Anti-human VSIG4 antibody and medical application thereof

Abstract

The invention relates to an anti-human VSIG4 antibody and a medical application thereof. In particular, the present invention relates to a novel antibody that specifically binds to human VSIG4 or an antigen-binding fragment thereof, and a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof. Furthermore, the invention relates to the use of these antibodies or antigen-binding fragments thereof in the treatment and / or prevention of diseases, in particular tumors.

Description

Technical Field

[0001] The present invention generally relates to the fields of tumor therapy and molecular immunology. In particular, the present invention relates to novel antibodies or antigen-binding fragments thereof that specifically bind to human VSIG4 and compositions containing the antibodies or antigen-binding fragments thereof. In addition, the present invention relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof, vectors comprising the polynucleotides, and host cells comprising the nucleic acids or vectors. In addition, the present invention relates to the use of these antibodies or antigen-binding fragments thereof in the treatment and / or prevention of diseases, especially tumors. Background Art

[0002] V-set immunoglobulin-domain-containing 4 (VSIG4) is a member of the type 1 transmembrane Ig superfamily and exists in two alternatively spliced ​​forms. The gene is located in the pericentromeric region of the human X chromosome. Both human and mouse VSIG4 are located on Xq12 between the ferroportin and moesin on the X chromosome [16530040]. It is highly expressed in the liver, lungs, and placenta of human tissues, while the mouse VSIG4 (mVSIG4) gene encodes a single Ig V-type domain. Human VSIG4 has a long form, hVSIG4 (L), and a short form, hVSIG4 (S) [16530040]. The extracellular structure of hVSIG4 (L) contains a potential O-glycosylation site, while the intracellular domain contains two potential phosphorylation sites, S311 and T-333 [11004523]. The hVSIG4(S) intracellular domain contains two potential phosphorylation sites, S217 and T239[27045607]. hVSIG4(L) encodes the V-type and C2-type terminal Ig domains, and its short form encodes a single V-type Ig domain. At the same time, the cytoplasmic domains of human and mouse VSIG4 contain the AP-2 internalization motifs YARL and DSQALI, respectively. hVSIG4(S) has 67% sequence homology with mVSIG4, of which 83% is located in the IgV domain.

[0003] VSIG4 is a membrane protein that belongs to the complement receptor (CRIg) of the immunoglobulin superfamily. Under physiological conditions, VSIG4 is specifically expressed in macrophages such as the thymic medulla, peritoneum, alveoli, synovium, fat, heart, liver, placenta, and atherosclerotic foam cells [11004523 / 19017980 / 18752851 / 16882875 / 18727628 / 17016562 / 11997108]. VSIG4-positive macrophages mainly show anti-inflammatory, homeostatic, and clearance effects in inflammatory or immune diseases such as liver inflammation, rheumatoid arthritis, and lupus nephritis through complement and T cells [22711680 / 17548523 / 25988858]. VSIG4 can also mediate the clearance of C3b-opsonized pathogens such as Listeria monocytogenes and Staphylococcus aureus by binding to the complement component C3b (16530040). In addition to the complement system and cellular immunity, VSIG4 also plays an important role in regulating the production of certain pathways and certain substances, thereby regulating the development of the disease. VSIG4 is considered to be an important immune checkpoint molecule in cancer and can serve as an independent prognostic factor for tumors or a potential target for immunotherapy [28498255 / 32924090].

[0004] WO2021044014A1 discloses anti-VSIG4 antibodies that can bind to the long form hVSIG4 (L) and the short form hVSIG4 (S) of VSIG4. At present, there is still a need to develop new anti-VSIG4 antibodies that have high binding affinity to both the long form hVSIG4 (L) and the short form hVSIG4 (S) of VSIG4 and can effectively treat VSIG4-related diseases, especially tumors. Summary of the invention

[0005] The present invention provides a group of novel anti-VSIG4 antibodies, which can bind to both human VSIG4-ECD-hFc and human VSIG4-short-ECD-hFc proteins. They show high binding affinity to hVSIG4-ECD-hFc protein, with OD450nm>3.0; all antibodies (except mAb004, mAb009, and mAb019) show binding affinity to hVSIG4-short-Fc protein, with OD450nm>3.0. Therefore, mAb004, mAb009, and mAb019 only bind to the IgC2 domain of the hVSIG4-ECD-Fc protein, while other antibodies bind to the IgV domain of the hVSIG4-ECD-Fc protein.

[0006] Thus, in a first aspect, the present invention provides an anti-VSIG4 antibody or an antigen-binding fragment thereof, wherein:

[0007] i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively,

[0008] ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8 and 9, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively,

[0009] iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14 and 15, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17 and 18, respectively,

[0010] iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 19, 20 and 21, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 23 and 24, respectively,

[0011] v) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 26 and 27, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28, 29 and 30, respectively,

[0012] vi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 34, 35 and 36, respectively,

[0013] vii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 38 and 39, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 40, 41 and 42, respectively,

[0014] viii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 43, 44 and 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 46, 47 and 48, respectively,

[0015] ix) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 49, 50 and 51, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 52, 53 and 54, respectively,

[0016] x) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 55, 56 and 57, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 58, 59 and 60, respectively,

[0017] xi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 61, 62 and 63, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 64, 65 and 66, respectively,

[0018] xii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 67, 68 and 69, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 70, 71 and 72, respectively,

[0019] xiii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 73, 74 and 75, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 76, 77 and 78, respectively,

[0020] xiv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 79, 80 and 81, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 82, 83 and 84, respectively, or

[0021] xv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 85, 86 and 87, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 88, 89 and 90, respectively; wherein the CDR sequences of i) to xv) are determined according to the Kabat numbering system; or

[0022] xvi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 136, 137 and 138, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 139, 140 and 141, respectively, or

[0023] xvii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 142, 143 and 144, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 145, 146 and 147, respectively, wherein the CDR sequences of xvi) to xvii) are determined according to the IMGT numbering system.

[0024] In specific embodiments, the anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention has at least one of the following activities:

[0025] 1) Binds to human, mouse, and cynomolgus monkey VSIG4 with high affinity;

[0026] 2) binds to both hVSIG4-ECD-hFc and hVSIG4-short-Fc;

[0027] 3) No binding activity to A33 and JAM-A proteins;

[0028] 4) Blocking the interaction between hVSIG4-his and hC3b with high affinity, and

[0029] 5) Antitumor activity in vitro and in vivo.

[0030] In another aspect, the present invention further provides a polynucleotide encoding the anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention.

[0031] In another aspect, the present invention further provides a vector comprising the polynucleotide.

[0032] In another aspect, the present invention further provides a cell comprising the vector.

[0033] On the other hand, the present invention further provides a pharmaceutical composition for treating cancer, comprising an antibody or an antigen-binding fragment thereof that specifically binds to VSIG4, and a pharmaceutical excipient, diluent or carrier.

[0034] In addition, the present invention further provides a method for treating cancer, comprising administering a therapeutically effective amount of the anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising the same, to a subject in need thereof.

[0035] On the other hand, the present invention provides use of the anti-VSIG4 antibody or antigen-binding fragment thereof, or pharmaceutical composition according to the present invention in the preparation of a medicament for treating a disease or condition, wherein the disease or condition is selected from cancer.

[0036] In a specific embodiment, the cancer of the present invention is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer (such as leukemia, lymphoma or myeloma), laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The binding of VSIG4 mouse antibody to hVSIG4-Fc and hVSIG4-short-Fc detected by ELISA is shown.

[0038] Figure 2A and Figure 2B The chimeric antibody is shown to bind to A33 and JAM-A proteins. Figure 2A The binding of VSIG4 chimeric antibody to A33 protein detected by ELISA is shown; Figure 2B The binding of VSIG4 chimeric antibody to JAM-A protein detected by ELISA is shown.

[0039] Figures 3A to 3C Binding of the chimeric antibody to CHOK1-human / cynomolgus monkey / mouse VSIG4 cells is shown. Figure 3A Shows the binding of VSIG4 chimeric antibody to CHOK1-human VSIG4 cells detected by flow cytometry; Figure 3B Shows the binding of VSIG4 chimeric antibody to CHOK1-cynomolgus monkey VSIG4 cells detected by flow cytometry; Figure 3C Shown is the binding of VSIG4 chimeric antibody to CHOK1-mouse VSIG4 cells detected by flow cytometry.

[0040] Figures 4A to 4B Experiment showing receptor blockade of chimeric antibodies with hC3b.Figure 4A The binding of VSIG4 chimeric antibody to hVSIG4-his protein detected by RBA competition assay is shown; Figure 4B Shows the competition binding experiment between VSIG4 chimeric antibody, C3b and human VSIG4 recombinant protein.

[0041] Figures 5A to 5E Binding of humanized antibodies to CHOK1-human / cynomolgus monkey / mouse VSIG4 cells is shown. Figure 5A Shows the binding of Hu004 series antibodies to CHOK1-human VSIG4 cells; Figure 5B Shows the binding of Hu023 series antibodies to CHOK1-human VSIG4 cells; Figure 5C Shows the binding of Hu023 series antibodies to CHOK1-cynomolgus monkey VSIG4 cells; Figure 5D Shows the binding of Hu004 series antibodies to CHOK1-cynomolgus monkey VSIG4 cells; Figure 5E Binding of Hu023 series antibodies to CHOK1-mouse VSIG4 cells is shown.

[0042] Figure 6 Showing the effect of humanized antibodies on T cell tumor killing.

[0043] Figures 7A to 7C Showing the effect of humanized antibodies on tumor growth in mice. Figure 7A Shows the changes in mouse body weight over time after antibody administration, with the abscissa indicating the number of days after antibody injection; Figure 7B The changes in tumor volume of mice over time after antibody administration are shown, with the abscissa representing the number of days after antibody injection; Figure 7C Shown are the changes in tumor weights of mice after antibody injection compared to isotype controls at day 21 after injection. DETAILED DESCRIPTION

[0044] In a specific embodiment, the present invention provides an anti-VSIG4 antibody or an antigen-binding fragment thereof that specifically binds to VSIG4, comprising:

[0045] HCDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79 and 85,

[0046] HCDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80 and 86,

[0047] HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81 and 87,

[0048] LCDR1 comprises an amino acid sequence shown in any one of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82 and 88,

[0049] LCDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83 and 89, and

[0050] LCDR3 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84 and 90,

[0051] The above CDR sequences are determined according to the Kabat numbering system.

[0052] In a specific embodiment, the present invention provides an anti-VSIG4 antibody or an antigen-binding fragment thereof that specifically binds to VSIG4, comprising:

[0053] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 136 or 142,

[0054] HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 137 or 143,

[0055] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 138 or 144,

[0056] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 139 or 145,

[0057] LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 140 or 146, and

[0058] LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 141 or 147,

[0059] The above CDR sequences are determined according to the IMGT numbering system.

[0060] In a specific embodiment, the present invention provides an anti-VSIG4 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:

[0061] i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively,

[0062] ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8 and 9, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively,

[0063] iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14 and 15, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17 and 18, respectively,

[0064] iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 19, 20 and 21, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 23 and 24, respectively,

[0065] v) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 26 and 27, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28, 29 and 30, respectively,

[0066] vi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 34, 35 and 36, respectively,

[0067] vii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 38 and 39, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 40, 41 and 42, respectively,

[0068] viii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 43, 44 and 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 46, 47 and 48, respectively,

[0069] ix) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 49, 50 and 51, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 52, 53 and 54, respectively,

[0070] x) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 55, 56 and 57, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 58, 59 and 60, respectively,

[0071] xi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 61, 62 and 63, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 64, 65 and 66, respectively,

[0072] xii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 67, 68 and 69, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 70, 71 and 72, respectively,

[0073] xiii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 73, 74 and 75, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 76, 77 and 78, respectively,

[0074] xiv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 79, 80 and 81, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 82, 83 and 84, respectively, or

[0075] xv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 85, 86 and 87, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 88, 89 and 90, respectively.

[0076] In a specific embodiment, the anti-hVSIG4 antibody provided by the present invention is a mouse antibody. In a specific embodiment, the anti-hVSIG4 antibody further comprises a light chain framework region (FR region) or a light chain constant region of a mouse κ, λ chain or a variant thereof. In some embodiments, the anti-hVSIG4 antibody further comprises a heavy chain framework region (FR region) or a heavy chain constant region of a mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0077] In a specific embodiment, the antigen-binding fragment provided by the present invention is selected from Fab, Fab', (Fab')2, Fv, scFv (sc means single chain), Bis-scFv, scFv-Fc fragment, Fab2, Fab3, miniantibodies, diabodies, triabodies, tetrabodies and nanoantibodies.

[0078] In a specific embodiment, an anti-VSIG4 antibody or an antigen-binding fragment thereof is provided, wherein the anti-VSIG4 antibody or the antigen-binding fragment thereof comprises:

[0079] 1) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:91, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:92;

[0080] 2) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:93, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:94;

[0081] 3) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:95, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:96;

[0082] 4) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:97, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:98,

[0083] 5) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:99, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:100;

[0084] 6) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 101, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 102;

[0085] 7) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 103, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 104;

[0086] 8) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 105, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 106,

[0087] 9) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 107, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 108;

[0088] 10) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 109, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 110;

[0089] 11) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 111, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 112;

[0090] 12) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 113, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 114,

[0091] 13) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 115, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 116;

[0092] 14) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 117, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 118; or

[0093] 15) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:119, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:120.

[0094] In a specific embodiment, an anti-VSIG4 antibody or an antigen-binding fragment thereof is provided, wherein the anti-VSIG4 antibody or the antigen-binding fragment thereof comprises:

[0095] 1) a heavy chain variable region VH as shown in SEQ ID NO:91, and a light chain variable region VL as shown in SEQ ID NO:92;

[0096] 2) a heavy chain variable region VH as shown in SEQ ID NO:93, and a light chain variable region VL as shown in SEQ ID NO:94;

[0097] 3) a heavy chain variable region VH as shown in SEQ ID NO:95, and a light chain variable region VL as shown in SEQ ID NO:96;

[0098] 4) a heavy chain variable region VH as shown in SEQ ID NO:97, and a light chain variable region VL as shown in SEQ ID NO:98;

[0099] 5) the heavy chain variable region VH shown in SEQ ID NO:99, and the light chain variable region VL shown in SEQ ID NO:100;

[0100] 6) the heavy chain variable region VH shown in SEQ ID NO: 101, and the light chain variable region VL shown in SEQ ID NO: 102;

[0101] 7) the heavy chain variable region VH shown in SEQ ID NO: 103, and the light chain variable region VL shown in SEQ ID NO: 104;

[0102] 8) the heavy chain variable region VH shown in SEQ ID NO: 105, and the light chain variable region VL shown in SEQ ID NO: 106;

[0103] 9) the heavy chain variable region VH shown in SEQ ID NO: 107, and the light chain variable region VL shown in SEQ ID NO: 108;

[0104] 10) a heavy chain variable region VH as shown in SEQ ID NO: 109, and a light chain variable region VL as shown in SEQ ID NO: 110;

[0105] 11) a heavy chain variable region VH as shown in SEQ ID NO: 111, and a light chain variable region VL as shown in SEQ ID NO: 112;

[0106] 12) a heavy chain variable region VH as shown in SEQ ID NO: 113, and a light chain variable region VL as shown in SEQ ID NO: 114;

[0107] 13) the heavy chain variable region VH shown in SEQ ID NO: 115, and the light chain variable region VL shown in SEQ ID NO: 116;

[0108] 14) the heavy chain variable region VH shown in SEQ ID NO: 117, and the light chain variable region VL shown in SEQ ID NO: 118; or

[0109] 15) The heavy chain variable region VH shown in SEQ ID NO:119, and the light chain variable region VL shown in SEQ ID NO:120.

[0110] In a specific embodiment, the present invention provides a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof, wherein the anti-VSIG4 antibody or the antigen-binding fragment thereof comprises:

[0111] A heavy chain variable region VH comprising an amino acid sequence as shown in any one of SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of the amino acid sequences shown therein; and

[0112] A light chain variable region VL, which comprises the amino acid sequence shown in any one of SEQ ID NO:127 and SEQ ID NO:128, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one of them.

[0113] In a specific embodiment, the present invention provides a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof, wherein the anti-VSIG4 antibody or the antigen-binding fragment thereof comprises:

[0114] 1) a heavy chain variable region VH as shown in SEQ ID NO: 121, and a light chain variable region VL as shown in SEQ ID NO: 127;

[0115] 2) a heavy chain variable region VH as shown in SEQ ID NO: 121, and a light chain variable region VL as shown in SEQ ID NO: 128;

[0116] 3) a heavy chain variable region VH as shown in SEQ ID NO: 122, and a light chain variable region VL as shown in SEQ ID NO: 127;

[0117] 4) a heavy chain variable region VH as shown in SEQ ID NO: 122, and a light chain variable region VL as shown in SEQ ID NO: 128;

[0118] 5) the heavy chain variable region VH shown in SEQ ID NO: 123, and the light chain variable region VL shown in SEQ ID NO: 127;

[0119] 6) the heavy chain variable region VH shown in SEQ ID NO: 123, and the light chain variable region VL shown in SEQ ID NO: 128;

[0120] 7) the heavy chain variable region VH shown in SEQ ID NO: 124, and the light chain variable region VL shown in SEQ ID NO: 127;

[0121] 8) the heavy chain variable region VH shown in SEQ ID NO: 124, and the light chain variable region VL shown in SEQ ID NO: 128;

[0122] 9) the heavy chain variable region VH shown in SEQ ID NO: 125, and the light chain variable region VL shown in SEQ ID NO: 127;

[0123] 10) a heavy chain variable region VH as shown in SEQ ID NO: 125, and a light chain variable region VL as shown in SEQ ID NO: 128;

[0124] 11) the heavy chain variable region VH shown in SEQ ID NO: 126, and the light chain variable region VL shown in SEQ ID NO: 127; or

[0125] 12) The heavy chain variable region VH shown in SEQ ID NO:126, and the light chain variable region VL shown in SEQ ID NO:128.

[0126] In another aspect, the present invention provides a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof, wherein the anti-VSIG4 antibody or the antigen-binding fragment thereof comprises:

[0127] A heavy chain variable region VH comprising an amino acid sequence as shown in any one of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any of the amino acid sequences shown therein; and

[0128] A light chain variable region VL, which comprises the amino acid sequence shown in any one of SEQ ID NO:133, SEQ ID NO:134 and SEQ ID NO:135, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any of the amino acid sequences shown therein.

[0129] In a specific embodiment, the present invention provides a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof, wherein the anti-VSIG4 antibody or the antigen-binding fragment thereof comprises:

[0130] 1) a heavy chain variable region VH as shown in SEQ ID NO: 129, and a light chain variable region VL as shown in SEQ ID NO: 133;

[0131] 2) a heavy chain variable region VH as shown in SEQ ID NO: 129, and a light chain variable region VL as shown in SEQ ID NO: 134;

[0132] 3) a heavy chain variable region VH as shown in SEQ ID NO: 129, and a light chain variable region VL as shown in SEQ ID NO: 135;

[0133] 4) a heavy chain variable region VH as shown in SEQ ID NO: 130, and a light chain variable region VL as shown in SEQ ID NO: 133;

[0134] 5) a heavy chain variable region VH as shown in SEQ ID NO: 130, and a light chain variable region VL as shown in SEQ ID NO: 134;

[0135] 6) the heavy chain variable region VH shown in SEQ ID NO: 130, and the light chain variable region VL shown in SEQ ID NO: 135;

[0136] 7) the heavy chain variable region VH shown in SEQ ID NO: 132, and the light chain variable region VL shown in SEQ ID NO: 133;

[0137] 8) the heavy chain variable region VH shown in SEQ ID NO: 132, and the light chain variable region VL shown in SEQ ID NO: 134; or

[0138] 9) the heavy chain variable region VH shown in SEQ ID NO:132, and the light chain variable region VL shown in SEQ ID NO:135.

[0139] the term

[0140] In order to make the present disclosure more easily understood, 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 those skilled in the art to which the present disclosure belongs.

[0141] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0142] The term "antibody" as described in the present disclosure refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have a κ chain or a λ chain.

[0143] The sequences of about 110 amino acids near the N-terminus of the heavy and light chains of antibodies vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.

[0144] "Complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are numbered sequentially from the N-terminus and are usually referred to as CDR1, CDR2, and CDR3. The CDRs located in the variable domain of the heavy chain of an antibody are also referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, its CDR sequence can be determined by a variety of schemes known in the art, such as: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The CDRs of the antibody were defined using the International Immunogenetics Information System (IMGT) database (International Immunogenetics Information System, World Wide Web imgt.cines.fr / ), and North's CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

[0145] For example, the different definition ranges of the CDR regions numbered by Kabat and Chothia et al. are used.

[0146] As used herein, the term "antigen-binding fragment" refers to a molecule different from an intact antibody, which comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies (dAb); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies (single-domain antibodies); antigen-binding fragments of bivalent or bispecific antibodies; camelid antibodies; and other fragments that exhibit the desired ability to bind to an antigen (e.g., VSIG4).

[0147] The term "murine antibody" in the present disclosure refers to a monoclonal antibody against human VSIG4 prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with the antigen of VSIG4, and then the hybridoma expressing the antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present disclosure, the murine VSIG4 antibody or its antigen-binding fragment may further comprise the light chain constant region of murine κ, λ chain or its variants, or further comprise the heavy chain constant region of murine IgG1, IgG2, IgG3 or IgG4 or its variants.

[0148] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody of a first species (such as mouse) with the constant region of an antibody of another species (such as human), which can reduce the immune response induced by heterologous antibodies. To establish a mouse-human chimeric antibody, it is necessary to select a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of human antibodies. The mouse variable region genes and human constant region genes are connected into chimeric genes and inserted into human vectors, and finally the chimeric antibody molecules are expressed in eukaryotic or prokaryotic industrial systems. The constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG1 or IgG2 heavy chain constant region.

[0149] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. It can overcome the strong immune response induced by chimeric antibodies due to carrying a large amount of foreign protein components. In order to avoid a decrease in activity while reducing immunogenicity, the variable region of the antibody can be subjected to minimal reverse mutation to maintain activity.

[0150] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. An epitope usually includes at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis, etc. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, etc.

[0151] The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in ligand binding affinity when in contact with an anti-VSIG4 antibody compared to a ligand not in contact with an anti-VSIG4 antibody.

[0152] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecμlar Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity. Common conservative substitutions of amino acids are as follows:

[0153] Table 1

[0154]

[0155]

[0156] As used herein, the term "multispecific antibody" refers to a protein molecule that is capable of specifically binding to two or more target antigens or target antigen epitopes, comprising an antibody or an antigen-binding fragment of an antibody (such as a single-chain antibody). The term encompasses, for example, bispecific antibodies, trispecific antibodies, etc.

[0157] As used herein, the term "cytotoxic agent" refers to a chemical molecule that has a strong activity of destroying the normal growth of target cells (e.g., tumor cells). In principle, cytotoxic agents can kill cells at sufficiently high concentrations, but due to the lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, leading to serious side effects. The term includes small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals, radioactive isotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0158] An "effective amount" includes an amount sufficient to improve or prevent a medical symptom or condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular described or veterinary subject may vary depending on factors such as the condition to be treated, the described general health, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0159] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid residue (for example, if every position of the two DNA molecules is occupied by adenine), then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.

[0160] The following examples are used to further describe the present invention, but the present invention is not limited to the scope of the examples. The experimental methods in the examples of the present invention that do not specify specific conditions are usually carried out according to conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents that do not specify the specific source are conventional reagents purchased from the market.

[0161] Example

[0162] Example 1. Screening of monoclonal antibodies (mAbs) with high specificity and affinity for human / cynomolgus monkey / mouse VSIG4 by hybridoma technology.

[0163] Four groups of mice (3 groups of SJL mice and 1 group of Balb / c mice) were immunized with different VSIG4 immunogens (HEK293F-hVSIG4 cells and hVSIG4 long / short isotype Fc proteins). The serum antibody titers of the immunized mice were detected by FACS and ELISA. Among them, FACS was detected by CHOK1-hVSIG4 and CHOK1-blank control cells, and ELISA was detected by hVSIG4-ECD-hFc protein. According to the results of FACS and ELISA, mice with high immune titers were selected, their spleen cells were obtained, and fused with myeloma SP2 / 0 cells. In this embodiment, a total of 3 fusion cells were screened, and the positive hybridoma clones obtained by Acumen were transferred to 24-well plates for culture, and rescreened by ELISA / FACS combination, cellular level VSIG4-Siglec-7 blocking experiment and PBMC proliferation experiment, and positive clones were selected for subcloning. Subsequently, the primary screening and rescreening methods were used to detect again, and finally the positive monoclonal clones were selected for antibody production and frozen storage.

[0164] The purified mouse monoclonal antibodies were tested for binding to CHOK1-human / cynomolgus monkey / mouse VSIG4 cells by FACS, and all mouse antibodies were epitope grouped by competitive ELISA.

[0165] The CDR sequences of the obtained mouse monoclonal antibodies are shown in Table 2 below:

[0166] Table 2. CDR sequences of mouse monoclonal antibodies

[0167]

[0168]

[0169]

[0170]

[0171] The above CDR sequences are determined according to the Kabat numbering system.

[0172] Table 3. CDR sequences of mouse monoclonal antibodies

[0173]

[0174] The above CDR sequences are determined according to the IMGT numbering system.

[0175] The heavy chain and light chain variable regions of the above-mentioned mouse monoclonal antibody are as follows, wherein the underlined portion represents the CDR region:

[0176] 67F11D3

[0177] Heavy chain variable region

[0178] EVQLQQSGPELVKPGASVKMSCKASGYTFT DYNMH WVKQSHGKSLEWIG YIIPNSGDADYNQKFKD KATLTVNKSSSTAYMELRRSLTSEDSTVYYCTR SAGGHFDY WGQGTTLTVSS (SEQ ID NO:91)

[0179] Light chain variable region

[0180] EVVLTQSPTTMAAFPGEKITITC SASSSISSNYLH WYQQKPGFSPKLLIY RTSNLAS GVPARFSGSGSGTSYSLTIGTMEAEDVATYYC QQGSNIPRT FGGGTKLEIK (SEQ ID NO:92)

[0182] 7D12B1

[0183] Heavy chain variable region

[0184] QVQLRQPGAELVKPGASVKMSCKASGDTFT TYWIT WVKQRPGQGLEWIG DTYPGSGSTNYNEKFKS KATLTVDTSSSTAYMQLSSLTSEDSAVYYCAR SRDYYDSSYPYWYFDV WGTGTTVIVSS (SEQ ID NO:93)

[0185] Light chain variable region

[0186] DIQMTQTTSSLSASLGDRVTISC SASQGISNYLN WYQQKPDGTVKLLIY YTSSLHS GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QQYSKLPYT FGGGTKLEIK (SEQ ID NO:94)

[0188] 33A6B10

[0189] Heavy chain variable region

[0190] QVQLKESGPGLVAPSQSLSITCTVSGFSLTRNGIS WIRQPPGKGLEWLG VIWGDGSAHYNSALIS RLNITKDNSKSQVFLKLSSLQTDDTATYYCAN LYR WGQGTSLIVSS (SEQ ID NO:95)

[0192] Light chain variable region

[0193] NIMMTQSPKSLSMSVGERVTLSC KASENVGTYVS WYQQKPEQSPKVLIY GASNRYT GVPDRFTGSGSATDFTLTISSVQAEDLADYFC GQSSRYPYT FGGGTKLEMK (SEQ ID NO:96)

[0195] 16E3B10(004)

[0196] Heavy chain variable region

[0197] QVQLQQPGAELVRPGSSVKLSCKASGYTFT IYWMD WVKQRPGQGLEWIG NINPSDSETHYNQKFKD KATLTVDKSSNTAYMQLSSLTSEDSAVYYCTR PGQAGGFAY WGQGTLVTVSA (SEQ ID NO:97) Light chain variable region

[0199] DIQMTQSPASLSVSVGETVTITC RASENIYSNLG WYQQKQGKSPQLLVY AATNLAD GVPSRFSGSGSGTQFSLKINSLQSEDFGTYYC QHFWVTPWT FGGGTNLEIK (SEQ ID NO:98)

[0201] 27G7B7

[0202] Heavy chain variable region

[0203] QVQLQQSGAELAKPGASVKLSCKASGYTFT SYWIH WVKQRPGQGLEWIG YISPNNYYTEYNQKFKD KATLTADRSSSTAYMQLSSLTFEDSAVYYCTR YDGYYVRNLDY WGQGTTLTVSS (SEQ ID NO:99)

[0205] Light chain variable region

[0206] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPLT FGAGTKLELK (SEQ ID NO: 100)

[0208] 20G4D11

[0209] Heavy chain variable region

[0210] QVQLQQSGAELAKPGASVKLSCKASGYTFT SYWMH WIKQRPGQGLEWIG YINPNSGFGEYNWKFKH KATMTADKTSRTAYMQLSSLTYEDSAVYYCAR YDGYYVRHLDY WGQGTTLTVSS (SEQ ID NO: 101)

[0212] Light chain variable region

[0213] DAVMTQTPLSLPVSLGDQASISC RSSQSLEHSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPLT FGAGTKLELK (SEQ ID NO: 102)

[0215] 39B11G3

[0216] Heavy chain variable region

[0217] EVKLVESGGVLVQPGGSLKLSCAASGFTFS DYGMA WVRQAPRKGPEWVA FISNLAYSIYYADTVTG RFTISRENAKNTLYLEMSSLRSEDTAIYYCAR LYDGYYADALDY WGQGTSVTVSS (SEQ ID NO: 103)

[0219] Light chain variable region

[0220] EIVLTQSPTTMAASPGEKITITC SASSSLNSDYLH WYQQKPGFSPKFLIY GTSNLAS GVPARFRGSGSGTSYSLTIVTMEAEDVATYYC HQGSSLPLT FGAGTKLELK (SEQ ID NO: 104)

[0222] 8A6E1

[0223] Heavy chain variable region

[0224] EVKLVESGGGLVQPGGSLKLSCAASGFTFS DYGMA WVRQAPRKGPEWVA FISNLAYSIYYADTVTG RFTISRENAKNTLYLEMSSLRSEDTAMYYCAR GGYYYAMDY WGQGTSVTVSS (SEQ ID NO: 105)

[0226] Light chain variable region

[0227] DIQMTQSPASLSASVGETVTITC RASENIYSYLA WYQQKQGKSPQLLVY NAKTLAE GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QHYYGTPLT FGAGTKLELK (SEQ ID NO: 106)

[0229] 15D8G7

[0230] Heavy chain variable region

[0231] EVQLQQSGAELVRPGASVKLSCTASGFNIK DDFMH WVKQRPEQGLEWIG WIDPENGDTEYASKFQG KATITADTSSNTAYLQLSSLTSEDTAVYYCTK TTVVARYFDY WGQGTTLTVSS (SEQ ID NO: 107)

[0233] Light chain variable region

[0234] DILLTQSPAILSVSPGERVSFSC RASQSIGTSIH WYQRRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIGDYYC QQTNSWPTT FGSGTKLEIK (SEQ ID NO: 108)

[0236] 22G5C4

[0237] Heavy chain variable region

[0238] EVKVEESGGGLVQPGGSMKLSCVASGVTFS NYWMNWVRQSPEKGLEWVAQIRLKSDNSATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTG RGYDYDGFYFDY WRQGTTLTVSS (SEQ ID NO: 109)

[0240] Light chain variable region

[0241] DIVITQDELSNPVTSGESVSISC RSSKSLLYKDGKTYLN WFLQRPGQSPQLLIY LMSTRAS GVSDRFSGSGSGTDFTLEISRVKAEDVGVYYC QQLVEYPLT FGAGTKLELK (SEQ ID NO: 110)

[0243] 11E7E7

[0244] Heavy chain variable region

[0245] QVQLKESGPGLVAPSQSLSITCTVSGFSLT TNGVS WVRQPPGKGLEWLG VIWGDGTTSYHSALIS RLSISKDNSKSQVFLKLNSLQSADTATYYCAK GPY WGQGTLVTVSA (SEQ ID NO: 111)

[0247] Light chain variable region

[0248] ENVLTQSPAIMSASPGEKVTMTC RASSSVSSSYLH WYQQKSGASPKVWIY STSNLAS GVPARFSGSGSGTSYSLTISSVETEDAATYYC LQYSGYPYT FGGGTKLEIK (SEQ ID NO: 112)

[0250] 58F8-2E9

[0251] Heavy chain variable region

[0252] EVQLQQSGPELVKPGASVKISCKASGYTFT DYYMN WVKQSHGKSLEWIG DINPNNGGTNYNQKFKD KATLTVDKSSSTAYMELRRSLTSEDSAVYYCAR NGNYHRYFDY WGQGTTLTVSS (SEQ ID NO: 113)

[0254] Light chain variable region

[0255] QIVLSQSPAILSASPGEKVTMTC RASSSVRYMY WYQQKPGSSPKPWIY ATSNLAS GVPTRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPRT FGGGTKLEIK (SEQ ID NO: 114)

[0257] 56E1B3

[0258] Heavy chain variable region

[0259] QVQLQQSGPELVKPGASVKISCKASGYSFT RYYIH WVKQRPGQGLEWIG WIYPGNGNTKYNEKFKD KATLTADTSSSTAYMQLSSQTSEDSAVYYCAR EGYVYEDAMDY WGQGTSVTVSS (SEQ ID NO: 115)

[0261] Light chain variable region

[0262] QIVLSQSPAILSASPGEKVTMTC RASSSVDYIH WYQQKPGSSPKPWIY ATSNLAS GVPARFSGSGSGTSYSLTIGRVEAEDAATYYC QQWSSNPRT FGGGTKLEIK (SEQ ID NO: 116)

[0264] 46D2E1

[0265] Heavy chain variable region

[0266] EVRLQQSGAELVRPGASVKLSCTASGFNIK DDFMY WVKQRPEQGLEWLG WIDPETGDTEYASKFQG KATITADTISNTAYLQLSSLTSEDTAVYYCTS WLLREY WGQGTTLTVSS (SEQ ID NO: 117)

[0268] Light chain variable region

[0269] QIVLTQSPAIMSASPGEKVTMTC SASSSISYMH WYQQKPGTSPKKWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC HQRSSYPYT FGGGTKLEIK (SEQ ID NO: 118)

[0271] 73G11G3

[0272] Heavy chain variable region

[0273] EVKLVESEGGLVQPGSSMKLSCTASGFTFS DYYMA WVRQVPEKGLEWVA NINYDGSIIYYLDSLKS RFIISRDNAKNILYLQMSSLKPEDTATYHCAR GGSGYVHFDY WGQGTTLTVSS (SEQ ID NO: 119)

[0275] Light chain variable region

[0276] YIQMTQTTSSLSASLGDRVTISC RASQDISNYLN WYQQKPDGTIKLLIY YTSKLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGFTFPRT FGGGTNLEII (SEQ ID NO: 120)

[0278] Positive control antibody A2 is a humanized antibody from WO2020 / 069507.

[0279] Example 2. Protein production steps and cell line construction

[0280] 1) Protein production

[0281] Plasmids (human VSIG4-ECD-hFc: human VSIG4 (Arg20-Pro283) with a linker (IEGRMD (SEQ ID NO: 150)) and a C-terminal Fc (Ref seq: Q9Y279); human VSIG4 short isoform hFc: human VSIG4 (Arg20-Pro189) with a linker (IEGRMD) and a C-terminal Fc (Ref seq: Q9Y279-3); human VSIG4-L-ECD-his: human VSIG4 (Arg20-Pro283) with a linker (IEGRMD) and a C-terminal his tag (Ref seq: Q9Y279); human Siglec-7-hFc: human Siglec-7 (Gln19-Gly357) with a linker (DIEGRMD) and a C-terminal Fc (Ref seq: Human Siglec-7 (Gln19-Gly357) with a linker (DIEGRMD (SEQ ID NO: 151)) and C-terminal Fc); The positive control antibody A2 (humanized antibody from WO2020 / 069507) was added to CD FortiCHO medium and filtered (0.22 μm). PEI diluted as shown in the table above was added to the DNA solution, mixed quickly, and incubated at room temperature for 20 minutes.

[0282] Pour the DNA-PEI mixture into HEK293 cells and culture them in a shaker at 37°C, 8% CO2, and 130 rpm / min. Add 50 mL / L 10% peptone 24 hours after transfection and continue to culture in a shaker at 37°C, 8% CO2, and 130 rpm / min. Seven days after transfection, collect the culture fluid for purification.

[0283] The protein was purified by Ni-Excel column or Superdex 75 column; the target protein was collected and concentrated; the concentrated protein was subjected to SDS-PAGE analysis, endotoxin detection and SEC-HPLC analysis.

[0284] The ELISA test results showed that human VSIG4-ECD-hFc, human VSIG4-ECD-His and glycosylated hVSIG4-ECD-his proteins could all bind strongly to the positive control antibody A2, human VSIG4 short isoform hFc protein could bind to anti-VSIG4 polyclonal antibody, and Siglec-7-hFc protein could bind to CHOK1-hVSIG4 cells. The above reagents can be used for subsequent immune and antibody detection.

[0285] 2) Cell line construction

[0286] The target plasmids PLVX-CMV-human VSIG4-IRES-puro, PLVX-CMV-mouse VSIG4-IRES-puro and PLVX-CMV-cynomolgus monkey VSIG4-IRES-puro were all obtained from CP. Using 293T as the host cell, the target plasmid was transferred to 293T cells by transient transfection. The cell culture supernatant was collected 48h and 72h after transfection, which was the target virus and concentrated to 1E8 EU / mL. The CHOK1 and 293F cells cultured in advance were digested and plated to 6-well cell culture plates at 1E5 / well to allow the cells to adhere overnight. The viruses obtained in the above steps were added to the cells in the 6-well plate that had been attached in advance at increasing concentrations (100μL, 200μL): CHOK1 cells were added with human / cynomolgus monkey / mouse VSIG4 plasmids, and 293F cells were added with human VSIG4 plasmids. After infection, the cells are tested for antigen expression and subcloned using a flow cytometer. Monoclones after subcloning are selected for antigen expression, and positive monoclones are expanded and cultured as cell lines for later experiments.

[0287] Lentiviral plasmids were used to transfect 293T cells for the production and packaging of specific lentiviruses, and CHOK1 and 293F cells were infected respectively to obtain stable cell lines with specific and high expression of VSIG4 (CHOK1-human VSIG4, CHOK1-cynomolgus monkey VSIG4, CHOK1-mouse VSIG4 and 293F-human VSIG4), which were used for subsequent hybridoma screening and antibody identification.

[0288] 3) Screening results of mouse antibody subclones

[0289] Table 4. FACS screening results

[0290]

[0291]

[0292] Table 5. RBA and ELISA screening

[0293]

[0294] Table 6. PBMC proliferation screening

[0295]

[0296]

[0297] The results showed that the MFI of 33A6B10 (mAb002), 27G7B7 (mAb005), 20G4D11 (mAb007), 20F11A4 (mAb010), 15D8G7 (mAb015), and 11E7E7 (mAb020) binding to CHOK1-human VSIG4 were higher than the control A2 antibody; the MFI of 56E1B3 (mAb027) and 46D2E1 (mAb028) binding to CHOK1-human VSIG4 were higher than the control A2 antibody, and all subclones promoted CD8+T cell proliferation more than the control A2 antibody. At the same time, in the F0720 fusion subclone, except 8A6E1 (mAb012), other monoclonal antibodies can effectively block the binding of Siglec-7 to 293F-human VSIG4 cells. The results showed that the monoclonal antibodies produced were of high purity and quality and were suitable for subsequent experiments.

[0298] Example 3. Purification and identification of mouse antibodies

[0299] After the antibodies were purified by Mabselect SuRe, the binding activity of mouse antibodies to proteins was detected by ELISA.

[0300] In brief, human VSIG4-ECD-hFc and human VSIG4-short-hFc proteins were diluted to 1 μg / ml with PBS, added to ELISA microplates at 100 μL / well, and incubated at 4°C overnight. Blocked with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4, the percentage is mass percentage) at 37°C for 2h. Add 20nM antibody, 100 μL / well, and incubate at 37°C for 1h. Wash the plate 3 times, add secondary antibody anti-mouse IgG (Fab specific)-HRP (Sigma, A3682, 1:5000 dilution), 100 μL / well, and incubate at 37°C for 1h. Wash the plate 3 times, add 100 μL / well TMB colorimetric solution (Huzhou Yingchuang Biotechnology, EL0009), incubate at 37°C for 10min, and then add 50 μL 1N hydrochloric acid to terminate the color reaction. Read the OD450nm value with an ELISA plate reader.

[0301] The results are shown in Figure 1 middle.

[0302] Figure 1The results showed that all antibodies had strong binding affinity with hVSIG4-ECD-hFc protein, OD450nm>3.0. Compared with the control A2 antibody, all antibodies except mAb004 had strong binding affinity with hVSIG4-short-Fc protein, OD450nm>3.0. mAb004, mAb009 and mAb019 antibodies could bind to the IgC2 domain of hVSIG4-ECD-Fc protein.

[0303] Example 4. Preparation of chimeric antibodies and their binding activity

[0304] Chimeric antibodies were produced based on the selected mouse antibodies, and their binding to CHOK1-human / cynomolgus monkey / mouse VSIG4 cells was detected by FACS, their binding to A33 (glycoprotein A33) and JAM-A (Junctional adhesion molecule A) proteins was detected by ELISA, and the blocking experiment of hVSIG4-his / hC3b was detected by ELISA.

[0305] Preparation of chimeric antibodies

[0306] The constant region sequences used in chimeric and humanized antibodies are shown below:

[0307] Heavy chain constant region

[0308] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 148

[0309] Light chain constant region

[0310] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 149

[0311] 4.1 ELISA method to detect the binding activity of chimeric antibodies to A33 and JAM-A proteins

[0312] Briefly, A33 or JAM-A was diluted to 1 μg / ml with PBS, added to ELISA microplate at 100 μL / well, and incubated at 4°C overnight. Block with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4) at 4°C overnight. After washing the plate, add 100 μL of the diluted antibody to be tested and incubate at 37°C for 1 hour. Wash the plate 3 times, add secondary antibody (1:5000 dilution), 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate 3 times, add 100 μL / well TMB colorimetric solution (Huzhou Yingchuang Biotechnology, EL0009), incubate at 37°C for 10 minutes, and then add 50 μL 1N hydrochloric acid to terminate the color development reaction. Read the OD450nm value using an ELISA plate reader. The results are shown in Figure 2A and Figure 2B middle.

[0313] A33 and JAM-A proteins are on the same evolutionary tree branch as VSIG4 proteins, so it is necessary to verify whether anti-VSIG4 antibodies specifically bind to VSIG4. ELISA binding experiments showed that all chimeric antibodies showed no binding activity to A33 and JAM-A, indicating that these chimeric antibodies specifically bind to VSIG4 but not to A33 and JAM-A.

[0314] 4.2 FACS detection of binding between chimeric antibodies and cells

[0315] According to the SOP of cell culture, CHOK1-human / cynomolgus monkey / mouse VSIG4 stable cell lines and CHOK1-blank cells were cultured and treated. The digested cells were centrifuged at 300g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed twice with FACS buffer (PBS + 2% FBS). 1-2E5 cells / well were placed in a 96-well round-bottom plate (Corning, Cat. No. 3799), the supernatant was discarded after centrifugation, and the gradient dilution of antibodies was added, with a starting concentration of 200nM, 5-fold dilution, 8 concentration points including 0 point, 100μl / well, mixed, and incubated at 4℃ for 1h. Centrifuge at 4℃, 300g for 5min, discard the supernatant solution, wash the cells twice with FACS buffer, add the corresponding secondary antibody solution, 100μl / well, and incubate at 4℃ for 1h. Centrifuge at 4℃, 300g for 5min, discard the supernatant solution, and wash the cells twice with FACS buffer. Finally, the cells were resuspended in FACS buffer, the signal value was detected on a BD flow cytometer, and the curve was fitted using GraphPad Prism6 to calculate the EC 50 value.

[0316] The results are shown in Figures 3A - 3C middle.

[0317] All chimeric antibodies showed binding to CHOK1-human / cynomolgus monkey VSIG4 cells, among which cAb002, 005, 007, 020, 028 had significantly higher MFI values ​​than control antibody A2. In addition, cAb012, 015, 017-1,023, 030 showed binding to CHOK1-mouse VSIG4 cells.

[0318] 4.3 ELISA detection of receptor ligand blocking experiment

[0319] Pre-experimental plan

[0320] Dilute human C3b to 2.5 μg / mL with PBS, add 100 μL / well to ELISA microplate, and incubate overnight at 4°C. Block with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4, the percentage is mass percentage) at 4°C overnight. After washing the plate, add diluted hVSIG4-His antibody, the starting concentration is 30 μg / mL, 3-fold dilution, 12 concentration points (including 0 point), 100 μl / well, and incubate at 37°C for 1h. Wash the plate 3 times, add secondary antibody (1:5000 dilution), 100 μL / well, and incubate at 37°C for 1h. Wash the plate 3 times, add 100 μL / well TMB colorimetric solution (Huzhou Yingchuang Biotechnology, EL0009), incubate at 37°C for 10min, and then add 50 μL 1N hydrochloric acid to terminate the color reaction. Read the OD450nm value with an ELISA plate reader.

[0321] Receptor ligand blocking assay

[0322] Dilute human C3b to 2.5 μg / mL with PBS, add 100 μL / well to ELISA microplate, and incubate overnight at 4°C. Block with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4, the percentage is mass percentage) at 4°C overnight. After washing the plate, add the diluted chimeric antibody first, the starting concentration is 100nM, 5-fold dilution, 8 concentration points (including 0 point), 50 μL / well, and then add 50 μL of hVSIG4-His diluted to 5 μg / mL to each well, incubate at 37°C for 1h. Wash the plate 3 times, add the secondary antibody Anti-hisHRP (1:5000 dilution), 100 μL / well, and incubate at 37°C for 1h. The plate was washed three times, 100 μL / well TMB colorimetric solution (Huzhou Yingchuang Biotechnology, EL0009) was added, incubated at 37°C for 10 min, and 50 μL 1N hydrochloric acid was added to terminate the colorimetric reaction. The OD450nm value was read using an ELISA plate reader.

[0323] The experiment shows Figures 4A to 4B middle.

[0324] When the chimeric antibody concentration was 100 nM, seven chimeric antibodies (cAb001, 011, 015, 017, 023, 027, 030) could block the interaction between hVSIG4-his and hC3b.

[0325] Example 5. Preparation of humanized antibodies and their binding activity

[0326] Based on the results of chimeric antibody detection, two chimeric antibodies (cAb004 and cAb023) were selected for humanized design, and the binding of all humanized variants to CHOK1-human / cynomolgus monkey / mouse VSIG4 cells was detected by FACS method.

[0327] Preparation of humanized antibodies

[0328] The epitope difference between the cAb023 antibody in this project and the reference antibody A2 is quite large, and it is used as the main candidate molecule for this project to carry out subsequent antibody humanization work.

[0329] Specifically, the heavy chain variable region and light chain variable region of the antibody were compared with the available human IgG gene sequence database to identify the best matching human germline Ig gene sequence. Specifically, the heavy chain sequence selected human germline IgG heavy chain 023VH2 (IGHV1-2*01); the light chain sequence selected human germline IgG light chain: 023VL3 (IGKV3-11*01). The heavy chain CDR of cAb023 and the light chain CDR of cAb023 were transplanted to the matching framework sequence, and the resulting VH and VL amino acid sequences were SEQ ID NO: 130 and SEQ ID NO: 135, respectively. The heavy chain constant region and light chain constant region of the antibody used were SEQ ID NO: 148 and SEQ ID NO: 149, respectively.

[0330] The designed heavy and light chain plasmids were combined, and the new antibody molecule was named Hu023-06 (VH2-VL3). Based on the above disclosed information, those skilled in the art can fully know the complete amino acid sequences of the heavy and light chains of the relevant humanized antibodies.

[0331] Sequences of the heavy chain variable region VH and light chain variable region VL of the humanized antibody:

[0332] 004VH

[0333] QVQLQQPGAELVRPGSSVKLSCKAS GYTFTIYW MDWVKQRPGQGLEWIGN INPSDSET HYNQKFKDKATLTVDKSSNTAYMQLSSLTSEDSAVYYC TRPGQAGGFAY WGQGTLVTVSA

[0334] (SEQ ID NO:97)

[0335] 004VH1(IGHV1-46*01)

[0336] QVQLVQSGAEVKKPGASVKVSCKAS MISCELLANEOUS MDWVKQAPGQGLEWIGN INPSDSET HYNQKFKDKATLTVDKSTSTAYMELSSLRSEDTAVYYC TRPGQAGGFAY WGQGTLVTVSS

[0337] (SEQ ID NO:121)

[0338] 004VH2(IGHV1-46*01)

[0339] QVQLVQSGAEVKKPGASVKVSCKAS MISCELLANEOUS MDWVRQAPGQGLEWIGN INPSDSET HYNQKFKDKATLTVDKSTSTAYMELSSLRSEDTAVYYC TRPGQAGGFAY WGQGTLVTVSS

[0340] (SEQ ID NO:122)

[0341] 004VH3(IGHV1-46*01)

[0342] QVQLVQSGAEVKKPGASVKVSCKAS MISCELLANEOUS MDWVRQAPGQGLEWIGN INSPECTION HYNQKFKDKATLTVDKSTSTAYMELSSLRSEDTAVYYC TRPGQAGGFAY WGQGTLVTVSS

[0343] (SEQ ID NO:123)

[0344] 004VH4(IGHV1-46*01)

[0345] QVQLVQSGAEVKKPGASVKVSCKAS MISCELLANEOUS MDWVRQAPGQGLEWIGN INPSDSET HYNQKFKDKATLTVDTSTSTVYMELSSLRSEDTAVYYC TRPGQAGGFAY WGQGTLVTVSS

[0346] (SEQ ID NO:124)

[0347] 004VH5(IGHV1-46*01)

[0348] QVQLVQSGAEVKKPGASVKVSCKAS MISCELLANEOUS MDWVRQAPGQGLEWIGN INPSDSET HYNQKFKDKATLTRDTSTSTVYMELSSLRSEDTAVYYC TRPGQAGGFAY WGQGTLVTVSS

[0349] (SEQ ID NO:125)

[0350] 004VH6(IGHV1-46*01)

[0351] QVQLVQSGAEVKKPGASVKVSCKAS MISCELLANEOUS MDWVRQAPGQGLEWMGNI NPSDSET HYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYC TRPGQAGGFAY WGQGTLVTVSS

[0352] (SEQ ID NO:126)

[0353] 004VL

[0354] DIQMTQSPASLSVSVGETVTITCRAS ENIYSN LGWYQQKQGKSPQLLVY AAT NLADGVPSRFSGSGSGTQFSLKINSLQSEDFGTYYC QHFWVTPWT FGGGTNLEIK

[0355] (SEQ ID NO:98)

[0356] 004VL1(IGKV1-39*01)

[0357] DIQMTQSPSSLSASVGDRVTITCRAS ENIYSN LGWYQQKPGKAPKLLVY AAT NLADGVPSRFSGSGSGTQFTLTISSLQPEDFATYYC QHFWVTPWT FGQGTKVEIK

[0358] (SEQ ID NO:127)

[0359] 004VL2(IGKV1-39*01)

[0360] DIQMTQSPSSLSASVGDRVTITCRAS ENIYSN LGWYQQKPGKAPKLLVY AAT NLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHFWVTPWT FGQGTKVEIK

[0361] (SEQ ID NO:128)

[0362] 023VH

[0363] EVQLQQSGPELVKPGASVKMSCKAS RARE MHWVKQSHGKSLEWIGY IIPNSGDA DYNQKFKDKATLTVNKSSSTAYMELRSLTSEDSTVYYC TRSAGGHFDYWGQGTTLTVSS

[0364] (SEQ ID NO:91)

[0365] 023VH1(IGHV1-2*01)

[0366] QVQLVQSGAEVKKPGASVKVSCKAS RARE MHWVRQAPGQGLEWIGY IIPNSGDA DYNQKFKDKATLTVDKSISTAYMELSRLRSDDTVVYYC TRSAGGHFDY WGQGTLVTVSS

[0367] (SEQ ID NO:129)

[0368] 023VH2(IGHV1-2*01)

[0369] QVQLVQSGAEVKKPGASVKVSCKAS RARE MHWVRQAPGQGLEWIGY IIPNSGDA DYNQKFKDKATLTVDTSISTAYMELSRLRSDDTVVYYC TRSAGGHFDY WGQGTLVTVSS

[0370] (SEQ ID NO:130)

[0371] 023VH3(IGHV1-2*01)

[0372] QVQLVQSGAEVKKPGASVKVSCKAS RARE MHWVRQAPGQGLEWMGYI IPNSGDA DYNQKFKDKATLTRDTSISTAYMELSRLRSDDTVVYYC TRSAGGHFDY WGQGTLVTVSS

[0373] (SEQ ID NO:131)

[0374] 023VH4(IGHV1-2*01)

[0375] QVQLVQSGAEVKKPGASVKVSCKAS RARE MHWVRQAPGQGLEWMGYI IPNSGDA DYNQKFKDRVTSTRDTSISTAYMELSRLRSDDTVVYYC TRSAGGHFDY WGQGTLVTVSS

[0376] <h2 style=";text-align:left;direction:ltr">(SEQ ID NO:132)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0377] <h2 style=";text-align:left;direction:ltr"> 023VL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0378] <h2 style=";text-align:left;direction:ltr"> EVVLTQSPTTMAAFPGEKITITCSAS<h2 style=";text-align:left;direction:ltr"> SSISSNY <h2 style=";text-align:left;direction:ltr"> LHWYQQKPGFSPKLLIY<h2 style=";text-align:left;direction:ltr"> RTS <h2 style=";text-align:left;direction:ltr"> NLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYC<h2 style=";text-align:left;direction:ltr"> QQGSNIPRT <h2 style=";text-align:left;direction:ltr"> FGGGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0379] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO:92)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0380] <h2 style=";text-align:left;direction:ltr"> 023VL1(IGKV3-11*01)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0381] <h2 style=";text-align:left;direction:ltr"> EVVLTQSPATLSLSPGERATLSCSAS<h2 style=";text-align:left;direction:ltr"> SSISSNY <h2 style=";text-align:left;direction:ltr"> LHWYQQKPGQAPRLLIY<h2 style=";text-align:left;direction:ltr"> RTS <h2 style=";text-align:left;direction:ltr"> NLASGIPARFSGSGSGTSYTLTISSLEPEDFAVYYC<h2 style=";text-align:left;direction:ltr"> QQGSNIPRT <h2 style=";text-align:left;direction:ltr"> FGQGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0382] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO:133)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0383] <h2 style=";text-align:left;direction:ltr"> 023VL2(IGKV3-11*01)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0384] <h2 style=";text-align:left;direction:ltr"> EVVLTQSPATLSLSPGERATLSCSAS<h2 style=";text-align:left;direction:ltr"> SSISSNY <h2 style=";text-align:left;direction:ltr"> LHWYQQKPGQAPRLLIY<h2 style=";text-align:left;direction:ltr"> RTS <h2 style=";text-align:left;direction:ltr"> NLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYC<h2 style=";text-align:left;direction:ltr"> QQGSNIPRT <h2 style=";text-align:left;direction:ltr"> FGQGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0385] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO:134)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0386] <h2 style=";text-align:left;direction:ltr"> 023VL3(IGKV3-11*01)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0387] <h2 style=";text-align:left;direction:ltr"> EIVLTQSPATLSLSPGERATLSCSAS<h2 style=";text-align:left;direction:ltr"> SSISSNY <h2 style=";text-align:left;direction:ltr"> LHWYQQKPGQAPRLLIY<h2 style=";text-align:left;direction:ltr"> RTS <h2 style=";text-align:left;direction:ltr"> NLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<h2 style=";text-align:left;direction:ltr"> QQGSNIPRT <h2 style=";text-align:left;direction:ltr"> FGQGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0388] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO:135)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0389] Table 7. Combinations of heavy chain variable regions and light chain variable regions of humanized antibodies

[0390] VH VL Hu004-01 VH1 VL1 Hu004-02 VH1 VL2 Hu004-03 VH2 VL1 Hu004-04 VH2 VL2 Hu004-05 VH3 VL1 Hu004-06 VH3 VL2 Hu004-07 VH4 VL1 Hu004-08 VH4 VL2 Hu004-09 VH5 VL1 Hu004-10 VH5 VL2 Hu004-11 VH6 VL1 Hu004-12 VH6 VL2

[0391] Table 8. Combinations of heavy chain variable regions and light chain variable regions of humanized antibodies

[0392] VH VL Hu023-01 VH1 VL1 Hu023-02 VH1 VL2 Hu023-03 VH1 VL3 Hu023-04 VH2 VL1 Hu023-05 VH2 VL2 Hu023-06 VH2 VL3 Hu023-07 VH4 VL1 Hu023-08 VH4 VL2 Hu023-09 VH4 VL3

[0393] FACS method to detect the binding activity of humanized antibodies to cells

[0394] According to the SOP of cell culture, CHOK1-human / cyno / mouse VSIG4 stable cell line cells were cultured and treated. The digested cells were centrifuged at 300g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed twice with FACS buffer (PBS + 2% FBS). 1-2E5 cells / well were placed in a 96-well round-bottom plate (Corning, Cat. No. 3799), the supernatant was discarded after centrifugation, and the gradient dilution of antibodies was added, with a starting concentration of 200nM, 5-fold dilution, 8 concentration points (including 0 point), 100μl / well, mixed, and incubated at 4℃ for 1h. Centrifuge at 4℃, 300g for 5min, discard the supernatant solution, wash the cells twice with FACS buffer, add the corresponding secondary antibody solution, 100μl / well, and incubate at 4℃ for 1h. Centrifuge at 4℃, 300g for 5min, discard the supernatant solution, and wash the cells twice with FACS buffer. Finally, the cells were resuspended in FACS buffer, the signal value was detected on a BD flow cytometer, and the curve was fitted using GraphPad Prism6 to calculate the EC 50 value.

[0395] The results are as follows Figure 5A to Figure 5E shown.

[0396] The results showed that all variants of humanized antibodies Hu004 and Hu023 bound to CHOK1-human / cynomolgus monkey VSIG4 cells, and all variants of Hu023 bound to CHOK1-mouse VSIG4 cells, but none of them reached a saturation platform.

[0397] Test Example 1. Effect of humanized antibodies on T cell tumor killing

[0398] First, add 50 μL of human CD3 antibody at a concentration of 5 μg / ml to each well of a 96-well plate and coat overnight at 4°C. Then add 400,000 T cells and incubate at 37°C for 3 days. Centrifuge and discard the supernatant, collect 100,000 activated T cells and mix with 10,000 tumor cells 8505C, and add 10 μg / ml humanized antibody and incubate at 37°C for 4 hours. Centrifuge and collect the supernatant, take 50 μL of the supernatant and add it to a 96-well plate, then add 50 μL of LDH reaction substrate and incubate at 37°C for 1 hour. Then add 50 μL of stop solution to stop the color reaction, and detect the OD value with an enzyme marker.

[0399] The results are as follows Figure 6 shown.

[0400] The results showed that the humanized antibodies Hu004 and Hu023 can both enhance the ability of T cells to kill tumor cells. Among them, the most potent antibody Hu023-06 (VH2-VL3) was selected for in vivo animal experiments.

[0401] Test Example 2. Effect of humanized antibodies on tumor growth in vivo

[0402] First, 5 million anaplastic thyroid cancer cell line 8505C cells (derived from the German DSMZ cell bank, Cat#ACC-219) were inoculated subcutaneously into 5-week-old immunodeficient NSG mice. Three days later, 10 million PBMC cells were reinfused into the tail vein. On the 7th day, Hu004-09 (VH5-VL1) and Hu023-06 (VH2-VL3) antibodies were injected intraperitoneally at a concentration of 10 mg / kg. The injections were performed every 2 days and the weight of the mice and the size of the tumor were recorded. The results are shown in Figure 2. Figure 7A to Figure 7C shown.

[0403] The anti-tumor activity of humanized antibodies was investigated using a tumor immune double humanization model. The results showed that Hu023-06 (VH2-VL3) antibody can effectively inhibit the growth of ATC tumors in vivo and reduce tumor weight.

Claims

1. An anti-VSIG4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively, ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8 and 9, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively, iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14 and 15, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17 and 18, respectively, iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 19, 20 and 21, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 23 and 24, respectively, v) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 26 and 27, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28, 29 and 30, respectively, vi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 31, 32 and 33, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 34, 35 and 36, respectively, vii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 38 and 39, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 40, 41 and 42, respectively, viii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 43, 44 and 45, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 46, 47 and 48, respectively, ix) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 49, 50 and 51, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 52, 53 and 54, respectively, x) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 55, 56 and 57, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 58, 59 and 60, respectively, xi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 61, 62 and 63, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 64, 65 and 66, respectively, xii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 67, 68 and 69, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 70, 71 and 72, respectively, xiii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 73, 74 and 75, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 76, 77 and 78, respectively, xiv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 79, 80 and 81, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 82, 83 and 84, respectively, or xv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 85, 86 and 87, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 88, 89 and 90, respectively; wherein the CDR sequences of i) to xv) are determined according to the Kabat numbering system; or xvi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 136, 137 and 138, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 139, 140 and 141, respectively, or xvii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 142, 143 and 144, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 145, 146 and 147, respectively, wherein the CDR sequences of xvi) to xvii) are determined according to the IMGT numbering system. 2 . The anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1 , wherein the anti-VSIG4 antibody is selected from the group consisting of a murine antibody, a chimeric antibody and a humanized antibody.

3. An anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, scFv (sc means single chain), Bis-scFv, scFv-Fc fragment, Fab2, Fab3, miniantibody, diabody, triabody, tetrabody and nanobody.

4. The anti-VSIG4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-VSIG4 antibody or antigen-binding fragment thereof comprises: 1) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:91, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:92; 2) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:93, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:94; 3) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:95, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:96; 4) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:97, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:98, 5) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:99, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:100; 6) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 101, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 102; 7) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 103, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 104; 8) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 105, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 106, 9) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 107, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 108; 10) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 109, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 110; 11) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 111, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 112; 12) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 113, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 114, 13) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 115, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 116; 14) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 117, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 118; or 15) a heavy chain variable region VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:119, and a light chain variable region VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

120.

5. The anti-VSIG4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-VSIG4 antibody or antigen-binding fragment thereof comprises: 1) a heavy chain variable region VH as shown in SEQ ID NO:91, and a light chain variable region VL as shown in SEQ ID NO:92; 2) a heavy chain variable region VH as shown in SEQ ID NO:93, and a light chain variable region VL as shown in SEQ ID NO:94; 3) a heavy chain variable region VH as shown in SEQ ID NO:95, and a light chain variable region VL as shown in SEQ ID NO:96; 4) a heavy chain variable region VH as shown in SEQ ID NO:97, and a light chain variable region VL as shown in SEQ ID NO:98; 5) the heavy chain variable region VH shown in SEQ ID NO:99, and the light chain variable region VL shown in SEQ ID NO:100; 6) the heavy chain variable region VH shown in SEQ ID NO: 101, and the light chain variable region VL shown in SEQ ID NO: 102; 7) the heavy chain variable region VH shown in SEQ ID NO: 103, and the light chain variable region VL shown in SEQ ID NO: 104; 8) the heavy chain variable region VH shown in SEQ ID NO: 105, and the light chain variable region VL shown in SEQ ID NO: 106; 9) the heavy chain variable region VH shown in SEQ ID NO: 107, and the light chain variable region VL shown in SEQ ID NO: 108; 10) a heavy chain variable region VH as shown in SEQ ID NO: 109, and a light chain variable region VL as shown in SEQ ID NO: 110; 11) a heavy chain variable region VH as shown in SEQ ID NO: 111, and a light chain variable region VL as shown in SEQ ID NO: 112; 12) a heavy chain variable region VH as shown in SEQ ID NO: 113, and a light chain variable region VL as shown in SEQ ID NO: 114; 13) the heavy chain variable region VH shown in SEQ ID NO: 115, and the light chain variable region VL shown in SEQ ID NO: 116; 14) the heavy chain variable region VH shown in SEQ ID NO: 117, and the light chain variable region VL shown in SEQ ID NO: 118; or 15) The heavy chain variable region VH shown in SEQ ID NO:119, and the light chain variable region VL shown in SEQ ID NO:

120.

6. The anti-VSIG4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-VSIG4 antibody or antigen-binding fragment thereof comprises: A heavy chain variable region VH comprising an amino acid sequence as shown in any one of SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of the amino acid sequences shown therein; and A light chain variable region VL, which comprises the amino acid sequence shown in any one of SEQ ID NO:127 and SEQ ID NO:128, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one of them.

7. The anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 6, wherein the anti-VSIG4 antibody or antigen-binding fragment thereof comprises: 1) a heavy chain variable region VH as shown in SEQ ID NO: 121, and a light chain variable region VL as shown in SEQ ID NO: 127; 2) a heavy chain variable region VH as shown in SEQ ID NO: 121, and a light chain variable region VL as shown in SEQ ID NO: 128; 3) a heavy chain variable region VH as shown in SEQ ID NO: 122, and a light chain variable region VL as shown in SEQ ID NO: 127; 4) a heavy chain variable region VH as shown in SEQ ID NO: 122, and a light chain variable region VL as shown in SEQ ID NO: 128; 5) the heavy chain variable region VH shown in SEQ ID NO: 123, and the light chain variable region VL shown in SEQ ID NO: 127; 6) the heavy chain variable region VH shown in SEQ ID NO: 123, and the light chain variable region VL shown in SEQ ID NO: 128; 7) the heavy chain variable region VH shown in SEQ ID NO: 124, and the light chain variable region VL shown in SEQ ID NO: 127; 8) the heavy chain variable region VH shown in SEQ ID NO: 124, and the light chain variable region VL shown in SEQ ID NO: 128; 9) the heavy chain variable region VH shown in SEQ ID NO: 125, and the light chain variable region VL shown in SEQ ID NO: 127; 10) a heavy chain variable region VH as shown in SEQ ID NO: 125, and a light chain variable region VL as shown in SEQ ID NO: 128; 11) the heavy chain variable region VH shown in SEQ ID NO: 126, and the light chain variable region VL shown in SEQ ID NO: 127; or 12) The heavy chain variable region VH shown in SEQ ID NO:126, and the light chain variable region VL shown in SEQ ID NO:

128.

8. The anti-VSIG4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-VSIG4 antibody or antigen-binding fragment thereof comprises: A heavy chain variable region VH comprising an amino acid sequence as shown in any one of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any of the amino acid sequences shown therein; and A light chain variable region VL, which comprises the amino acid sequence shown in any one of SEQ ID NO:133, SEQ ID NO:134 and SEQ ID NO:135, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any of the amino acid sequences shown therein.

9. The anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 8, wherein the anti-VSIG4 antibody or antigen-binding fragment thereof comprises: 1) a heavy chain variable region VH as shown in SEQ ID NO: 129, and a light chain variable region VL as shown in SEQ ID NO: 133; 2) a heavy chain variable region VH as shown in SEQ ID NO: 129, and a light chain variable region VL as shown in SEQ ID NO: 134; 3) a heavy chain variable region VH as shown in SEQ ID NO: 129, and a light chain variable region VL as shown in SEQ ID NO: 135; 4) a heavy chain variable region VH as shown in SEQ ID NO: 130, and a light chain variable region VL as shown in SEQ ID NO: 133; 5) a heavy chain variable region VH as shown in SEQ ID NO: 130, and a light chain variable region VL as shown in SEQ ID NO: 134; 6) the heavy chain variable region VH shown in SEQ ID NO: 130, and the light chain variable region VL shown in SEQ ID NO: 135; 7) the heavy chain variable region VH shown in SEQ ID NO: 132, and the light chain variable region VL shown in SEQ ID NO: 133; 8) the heavy chain variable region VH shown in SEQ ID NO: 132, and the light chain variable region VL shown in SEQ ID NO: 134; or 9) the heavy chain variable region VH shown in SEQ ID NO:132, and the light chain variable region VL shown in SEQ ID NO:

135.

10. An anti-VSIG4 antibody or an antigen-binding fragment thereof according to any one of claims 1-9, wherein the anti-VSIG4 antibody or an antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and / or a light chain constant region of a human κ or λ chain or a variant thereof; preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 148; and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

149.

11. An isolated nucleic acid encoding the anti-VSIG4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A vector comprising the isolated nucleic acid of claim 11.

13. A host cell comprising the isolated nucleic acid of claim 11 or the vector of claim 12, preferably, the host cell is a prokaryotic cell or a eukaryotic cell, preferably, the cell is selected from bacteria, fungi, viruses, yeast, mammalian cells, more preferably, selected from Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells.

14. A pharmaceutical composition comprising the anti-VSIG4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutical excipient, diluent or carrier.

15. Use of an anti-VSIG4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10, or a pharmaceutical composition according to claim 14, in the preparation of a medicament for treating a disease or condition, wherein the disease or condition is selected from cancer, preferably, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer (such as leukemia, lymphoma or myeloma), laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer.

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