Anti-siglec-15 antibodies and uses thereof

Abstract

The application belongs to the field of biological medicine, and relates to an anti-Siglec-15 antibody, which comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3. The anti-Siglec-15 antibody or an antigen binding fragment thereof has high affinity to the antigen, can effectively reverse the proliferation inhibition effect of a Siglec-15 Fc fusion protein on T cells, and has a good drug development prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to an anti-Siglec-15 antibody. BACKGROUND

[0002] Sialic acid-binding immunoglobulin-like lectin (Siglec) is a classic immunoglobulin-like lectin, which belongs to a type I membrane protein that penetrates once, has very typical and conservative structural characteristics, and has a transmembrane region composed of 2-17 extracellular Ig domains, and an N-terminal composed of a V-set Ig domain binding to sialic acid and a certain number of C2-set Ig domains. At present, 15 human and 9 mouse Siglec molecules have been found, which are mainly specifically expressed on the surface of myeloid cells and immune cells, can recognize sugar chain structures containing sialic acid, mediate the interaction between cells and cells or pathogens, and play an important role in innate immunity and adaptive immunity.

[0003] In recent years, studies have shown that members of the Siglec family are involved in the regulation of immune cell activation, proliferation and apoptosis. At the same time, members of the Siglec family are also involved in the regulation of immune tolerance, and play an important immune regulatory role in autoimmune diseases, inflammatory reactions and tumor occurrence. Therefore, more and more therapeutic drugs targeting Siglecs or glycosylated ligands have been developed and used for the treatment of lymphoma, leukemia and autoimmune diseases and other Siglecs-related diseases.

[0004] Siglec-15 is expressed in some myeloid cells (such as macrophages) under physiological conditions, and in the tumor microenvironment, the expression of Siglec-15 is up-regulated in M2-type macrophages and in various human tumor cells including lung cancer, ovarian cancer and head and neck cancer, and it is involved in inhibiting antigen-specific T cell response by recognizing unknown ligands on the surface of T cells. Siglec-15, as a new immune regulatory target, inhibits immune response in the tumor microenvironment, and anti-Siglec-15 antibody is a new way to activate immune cells in the tumor microenvironment. SUMMARY

[0005] The purpose of the present application is to provide an anti-Siglec-15 antibody that can specifically bind to Siglec-15, thereby providing a new choice for drug development.

[0006] In a first aspect, the present application provides an anti-Siglec-15 antibody or an antigen-binding fragment thereof that binds to Siglec-15 or a fragment thereof, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three CDRs, VH CDR1, VH CDR2 and VH CDR3, respectively, and the light chain variable region comprising three CDRs, VL CDR1, VL CDR2 and VL CDR3, respectively; wherein the VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, 10, 18, 26, 34 or 42, the VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, 11, 19, 27, 35 or 43, the VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, 12, 20, 28, 36 or 44, the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, 14, 22, 30, 38 or 46, the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, 15, 23, 31, 39 or 47, and the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8, 16, 24, 32, 40 or 48.

[0007] In preferred embodiments, the antibody or the antigen-binding fragment thereof comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0008] the amino acid sequence of VH CDR1 is set forth in SEQ ID NO: 2;

[0009] the amino acid sequence of VH CDR2 is set forth in SEQ ID NO: 3;

[0010] the amino acid sequence of VH CDR3 is set forth in SEQ ID NO: 4;

[0011] the amino acid sequence of VL CDR1 is set forth in SEQ ID NO: 6;

[0012] the amino acid sequence of VL CDR2 is set forth in SEQ ID NO: 7;

[0013] the amino acid sequence of VL CDR3 is set forth in SEQ ID NO: 8.

[0014] In preferred embodiments, the antibody or the antigen-binding fragment thereof comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0015] the amino acid sequence of the VH CDR1 is set out in SEQ ID NO: 10;

[0016] the amino acid sequence of the VH CDR2 is set out in SEQ ID NO: 11 ;

[0017] the amino acid sequence of the VH CDR3 is set out in SEQ ID NO: 12;

[0018] the amino acid sequence of the VL CDR1 is set out in SEQ ID NO: 14;

[0019] the amino acid sequence of the VL CDR2 is set out in SEQ ID NO: 15;

[0020] the amino acid sequence of the VL CDR3 is set out in SEQ ID NO: 16.

[0021] In preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3; wherein,

[0022] the amino acid sequence of the VH CDR1 is set out in SEQ ID NO: 18;

[0023] the amino acid sequence of the VH CDR2 is set out in SEQ ID NO: 19;

[0024] the amino acid sequence of the VH CDR3 is set out in SEQ ID NO: 20;

[0025] the amino acid sequence of the VL CDR1 is set out in SEQ ID NO: 22;

[0026] the amino acid sequence of the VL CDR2 is set out in SEQ ID NO: 23;

[0027] the amino acid sequence of the VL CDR3 is set out in SEQ ID NO: 24.

[0028] In preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3; wherein,

[0029] the amino acid sequence of the VH CDR1 is set out in SEQ ID NO: 26;

[0030] the amino acid sequence of the VH CDR2 is set out in SEQ ID NO: 27;

[0031] the amino acid sequence of the VH CDR3 is set out in SEQ ID NO: 28;

[0032] the amino acid sequence of the VL CDR1 is set out in SEQ ID NO: 30;

[0033] the amino acid sequence of the VL CDR2 is set out in SEQ ID NO: 31 ;

[0034] the amino acid sequence of the VL CDR3 is set out in SEQ ID NO: 32.

[0035] In preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3; wherein,

[0036] the amino acid sequence of the VH CDR1 is set out in SEQ ID NO: 34;

[0037] the amino acid sequence of the VH CDR2 is set out in SEQ ID NO: 35;

[0038] the amino acid sequence of the VH CDR3 is set out in SEQ ID NO: 36.

[0039] the amino acid sequence of the VL CDR1 is set out in SEQ ID NO: 38;

[0040] the amino acid sequence of the VL CDR2 is set out in SEQ ID NO: 39;

[0041] the amino acid sequence of the VL CDR3 is set out in SEQ ID NO: 40.

[0042] In preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3; wherein,

[0043] the amino acid sequence of the VH CDR1 is set out in SEQ ID NO: 42;

[0044] the amino acid sequence of the VH CDR2 is set out in SEQ ID NO: 43;

[0045] the amino acid sequence of the VH CDR3 is set out in SEQ ID NO: 44.

[0046] the amino acid sequence of the VL CDR1 is set out in SEQ ID NO: 46;

[0047] the amino acid sequence of VL CDR2 is set out in SEQ ID NO: 47;

[0048] the amino acid sequence of VL CDR3 is set out in SEQ ID NO: 48.

[0049] In preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1, a VH CDR2, and a VH CDR3, and a VL CDR1, a VL CDR2, and a VL CDR3; wherein,

[0050] the amino acid sequence of VH CDR1 is set out in SEQ ID NO: 2;

[0051] the amino acid sequence of VH CDR2 is set out in SEQ ID NO: 3;

[0052] the amino acid sequence of VH CDR3 is set out in SEQ ID NO: 4;

[0053] the amino acid sequence of VL CDR1 is set out in SEQ ID NO: 38;

[0054] the amino acid sequence of VL CDR2 is set out in SEQ ID NO: 39;

[0055] the amino acid sequence of VL CDR3 is set out in SEQ ID NO: 40.

[0056] In certain preferred embodiments, the 3 CDRs contained within the heavy chain variable region, and / or the 3 CDRs contained within the light chain variable region, are defined by the Kabat or Chothia numbering system.

[0057] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is set out in SEQ ID NO: 1, 9, 17, 25, 33, 41, or 49, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the above sequences.

[0058] In some embodiments, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is set out in SEQ ID NO: 5, 13, 21, 29, 37, 45, or 50, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the above sequences.

[0059] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 1 ; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 5 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 5.

[0060] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 13.

[0061] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 17; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 21 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 21.

[0062] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 25 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 25; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 29 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 29.

[0063] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 33, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 33; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 37, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 37.

[0064] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 41, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 41; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 45, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 45.

[0065] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 49, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 49; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 50, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 50.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof of the present application can further comprise one or more of a heavy chain constant region, a light chain constant region, an Fc region. In further preferred embodiments, the light chain constant region is a lambda chain or kappa chain constant region. In some preferred embodiments, the antibody or antigen-binding fragment thereof is of the IgGl, IgG2, IgG3, or IgG4 type.

[0067] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody or antigen-binding fragment thereof.

[0068] In a preferred embodiment, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO: 51, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO: 52.

[0069] In a second aspect, the present application provides a biological material, which is:

[0070] 1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present application; in a preferred embodiment, the nucleic acid molecule encodes the heavy chain variable region and / or the light chain variable region of the antibody or antigen-binding fragment thereof;

[0071] 2) a vector comprising the nucleic acid molecule of the present application;

[0072] 3) a host cell or microorganism comprising the nucleic acid molecule or vector of the present application;

[0073] 4) an expression product, expression supernatant or expression suspension of 3) above.

[0074] A person skilled in the art can easily obtain a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the amino acid sequence of the antibody or antigen-binding fragment thereof, select and prepare a vector, host cell or microorganism comprising the nucleic acid molecule, and know how to culture such host cell or microorganism to obtain the corresponding expression product, suspension, supernatant, etc., to obtain the corresponding antibody. This is all routine technical means in the art.

[0075] In a third aspect, a composition comprising the antibody or antigen-binding fragment thereof of the present application is provided; preferably, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0076] In a fourth aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present application is provided, comprising: culturing the host cell described above to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment thereof.

[0077] In a fifth aspect, the use of the antibody or antigen-binding fragment thereof of the present application or the biological material of the present application or the composition of the present application in the preparation of a medicament for treating tumors is provided.

[0078] In the present application, the tumor is a Siglec-15-expressing tumor; further preferably, the tumor is various hematological tumors and solid tumors, such as leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, prostate cancer, ovarian cancer, thyroid cancer, glioma.

[0079] In a seventh aspect, there is provided use of the antibody or antigen-binding fragment thereof of the present application or the biomaterial of the present application or the composition of the present application in the preparation of a preparation for blocking the inhibition of T cell proliferation by Siglec-15 protein.

[0080] The anti-Siglec-15 antibody or antigen-binding fragment thereof of the present application has high affinity for its antigen, can effectively reverse the inhibition of T cell proliferation by Siglec-15 Fc fusion protein in vitro experiments, can play a tumor killing function by effectively promoting T cell proliferation, can effectively inhibit the growth of tumors, and has a good drug development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0081] Figure 1 ELISA results of the chimeric antibody on human Siglec-15 protein.

[0082] Figure 2 FACs results of the chimeric antibody on human Siglec-15 stably transfected cells.

[0083] Figure 3 T cell activation activity of the chimeric antibody on donor A-derived PBMC.

[0084] Figure 4 T cell activation activity of the chimeric antibody on donor B-derived PBMC.

[0085] Figure 5 Epitope competition experiment of the chimeric antibody.

[0086] Figure 6 ELISA results of the candidate humanized antibody on human Siglec-15 protein.

[0087] Figure 7 FACs results of the candidate humanized antibody on human Siglec-15 stably transfected cells.

[0088] Figure 8 T cell activation activity of the candidate humanized antibody B609415 on donor A-derived PBMC.

[0089] Figure 9 ELISA results of the candidate humanized antibody B609415 on human Siglec-1 / 2 / 4 protein and monkey and mouse Siglec-15 protein. DETAILED DESCRIPTION

[0090] Definitions

[0091] In the present application, the term "antibody" refers to an immunoglobulin that can specifically recognize and bind to an antigen, and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or antibody fragments.

[0092] The term "variable region" refers to the domain of an antibody heavy or light chain that recognizes and specifically binds to an epitope of an antigen.

[0093] The CDR regions, or "complementarity determining regions," are regions in an antibody variable region that are hypervariable in sequence and form structurally defined loops and / or contain amino acid residues that contact antigen. The CDRs are primarily responsible for binding of an antibody to an epitope of an antigen and determine the specificity of the antibody. The specific amino acid sequences of the CDRs in a given heavy or light chain variable region amino acid sequence are identified using any of a number of well-known numbering schemes, including, for example, Kabat, Contact, AbM, and Chothia, or combinations thereof.

[0094] Antibodies of the Invention

[0095] The present application provides anti-Siglec-15 antibodies with high affinity to human Siglec-5 protein, which can effectively reverse the proliferation inhibition of T cells by Siglec-15 Fc fusion protein in vitro experiments, and further play an effective tumor killing function.

[0096] The anti-Siglec-15 antibodies or antigen-binding fragments thereof of the present application comprise substitutions, insertions, or deletions. The anti-Siglec-15 antibodies of the present application include modifications to the light chain variable region, the heavy chain variable region, the light chain, or the heavy chain, after which the amino acid sequence is different from the amino acid sequence derived from the antibody. For example, the amino acid sequence derived from the same specified protein can be similar to the starting sequence, for example, having a certain percentage of identity, for example, it can have a percentage of identity of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% to the starting sequence.

[0097] In the present application, "identity" refers to the percentage of bases (or amino acids) that are the same in the two sequences being compared when the sequences are aligned. The alignment and percentage homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP.

[0098] In certain embodiments, amino acid modifications can be introduced into the Fc region of an antibody provided herein, either at one or more amino acid positions, to thereby generate Fc variants. An Fc variant can comprise a human Fc region sequence comprising an amino acid modification at one or more amino acid positions.

[0099] “Antibodies or antigen-binding fragments thereof” suitable for use in the present application include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunized antibodies, or Fab fragments, Fab’ fragments, F(ab’)2 fragments, single-chain antibodies, nanobodies, and epitope-binding fragments of any of the above.

[0100] In some embodiments, the antibodies of the present application can be monospecific, bispecific, or multispecific. Anti-Siglec-15 antibodies can be linked to another antibody or antibody fragment to generate a bispecific or multispecific antibody with a second or more binding specificities.

[0101] In certain embodiments, antibodies can be further modified to add functional components, moieties suitable for antibody derivatization include, but are not limited to, the following examples, PEG, dextran, proteins, lipids, therapeutic agents, or toxins. Antibodies can be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or linkage to other proteins, etc.

[0102] Methods of tumor treatment and antibody uses

[0103] The anti-Siglec-15 antibodies and antigen-binding fragments thereof and pharmaceutical compositions comprising the same provided by the present application can be used for the diagnosis, prognosis, treatment, or inhibition of cancer. The present application relates to methods of treating a subject for cancer by administering to a subject in need thereof an anti-Siglec-15 antibody or fragment thereof of the present application. Therapeutic compounds of the present application include, but are not limited to, the antibodies described herein (including variants and derivatives described herein) and nucleic acids or polynucleotides encoding the antibodies described herein (including variants and derivatives described herein).

[0104] The antibodies of the present application can be administered by any suitable means, including but not limited to intraperitoneal, intravenous, subcutaneous, intranasal, intramuscular injection. The antibodies and variants or compositions thereof can be administered by any convenient route, for example by injection, by absorption through epithelial or mucocutaneous membranes.

[0105] Anti-Siglec-15 antibody sequences under the present invention

[0106] Table 1: VH, VH CDR1, VH CDR2, VH CDR3, VL, VL CDR1, VL CDR2, VL CDR3 amino acid sequence numbers for antibodies of the present application

[0107]

[0108]

[0109] Examples

[0110] Example 1: Preparation of anti-Siglec-15 mouse monoclonal antibodies

[0111] BALB / c or SJL mice (6-8 weeks old, Shanghai Slac Laboratory Animal Co. Ltd.) were immunized with 50 μg of commercial human Siglec-15 Fc fusion protein (AcroBiosystems, Cat. No. SG5-H5253) for the first time, and then immunized with 25 μg of the above-mentioned fusion protein on the 14th day and the 35th day. On the 56th day after immunization, 25 μg of the above-mentioned fusion protein was used for booster immunization, and the obtained mice were used for fusion.

[0112] The serum titer of the immunized mice was detected by enzyme-linked immunosorbent assay (ELISA). Human Siglec-15 His protein (AcroBiosystems, SG5-H52H3) was diluted with PBS to 1 μg / ml, 100 μl / well was added to the enzyme-labeled plate, and was coated at 4°C overnight. Blocking solution (2% BSA-PBS buffer) was used for blocking at 37°C for 1 h, and the plate was washed with 1x PBST for 3 times. Mouse serum diluent (1:100; 1:1k; 1:10k) was added to the plate and incubated at 37°C for 2 h. Anti-mouse Fab-HRP (Sigma, A3862, 1:10,000) was added to the plate after washing with 1x PBST for 3 times, and was reacted at 37°C for 1 h. After washing the plate for 3 times, TMB solution was added, and the reaction was stopped after reaction at room temperature for 5 min. The OD450 absorbance value was detected by enzyme-labeled instrument. After immunization, the myeloma cell SP2 / 0 (ATCC) suspension and the spleen cell suspension of the immunized mice were prepared, counted by a blood cell counting plate, and mixed at a ratio of 1:3. The cell fusion was performed by an electrofusion instrument. HAT screening medium was added to the fusion tube, mixed and diluted, and then 100 μl / well was added to a 96-well cell culture plate. After being cultured in a 5% CO2 37°C incubator for 10-14 days, the hybridoma supernatant was detected by ELISA. Multiple different hybridomas were identified, and the mAb006, mAb009, mAb010, mAb044, mAb060 and mAb070 clones were selected for further analysis. Finally, the amino acid sequences of the CDR region, the heavy chain variable region and the light chain variable region of the antibody were analyzed, and the specific sequences are shown in Table 1.

[0113] Example 2: Affinity detection of mouse chimeric antibodies

[0114] The variable region genes of clones mAb006, mAb009, mAb010, mAb044, mAb060 and mAb070 were chimerized with human IgG constant region genes (heavy chain constant region sequence is SEQ ID NO: 51; light chain constant region sequence is SEQ ID NO: 52) and cloned into expression vectors, and named ch006, ch009, ch010, ch044, ch060 and ch070, respectively. After one week of transient expression in HEK293 cells, the culture supernatant was harvested and purified by protein A affinity, and the affinity of the candidate antibodies to human Siglec-15 protein (Acrobiosystems, SG5-H52H3) and stably transfected human Siglec-15 cells (Human Siglec-15-8D2, Rizhao Chemical, cell clone number 8D2) was detected by ELISA and FACs experiments.

[0115] The human Siglec-15 protein was diluted with PBS to 2 μg / ml, and after incubating the coated enzyme-labeled plate at 4°C overnight, it was washed with PBST for three times. 100 μl of BSA blocking solution (2% BSA in PBST solution) was added to each well, and incubated at 37°C for 1 h. After washing with PBST for three times, 100 μl of gradient concentration diluted candidate antibodies and control antibodies (negative control is hlgG1; positive control is NC318-5G12, antibody heavy and light chain variable region sequences are shown in sequences 7 and 18 of patent US62451271P0) were added, with a starting concentration of 100 nM, 5-fold gradient dilution, a total of 8 concentration gradients. After incubation at 37°C for 1.5 h, washing with PBST for three times, adding anti-human IgG Fc-HRP antibody (Sigma, A0170) and incubating at 37°C for 1 h. Washing with PBST for three times, adding TMB developing solution (Biotechnology, TMB-S-003), and incubating at 37°C for 0.5 h in the dark. Adding 100 μl of stop solution, and after color development, measuring the OD value at 450 nm with a microplate reader. The ELISA results are shown in Figure 1 As shown in the figure, the negative control hlgG1 irrelevant antibody has no binding signal, while the candidate murine chimeric antibodies can efficiently bind to human Siglec-15 protein.

[0116] Human Siglec-15 stably transfected cells (human Siglec-15-8D2) were collected, and the cells were diluted to 1.5 x 10 6Cells / ml, take 100 μl cells to the staining plate, 300g centrifugal 5min, discard the supernatant, add 100 μl gradient concentration diluted candidate antibody and control antibody (negative control is hlgG1, positive control is NC318-5G12), incubate at 4℃ for 1h. Wash three times with FACS buffer, add 100 μl fluorescent secondary antibody (Life Technology, A21202), incubate at 4℃ for 1h. After washing three times with FACS buffer, carry out flow cytometry. As shown in Figure 2 The negative control IgG1 has no binding signal with human Siglec-15 stable cell line (human Siglec-15-8D2) in FACs detection, and the relevant candidate antibodies all show high affinity.

[0117] Example 3 Evaluation of T cell activation function of murine chimeric antibody

[0118] Dilute anti-CD3 antibody OKT3 (Rui Zhi Chemical, 180327005) to 0.05 μg / ml with PBS, add 100 μl per well in a 96-well plate, shake at 400 rpm for 30 min, then incubate at 4℃ overnight. Quickly thaw the PBMC, mix well with 10 ml PBMC cell culture medium (RPMI1640 culture medium containing 10% FBS) (Gibco, A1049), centrifuge at 400g for 5 min, mix well with 10 ml PBMC cell culture medium, inoculate in a 10 cm culture dish, incubate at 37℃ in 5% CO2 overnight.

[0119] Dilute anti-Siglec-15 antibodies (ch006, ch009, ch010, ch044, ch060, ch070 and NC318-5G12) to 20 μg / ml, 6.7 μg / ml and 2.2 μg / ml respectively with PBMC cell culture medium, dilute human Siglec-15 ECD-hFc (Biointron, 0190709A22) to 3 μg / ml, mix equal volumes of antibody and human Siglec-15 ECD-hFc protein in a sterile deep well plate, incubate at room temperature for 1 hour. Wash the anti-CD3 pre-coated plate twice with PBS, then add 100 μl of the mixture per well, incubate at 37℃ for 1 hour. Collect PBMC, centrifuge at 400g for 5 min, suspend in PBS to a cell density of 2×10 6 Cells / ml, add 0.5 μM CFSE to a final concentration, incubate at room temperature for 8 min, then add an equal amount of fetal bovine serum and incubate at room temperature for 8 min, centrifuge at 400g for 5 min, discard the supernatant. Wash once with PBS, wash twice with PBMC cell culture medium, then resuspend the cells in the culture medium to a cell density of 2×10 6PBMCs were stained with CFSE at 1 x 106cells / ml, 100 μl / well, and incubated in a 37 °C incubator for about 65 h. The PBMC cells were removed and the wells were washed once with 100 μl PBS and the residual cells were collected and centrifuged at 400 g for 5 min. The PBMCs were resuspended and incubated at room temperature for 30 min with APC-labeled mouse anti-human CD8 antibody (BD, 555369) and BV421-labeled mouse anti-human CD4 antibody (BD, 562424) diluted 1:1000 in FACS buffer. The OKT3-unstimulated control groups were treated with anti-CD8 antibody only, anti-CD4 antibody only, and no antibody. After the reaction, the cells were washed twice with 200 μl / well FACS buffer, centrifuged at 400 g for 5 min, and the supernatant was discarded. The cells were resuspended in 100 μl FACS buffer and analyzed by flow cytometry to detect the proportion of CD4+T and CD8+T cells in the PBMC cells. The results are shown in FIGS. 13A and 13B, which show that ch006, ch009, ch010, ch044, ch060, ch070, and NC318-5G12 can significantly reverse the inhibition of T cell proliferation by Siglec-15 protein in PBMC cells from two donors in vitro. Figure 3 and Figure 4 The results are shown in FIGS. 13A and 13B, which show that ch006, ch009, ch010, ch044, ch060, ch070, and NC318-5G12 can significantly reverse the inhibition of T cell proliferation by Siglec-15 protein in PBMC cells from two donors in vitro.

[0120] Example 4. Epitope binning of candidate antibodies

[0121] Each of the antibodies to be tested (ch006, ch009, ch010, ch044, ch060, ch070, and NC318-5G12) was diluted to 1 μg / ml and added to the coated plate at 50 μl / well. After incubation at 37 °C for 1.5 h, the plate was washed 3 times with PBST. After incubation at 37 °C for 2 h with 100 μl blocking solution (PBST containing 1% BSA) and washing the plate 3 times with PBST, 50 μl of biotin-human Siglec-15 His protein (Acro, SG5-H82E9) diluted at a gradient concentration (starting concentration of 1 μg / ml, diluted at a gradient of 5 times) was added, and the plate was incubated at 37 °C for 1 h. After washing the plate 3 times with PBST, 50 μl of SA-HRP (Sigma, S2438) was added to each well, and the plate was incubated at 37 °C for 0.5 h. After washing 6 times with PBST, 100 μl of substrate TMB (innoReagents, EL0009) was added to each well, and the plate was incubated at 37 °C for 15 min. After adding 50 μl of stop solution to each well, the color was developed, and the OD value was measured at 450 nm using a microplate reader. The concentration of biotin-human Siglec-15 His protein that bound to each antibody at an EC80 rate was calculated.

[0122] The reference antibody NC318-5G12 was diluted to 1 pg / ml, 50 mΐ per well was added to the coated plate, after incubation at 37°C for 1.5 h, the plate was washed 3 times with PBST. 100 mΐ of blocking solution was added and incubated at 37°C for 2 h, then the plate was washed 3 times with PBST. Biotin-human Siglec-15 His and the test competitive antibody were mixed in equal proportions to a final concentration of EC80 value concentration and 20 pg / ml respectively, after incubation at 37°C for 1 h, 100 mΐ per well was added to the coated well, incubated at 37°C for 1 h, then washed 3 times with PBST. 100 mΐ of SA-HRP (1:5000 dilution) was added to each well, and incubated at 37°C for 0.5 h. After washing the plate 6 times with PBST, 100 mΐ of substrate TMB was added, and incubated at 37°C for 15 min. After adding 50 mΐ of stop solution to each well, the color was developed and the OD value was measured at 450 nm with a microplate reader. The binding inhibition of the competitive antibody to the coated antibody was calculated to classify each candidate antibody. The results are shown in Table 1. Figure 5 It can be seen that the candidate molecules ch044 and ch070 have high competition efficiency with the reference antibody NC318-5G12, indicating that ch044 and ch070 are similar to the epitope of the reference antibody; while ch006, ch009, ch010 and ch060 have low competition rate with the reference antibody, indicating that they are different from the epitope of the reference antibody.

[0123] Example 5 Humanization of the candidate antibody

[0124] In this project, the epitopes of ch006, ch009, ch010 and ch060 antibodies are quite different from the reference antibody NC318-5G12, so they are selected as the main candidate molecules for further research. After sequence analysis, the sequence homology of the related antibodies is high, and finally ch006-VH is selected as the heavy chain variable region and ch060-VL is selected as the light chain variable region for combination to carry out the subsequent antibody humanization work.

[0125] Specifically, the heavy chain variable region and the light chain variable region of the antibody are compared with the available human IgG gene sequence database to identify the best matching human Ig gene sequence. Specifically, the heavy chain sequence selects human IgG heavy chain IGHV1-2*02; the light chain sequence selects human IgG light chain: IGKV4-1*01. The CDR regions of the heavy chain of ch006 and the light chain of ch060 are transplanted into the matching framework sequence, and the obtained VH and VL amino acid sequences are sequence 49 and sequence 50 respectively, and the antibody heavy chain constant region and light chain constant region used are sequence 51 and sequence 52 respectively.

[0126] The designed heavy and light chain plasmids are combined, and the new antibody molecules are named B609415 respectively. Those skilled in the art can completely know the complete amino acid sequences of the heavy chain and light chain of the related humanized antibody according to the information disclosed above.

[0127] Example 6 Affinity and activity detection of humanized antibodies

[0128] As described above, the humanized antibody B609415 obtained by humanization design of chimeric antibodies ch006 and ch060 was obtained by protein A affinity purification after HEK293 transient expression. The affinity of humanized antibody, ch006 murine chimeric antibody and control antibody NC318-5G12 to human Siglec-15 protein (Acrobiosystems, SG5-H52H3) and stably transfected human Siglec-15 cells (Ruiyi Chemical, cell clone number 8D2) was detected by ELISA and FACs experiments, and the experimental method was the same as that in Example 2. The results showed that Figure 6 、 Figure 7 The results showed that the related humanized antibodies had high affinity to human Siglec-15 protein and stably transfected human Siglec-15 cells, and had no significant difference with the reference drug NC318-5G12.

[0129] The experimental method of T cell activation of the antibody was the same as that described in Example 3, and the results were as shown in Figure 8 The results showed that the humanized antibody B609415 efficiently reversed the proliferation inhibition of Siglec-15 protein on CD4+T cells and CD8+T cells, and its functional activity was slightly better than that of the reference antibody NC318-5G12.

[0130] Example 7 Specific binding of humanized antibodies

[0131] The specific binding detection experiment was carried out on the candidate molecule B609415. Among them, the affinity of ELISA method to murine Siglec-15 protein (Acro, SG5-M52H7), monkey Siglec-15 protein (Acro, SG5-C52H6), and homologous human Siglec-1 protein (R&D, 5197-SL-050), human Siglec-2 protein (R&D, 10191-SL-050), human Siglec-4 protein (R&D, 8940-MG-050) was detected.

[0132] The related experimental method was shown in Example 2, and the results were as shown in Figure 9 The results showed that the humanized antibody B609415 had no non-specific binding to the Siglec-1 / 2 / 4 proteins of the human Siglec family, indicating that the recognition of the candidate antibody to Siglec-15 had high specificity; at the same time, the molecule maintained high affinity to stably transfected murine and monkey Siglec-15 proteins, which could provide reference basis for the selection of subsequent pharmacodynamic model and monkey toxicology experimental mode animals.

[0133] Sequence Listing

[0134] 1 : mAb006-VH

[0135] QVQLQQSGPELVKPGTSVKISCKASGYSFTDYFINWMKQRPGQGLEWIGWIFPGSGDTYYNEKFKGKATLTVDNSSSTAYMLLSSLTSEDSAVFFCARTGDYFDYWGQGTTLTVSS

[0136] 2: mAb006-VH CDR1

[0137] DYFIN

[0138] 3: mAb006-VH CDR2

[0139] WIFPGSGDTYYNEKFKG

[0140] 4: mAb006-VH CDR3

[0141] TGDYFDY

[0142] 5: mAb006-VL

[0143] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPWTFGGGTKLEIK

[0144] 6: mAb006-VL CDR1

[0145] RSSQSLVHSNGNTYLH

[0146] 7: mAb006-VL CDR2

[0147] KVSNRFS

[0148] 8: mAb006-VL CDR3

[0149] SQSTHVPPWT

[0150] 9: mAb009-VH

[0151] QVQLQQSGPELVKPGASVKISCKASGYTFTDYYFNWVKQRPGQGLEWIGWIFPGSGDAYYNANFKGQATLTVDNSSSTAYMLLSSLTSEDSAVYLCARSGDYFDYWGQGTTLTVSS

[0152] 10: mAb009-VH CDR1

[0153] DYYFN

[0154] 11: mAb009-VH CDR2

[0155] WIFPGSGDAYYNANFKG

[0156] 12: mAb009-VH CDR3

[0157] SGDYFDY

[0158] 13: mAb009-VL

[0159] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0160] 14: mAb009-VL CDR1

[0161] RSSQSLVHSNGNTYLH

[0162] 15: mAb009-VL CDR2

[0163] KVSNRFS

[0164] 16: mAb009-VL CDR3

[0165] SQSTHVPWT

[0166] 17: mAb010-VH

[0167] QVQLQQSGPELVKPGTSVKISCKASGYTFTDYYINWVKQRPGQRLEWIGWIFPGSGDTYYNETFKGKATLNVDNSSSTAYMLLSSLTSEDSAVYFCARWGDFDDYWGQGTTLTVSS

[0168] 18: mAb010-VH CDR1

[0169] DYYIN

[0170] 19: mAb010-VH CDR2

[0171] WIFPGSGDTYYNETFKG

[0172] 20: mAb010-VH CDR3

[0173] WGDFDDY

[0174] 21: mAb010-VL

[0175] DVVMTQTPLSLPVSLGDQTSISCRSSQSLVHNNGNTYLHWYLQKPGQSPKLLIFKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSSHVPLTFGAGTKLELK

[0176] 22: mAb010-VL CDR1

[0177] RSSQSLVHNNGNTYLH

[0178] 23: mAb010-VL CDR2

[0179] KVSNRFS

[0180] 24: mAb010-VL CDR3

[0181] SQSSHVPLT

[0182] 25: mAb044-VH

[0183] EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEKGLEWVAYISSGSSTIYFADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRDDYHAMDFWGQGTSVTVSS

[0184] 26: mAb044-VH CDR1

[0185] DYGIH

[0186] 27: mAb044-VH CDR2

[0187] YISSGSSTIYFADTVKG

[0188] 28: mAb044-VH CDR3

[0189] RDDYHAMDF

[0190] 29: mAb044-VL

[0191] DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAAAWYQQKPGQSPKLLIYSASYRYTGVPDRFNGSGSGTDFTFTISTVQAEDLAVYYCQQHDSPPYTFGGGTKLEIK

[0192] 30: mAb044-VL CDR1

[0193] KASQDVSTAAA

[0194] 31: mAb044-VL CDR2

[0195] SASYRYT

[0196] 32: mAb044-VL CDR3

[0197] QQHDSPPYT

[0198] 33: mAb060-VH

[0199] QVQLQQSGPELVKPGASVKISCKASDYTFTDYYINWVKQRPGQGLEWIGWIFPGSGSTYYNEKFEGKATLTVDNSSSTACMLLSSLTSEDSAVYFCARTGDYFDYWGQGTTLTVSS

[0200] 34: mAb060-VH CDR1

[0201] DYYIN

[0202] 35: mAb060-VH CDR2

[0203] WIFPGSGSTYYNEKFEG

[0204] 36: mAb060-VH CDR3

[0205] TGDYFDY

[0206] 37: mAb060-VL

[0207] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0208] 38: mAb060-VL CDR1

[0209] RSSQSLVHSNGNTYLH

[0210] 39: mAb060-VL CDR2

[0211] KVSNRFS

[0212] 40: mAb060-VL CDR3

[0213] SQSTHVPWT

[0214] 41: mAb070-VH

[0215] EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMNWVRQGPEKGLEWVALITSGSSAIYFADTVKGRFTISGDNAKNTLFLQMTSLRSEDTAIYYCTRRDDYHAMDFWGQGTSVTVSS

[0216] 42: mAb070-VH CDR1

[0217] DYGMN

[0218] 43: mAb070-VH CDR2

[0219] LITSGSSAIYFADTVKG

[0220] 44: mAb070-VH CDR3

[0221] RDDYHAMDF

[0222] 45: mAb070-VL

[0223] DIVMTQSHKFMSTSVGDRVSVTCKASQHVSTAVAWYQQKPGQSPKLLIFSASYRYTGVPDRFNGSGYGTDFTFTISSVQAEDLAVYYCQQHDTTPYTFGGGTKLEIN

[0224] 46: mAb070-VL CDR1

[0225] KASQHVSTAVA

[0226] 47: mAb070-VL CDR2

[0227] SASYRYT

[0228] 48: mAb070-VL CDR3

[0229] QQHDTTPYT

[0230] 49: B609415-VH

[0231] QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYFINWVRQAPGQGLEWMGWIFPGSGDTYYNEKFKGRVTMTVDNS TS TAYMELSSLRSEDTAVYYCARTGDYFDYWGQGTLVTVSS

[0232] 50: B609415-VL

[0233] DVVMTQSPDSLAVSLGERATINCRSSQSLVHSNGNTYLHWYQQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDLAVYFCSQSTHVPWTFGGGTKVEIK

[0234] 51: Heavy chain constant region

[0235] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0236] 52: Light chain constant region

[0237] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An anti-Siglec-15 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three CDRs, namely VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprising three CDRs, namely VL CDR1, VL CDR2, and VL CDR3; wherein, The amino acid sequence of VH CDR1 is shown in SEQ ID NO:2; The amino acid sequence of VH CDR2 is shown in SEQ ID NO:3; The amino acid sequence of VH CDR3 is shown in SEQ ID NO:4; The amino acid sequence of VL CDR1 is shown in SEQ ID NO:38; The amino acid sequence of VL CDR2 is shown in SEQ ID NO:39; The amino acid sequence of VL CDR3 is shown in SEQ ID NO:40; or The amino acid sequence of VH CDR1 is shown in SEQ ID NO:2; The amino acid sequence of VH CDR2 is shown in SEQ ID NO:3; The amino acid sequence of VH CDR3 is shown in SEQ ID NO:4; The amino acid sequence of VL CDR1 is shown in SEQ ID NO:6; The amino acid sequence of VL CDR2 is shown in SEQ ID NO:7; The amino acid sequence of VL CDR3 is shown in SEQ ID NO:

8.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The amino acid sequence of the heavy chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:49; the amino acid sequence of the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:

5.

3. The antibody or its antigen-binding fragment according to claim 1, wherein, The amino acid sequence of the heavy chain variable region has at least 95% sequence identity with the sequence shown in SEQ ID NO:49; the amino acid sequence of the light chain variable region has at least 95% sequence identity with the sequence shown in SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region has at least 95% sequence identity with the sequence shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region has at least 95% sequence identity with the sequence shown in SEQ ID NO:

5.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 96% sequence identity with the sequence shown in SEQ ID NO:49; and the amino acid sequence of the light chain variable region has at least 96% sequence identity with the sequence shown in SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region has at least 96% sequence identity with the sequence shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region has at least 96% sequence identity with the sequence shown in SEQ ID NO:

5.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 97% sequence identity with the sequence shown in SEQ ID NO:49; and the amino acid sequence of the light chain variable region has at least 97% sequence identity with the sequence shown in SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region has at least 97% sequence identity with the sequence shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region has at least 97% sequence identity with the sequence shown in SEQ ID NO:

5.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 98% sequence identity with the sequence shown in SEQ ID NO:49; and the amino acid sequence of the light chain variable region has at least 98% sequence identity with the sequence shown in SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region has at least 98% sequence identity with the sequence shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region has at least 98% sequence identity with the sequence shown in SEQ ID NO:

5.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 99% sequence identity with the sequence shown in SEQ ID NO:49; and the amino acid sequence of the light chain variable region has at least 99% sequence identity with the sequence shown in SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region has at least 99% sequence identity with the sequence shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region has at least 99% sequence identity with the sequence shown in SEQ ID NO:

5.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:50; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein, The antibody or its antigen-binding fragment further comprises one or more of a heavy chain constant region, a light chain constant region, and an Fc region; the light chain constant region is a λ chain or a κ chain constant region, and the antibody or its antigen-binding fragment is of type IgG1, IgG2, IgG3, or IgG4.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein, The antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, the amino acid sequence of the heavy chain constant region being shown in SEQ ID NO:51, and the amino acid sequence of the light chain constant region being shown in SEQ ID NO:

52.

11. A biomaterial, comprising: 1) A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 10; 2) A vector comprising the nucleic acid molecule described in 1); 3) A host cell or microorganism containing the nucleic acid molecule described in 1) or the vector described in 2); 4) The expression product of 3) above, wherein the expression product is the antibody or its antigen-binding fragment as described in any one of claims 1 to 10.

12. The biomaterial according to claim 11, wherein, The nucleic acid molecule encodes the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment.

13. A composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10.

14. The composition according to claim 13, wherein, The composition is a pharmaceutical composition and also contains a pharmaceutically acceptable carrier.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising: The host cells of claim 11 or 12 are cultured to express the antibody or its antigen-binding fragment, and the antibody or its antigen-binding fragment is isolated.

16. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the biological material according to claim 11 or 12, or the composition according to claim 13 or 14, in the preparation of a medicament for treating tumors, wherein the tumor is a Siglec-15 positive tumor, and the tumor is lung cancer, ovarian cancer, or head and neck cancer.

Images