence thereof.

[0124] The term "heavy chain constant region" in this document refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in antibody-antigen binding but exhibits effector functions, such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domains of the antibody. A "heavy chain constant region" contains at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. "Heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter includes only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domains.

[0125] The term "light chain constant region" in this article refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0126] The term "VH" in this article refers to the variable region of the immunoglobulin heavy chain (including the heavy chain of Fv, scFv, or Fab) of an antibody. The term "VL" refers to the variable region of the immunoglobulin light chain (including the light chain of Fv, scFv, dsFv, or Fab).

[0127] The term "mouse antibody" in this disclosure refers to a monoclonal antibody against human CD24 prepared in accordance with the knowledge and skills in the art. Preparation involves injecting a test subject (mouse) with CD24 antigen, followed by isolation and expression of an antibody having the desired sequence or functional characteristics. In some embodiments of this disclosure, the mouse CD24 antibody or its antigen-binding fragment may further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3, IgG4, or a mutant sequence thereof, or further comprise a light chain constant region of mouse κ chain, λ chain, or a mutant sequence thereof.

[0128] The term "chimeric antibody" used in this paper refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. To establish a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established. Then, the variable region gene is cloned from mouse hybridoma cells, and the constant region gene of a human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of the invention, the antibody light chain of the chimeric antibody further includes the light chain constant region of a human κ, λ chain, or a mutant sequence thereof. The antibody heavy chain of the chimeric antibody further includes the heavy chain constant region of a human IgG1, IgG2, IgG3, or IgG4, or a mutant sequence thereof, preferably including the heavy chain constant region of a human IgG1, or a mutant sequence thereof, and the light chain constant region of a human κ chain, or a mutant sequence thereof.

[0129] The term "humanized antibody" in this document refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance immune responses. In a preferred embodiment of the invention, the variable region of the humanized antibody is derived from mouse antibodies, and the constant region is derived from the heavy chain constant region of human IgG1 or a mutant sequence thereof, and the light chain constant region of the human κ chain or a mutant sequence thereof.

[0130] The term "antigen-binding fragment" in this article refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibody fragments can be Fab, F(ab')2, scFv, SMIP, biantibodies, triantibodies, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by disulfide bonds in a hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of an antibody arm; (v) a dAb containing VH and VL domains; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of either a VH or VL domain; (viii) a separate complementarity-determining region (CDR); and (ix) a combination of two or more separate CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by independent genes, these two domains can be joined using recombination methods via a linker that enables the creation of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments can be generated by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.

[0131] The term "monoclonal antibody" in this article refers to an antibody derived from a single clonal cell line, which can be eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be recombined using various techniques, including hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies, such as murine monoclonal antibodies, chimeric monoclonal antibodies, and humanized monoclonal antibodies.

[0132] The term "mutated sequence" in this document refers to a nucleotide and amino acid sequence that, when modified by appropriate substitutions, insertions, or deletions, exhibits a different percentage of sequence identity with the nucleotide and amino acid sequences of this invention. The sequence identity described in this invention can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Sequence comparisons and identity percentage determinations between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithm available on the National Center for Biotechnology Institute website.

[0133] The term "polymerase chain reaction" or "PCR" as used herein refers to a procedure or technique for amplifying trace amounts of specific portions of nucleic acids, RNA, and / or DNA as described in U.S. Patent No. 4,683,195. Sequence information from the ends or outside the target region is typically required to design oligonucleotide primers; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified. The 5' nucleotides of both primers may be congruent to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA, etc. (References include Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol. 51:263; Erlich et al. (1989) PCR TECHNOLOGY (Stockton Press, NY). The PCR used herein is considered one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying nucleic acid test samples, which involves using known nucleic acids as primers and a nucleic acid polymerase to amplify or produce specific portions of nucleic acids.)

[0134] The term "multispecific" in this article refers to having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Therefore, terms such as "bispecific," "trispecific," and "quadrispecific" refer to the number of different epitopes that an antibody / antigen binding molecule can bind to.

[0135] The term "affinity" in this article refers to the binding strength between the antigen-binding site of an antibody and the corresponding antigenic determinant. It is the inherent binding force between the antigen and the antibody, representing the tightness of the specific binding between them. The magnitude of antibody affinity is generally expressed by the affinity constant "KD" (sec⁻¹). The term "KD" (M) used in this invention refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, KD = kd / ka.

[0136] The term "pharmaceutical composition" as used herein refers to a mixture containing one or more CD24 antibodies or their antigen-binding fragments described herein, along with other chemical components, such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0137] The terms “subject,” “object,” and “patient” refer to an organism that receives treatment for a specific disease or condition, such as cancer or an infectious disease, as described herein. Examples of objects and patients include mammals receiving treatment for diseases or conditions, such as proliferative disorders like cancer or infectious diseases, including humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk, and yaks), sheep, and horses.

[0138] The term "treatment" refers to the administration of oral or topical therapeutic agents, such as compositions comprising any of the anti-CD24 antibodies of the present invention or antigen fragments thereof, to a patient for the purpose of preventing, slowing (reducing) the progression of undesirable physiological changes or lesions, such as proliferative disorders (e.g., cancer or infectious diseases), to a patient. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of disease status (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Subjects requiring treatment include those who already have the condition or disease, those susceptible to the condition or disease, or those who intend to prevent the condition or disease. When the terms slowing, reducing, weakening, mitigating, or alleviating are used, they also include elimination, disappearance, and non-occurrence.

[0139] The term "effective dose" in this document refers to the amount of a therapeutic agent, when administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective dose" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, consecutively, or simultaneously.

[0140] The term "appropriate conditions" in this article refers to conditions suitable for culturing various host cells, including eukaryotic and prokaryotic cells.

[0141] The term "cancer" in this article refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers.

[0142] The term "tumor" in this article refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used in this article.

[0143] The term "antitumor agent" in this article refers to antitumor drugs, which are a class of drugs used to treat tumor diseases, including chemotherapy drugs and biological agents.

[0144] The term "EC" in this article 50 "" refers to the half-maximum effective concentration, which includes the antibody concentration that induces a half-range response between baseline and maximum after a specified exposure time. 50 Essentially representing 50% of the antibody concentration where its maximum effect is observed, it can be measured using methods known in the art.

[0145] The term "ADCC" in this article refers to antibody-dependent cell-mediated cytotoxicity, which mainly refers to the process by which immune effector cells are activated and kill tumor target cells when antibodies bind to tumor cell surface antigens at antigen-binding sites and bind to FcRs on the surface of immune effector cells at Fc sites.

[0146] The term "ADCP" in this article refers to antibody-dependent cell-mediated phagocytosis, an important mechanism for recognizing and mediating the action of therapeutic antibodies on tumor cells. The mechanism is based on the phagocytosis of target cells (e.g., tumor cells) by effector cells with phagocytic potential (macrophages, monocytes). Antibodies bind to target cells by recognizing their antigens, and subsequently, phagocytes bind to their Fc fragments on the target cells. Once bound to the Fc fragment of the phagocyte, the target cell is phagocytosed. The phagosomes fuse with lysosomes and are degraded. This process also leads to the production of soluble factors by effector cells, which help initiate and drive the immune response.

[0147] The control antibodies 47L1 and 47H1 of the present invention refer to antibodies composed of light and heavy chain variable region fragments that bind to the target antigen and human light chain constant region kappa chain and heavy chain constant region IgG1, respectively, as positive control antibodies.

[0148] In this invention, NC refers to IgG1 Fc as a negative control.

[0149] The MDA-MB-68 human breast cancer cells, HT-29 human colon cancer cells, and OVCAR-3 human ovarian cancer cells of the present invention naturally express human CD24 protein on their cell surface, which can be used to detect the affinity and phagocytic activity of anti-CD24 antibodies for human CD24 antigen.

[0150] The methods for producing and purifying antibody-antigen binding fragments according to the present invention are conventional techniques in the art (see Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15). Amino acid sequencing can also be performed using conventional methods.

[0151] The present invention is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention. Experimental methods in the embodiments of the present invention that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents and consumables that do not specify their source are commercially available, conventional reagents.

[0152] Example 1: Preparation and quality control of human, monkey, and mouse CD24 antigens and positive control antibodies

[0153] 1.1 Molecular construction of antigen and control antibody expression vectors

[0154] Uniprot was used to query the amino acid sequences of functional regions of human CD24 (P25063, 27-59aa), cynomolgus monkey CD24 (I7GKK1-1, 27-57aa), rhesus monkey CD24 (A0A5F8ARC0, 27-54aa), and mouse CD24 (P24807, 27-53aa). The light and heavy chain sequences of the CD24 positive control antibody were obtained by searching the patent (patent number: WO2023 / 061064A1) (SEQ ID NO:11, SEQ ID NO:10 in WO2023 / 061064A1).

[0155] Suzhou Genewise Biotechnology Co., Ltd. was commissioned to synthesize the gene sequences of human CD24 (P25063, 27-59aa), cynomolgus monkey CD24 (I7GKK1-1, 27-57aa), rhesus monkey CD24 (A0A5F8ARC0, 27-54aa), and mouse CD24 (P24807, 27-53aa) and recombinant them into pcDNA3.4 expression vectors containing signal peptides and human IgG1 Fc. These sequences were named plasmids A15, A19, A20, and A25, respectively, with sequence codes SEQ ID NO.86, SEQ ID NO.87, SEQ ID NO.88, and SEQ ID NO.89, respectively. Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to synthesize the light chain variable region (VL) (Sequence No.: SEQ ID NO: 11 in patent No.: WO2023 / 061064A1) + human Kappa light chain constant region (CL) sequence of the control antibody (Table 1), and the heavy chain variable region (VH) (Sequence No.: SEQ ID NO: 10 in patent No.: WO2023 / 061064A1) + human IgG1 heavy chain constant region (CH) sequence of the control antibody (Table 1). These sequences were then recombined into a pcDNA3.4 expression vector containing a signal peptide, named 47L1 and 47H1, with sequence codes SEQ ID NO. 91 and SEQ ID NO. 90, respectively. The negative control antibody human IgG1 Fc sequence is encoded as SEQ ID NO. 92. The above expression vectors were prepared using an endotoxin-free plasmid mini-extraction kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP118-03) and sterilized by filtration using a 0.22 μM filter membrane (NEST brand, catalog number: 331011).

[0156] Table 1. Amino acid sequences of antigen and control antibody Note: * indicates the stop codon and is not translated.

[0157] 1.2 Expression and purification of antigen and control antibody

[0158] Using ExpiFectamine TMThe expression vectors A15, A19, A20, A25, 47H1 / 47L1 (co-transfected), and NC-VH were transiently transfected into Expi-293F cells (Thermo, A29127) using the 293Transfection Kit (Thermo, A14524). Cells were then cultured for 5 days using Expi293™ Expression Medium (Thermo, A1435101) on a cell culture shaker (Suzhou Jiemei Electronics Co., Ltd., model: IS-6CM5) at 37°C, 8% CO2, 85% relative humidity, 100 rpm / min, and a rotation radius of 55 mm. Cell components were removed by centrifugation, yielding a culture supernatant containing the aforementioned antigens and control antibodies.

[0159] The culture supernatant was subjected to affinity chromatography using a protein purification system (Suzhou Insys Intelligent Technology Co., Ltd., model: Auto Pre25 DM402) and a Protein A affinity chromatography column (Suzhou Saifen Technology Co., Ltd., catalog number: 4F86574). The column was washed with 5 column volumes (CV) of deionized water, then with equilibration buffer (20 mM Pi, 500 mM NaCl, pH = 7.4 ± 0.1) for 5 CV. The culture supernatant was then loaded onto the affinity chromatography column, followed by washing with equilibration buffer for 6 CV, elution buffer for 10 CV, elution buffer (0.1 M Glycine, pH = 3.0 ± 0.1), deionized water for 5 CV, 0.2 M NaOH for 5 CV, pure water for 5 CV, and 20% ethanol for 5 CV. The retention time for each chromatographic step was 1 min.

[0160] The affinity chromatography eluent was replaced with buffer using a protein purification system (Suzhou Insys Intelligent Technology Co., Ltd., model: Auto Pre25 DM402) and a dextran gel chromatography column (Suzhou Nanomicro Technology Co., Ltd., catalog number: 60011-003401-2100). The dextran gel chromatography column was washed with equilibration buffer (PBS, pH=7.2) for 0.5 CV. 10 mL of affinity chromatography eluent was loaded onto the dextran gel chromatography column, and the column was eluted with equilibration buffer (PBS, pH=7.2) for 1.5 CV. 50 mAU-UVmax-50 mAU was collected. The retention time for all chromatographic steps was 7.5 min. Antigens and antibodies were named according to Table 2 below:

[0161] Table 2. Comparison of Antigen (Control Antibody) and Expression Vector Names

[0162] 1.3 Molecular weight and purity of antigen and control antibody

[0163] To evaluate whether the molecular weights of the antigen and control antibody met expectations, the antibody was premixed with 5× reducing loading buffer (Beyotime Biotechnology Co., Ltd., catalog number: P0286) and subjected to SDS-PAGE electrophoresis (120V, 45min) using a high-resolution gradient gel (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: P7311040). After the gel was stained with Coomassie Brilliant Blue R-250 staining solution and destained, it was imaged using a chemiluminescence imaging system (Hangzhou Shenhua Technology Co., Ltd., catalog number: SH-523).

[0164] The results showed that after reducing SDS-PAGE protein electrophoresis, the molecular weights of antigens AF01, AF02, AF03, and AF04 were all within the range of 40kDa to 55kDa, consistent with the theoretical molecular weights; and they exhibited a diffuse morphology, consistent with the electrophoretic characteristics of glycosylated proteins (Figure 1). The positive control antibody PC had a light chain molecular weight of approximately 25kDa and a heavy chain molecular weight of approximately 55kDa; the negative control antibody NC had a molecular weight of approximately 30kDa, all consistent with the theoretical molecular weights.

[0165] 1.4 Purity of antigen and control antibody

[0166] The purity of the chimeric antibody was analyzed by size exclusion chromatography using a Thermo Scientific Vanquish C column (TSK gel G3000 SWXL (7.8 mm ID × 30 cm, 5 μm)). The antigen and control antibody were diluted to 0.5 mg / mL with PBS, and 100 μL of each solution was pipetted into a lined tube, which was then placed in a sample vial and positioned on the autosampler rack. The following chromatographic parameters were set: flow rate 0.6 mL / min; isogradient elution for 35 min; column temperature 25 °C; detection wavelength 280 nm; autosampler temperature: 5 °C; injection volume: 10 μL. The mobile phase (0.1 M disodium hydrogen phosphate and 0.1 M sodium sulfate aqueous solution (pH 6.7)) was used. The system was equilibrated, the detector was preheated, and the sequence was injected after the baseline stabilized.

[0167] The results showed that the purity of antigens AF01, AF02, AF03, AF04 and control antibodies PC and NC were all greater than 90% (Table 3).

[0168] Table 3. HPLC-SEC purity analysis of antigen and control antibody

[0169] 1.5 Activity of positive control antibody

[0170] To investigate whether the prepared positive control antibody could bind to the human CD24 antigen, the concentration of the human CD24-His (KACTUS, catalog number: CD2-HM124) antigen was diluted to 0.5 μg / mL with PBS and added to 100 μL per well of a 96-well ELISA plate (Thermo Scientific, catalog number: 468667). The plate was sealed with sealing film (Sangon Biotech (Shanghai) Co., Ltd., catalog number: F601418) and incubated overnight at 4°C in the dark. The next day, the plate was washed three times with a plate washer, and blocking buffer (PBS containing 5% skim milk powder) was added to each well (200 μL). The plate was then sealed with sealing film and incubated at 37°C in a shaker in the dark for 1 hour. First, dilute the chimeric antibody to 33.33 nM with PBS, then perform a 4-fold serial dilution, resulting in a total of 7 concentration gradients. Add 100 μL to each well of the sealed ELISA plate. Seal the ELISA plate with a sealing film and incubate it at 37°C in the dark for 1 hour. Discard the chimeric antibody from the ELISA plate and wash it 3 times with a plate washer. Dilute goat anti-human IgG Fc-HRP (Thermo Scientific, catalog number: 31413) with PBS at a ratio of 1:2000 and add 100 μL to each well of the ELISA plate. Seal the ELISA plate with a sealing film and incubate it at 37°C in the dark for 1 hour. Discard the secondary antibody from the ELISA plate, wash the plate three times with a plate washer, and add 100 μL of TMB solution (Solepro Technology Co., Ltd., catalog number: PR1200) to each well. After 4 min of color development, add 100 μL of stop solution (1M H2SO4 solution) to each well, shake thoroughly to stop the color development reaction. Place the ELISA plate in a microplate reader and read the OD value. 450 Values ​​(see Table 4, Figure 2).

[0171] The results showed that the positive control antibody PC could bind to human CD24 antigen, EC 50 =168.5nM.

[0172] Table 4. OD of control antibody PC binding to human CD24-His antigen 450 absorbance

[0173] Example 2: Construction of a stable overexpression cell line

[0174] 2.1 Construction of stable monoclonal cell lines of human and cynomolgus monkey CD24 overexpressing HEK293

[0175] The amino acid sequences of human CD24 (P25063, 1-80aa) and cynomolgus monkey CD24 (I7GKK1-1, 1-137aa) proteins were determined using Uniprot. Genes were synthesized and recombined into the pIRES2-EGFP vector by Suzhou Genewiz Biotechnology Co., Ltd., and named plasmids A9 (human CD24) and A23 (cynomolgus monkey CD24), respectively. The plasmids were prepared using an endotoxin-free plasmid mini-prep kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP118-03) and sterilized by filtration through a 0.22 μM filter membrane (NEST brand, catalog number: 331011).

[0176] HEK 293 cells (Ningbo Mingzhou Biotechnology Co., Ltd., catalog number: MZ-8311) were cultured to the logarithmic growth phase, and the target gene plasmid was transfected into HEK 293 cells using PEI (Yisheng Biotechnology (Shanghai) Co., Ltd., 40816ES03). Forty-eight hours after transfection, G418 solution (Yisheng Biotechnology (Shanghai) Co., Ltd., 60220ES08) at a final concentration of 1800 μg / mL was added for stress selection culture. GFP expression was observed using a fluorescence microscope (OLYMPUS brand, model: CKX53). When green cells reached 70-90%, cells were collected and single-cell sorted using a flow cytometer (Beckman Co., Ltd., model: CytoFLEX SRT) 488 (FITC) fluorescence channel.

[0177] After sorting, the monoclonal cells were expanded in the following order: 96-well plates → 24-well plates → 6-well plates. The expression level of human CD24 in the cell lines was analyzed by flow cytometry using the positive control antibody PC (Thermo Scientific, model: Attune NxT), yielding a 293 monoclonal cell line overexpressing human CD24 (hCD24-293). The expression level of GFP in the cell lines was analyzed by flow cytometry, yielding a 293 monoclonal cell line overexpressing cynomolgus monkey CD24 (cynoCD24-293). The monoclonal cell lines were expanded to 10 cm culture dishes and cryopreserved in liquid nitrogen.

[0178] 2.2 Construction of a stable monoclonal cell line of human CD24-overexpressing MC38

[0179] The amino acid sequence of human CD24 (P25063, 1-80aa) protein was determined using Uniprot, and the gene was synthesized and recombined into the pCDH-CMV-MCS-EF1-GFP+Puro (HonorGene brand, catalog number: HG-VMS0751) vector by Suzhou Genewise Biotechnology Co., Ltd., and named plasmid A26. The plasmid was prepared using an endotoxin-free plasmid mini-prep kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP118-03) and sterilized by filtration through a 0.22 μM filter membrane (NEST brand, catalog number: 331011).

[0180] HEK 293 cells (Ningbo Mingzhou Biotechnology Co., Ltd., catalog number: MZ-8311) were cultured to the logarithmic growth phase. The target gene plasmid, lentiviral packaging plasmid psPAX2 (HonorGene brand, catalog number: HG-VMS0649), and pMD2.G (HonorGene brand, catalog number: HG-VMS0648) were co-transfected into HEK 293 cells using a PEI (Yisheng Biotechnology (Shanghai) Co., Ltd., 40816ES03). Cell supernatants were collected at 48 and 72 hours post-transfection.

[0181] MC38 cells (BNCC brand, catalog number: SCSP-5431) were cultured to the logarithmic growth phase. HEK293 cell supernatant was added to MC38 cell culture medium at a ratio of 1:10. After 48 hours of culture, puromycin (Solarbio brand, catalog number: P8230) was added for stress selection. GFP expression was observed using a fluorescence microscope (OLYMPUS brand, model: CKX53). When green cells reached 70-90%, cells were collected and single-cell sorted using a flow cytometer (Beckman Company, model: CytoFLEX SRT) with 488 (FITC) fluorescence channels.

[0182] After sorting, the monoclonal cells were expanded in the order of 96-well plates → 24-well plates → 6-well plates. The expression level of human CD24 in the cell lines was analyzed by flow cytometry using the positive control antibody PC (Thermo Scientific, model: Attune NxT), yielding a human CD24-overexpressing MC38 monoclonal cell line (hCD24-MC38). The monoclonal cell line was expanded to 10 cm culture dishes and cryopreserved in liquid nitrogen.

[0183] Example 3: Discovery of human CD24-targeted mouse hybridoma antibodies

[0184] 3.1 Mouse Immunization

[0185] Twelve SPF-grade CD1 mice, ear-tagged as 64#-75#, were selected by Aikon Biotechnology. Mice numbered 65# were excluded due to failing pre-experimental veterinary quarantine, leaving 11 mice. These mice were immunized with hCD24-MC38 cells. 5×10 6 Cells were mixed with complete Freund's adjuvant (Sigma brand, catalog number: F5881) and injected into the peritoneal cavity of mice. Subsequently, immunizations were performed every two weeks using incomplete Freund's adjuvant (Sigma brand, catalog number: F5506) in the same manner, for a total of five immunizations. One week after the third, fourth, and fifth immunizations, 100 μL of blood was collected from the orbital sinus, and the plasma was centrifuged for serum titer determination.

[0186] 3.2 Serum titer detection

[0187] The titer of human CD24 antibody in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA).

[0188] Dilute AF01 to 1 μg / mL with PBS and add 100 μL to each well of a 96-well microplate (Thermo Scientific, catalog number: 468667). Seal the plate with sealing film and incubate overnight at 4°C in the dark. The next day, wash the plate three times with a plate washer, and block it with PBS containing 3% BSA (Beijing Solarbio Science & Technology Co., Ltd., catalog number: PR1200), adding 200 μL to each well. Seal the plate with sealing film and incubate at 37°C in the dark for 1 hour. Dilute the serum 1:1000 with PBS, then perform five-fold serial dilutions, resulting in seven concentration gradients, and add 100 μL to each well of the blocked microplate. Seal the plate with sealing film and incubate at 37°C in the dark for 1 hour. Discard the chimeric antibody from the ELISA plate and wash the plate three times with a plate washer. Dilute HRP Anti-Mouse IgG H&L (Thermo Scientific, A16078) with PBS at a ratio of 1:2000 and add 100 μL to each well of the ELISA plate. Seal the plate with a sealing film and incubate it in a 37°C incubator in the dark for 1 hour. Discard the secondary antibody from the ELISA plate, wash the plate three times with a plate washer, and add 100 μL of TMB solution (Soluble Biotech, Beijing, catalog number: PR1200) to each well. After 4 minutes of color development, add 100 μL of stop solution (1M H2SO4 solution) to each well and mix thoroughly to stop the color development reaction. Place the ELISA plate in a microplate reader and read the OD value. 450 value.

[0189] The results showed that after the fifth immunization, the serum titer of some mice reached 1:125000 (OD). 450>0.2), serum titer meets fusion standard (Table 5).

[0190] Table 5. Serum ELISA titer of mice after the fifth immunization

[0191] The binding of antibodies to human CD24-positive cells in mouse serum was detected by flow cytometry (FACS).

[0192] HEK293, hCD24-293, and MDA-MB-468 human breast cancer cells (Chinese Academy of Sciences Cell Bank, catalog number: THu136) were cultured in T75 culture flasks until they reached the logarithmic growth phase. The supernatant was discarded, and the cells were rinsed with sterile ice-cold PBS. 1 mL of 0.25% trypsin containing EDTA was added, and the cells were digested at room temperature. After the cells detached from the bottom of the culture dish, 5 mL of complete culture medium was added, and the cells were thoroughly mixed by pipetting using a sterile pipette. The cells were then transferred to a 50 mL sterile centrifuge tube. 25 mL of FACS Buffer (PBS containing 0.2% BSA) was added to the centrifuge tube using a sterile pipette, and the cells were gently resuspended by pipetting. The cells were centrifuged at 300g for 4 min at 4°C, and the supernatant was discarded. This washing process was repeated twice. The cells were resuspended again by adding FACS Buffer, and the cell concentration was adjusted to 2 × 10⁶ cells / mL. 6 Add 100 μL of cell suspension to each well of a U-bottom 96-well plate. Dilute serum 1:1000 with FACS Buffer (PBS containing 0.2% BSA) and add 100 μL to each well of the U-bottom 96-well plate. Mix well and incubate at 4°C in the dark for 1 hour. Centrifuge at 300g for 4 min at 4°C and discard the supernatant. Repeat the washing process 3 times. Dilute Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Thermo Fisher Scientific, catalog number: A21235) 1:1000 to the working concentration with FACS Buffer (PBS containing 0.2% BSA) and add 100 μL to each well of the U-bottom 96-well plate. Incubate at 4°C in the dark for 1 hour. Centrifuge at 300g for 4 min at 4°C and discard the supernatant. Repeat the washing process 3 times. Using a multichannel pipette, add 200 μL of FACS Buffer (PBS containing 0.2% BSA) to each well of a U-bottom 96-well plate, resuspend the cells by pipetting, and detect the average fluorescence intensity of the APC channels using flow cytometry.

[0193] The results showed that after the fifth immunization, compared with the negative control (NC), the serum of mice diluted 1:1000 all showed detectable mean fluorescence intensity (MFI) signals binding to MDA-MB-468 and hCD24-293 cells, indicating the production of human CD24 antibodies in the serum. Compared with HEK293 cells, the mouse serum showed stronger binding to hCD24-293, indicating that it was not a non-specific binding signal (Table 6). The immune titers of the mice all met expectations, and hybridoma fusion and antibody screening could be performed.

[0194] Table 6. Mean fluorescence intensity (MFI) values ​​of mouse serum FACS titer after the fifth immunization

[0195] 3.3 Hybridoma Fusion

[0196] Mice #64 were selected for antigen shock immunization (without adjuvant). Three days after immunization, the mice were euthanized, and the spleens were obtained under aseptic conditions. A single-cell suspension of B cells was prepared using a sterile sieve. After lysing red blood cells, the suspension was mixed with non-secreting SP2 / 0 myeloma cells at a 1:1 volume ratio, and cell fusion was performed using a BTX cell electrofusion instrument. After electrofusion, all cells were immediately suspended in DMEM complete medium (containing 20% ​​FBS and HAT selection reagent) and seeded into 96-well cell culture plates, for a total of 25 plates. Approximately 10 days after fusion, the medium was changed to HT medium. After 2 days of culture, the hybridoma supernatant was used to detect the binding ability of the antibody to the human CD24 antigen.

[0197] 3.4 Screening of hybridoma mother clones

[0198] Following the method described in Example 3.2, hybridoma parent clones were screened using an enzyme-linked immunosorbent assay (ELISA). The hybridoma supernatant was not diluted in this assay. A total of 2400 hybridoma parent clones were tested (25 plates, 96 clones per plate), and 166 ELISA-positive parent clones were obtained.

[0199] Following the method described in Example 3.2, flow cytometry (FACS) was used to further screen hybridoma parent clones that tested positive for ELISA. In this assay, the hybridoma supernatant was not diluted. A total of 166 hybridoma parent clones were analyzed, and 11 FACS-positive parent clones were obtained.

[0200] 3.5 Hybridoma subcloning screening

[0201] Eleven hybridoma mother clones that tested positive for both ELISA and FACS were subcloned, with each mother clone plated on one subcloning plate (96 clones). Hybridoma cell lines in good growth condition were collected and prepared into single-cell suspensions. Cells were counted using a hemocytometer, and 100 cells were taken and diluted to 10 mL. 100 μL of the cell suspension was added to each well of a 96-well plate, bringing the final volume to 200 μL. The plates were incubated at 37°C and labeled. After 3 days of continuous culture, single clones were selected under a microscope. On day 7, the culture medium was replaced with fresh medium. On day 8, affinity was determined by ELISA and FACS according to the method described in Example 3.2. Subcloning was repeated twice more until single clones were formed.

[0202] The results showed that, after the above screening, a total of 6 hybridoma monoclonal antibodies were obtained, named MAB01, MAB02, MAB03, MAB04, MAB05, and MAB06, respectively.

[0203] 3.6 Hybridoma antibody sequencing

[0204] Sequencing was commissioned to Suzhou Aikon Technology Co., Ltd., with 1×10⁶ samples taken from each hybridoma monoclonal sample. 6 Total RNA was extracted from hybridoma cells using an RNA extraction kit. The total RNA was then reverse transcribed into cDNA using a reverse transcription kit. The heavy and light chain variable region gene sequences of the antibody were amplified by PCR and constructed into a T vector. Clones were selected for sequencing. The final amino acid sequences (Table 7) and nucleotide sequences (Table 8) of the light and heavy chain variable regions of the mouse hybridoma antibody were obtained. The CDR region sequences were analyzed using KABAT, IMGT, and Chothia methods, respectively (Table 9).

[0205] Table 7. Human CD24 mouse information

[0206] Table 8. Nucleotide sequence information of the variable regions of the light and heavy chains of human CD24 mouse hybridoma antibodies.

[0207] Table 9. Amino acid sequence information of light and heavy chain CDRs of human CD24-mouse hybridoma antibodies

[0208] Example 4: Preparation and Quality Control of Human-Mouse Chimeric Antibodies

[0209] 4.1 Molecular construction of chimeric antibody expression vector

[0210] Aikon Biotechnology was commissioned to construct human-mouse chimeric antibody expression vectors from the screened positive hybridoma antibodies. The mouse hybridoma antibody light chain variable region (VL) sequence was recombined into a pcDNA3.4 expression vector containing a signal peptide and a human Kappa light chain constant region (CL) sequence, and the mouse hybridoma antibody heavy chain variable region (VH) sequence was recombined into a pcDNA3.4 expression vector containing a signal peptide and a human IgG1 heavy chain constant region (CH). The expression vectors were prepared using an endotoxin-free plasmid large-scale extraction kit (Hangzhou Beiwo Medical Technology Co., Ltd., catalog number: BW-PD1520-02) and filtered sterilized using a 0.22 μM filter membrane (NEST brand, catalog number: 331011).

[0211] 4.2 Expression and purification of chimeric antibodies

[0212] The chimeric antibody was expressed and purified according to the method in Example 1.2.

[0213] The names of the chimeric antibodies and their corresponding expression vectors are shown below (Table 10):

[0214] Table 10. Comparison of chimeric antibodies and expression vectors

[0215] 4.3 Analysis of chimeric antibody molecular weight using SDS-PAGE protein electrophoresis

[0216] The molecular weights of the light and heavy chains of the chimeric antibody were evaluated according to the method described in Example 1.3.

[0217] The results showed that after SDS-PAGE protein electrophoresis, the molecular weights of the light chains of the six chimeric antibodies were all in the range of 25kDa to 30kDa, and the molecular weights of the heavy chains were all in the range of 45kDa to 55kDa (Figure 3), which were consistent with the theoretical molecular weights.

[0218] 4.4 Size Exclusion Chromatography (HPLC-SEC) Method for Analyzing the Purity of Chimeric Antibodies

[0219] The purity of the chimeric antibody was evaluated according to the method in Example 1.3.

[0220] The results showed that the purity of all six chimeric antibodies was high (greater than 90%). Except for CMAB02, which had a purity of 95.08%, the purity of the other chimeric antibodies was 100% (Table 11).

[0221] Table 11. HPLC-SEC purity analysis of chimeric antibodies

[0222] Example 5: Evaluation of the binding affinity of chimeric antibodies to human, monkey, and mouse CD24 antigens using enzyme-linked immunosorbent assay (ELISA).

[0223] 5.1 Binding affinity of chimeric antibodies to human CD24 antigen

[0224] To evaluate the binding affinity of the chimeric antibody to human CD24 antigen, the concentration of human CD24-His (Cusabio brand, catalog number: CSB-MP004902HU) antigen was diluted to 0.5 μg / mL with PBS, and the concentration of human CD24-Fc (AF01) antigen was diluted to 2 μg / mL; 100 μL was added to each well of a 96-well ELISA plate; the plate was sealed with sealing film and incubated overnight at 4°C in the dark. The next day, the ELISA plate was washed three times with a plate washer, and 200 μL of blocking buffer (PBS containing 5% skim milk powder) was added to each well; the plate was sealed with sealing film and incubated for 1 hour in a 37°C incubator in the dark. The chimeric antibody was first diluted to 66.7 nM with PBS, then serially diluted 4-fold to obtain a total of 7 concentration gradients, and added to each well of the sealed ELISA plate (100 μL). The ELISA plate was sealed with a sealing film and incubated at 37°C in a light-protected shaker for 1 hour. The chimeric antibody was then discarded, and the ELISA plate was washed 3 times with a plate washer. Dilute goat anti-human IgG Fc-HRP (Thermo Fisher Scientific, catalog number: 31413) at a ratio of 1:2000 with PBS and add 100 μL to each well of an ELISA plate coated with human CD24-His antigen. Dilute goat anti-human Fab-HRP (Thermo Fisher Scientific, catalog number: A56868) at a ratio of 1:2000 with PBS and add 100 μL to each well of an ELISA plate coated with human CD24-Fc(AF01) antigen. Seal the ELISA plate with a sealing film and incubate it in a 37°C incubator in the dark for 1 hour. Discard the secondary antibody from the ELISA plate, wash the plate three times with a plate washer, and add 100 μL of TMB (Solebio Technology Co., Ltd., catalog number: PR1200) solution to each well. After 4 minutes of color development, add 100 μL of stop solution (1M H₂SO₄ solution) to each well, and shake thoroughly to terminate the color development reaction. Place the ELISA plate in a microplate reader and read the OD value. 450 value.

[0225] The results showed that the chimeric antibody CMAB02 was almost undetectable in its binding to the human CD24-His antigen, EC 50 Unable to calculate; all other antibodies showed detectable binding activity. Compared to the positive control antibody PC, the chimeric antibodies CMAB01 and CMAB05 showed slightly weaker binding to the human CD24-His antigen, with CMAB01 having a lower EC50. 50 =5.8150nM, EC of CMAB05 50=2.9090 nM; chimeric antibodies CMAB03, CMAB04, and CMAB06 exhibit strong binding affinity to human CD24-His antigen (Table 12, Figure 4), EC 50 All were smaller than the EC50 of the positive control antibody PC. 50 =1.08nM (Table 12);

[0226] Chimeric antibodies CMAB01 and CMAB02 have weak affinity for human CD24-Fc antigen, EC 50 The concentrations were 31.12 nM and 73.84 nM, respectively. The chimeric antibodies CMAB03, CMAB04, CMAB05, and CMAB06 also showed stronger binding affinity to the human CD24-Fc antigen (Table 13, Figure 5). 50 All were significantly greater than the EC50 of the positive control antibody PC. 50 = 4.99nM (Table 13).

[0227] Table 12. OD of chimeric antibody binding to human CD24-His 450 absorbance

[0228] Table 13. OD of chimeric antibody binding to human CD24-Fc(AF01) 450 absorbance

[0229] 5.2 Binding affinity of chimeric antibodies to monkey CD24 antigen

[0230] The binding affinity of the chimeric antibody to cynomolgus monkey CD24-His (ACRO Biosystems brand, catalog number: CD4-C52H4), cynomolgus monkey CD24-Fc (AF02), and rhesus monkey CD24-Fc (AF03) antigens was evaluated according to the method in Example 5.1.

[0231] The results showed that, similar to the positive control antibody PC, the chimeric antibodies did not bind to CD24 in cynomolgus monkeys (Tables 14 and 15) or CD24 in rhesus monkeys (Table 16), EC 50 None of them can be calculated.

[0232] Table 14. OD of chimeric antibody binding to CD24-His in cynomolgus monkeys 450 absorbance

[0233] Table 15. OD of chimeric antibody binding to cynomolgus monkey CD24-Fc(AF02) 450 absorbance

[0234] Table 16. OD of chimeric antibody binding to rhesus monkey CD24-Fc(AF03) 450 absorbance

[0235] 5.3 Binding affinity of chimeric antibodies to mouse CD24 antigen

[0236] The binding affinity of the chimeric antibody to mouse CD24-His (ACRO Biosystems brand, catalog number: CD4-M52H4) and mouse CD24-Fc (AF04) antigens was evaluated according to the method in Example 5.1.

[0237] The results showed that, similar to the positive control antibody PC, the chimeric antibodies did not bind to the mouse CD24 antigen (Tables 17 and 18), EC 50 None of them can be calculated.

[0238] Table 17. OD of chimeric antibody binding to mouse CD24-His 450 absorbance

[0239] Table 18. OD of chimeric antibody binding to mouse CD24-Fc(AF04) 450 absorbance

[0240] Example 6: Evaluation of the binding affinity of chimeric antibodies to human, monkey, and mouse CD24 antigens using biomembrane interference (BLI) method.

[0241] 6.1 Binding affinity of chimeric antibodies to human CD24 antigen

[0242] The binding affinity of the chimeric antibody to human CD24 antigen (KACTUS brand, catalog number: CD2-HM124) was detected and analyzed using a biomembrane interference molecular interaction analyzer (GatorBio Bio, Inc., model: Gator Prime). The detection buffer was a 10mM PBS (pH 7.4) solution containing 0.02% Tween (Beijing Solarbio Technology Co., Ltd., catalog number: T8220) and 0.2% BSA IgG Free (Jackson ImmunoResearch Laboratories, Inc., catalog number: 001-000-061). The concentration of human CD24 (KACTUS brand, catalog number: CD2-HM124) antigen was diluted to 5 μg / mL with the detection buffer, and the concentrations of the chimeric antibody were diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 0 nM. Antibody diluent, antigen diluent, and detection buffer were added to the corresponding wells of the detection plate. The detection plate temperature was set to 30℃, the light signal acquisition rate to 5.0 Hz, and the probe rotation speed to 1000 rpm / min. In each detection cycle, the Ni-NTA probe (GatorBio Bio, Inc., catalog number: 160016) was equilibrated in the detection buffer for 1 min, loaded with human CD24-His antigen solution for 2 min, equilibrated in the detection buffer for 1 min, bound to different concentrations of chimeric antibody for 5 min, and dissociated in the detection buffer for 5 min. The light signal value of the 0 nM well was used as a blank reference value, and the light signal values ​​of other concentrations were subtracted from the reference value. The binding-dissociation kinetic curve was fitted using Global mode (fitting detection values ​​of different concentrations together), and the dissociation rate constant Koff, binding rate constant Kon, and binding affinity KD were calculated.

[0243] The results showed that chimeric antibodies CMAB01, CMAB03, CMAB04, CMAB05, and CMAB06 exhibited strong binding affinity to human CD24 antigen, with a KD of 10. -9 Grade (1 nM), the chimeric antibody CMAB02 has weak binding affinity to both human CD24 antigen and KD is 10. -8 Level (10nM) (Table 19).

[0244] Table 19. Binding affinity (KD) of chimeric antibodies to human CD24 antigen

[0245] 6.2 Binding affinity of chimeric antibodies to cynomolgus monkey CD24 antigen

[0246] The binding affinity of the chimeric antibody (detection concentrations of 100 nM and 0 nM) to the cynomolgus monkey CD24-His (ACRO, catalog number: CD4-C52H4) antigen was evaluated according to the method in Example 6.1.

[0247] The results showed that none of the chimeric antibodies bound to the cynomolgus CD24 antigen, and the dissociation rate constant Koff and binding rate constant Kon could not be detected, so the binding affinity KD could not be calculated (Table 20).

[0248] Table 20. Binding affinity (KD) of chimeric antibodies to cynomolgus monkey CD24 antigen

[0249] 6.3 Binding affinity of chimeric antibodies to mouse CD24 antigen

[0250] The binding affinity of the chimeric antibody (detection concentrations of 100 nM and 0 nM) to the mouse CD24-His (ACRO, catalog number: CD4-M52H4) antigen was evaluated according to the method in Example 6.1.

[0251] The results showed that none of the chimeric antibodies bound to mouse CD24, and the dissociation rate constant Koff and binding rate constant Kon could not be detected, so the binding affinity KD could not be calculated (Table 21).

[0252] Table 21. Binding affinity (KD) of chimeric antibodies to mouse CD24 antigen

[0253] Example 7: Evaluation of the binding ability of chimeric antibodies to human and cynomolgus monkey CD24-positive cells by flow cytometry (FACS).

[0254] 7.1 Binding ability of chimeric antibodies to human CD24-positive cells

[0255] To evaluate the binding ability of chimeric antibodies to human CD24-positive cells, human CD24-overexpressing MC38 mouse colon cancer cells (hCD24-MC38), HT-29 human colon cancer cells (Chinese Academy of Sciences Cell Bank, catalog number: THu103), MDA-MB-468 human breast cancer cells (Chinese Academy of Sciences Cell Bank, catalog number: THu136), OVCAR-3 human ovarian cancer cells (Shanghai Pronoss Biotechnology Co., Ltd., catalog number: CL-0178), and HeLa human cervical cancer cells (Chinese Academy of Sciences Cell Bank, catalog number: THu187) were cultured in T75 culture flasks to the logarithmic growth phase. After the supernatant was discarded, the cells were rinsed with sterile ice-cold PBS, and 1 mL of 0.25% trypsin containing EDTA was added. The cells were digested at room temperature. After the cells detached from the bottom of the culture dish on their own, 5 mL of complete culture medium was added, and the cells were transferred to 50 mL sterile centrifuge tubes by pipetting. Using a sterile pipette, add 25 mL of FACS Buffer (PBS containing 0.2% BSA) to a centrifuge tube, gently pipette to resuspend the cells, centrifuge at 300g for 4 min at 4°C, and discard the supernatant. Repeat the washing process twice. Resuspend the cells in FACS Buffer, count the cells, and adjust the cell concentration to 2 × 10⁻⁶ cells / mL. 6 / mL, add 100μL of cell suspension to each well of a U-bottom 96-well plate. Dilute the chimeric antibody to working concentrations (23.3nM for hCD24-MC38, MDA-MB-468, OVCAR-3, and HeLa cells; 66.7nM and 13.3nM for HT-29 cells) with FACS Buffer (PBS containing 0.2% BSA) and add 100μL to each well of the U-bottom 96-well plate. Mix well and incubate at 4°C in the dark for 1 hour. Centrifuge at 300g for 4 min at 4°C and discard the supernatant. Repeat the washing process 3 times. Add Goat anti-Human IgG Fc and Alexa Fluor to FACS Buffer (PBS containing 0.2% BSA). TM Dilute 647 (Thermo Scientific, A55749) 1:2000 to the working concentration and add 100 μL to each well of a U-bottom 96-well plate. Incubate at 4°C in the dark for 1 hour. Centrifuge at 300g for 4 min at 4°C and discard the supernatant. Repeat the washing process 3 times. Using a multichannel pipette, add 200 μL of FACS Buffer (PBS containing 0.2% BSA) to each well of the U-bottom 96-well plate, resuspend the cells by pipetting, and analyze using flow cytometry.

[0256] The results showed that, compared with the negative control antibody PC, all six chimeric antibodies were able to bind to hCD24-MC38, MDA-MB-468, OVCAR-3, HeLa, and HT-29 cells; compared with the positive control, CMAB02 had slightly weaker binding ability to hCD24-MC38, MDA-MB-468, OVCAR-3, HeLa, and HT-29 cells, while the other chimeric antibodies had similar binding ability to cells (Table 22, Figures 6, 7, 8, 9, 10).

[0257] Table 22. Concentration of chimeric antibody binding to human CD24-positive cells - mean fluorescence intensity

[0258] 7.2 Binding ability of chimeric antibodies to CD24-positive cells of cynomolgus monkeys

[0259] The binding ability of the chimeric antibody to cynomolgus CD24-overexpressing HEK293 cells and HEK293 cells was evaluated according to the method in Example 7.1.

[0260] The results showed that, similar to the positive control antibody PC, there was no significant difference in the mean fluorescence intensity (MFI) of the chimeric antibody binding to cynoCD24-293 cells and HEK293 cells, indicating that the chimeric antibody did not bind to cynoCD24-293 cells (Table 23, Figures 11 and 12).

[0261] Table 23. Concentration of chimeric antibody binding to cynoCD24-293 and HEK293 cells - mean fluorescence intensity

[0262] Example 8: Humanization of human CD24 antibody

[0263] Chimeric antibodies CMAB01 and CMAB05 were selected, and their light and heavy chain variable regions were humanized. The Antibody Modeling Cascade module of Discovery Studio software was used to model the antibody variable region sequences. The Fr region template cutoff value was 10, Kabat annotation of the CDR loop region was selected, and the optimization level was set to High. After obtaining the antibody model, a CHARMm Polar H force field was applied to the protein in the Change Forcefield module. Antibody humanization was performed using the Predict Humanizing Mutations module. The corresponding antibody sequence and model were selected, Germline germline gene annotation site information was used, the threshold was adjusted to 50, CDR and reversion mutation residues were excluded in residue replacement, mutation energy was calculated and the cufoff was set to 0.5, and germline and high-frequency residue replacement were enabled. After the humanization calculation was completed, three different modified sequences were given: the first replaced all mouse residues with the human residues most conducive to stable structure; the second directly replaced mouse residues with human germline residues; and the third replaced mouse residues with human high-frequency residues. The Calculate Developability Indices module was used to assess the developability and aggregation trends of humanized antibodies.

[0264] For CMAB05, the humanization of the heavy chain variable region involves positions 3, 5, 13, 18, 19, 23, 40, 42, 71, 79, 80, 82, 87, 89, 90, 94, 95, 107, 110, and 111 in the sequence, specifically involving mutations at one or more of the following sites: K3Q, Q5V, Q5L, R13K, R13Q, M18L, K19R, V23A, V23T, S40A, E42G, I71T, S79N, S80T, H82Y, N87S, R89T, A90S, G94A, I95V, P107R, T110M, and L111V; two humanized sequences were obtained through screening.

[0265] For CMAB05, the humanization of the light chain variable region involves positions 10, 12, 15, 42, 50, 66, 68, 88, 90, and 105 in the sequence, specifically involving one or a combination of mutations at one or more of the following sites: T10S, S12P, I15L, I15P, L42Q, K50Q, K66R, T68S, L88F, L88V, G105P, I90V, and G105Q. Three humanized sequences were obtained through screening.

[0266] For CMAB01, the humanization of the heavy chain variable region involves positions 1, 3, 5, 13, 16, 17, 20, 37, 48, 67, 68, 76, 79, 82, 83, 85, 86, 87, 88, 92, 109, 112, and 113 in the sequence, specifically involving E1Q, K3Q, V5L, A13K, Q16E, Q16G, S17T, I20L, V37I, and L48V. Three humanized sequences were obtained by screening for mutations at one or more of the following sites: L48I, L67F, L67V, S68T, S76N, F79Y, F79S, M82L, N83R, L85V, Q86R, Q86T, T87S, T87A, D88E, D88A, M92L, M92V, N92V, P109R, P109Q, S112L, and L113V.

[0267] For CMAB01, humanization of the light chain variable region involved positions 3, 9, 10, 11, 15, 17, 18, 21, 39, 40, 41, 42, 59, 69, 71, 78, 79, 82, 99, 103, and 105 in the sequence, specifically involving mutations at one or more of the following sites: V3Q, T9S, I10S, M11L, P15L, E17Q, E17D, K18R, M21I, S39P, S40G, T41K, S42P, S42A, G59S, S69E, S71T, E78Q, A79S, A79P, V82F, A99G, A99Q, L103V, and L105I. Three humanized sequences were obtained through screening. The codes and sequences of the humanized antibodies are shown in Table 24 below.

[0268] Table 24. Mutated amino acid sequences of the variable region of humanized antibodies.

[0269] Example 9: Preparation of Humanized Antibodies

[0270] 9.1 Molecular construction of humanized antibody expression vector

[0271] Genewiz Biotechnology was commissioned to synthesize the light and heavy chain variable region sequences of humanized antibodies. The antibody light chain variable region (HAB-VL) sequence was recombined into a pcDNA3.4 expression vector containing a signal peptide and the human Kappa light chain constant region (CL) sequence, and the antibody heavy chain variable region (HAB-VH) sequence was recombined into a pcDNA3.4 expression vector containing a signal peptide and the human IgG1 heavy chain constant region (CH1-CH2-CH3). The expression vectors were prepared using an endotoxin-free plasmid mini-extraction kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP118-03) and sterilized by filtration through a 0.22 μM filter membrane (NEST brand, catalog number: 331011).

[0272] 9.2 Expression and purification of humanized antibodies

[0273] Using ExpiFectamine TM The expression vector was transiently transfected into Expi-293F cells (Thermo, A29127) using the 293 Transfection Kit (Thermo, catalog number: A14524) according to the combination shown in Table 30 below, and Expi293 was used. TM Expression Medium (Thermo Scientific, catalog number: A1435101) was cultured continuously for 5 days using a cell culture shaker (IKA, MS3 digital) at 8% CO2, 37°C, 85% relative humidity, and a rotation speed of 1000 rpm / min. Cell components were removed by centrifugation to obtain a culture supernatant containing chimeric antibodies.

[0274] Add an appropriate amount of Protein A purification magnetic beads (GenScript Biotech Co., Ltd., catalog number: L00695) to the supernatant culture medium, incubate overnight at 4°C with rotational mixing, and then adsorb the magnetic beads using a magnetic separator (GenScript Biotech Co., Ltd., catalog number: L00722). Discard the supernatant, wash four times with an equal volume of PBST, and then elute the antibody with 0.1M glycine-hydrochloric acid buffer (pH 3.0±0.1) and neutralize with 1 / 10 volume of 1M Tris-HCl (pH 8.5±0.1). Ultrafilter the antibody using a 10kDa ultrafiltration tube (Hangzhou Kebote Filter Material Co., Ltd., catalog number: ULRC0100150P) at 4°C, replacing the buffer with PBS. After sterile filtration through a 0.22μM filter membrane, aliquot and store at -80°C. Measure the antibody concentration using a micro spectrophotometer.

[0275] The results showed that among the modified humanized antibodies of MAB05, the expression levels of humanized antibodies containing the expression vector (HAB-L1) (HAB01, HAB04) were lower than those of other antibodies (Table 25); among the humanized antibodies of MAB01, the expression levels of humanized antibodies containing the expression vector (HAB-L4) (HAB07, HAB10, HAB13) were lower than those of other antibodies (Table 25).

[0276] Table 25. Comparison Table of Humanized Antibodies and Their Expression Vectors

[0277] Example 10: Analysis of the molecular weight and purity of humanized antibodies

[0278] 10.1 Analysis of the molecular weight and purity of humanized antibodies using reducing and non-reducing SDS-PAGE protein electrophoresis.

[0279] To evaluate the molecular weight and purity of the humanized antibody, the antibody was premixed with 5× reducing loading buffer (Beyotime Biotechnology Co., Ltd., catalog number: P0286) and 5× non-reducing loading buffer (Sangon Biotech (Shanghai) Co., Ltd., catalog number: 1927DA0001), respectively. SDS-PAGE electrophoresis (120V, 45min) was performed using a high-resolution gradient gel (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: P7311040). After the gel was stained with Coomassie Brilliant Blue R-250 and destaining, gel imaging was performed using a chemiluminescence imaging system (Hangzhou Shenhua Technology Co., Ltd., catalog number: SH-523).

[0280] The results showed that after reducing SDS-PAGE protein electrophoresis, the molecular weights of the light chains of the 15 humanized antibodies were all in the range of 25kDa to 30kDa, and the molecular weights of the heavy chains were all in the range of 45kDa to 55kDa (Figure 13), which were consistent with the theoretical molecular weights. After non-reducing SDS-PAGE protein electrophoresis, the bands of the 15 humanized antibodies were uniform and the purity was high (Figure 14).

[0281] 10.2 Real-time quantitative PCR (Q-PCR) method for analyzing the melting point temperature (Tm value) of humanized antibodies

[0282] Using a real-time quantitative PCR system (Thermo Scientific, model: QuantStudio 5) and a protein thermal drift dye kit (Thermo Scientific, catalog number: 4461146), the melting temperature of the antibody was analyzed by continuously detecting the intensity change of the fluorescent dye binding to the antibody at temperatures ranging from 25℃ to 99℃. Protein Thermal Shift Dye (1000×) was diluted 125-fold with pure water to obtain Protein Thermal Shift Dye (8×), and the antibody solution was diluted with PBS to a protein solution of 160 μg / mL. In each well of an 8-tube qPCR tube (AXYGEN, Inc., catalog number: PCR-0208-C), 12.5 μL of the diluted antibody, 5 μL of Protein Thermal Shift Buffer, and 2.5 μL of Protein Thermal Shift Dye (8×) were added, making the total loading volume per well 20 μL and the total antibody volume 2 μg. The 8-tube tubes were then placed in the sample tray of the qPCR instrument. Set the temperature program according to the following parameters: Step 1: Heating rate is 1.6℃ / s, upper limit temperature is 25.0℃, and upper limit temperature holding time is 2min;

[0283] Step 2: The heating rate was 0.05℃ / s, the upper limit temperature was 99.0℃, and the upper limit temperature was maintained for 2 minutes. The melting curve was fitted using real-time quantitative PCR system software, and the protein melting point temperature (Tm) was calculated.

[0284] The results showed that the melting point (Tm) of all 15 humanized antibodies met the drug-likeness requirements (Tm > 60℃). Except for three humanized antibodies containing the light chain HAB-L4, HAB07 (Tm undetectable), HAB10 (Tm = 69.51℃), and HAB13 (Tm = 69.61℃), whose melting points were slightly lower (< 70℃), the melting points of the remaining humanized antibodies were all higher (≥ 70℃).

[0285] Table 26. Melting point temperature (Tm value) of humanized antibodies

[0286] Example 11 Evaluation of the binding ability of humanized antibodies to human CD24

[0287] 11.1 Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the binding affinity of humanized antibodies to human CD24 antigen.

[0288] The binding affinity of the humanized antibody to the human CD24 antigen (CUSABIO brand, catalog number: CSB-MP004902HU) was detected and analyzed according to the method in Example 5.1.

[0289] The results showed that, except for the humanized antibody HAB07 having a slightly weaker binding ability to human CD24-His antigen, EC 50 >10 nM (Table 27, Figure 15), the binding ability of the remaining humanized antibodies to human CD24-His antigen was the same as that of the positive control PC (EC). 50 =1.531nM) is on the same order of magnitude as 1nM < EC 50 <10 nM (Table 27, Figure 15).

[0290] Table 27. Concentration, absorbance, and EC50 of humanized antibodies and human CD24-His antigen 50 value

[0291] 11.2 Evaluation of binding affinity (KD) between humanized antibodies and human CD24 antigen using biomembrane interference (BLI) method

[0292] The binding affinity of the humanized antibody to the human CD24 antigen (KACTUS, catalog number: CD2-HM124) was detected and analyzed according to the method in Example 6.1.

[0293] The results showed that, except for the humanized antibodies HAB-H6 / HAB-L4, the binding affinity to human CD24 antigen was slightly weaker (KD = 10). -8 Apart from the grade (10nM), the other humanized antibodies all exhibited strong binding affinity to the human CD24 antigen, with a KD of 10. -9 Level (1nM) (Table 28).

[0294] Table 28. Binding affinity (KD) of humanized antibodies to human CD24 antigen

[0295] 11.3 Flow cytometry (FACS) was used to evaluate the binding ability of humanized antibodies to human CD24-positive cells.

[0296] The binding affinity of the humanized antibody (starting concentration 200 nM, 4-fold dilution, 8 concentration gradients) to HT-29 human colon cancer cells (Chinese Academy of Sciences Cell Bank, catalog number: THu103), MDA-MB-468 human breast cancer cells (Chinese Academy of Sciences Cell Bank, catalog number: THu136), and OVCAR-3 human ovarian cancer cells (Wuhan Pronoss Technology Co., Ltd., catalog number: CL-0178) was evaluated according to the method in Example 7.1.

[0297] The results showed that, similar to the positive control antibody, all 15 humanized antibodies were able to bind to MDA-MB-468, HT-29, and OVCAR-3 cells. (15 humanized antibodies (2nM < EC50)) 50 <5nM) and positive control antibody (EC) 50 =6.9 nM) showed similar binding affinities to MDA-MB-468 cells (Table 29, Figure 16). 15 humanized antibodies (1 nM < EC50) 50 <3nM) and positive control antibody (EC) 50 =1.69 nM) showed similar binding affinity to OVCAR-3 cells (Table 30, Figure 17). Compared with the positive control antibody (EC... 50 =1.84nM) compared to humanized antibodies HAB07-HAB15 (1nM < EC50) 50 <3nM) antibodies showed similar binding affinity to HT-29 cells, while humanized antibodies HAB01-HAB06 (EC) showed similar affinity. 50 The binding affinity of (>3nM) is slightly lower (Table 31, Figure 18).

[0298] Table 29. Mean fluorescence intensity (MFI) and EC50 of humanized antibodies binding to MDA-MB-468 cells 50 value

[0299] Table 30. Mean fluorescence intensity (MFI) and EC50 of humanized antibodies binding to OVCAR-3 cells 50 value

[0300] Table 31. Mean fluorescence intensity (MFI) and EC50 of humanized antibodies binding to HT-29 cells 50 value

[0301] Example 12: Analysis of the effect of humanized antibodies on the release of cytokines from human PBMCs using enzyme-linked immunosorbent assay (ELISA).

[0302] To investigate the effect of humanized antibodies on cytokine release from human peripheral blood mononuclear cells (PBMCs), PBMCs (Rubai Biotechnology) were resuspended in RPMI 1640 medium (Gibco, catalog number: C11995500BT) after centrifugation at 4°C, 300g, for 5 min. Cell counts were performed using a Thermo Countess III cell counter, and the concentration of PBMCs was adjusted to 2 × 10⁻⁶ cells / year. 6 Cells were cultured at a density of 100 μL / mL and seeded into 96-well cell culture plates (Corning brand, catalog number: 3599), 100 μL per well. Humanized antibody and positive control PC were diluted to working concentration (final concentration 100 nM, 10-fold dilution, 6 concentration gradients) and added to the 96-well plates, mixing thoroughly. The plates were incubated at 37°C in the dark for 24 hours. After centrifugation at 300 × g for 5 min at 4°C, the cell supernatant was transferred to a new 96-well plate. The supernatant was diluted 10-fold with PBS, and the secretion of human IFN-γ in the supernatant was detected using the Human IFN-gamma ELISA kit (Thermo Fisher Scientific, catalog number: 88-7316-88).

[0303] The results showed that the humanized antibodies had a weak ability to induce PBMCs to secrete human IFN-γ cytokines (Human IFN-γ < 1 ng / mL) (Table 32). Compared with the humanized antibodies HAB07–HAB15 of CMAB01, the humanized antibodies HAB01–HAB06 of CMAB05 had a slightly stronger ability to secrete human IFN-γ cytokines (Table 32).

[0304] Table 32. Humanized antibodies induce PBMCs to secrete human IFN-γ cytokines.

[0305] Example 13: Evaluation of antibody-mediated cytotoxicity (ADCC) of humanized antibodies using the reporter gene method.

[0306] To evaluate the humanized antibody-mediated cytotoxicity (ADCC) effect, MDA-MB-468 cells were used as target cells and CD16-Jurkat-NFAT reporter cells (Invivogen, catalog number: jktl-nfat-cd32) were used as effector cells. MDA-MB-468 cells were cultured to the logarithmic growth phase using DMEM medium (Gibco, C22400500BT) containing 10% fetal bovine serum (Gibco, catalog number: 10091148) and 1% penicillin & streptomycin (Gibco, catalog number: 15140122). RPMI was then used to treat the cells with 10% fetal bovine serum (Gibco, catalog number: 10091148), 1% penicillin & streptomycin (Gibco, catalog number: 15140122), 10 μg / mL Blasticidin (Invivogen, catalog number: ant-bl-05), and 100 μg / mL Zeocin (Invivogen, catalog number: ant-zn-05). CD16a-Jurkat-NFAT reporter cells (Invivogen, JKTL-Nfat-CD16) were cultured in T75 culture flasks to the logarithmic growth phase using 1640 medium (Gibco, catalog number: C11995500BT). The cells were then transferred to 50 mL sterile centrifuge tubes. Cell counts were performed using a Thermo Countess III cell counter. The MDA-MB-468 cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 The concentration of the CD16-Jurkat-NFAT reporter gene in cells was adjusted to 2.5 × 10⁶ cells / mL. 6 Adjust cell concentration to 2 × 10⁶ cells / mL 6 Cells / mL, humanized antibody and positive control PC diluted to working concentration (final concentration 120 nM, 3-fold dilution, 9 concentration gradients). Add 100 μL of MDA-MB-468 cell suspension, 80 μL of CD16a-Jurkat-NFAT reporter gene cell suspension, and 20 μL of antibody dilution buffer to each well of a 96-well cell culture plate (Corning brand, catalog number: 3599), mix well, and incubate at 37°C in the dark for 24 hours. Centrifuge at 300×g for 5 min at 4°C. Using a multichannel pipette, transfer 40 μL of the cell supernatant after incubation to each well of a 96-well detection plate (Biosharp brand, catalog number: BS-MP-96W). Add substrate QUANTI-Luc to each well. TM4. Add 50 μL of Reagent (Invivogen, catalog number: rep-qlc4lg1) and incubate at room temperature for 5 minutes. Place the test plate in a microplate reader (TECAN, catalog number: SPARK) and read the Luminescence value at 500 ms intervals.

[0307] The results showed that the humanized antibodies all produced antibody-dependent cytotoxicity (ADCC) in MDA-MB-68 cells. Compared with the positive control PC (ECG) 50 Compared to HAB07 (2.53 nM), the ADCC activity of humanized antibodies HAB07 and HAB13 was slightly weaker (EC). 50 >10nM), the ADCC activity of the other antibodies was also strong (EC). 50 <10 nM)(Table 33, Figure 19).

[0308] Table 33. Antibody-dependent cytotoxicity (ADCC) of humanized antibodies on MDA-MB-468 cells

[0309] Example 14: Evaluation of antibody-mediated phagocytosis (ADCP) of humanized antibodies using the reporter gene method.

[0310] To evaluate the ADCP effect mediated by humanized antibodies, MDA-MB-468 cells were used as target cells and CD32-Jurkat-NFAT reporter cells (Invivogen, catalog number: jktl-nfat-cd32) were used as effector cells. The ADCP effect of humanized antibodies on MDA-MB-468 cells was evaluated using the same method as in Example 14.

[0311] The results showed that the humanized antibodies all induced antibody-dependent phagocytosis (ADCP) in MDA-MB-68 cells. Compared with the positive control PC (EC) 50 Compared to HAB07 (58.46 nM), the ADCP activity of humanized antibodies HAB07, HAB10, and HAB13 was slightly weaker (EC50). 50 >100nM), the ADCP activity of the remaining humanized antibodies was also strong (EC). 50 <100 nM (Table 34, Figure 20).

[0312] Table 34. Antibody-dependent phagocytosis (ADCP) effect of humanized antibodies on MDA-MB-468 cells

[0313] Example 15: Evaluation of the antitumor efficacy of humanized antibodies against a subcutaneous tumor-bearing model of hCD24-MC38 mouse colon cancer cells.

[0314] The in vivo antitumor efficacy and toxicity of humanized antibodies were studied using a subcutaneous tumor-bearing model of human CD24-overexpressing MC38 mouse colon cancer cells (hCD24-MC38). hCD24-MC38 cells were resuscitated and cultured in DMEM complete medium (containing 10% FBS, 1% penicillin & streptomycin, 2 μg / ml Puromycin) at 37°C in a cell culture incubator containing 5% CO2. After 7 days of acclimatization in 6-8 week old female C57BL / 6J mice, hCD24-MC38 cells cultured to the logarithmic growth phase were harvested, filtered through a 70 μM filter, and counted using trypan blue staining to ensure cell viability was not less than 95% before inoculation. The cell density was adjusted to 5 × 10⁶ cells / mL with PBS. 6 Cells were injected subcutaneously into the right axilla of mice at a rate of 100 μL / mL, with each mouse receiving 100 μL of cell suspension. A total of 72 mice were inoculated. Tumors were observed every 2 days after inoculation, and tumor diameter and volume were measured. The average tumor volume in mice undergoing modeling reached 100–150 mm. 3 Forty-eight mice were selected and randomly divided into six groups of eight each, based on tumor volume, to ensure that the mean and standard deviation of tumor volume (while also considering body weight) were as close as possible to each group. Antibody purity analysis was performed before administration, showing that the purity of antibodies HAB02, HAB06, HAB11, HAB15, and the positive control PC was all above 95%. The day of grouping was designated D0, and administration began on D1. Tumor volume and body weight were measured every three days during the administration period, and the mice's behavior and activity were regularly observed, with adverse reactions recorded promptly. After the experiment, a time-mean tumor volume curve was plotted, and the tumor growth inhibition rate (TGI%) was calculated; a time-mouse body weight curve was also plotted. The specific experimental protocol is as follows (Table 35):

[0315] Table 35. Animal efficacy design of humanized antibodies

[0316] Formula for calculating tumor volume:

[0317] Where a is the long axis of the tumor, b is the short axis of the tumor, and V is the tumor volume.

[0318] Formula for calculating relative tumor volume (RTV):

[0319] Where Vn is the tumor volume on day n after grouping, and V0 is the tumor volume on the day of grouping.

[0320] Formula for calculating tumor volume inhibition rate:

[0321] Tumor volume inhibition rate RTV1 represents the average relative tumor volume of the model control group, and RTV2 represents the average relative tumor volume of the drug-treated group.

[0322] All data are expressed as mean ± SD. Tumor volume and mouse body weight data passed the Kolmogorov-Smirnov test for normality and showed homogeneity of variance (P > 0.05). Statistical analysis was performed using two-way ANOVA and Dunnett's multiple comparisons. The statistical significance level was set at P ≤ 0.05.

[0323] The results showed that at the end of the experiment (D16), the average tumor volume of mice in the HAB02, HAB06, HAB11, HAB15 treatment groups and the positive control PC group was significantly smaller than that in the blank control group (statistical differences were observed at D16, D13, D10, D7, and D10, respectively (P≤0.05)) (Table 36), and the tumor inhibition rates (TGI%) were 49.76%, 45.72%, 64.79%, 82.08%, and 73.40%, respectively (Table 37, Figure 21). Compared with the positive control PC, the tumor growth inhibition rate (TGI%) in the HAB15 treatment group was lower, indicating that HAB15 has a stronger anti-tumor efficacy.

[0324] From the first administration to the end of the experiment (D16), the average body weight of mice in each administration group did not decrease significantly (Table 38, Figure 22).

[0325] Table 36. Time-mean tumor volume in mice for each treatment group (mm) 3 )

[0326] Table 37. Time-mean tumor growth inhibition rate (TGI%) in mice for each treatment group

[0327] Table 38. Time-mean body weight of mice in each drug administration group (g)

Claims

1. A CD24 antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: 1) The heavy chain variable region (VH) comprises HCDR1, HCDR2, and HCDR3; HCDR1 comprises any sequence of SEQ ID NO.1–5, SEQ ID NO.24–28, or SEQ ID NO.42–46, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequence; HCDR2 comprises any sequence of SEQ ID NO.6–9, SEQ ID NO.29–32, or SEQ ID NO.47–50, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequence; and HCDR3 comprises any sequence of SEQ ID NO.10–12 or SEQ ID NO.33–35, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequence; and 2) The light chain variable region (VL) comprises LCDR1, LCDR2, and LCDR3; LCDR1 comprises any sequence of SEQ ID NO.13-15, SEQ ID NO.36-38, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequence; LCDR2 comprises any sequence of SEQ ID NO.16-20, SEQ ID NO.39-41, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequence; and LCDR3 comprises any sequence of SEQ ID NO.21-23, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequence; each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is encoded according to the KABAT, IMGT, or Chothia common analysis method.

2. The CD24 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region (VL) and a light chain variable region (VH), wherein: (1) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 has any of the HCDR1 sequences shown in Table 1 below or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown below; HCDR2 has any of the HCDR2 sequences shown below or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown below; and HCDR3 has any of the HCDR3 sequences shown below or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown below: Table 1 and, (2) The light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 has any of the LCDR1 sequences shown in Table 2 below, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown below; LCDR2 has any of the LCDR2 sequences shown below, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown below; and LCDR3 has any of the LCDR3 sequences shown below, or a sequence having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the sequences shown below: Table 2 3. A CD24 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), coded according to the KABAT analytical method, wherein, The heavy chain variable region (VH) contains HCDR1, HCDR2, and HCDR3 selected from any of the following sequences (1)-(6) or sequences having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequences: (1) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.1, 6 and 10 respectively; (2) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.2, 6 and 11 respectively; (3) The HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO.3, 7 and 12; (4) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.4, 8 and 12 respectively; (5) The HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO.5, 9, and 12, respectively; or (6) The HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.5, 8 and 12 respectively; Furthermore, according to the KABAT analysis method, the LCDR1, LCDR2, and LCDR3 contained in the light chain variable region (VL) are selected from any of the following sequences (7)-(11) or sequences having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the aforementioned sequences: (7) The LCDR1, LCDR2 and LCDR3 are respectively as shown in the sequences SEQ ID NO.13, 16 and 21; (8) The LCDR1, LCDR2 and LCDR3 are respectively as shown in the sequences SEQ ID NO.14, 17 and 22; (9) The LCDR1, LCDR2 and LCDR3 are respectively as shown in the sequences SEQ ID NO.15, 18 and 23; (10) The LCDR1, LCDR2, and LCDR3 are respectively in the sequences shown in SEQ ID NO.15, 19, and 23; or (11) The LCDR1, LCDR2 and LCDR3 are respectively as shown in the sequences SEQ ID NO.15, 20 and 23.

4. The CD24 antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or fragments thereof, and / or humanized antibodies or fragments thereof.

5. A CD24 antibody or its antigen-binding fragment according to claim 4, wherein the heavy chain variable region of the murine antibody or its antigen-binding fragment comprises a murine heavy chain FR region or its mutated sequence; and the light chain variable region of the murine antibody or its antigen-binding fragment comprises a murine light chain FR region or its mutated sequence.

6. A CD24 antibody or its antigen-binding fragment according to claim 5, wherein the CD24 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), characterized in that, 1) The heavy chain variable region (VH) is selected from any of the sequences in SEQ ID NO.51 to SEQ ID NO.56 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it; 2) The light chain variable region (VL) is selected from any of the sequences in SEQ ID NO.57 to SEQ ID NO.62 or the light chain variable region having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with the amino acid sequence.

7. A CD24 antibody or its antigen-binding fragment according to claim 5 or 6, characterized in that, The antibody comprises: a heavy chain variable region containing any of the nucleotide sequences SEQ ID NO: 63-68 or a nucleotide sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it; and a light chain variable region containing any of the nucleotide sequences SEQ ID NO: 69-74 or a light chain variable region having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it.

8. A CD24 antibody or its antigen-binding fragment according to claim 4, wherein the FR region of the heavy chain variable region of the humanized antibody or its antigen-binding fragment is subjected to a combination of 0 to multiple amino acid combinations of site-directed mutations based on a mouse sequence, preferably comprising any one of the mouse sequences SEQ ID NO. 51 and / or SEQ ID NO. 55 or their mutant sequences; and / or the FR region of the light chain variable region of the humanized antibody or its antigen-binding fragment is subjected to a combination of 0 to multiple amino acid combinations of site-directed mutations based on a mouse sequence, preferably comprising any one of the sequences SEQ ID NO. 57 and / or SEQ ID NO. 61 or their mutant sequences.

9. A CD24 antibody or its antigen-binding fragment according to claim 8, wherein: The FR region of the heavy chain variable region of the humanized antibody or its antigen-binding fragment has been site-directedly mutated at any of the following sites according to the sequence of SEQ ID NO. 51: E1Q, K3Q, V5L, A13K, Q16E, Q16G, S17T, I20L, V37I, L48V, L48I, L67F, L67V, S68T, S76N, F79Y, F79S, M82L, N83R, L85V, Q86R, Q86T, T87S, T87A, D88E, D88A, M92L, M92V, N92V, P109R, P109Q, S112L, L113V, or a combination of multiple such mutations; Site-directed mutations at any of the following sites in the sequence SEQ ID NO. 55: K3Q, Q5V, Q5L, R13K, R13Q, M18L, K19R, V23A, V23T, S40A, E42G, I71T, S79N, S80T, H82Y, N87S, R89T, A90S, G94A, I95V, P107R, T110M, L111V, or a combination of mutations at multiple sites; And / or site-directed mutations at any of the following sites in the FR region of the variable region of the light chain of the humanized antibody or its antigen-binding fragment, according to the sequence of SEQ ID NO. 57: V3Q, T9S, I10S, M11L, P15L, E17Q, E17D, K18R, M21I, S39P, S40G, T41K, S42P, S42A, G59S, S69E, S71T, E78Q, A79S, A79P, V82F, A99G, A99Q, L103V, L105I, or a combination of multiple such site mutations; and / or site-directed mutations at any of the following sites according to the sequence of SEQ ID NO. 61: T10S, S12P, I15L, I15P, L42Q, K50Q, K66R, T68S, L88F, L88V, G105P, I90V, G105Q, or a combination of multiple such mutations.

10. A CD24 antibody or its antigen-binding fragment according to claim 8 or 9, wherein the humanized antibody or its antigen-binding fragment is characterized in that, The heavy chain variable region is selected from any of the sequences in SEQ ID NO. 75 to SEQ ID NO. 79 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it; the light chain variable region is selected from any of the sequences in SEQ ID NO. 80 to SEQ ID NO. 85 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity with it.

11. The CD24 antibody or its antigen-binding fragment according to claim 10, characterized in that, in, The humanized CD24 antibody or its antigen-binding fragment comprises: a heavy chain amino acid sequence as shown in SEQ ID NO:75 and a light chain amino acid sequence as shown in SEQ ID NO:80; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:75 and a light chain amino acid sequence as shown in SEQ ID NO:81; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:75 and a light chain amino acid sequence as shown in SEQ ID NO:82; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:76 and a light chain amino acid sequence as shown in SEQ ID NO:80; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:76 and a light chain amino acid sequence as shown in SEQ ID NO:81; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:76 and a light chain amino acid sequence as shown in SEQ ID NO:82; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:77 and a light chain amino acid sequence as shown in SEQ ID NO:83; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:77 and a light chain amino acid sequence as shown in SEQ ID NO:84; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:77 and a light chain amino acid sequence as shown in SEQ ID NO:85; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:78 and a light chain amino acid sequence as shown in SEQ ID NO:83; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:78 and a light chain amino acid sequence as shown in SEQ ID NO:84; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:78 and a light chain amino acid sequence as shown in SEQ ID NO:85; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:79 and a light chain amino acid sequence as shown in SEQ ID NO:83; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:79 and a light chain amino acid sequence as shown in SEQ ID NO:84; Alternatively, it may contain a heavy chain amino acid sequence as shown in SEQ ID NO:79 and a light chain amino acid sequence as shown in SEQ ID NO:

85.

12. The CD24 antibody or antigen-binding fragment thereof according to any one of claims 4, 8 to 11, wherein the chimeric antibody or antigen fragment thereof, and / or the humanized antibody or antigen fragment thereof, comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or variants thereof, and / or a light chain constant region of human Kappa(k) or lambda(λ) chain or a mutant sequence thereof; preferably, the chimeric antibody or antigen fragment thereof, and / or the humanized antibody or antigen fragment thereof, comprises a heavy chain constant region of human IgG1 or a mutant sequence thereof and a light chain constant region of human Kappa chain or a mutant sequence thereof.

13. A CD24 antibody or its antigen-binding fragment according to any one of claims 1 to 12, characterized in that, The antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies.

14. A multispecific antigen-binding molecule, characterized in that, It comprises the CD24 antibody or its antigen-binding fragment as described in any one of claims 1 to 13.

15. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the CD24 antibody or antigen-binding fragment of any one of claims 1-13, or any combination thereof, or the multispecific antigen-binding molecule of claim 14.

16. An expression vector comprising the isolated nucleic acid molecule of claim 15.

17. A host cell, which is transformed with the expression vector of claim 16, wherein the host cell is selected from prokaryotic cells and eukaryotic cells; wherein the eukaryotic cell is a mammalian cell; preferably, the host cell is selected from HEK293.

18. A method for preparing the CD24 antibody or its antigen-binding fragment according to any one of claims 1 to 13, characterized in that, The host cells of claim 17 are cultured under appropriate conditions, and antibodies or antigen-binding fragments or multispecific antigen-binding molecules are isolated and purified from the host cells.

19. A pharmaceutical composition, characterized in that, The composition comprises an antibody or antigen-binding fragment according to any one of claims 1-13, a multispecific antigen-binding molecule according to claim 14, an isolated nucleic acid molecule according to claim 15, an expression vector according to claim 16, a host cell according to claim 17, or a product prepared by the method of claim 18; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient; preferably, the pharmaceutical composition further comprises an additional antitumor agent.

20. Use of any antibody or antigen-binding fragment of any one of claims 1-13, the multispecific antigen-binding molecule of claim 14, the isolated nucleic acid molecule of claim 15, the expression vector of claim 16, the host cell of claim 17, the product prepared by the method of claim 18, or the pharmaceutical composition of claim 19 in the preparation of a medicament for the prevention and / or treatment of CD24-mediated diseases; preferably, the CD24-mediated diseases are B-cell lymphoma, leukemia, ovarian cancer, breast cancer, glioma, small cell lung cancer, esophageal squamous cell carcinoma, liver cancer, pancreatic cancer, or prostate cancer.

21. A method for preventing and / or treating CD24-mediated diseases, comprising administering to a patient in need an effective amount of an antibody or antigen-binding fragment according to any one of claims 1-13, a multispecific antigen-binding molecule according to claim 14, an isolated nucleic acid molecule according to claim 15, an expression vector according to claim 16, a host cell according to claim 17, a product prepared by the method of claim 18, or a pharmaceutical composition according to claim 19; preferably, the CD24-mediated disease is B-cell lymphoma, leukemia, ovarian cancer, breast cancer, glioma, small cell lung cancer, esophageal squamous cell carcinoma, liver cancer, pancreatic cancer, or prostate cancer.

22. A kit comprising an antibody or antigen-binding fragment according to any one of claims 1-13, a multispecific antigen-binding molecule according to claim 14, an isolated nucleic acid molecule according to claim 15, an expression vector according to claim 16, a host cell according to claim 17, a product prepared by the method of claim 18, or a pharmaceutical composition according to claim 19; optionally, further comprising instructions for use.

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