Anti-VSIG4 and Anti-siglec-7 inhibitory antibody against intermolecular interaction between VSIG4 and siglec-7

Abstract

To create an antibody that inhibits an immune checkpoint function based on interaction between VSIG4 and Siglec-7 and exhibits antitumor activity, and to provide an anti-tumor immune response activator which includes the antibody as an active ingredient.SOLUTION: The present invention provides an anti-tumor immune response activator including an antibody that inhibits intermolecular interaction between VSIG4 and Siglec-7 or a functional fragment thereof.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] The present invention inhibits the molecular interaction between VSIG4 and Siglec-7 and activates anti-tumor immune responses. and anti-VSIG4 and anti-Siglec-7 antibodies, as well as anti-tumor immune response activators containing the same. Regarding. [Background technology]

[0002] V-set and immunoglobulin domain containing 4 (VSIG4) is an immunoglobulin superfamily It belongs to the immunoglobulin superfamily (IgSF) and has a structure similar to PD-L1. It has been reported that VSIG4 negatively regulates T cell activity by binding to the surface of T cells. High expression of VSIG4 is associated with poor prognosis in glioma, ovarian cancer, multiple myeloma, and gastric cancer. In addition, VSIG4 expression in pancreatic ductal carcinoma is negatively correlated with CD8+ T cell infiltration. Thus, VSIG4 functions as a ligand for the immune checkpoint that suppresses T cells, but its receptor in immune cells has not been elucidated. VSIG4 is a receptor for complement C3b, also known as CRIg (Complement receptor of the immunoglobulin superfamily), but it was thought to be different from the VSIG4 receptor in immune cells.

[0003] Recently, Bio-Techne has identified Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) as a candidate receptor for VSIG4 (Patent Document 1). Siglec-7 is an inhibitory receptor for sialic acid that is mainly expressed in monocytes, NK cells, and T cells. Therefore, it is believed that the interaction between VSIG4 and Siglec-7 controls the immune checkpoint, and it was predicted that anti-VSIG4 antibodies and anti-Siglec-7 antibodies that inhibit this interaction would be effective as immune checkpoint inhibitors against tumors that highly express VSIG4, including glioma, ovarian cancer, multiple myeloma, gastric cancer, and pancreatic cancer. However, in fact, an immune checkpoint based on the interaction between VSIG4 and Siglec-7 To date, no antibodies have been developed that both inhibit this function and activate antitumor immune responses. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US10752689B2 Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above, the present invention provides an immune checkpoint inhibitor based on the interaction between VSIG4 and Siglec-7. The objective of the present invention is to obtain an antibody having antitumor activity by inhibiting the tumor function, and to provide an antitumor immune response activator containing the antibody as an active ingredient. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems. It inhibits the molecular interaction between VSIG4 and Siglec-7, and inhibits the immune checkpoint inhibitor By obtaining anti-VSIG4 and anti-Siglec-7 antibodies that inhibit the tumor function and have antitumor activity, The inventors have succeeded in this research, and have found that such antibodies can be used as agents for activating anti-tumor immune responses. Based on this finding, the present invention has been completed. That is, the gist of the present invention relates to the following.

[0007] [1] A compound comprising an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. , anti-tumor immune response activators. [2] The antibody or functional fragment thereof is an anti-VSIG4 antibody or a functional fragment thereof, The anti-tumor immune response activator described in [1], which is / or an anti-Siglec-7 antibody or a functional fragment thereof. [3] The antibody or functional fragment thereof is an antibody having an amino acid sequence set forth in SEQ ID NO: 13 of VSIG4. The epitope is either the region contained in the VSIG4 or a region equivalent to the same region in other VSIG4 genes. The anti-tumor immune response activator according to [1] or [2], which is an antibody or a functional fragment thereof. [4] The anti-tumor immune response activator according to any one of [1] to [3], wherein the antibody or functional fragment thereof is selected from the following (1) to (4): (1) CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO:5; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO:6; A heavy chain variable region comprising CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:7; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 8; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 9; and a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 10, or a functional fragment thereof; (2) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 or a heavy chain variable region having 90% identical to said amino acid sequence; The amino acid sequence has the above sequence identity and has binding activity to VSIG4. A heavy chain variable region; A light chain variable region having the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having 90% or more sequence identity thereto, and having binding activity to VSIG4. an antibody or functional fragment thereof comprising a nucleotide sequence and a nucleotide sequence of a nucleotide sequence; (3) CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO: 14; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO: 15; A heavy chain variable region comprising a CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 17; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 18; and a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and (4) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 or a heavy chain variable region having 90% identical to said amino acid sequence; A heavy chain variable region that has an amino acid sequence having the above sequence identity and has binding activity to Siglec-7; An antibody or a functional fragment thereof comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence and having binding activity to Siglec-7. [5] To stimulate NK cell immune responses and / or stimulate T cell immune responses The anti-tumor immune response activator according to any one of [1] to [4] above. [6] An anti-tumor immune response activator according to any one of [1] to [5] for enhancing the cytotoxicity of NK cells and / or enhancing cytokine production by NK cells. [7] An antitumor pharmaceutical composition comprising the antitumor immune response activator described in any one of [1] to [6]. [8] An antibody or a functional fragment thereof that recognizes as an epitope a region contained in the amino acid sequence set forth in SEQ ID NO: 13 of VSIG4 or a region corresponding to the same region in other VSIG4. [9] An antibody or a functional fragment thereof selected from the following (1) to (4): (1) CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO:5; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO:6; A heavy chain variable region comprising CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:7; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 8; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 9; and a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 10. or a functional fragment thereof; (2) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 or a heavy chain variable region having 90% identical to said amino acid sequence; The amino acid sequence has the above sequence identity and has binding activity to VSIG4. A heavy chain variable region; A light chain variable region having the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having 90% or more sequence identity thereto, and having binding activity to VSIG4. an antibody or functional fragment thereof comprising a nucleotide sequence and a nucleotide sequence of a nucleotide sequence; (3) CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO: 14; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO: 15; A heavy chain variable region comprising a CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 17; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 18; and a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and (4) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 or a heavy chain variable region having 90% identical to said amino acid sequence; A heavy chain variable region that has an amino acid sequence having the above sequence identity and has binding activity to Siglec-7; An antibody or a functional fragment thereof comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence and having binding activity to Siglec-7.

[0008] The present invention can also have the following aspects.

[10] We developed an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. A method for activating an anti-tumor immune response, comprising administering to a subject; Antitumor immunity against antibodies or functional fragments thereof that inhibit the intermolecular interaction between VSIG4 and Siglec-7 Use for activating an immune response; Molecular interaction between VSIG4 and Siglec-7 for use in activating anti-tumor immune responses. Inhibitory antibodies or functional fragments thereof; Antitumor immunity against antibodies or functional fragments thereof that inhibit the intermolecular interaction between VSIG4 and Siglec-7 Use for the manufacture of an immune response activator; or The present invention provides a method for the preparation of an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. A method for producing an anti-tumor immune response activator, comprising:

[0009]

[11] We have developed an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. a method for preventing or treating a tumor (or cancer) comprising administering to an elephant; The antibody or its functional fragment that inhibits the molecular interaction between VSIG4 and Siglec-7 was administered to tumors (or Use for the prevention or treatment of cancer; Separation of VSIG4 and Siglec-7 for use in the prevention or treatment of tumors (or cancer) an antibody or a functional fragment thereof that inhibits the interaction between the molecules; Antitumor therapy using an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 Use for the manufacture of a pharmaceutical composition; or The present invention provides a method for the preparation of an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. A method for producing an antitumor pharmaceutical composition, comprising:

[0010]

[12] We developed an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. including, immune checkpoint inhibitors; An antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 is administered to a subject. a method for inhibiting an immune checkpoint comprising administering to a subject a therapeutically effective amount of Immunotherapy using antibodies or their functional fragments that inhibit the intermolecular interaction between VSIG4 and Siglec-7 Use for checkpoint inhibition; Molecular interaction of VSIG4 with Siglec-7 for use in immune checkpoint inhibition an antibody or a functional fragment thereof that inhibits the use of the Immunotherapy using antibodies or their functional fragments that inhibit the intermolecular interaction between VSIG4 and Siglec-7 Use for the manufacture of a checkpoint inhibitor; or The present invention provides a method for the preparation of an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. A method for producing an immune checkpoint inhibitor, comprising: Effect of the Invention

[0011] According to the present invention, there is provided an anti-VSIG4 antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7, inhibits the immune checkpoint function based on the interaction between VSIG4 and Siglec-7, and has antitumor activity. and anti-Siglec-7 antibodies are provided. Antibodies that inhibit the molecular interaction between VSIG4 and Siglec-7 inhibit the immune checkpoint function between VSIG4 and Siglec-7, have antitumor activity, and are useful as a treatment for tumors expressing VSIG4. The results are expected to be effective. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 shows the interaction between VSIG4 and Siglec family proteins. (A) Schematic diagram of VSIG4-COMP protein. The rat Cd4 domain 3+4, rat COMP pentamerization motif, FLAG, and 6×His tag are added to the C-terminus of the extracellular domain (ECD) of VSIG4. (B) Binding assay of VSIG4-COMP protein and purified proteins of all human Siglec family molecules was performed using ELISA. The figure shows the average absorbance ± standard deviation of three independent experiments. Siglec-13 is not conserved in humans and is a missing number. VSIG4 was shown to bind to Siglec-1, -4, and -7. (C) Purity of VSIG4-COMP and Siglec-7-Fc proteins used in the binding analysis was shown by silver staining (photograph in place of drawing). (D) VSIG4-COMP protein bound to the surface of HEK293 + vector and + Siglec-7 cells was quantified by flow cytometry, suggesting that more VSIG4 binds to Siglec-7 expressing cells. (E) Siglec-7-Fc protein bound to the surface of HEK293 + vector and + VSIG4 cells was quantified by flow cytometry, suggesting that more Siglec-7 binds to VSIG4 expressing cells. [Diagram 2]Figure 2 shows the sialic acid dependency of the interaction between VSIG4 and Siglec-7. (A) Sialylase was used to remove sialic acid from the VSIG4-COMP protein. The removal of sialic acid was confirmed using an anti-Siaα2-3 antibody (photograph in place of drawing). (B) Evaluation of the binding of VSIG4-COMP protein and Siglec-7-Fc protein using Alpha technology with or without sialidase treatment. A significant decrease in the binding signal was observed after removal of sialic acid. (C) Schematic diagram of candidate O-glycosylation sites in the extracellular domain of VSIG4. The seven predicted glycosylation sites are concentrated in two sites: one located between the two immunoglobulin-like loop structures IgV and IgC2 in VSIG4 (S139, S141, T144, T146, T147) and the other located near the membrane (T241, T245). SP: signal peptide, TM: transmembrane region. (D) Silver staining of VSIG4-COMP mutant proteins in which mutations were introduced into the candidate O-glycosylation sites of VSIG4 (photograph instead of drawing). (E) Binding assay of VSIG4-COMP mutants and Siglec-7-Fc using ELISA. Compared with wild-type (WT) VSIG4, the S141A, T144A, T147A, and T241A mutants showed equal or higher binding ability to Siglec-7, whereas the S139, T146A, and T245A mutants showed lower binding ability to Siglec-7, and the T146A mutant had the lowest binding ability, suggesting that sialic acid modification at the T146 residue is important for binding to Siglec-7. The graph shows the relative values ​​when the absorbance of wild-type (WT) VSIG4 was set to 1. [Diagram 3]Figure 3 shows the expression of Siglec-7 and VSIG4 in cell lines detected by flow cytometry. (A) Expression of Siglec-7 in NK cells. Human normal NK cells and the NK cell line KHYG-1 endogenously express Siglec-7, whereas no expression was observed in NK-92 cells, and Siglec-7-forced expressing cells were generated. (B) Expression of VSIG4 in cell lines. P31 / FUJ cells endogenously express VSIG4, and VSIG4 knockout (KO) cells were generated using the CRISPR / Cas9 method. On the other hand, no VSIG4 expression was observed in HEK293 and Jurkat cells, and VSIG4-forced expressing cells were generated. [Figure 4]Figure 4 shows that binding of VSIG4 to Siglec-7 reduces cytokine secretion from NK cells. (A) When KHYG-1 cells were co-cultured as effector cells with HEK293 + vector or + VSIG4 cells as target cells, forced expression of VSIG4 reduced IFN-γ secretion from KHYG-1 cells. (B) When KHYG-1 cells were co-cultured as effector cells with HEK293 + vector or + VSIG4 cells as target cells, cytokines in the culture medium were detected using a human cytokine antibody array kit. It was suggested that forced expression of VSIG4 reduced secretion of CCL1 / I-309, MIP-1α / MIP-1β, CCL5 / RANTES, GM-CSF, IL-8, and IL-10 from KHYG-1 cells. (C) Cytotoxicity was measured when NK-92 + vector or + Siglec-7 cells were co-cultured as effector cells with Jurkat + vector or + VSIG4 cells as target cells. A decrease in cytotoxicity was observed only when NK-92 + Siglec-7 cells were co-cultured with Jurkat + VSIG4 cells. (D) Cytotoxicity was measured when NK-92 + vector or + Siglec-7 cells were co-cultured as effector cells with P31 / FUJ or P31 / FUJ VSIG4-KO cells as target cells. Knockout of VSIG4 enhanced the cytotoxicity of NK-92 + Siglec-7 cells. [Diagram 5] Figure 5 shows that the binding of VSIG4 to Siglec-7 reduces the activity of T cell receptor signaling. (A) Siglec-7 expression was not observed in Jurkat NFAT-Luc cells, and a Siglec-7 expressing line was generated. (B) Jurkat NFAT-Luc + vector or +Siglec-7 cells were seeded on a plate immobilized with anti-CD3 antibody and IgG or VSIG4-Fc protein, and the activity of T cell receptor signaling was detected by luciferase. The VSIG4 protein reduced the luciferase activity in Jurkat NFAT-Luc + Siglec-7 cells. [Figure 6] Figure 6 shows the neutralizing activity of anti-VSIG4 antibodies (clones 2C7 and 2F12) against the interaction between VSIG4 and Siglec family molecules. The interaction of VSIG4 with known binding molecules C3b and Siglec-1, -4, and -7 was detected by ELISA, and it was examined whether the absorbance indicating the interaction decreased when anti-VSIG4 antibodies were added. The results showed that the 2C7 antibody has neutralizing activity against the interaction between VSIG4 and Siglec-1, -4, and -7. On the other hand, it did not inhibit the interaction between VSIG4 and C3b, suggesting that the interaction occurs in a region different from that of the Siglec family. It was also suggested that the 2F12 antibody has neutralizing activity against the interaction between VSIG4 and Siglec-7. [Figure 7] Figure 7 shows the neutralizing activity of anti-Siglec-7 antibody (clone 4G7) against the interaction between Siglec-7 and its ligand. The interaction between Siglec-7 and VSIG4 was detected by ELISA, and it was examined whether the absorbance indicating the interaction was reduced when anti-Siglec-7 antibody was added. The results showed that 4G7 antibody has neutralizing activity against the interaction between Siglec-7 and VSIG4. [Figure 8]Figure 8 shows the results of narrowing down the epitope of anti-VSIG4 antibodies (clone 2C7, 2F12). (A) First, the endogenous signal peptide sequence (amino acids 1-19 in SEQ ID NO: 2) of the full-length amino acid sequence of VSIG4 was replaced with an Igκ-leader signal peptide followed by an HA tag to create the HA-VSIG4 construct (a). Next, a series of deletion mutants (b, c, d) were created by sequentially deleting the first loop (amino acid residues 20-137), the amino acid sequence containing five potential O-glycosylation sites (amino acid residues 138-147), and the second loop (amino acid residues 148-231). (B) These were transiently introduced into 293FT cells, and expression on the cell surface was detected by flow cytometry using APC-labeled anti-HA antibody and FITC-labeled anti-VSIG4 (2C7, 2F12) antibody. As a result, the 2C7 antibody recognized full-length VSIG4 and VSIG4 lacking the first loop, but the reactivity was greatly reduced to VSIG4 lacking the first loop as well as amino acid residues 138-147, and did not recognize VSIG4 lacking both loops. The 2F12 antibody also bound to full-length VSIG4 and VSIG4 lacking the first loop, but did not bind to the mutant lacking amino acid residues 138-147 and both loops. This suggests that most of the epitopes of the anti-VSIG4 antibodies (clones 2C7 and 2F12) are contained between amino acid residues 138-147. [Figure 9]Figure 9 shows the effect of anti-VSIG4 (2C7) antibody and anti-Siglec-7 (4G7) antibody as immune checkpoint inhibitors. (A) When KHYG-1 cells were co-cultured as effector cells and HEK293 + vector or + VSIG4 cells as target cells, forced expression of VSIG4 reduced IFN-γ secretion from KHYG-1 cells, but IFN-γ secretion was restored by the addition of anti-VSIG4 (2C7) neutralizing antibody. (B) In the same experimental system as (A), IFN-γ secretion was restored by the addition of anti-Siglec-7 (4G7) neutralizing antibody. (C) When NK-92 + Siglec-7 cells were co-cultured as effector cells and Jurkat + vector or + VSIG4 cells as target cells, forced expression of VSIG4 reduced the cytotoxicity of NK-92 cells, but the cytotoxicity was restored by the addition of anti-VSIG4 (2C7) neutralizing antibody. [Figure 10] Figure 10 shows the amino acid sequences of the heavy and light chains of the anti-VSIG4 antibody (clone 2C7) produced and used in each Example. In the figure, the signal sequence is a signal peptide sequence present in the antibody molecule for extracellular secretion. [Figure 11] Fig. 11 shows the amino acid sequences of the heavy and light chains of the anti-Siglec-7 antibody (clone 4G7) produced and used in each Example. In the figure, the signal sequence is a signal peptide sequence that is endogenous to the antibody molecule for extracellular secretion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will be described below. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention. In this specification, when a numerical range is expressed as "lower limit to upper limit", the upper limit may be "lower than" or "less than", and the lower limit may be "higher than" or "greater than".

[0014] The present invention relates to an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 and is based on the inhibitory effect of such an immune checkpoint, and such an antibody or a functional fragment thereof activates an anti-tumor immune response based on immune checkpoint inhibition. (Hereinafter, the "antibody or a functional fragment thereof" may be collectively referred to as "antibody etc.".) That is, one aspect of the present invention relates to an antibody etc. that inhibits the intermolecular interaction between VSIG4 and Siglec-7 and an anti-tumor immune response activator based on immune checkpoint inhibition containing the same. Furthermore, one aspect of the present invention relates to an antibody etc. that inhibits the intermolecular interaction between VSIG4 and Siglec-7 Furthermore, the present invention can also be in the form of an immune checkpoint inhibitor containing such an antibody etc.

[0015] <An antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7> In the present invention, the "antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7" inhibits the intermolecular interaction between VSIG4 and Siglec-7, and inhibits the immune che ckpoint function based on the interaction between VSIG4 and Siglec-7, and is an antibody that activates an anti-tumor immune response. In one aspect, the antibody is (a) an anti-VSIG4 antibody (anti-VSIG4 neutralizing antibody) that binds to the VSIG4 protein and has an action of inhibiting the function of the VSIG4 protein, or (b) an anti-Siglec-7 antibody (anti-Siglec-7 neutralizing antibody) that binds to Siglec-7, which is a receptor of the VSIG4 protein, and has an action of inhibiting the function of the Siglec-7 protein.

[0016] Siglec-7 that interacts with VSIG4 is an immunosuppressive receptor expressed in immune cells etc and functions as an "immune checkpoint molecule". Its ligand, VSIG4, is a cancer ​It is expressed in cells and negatively regulates anti-tumor immune responses in immune cells. Antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 inhibit the immune checkpoint mechanism between VSIG4 and Siglec-7. More specifically, antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 bind, preferably specifically bind, to VSIG4 or Siglec-7 protein and inhibit its function. Antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 can bind to endogenous VSIG4 or Siglec-7 protein of the subject organism to which they are administered and inhibit its function. In a preferred embodiment, antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 can bind to VSIG4 protein expressed on the cell surface of cancer cells and the like and inhibit the function of VSIG4 protein as a ligand of immune checkpoint molecules. In another preferred embodiment, antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 can bind to Siglec-7 protein expressed on the cell surface of immune cells such as NK cells and T cells and inhibit the function of Siglec-7 protein as an immune checkpoint molecule.

[0017] VSIG4 and Siglec-7, which are targets of antibodies that inhibit the intermolecular interaction between VSIG4 and Siglec-7, may be derived from any animal, but are preferably derived from mammals, for example, from primates such as humans, chimpanzees, gorillas, etc., rodents such as mice, rats, guinea pigs, etc., cows, horses, pigs, sheep, goats, llamas, camels, dogs, cats, rabbits, etc. Preferably, they are derived from humans, mice, etc.

[0018] An example of the full-length amino acid sequence of the human VSIG4 protein is shown in SEQ ID NO: 2 (NP_009199.1). An example of the base sequence to be loaded is shown in SEQ ID NO: 1 (NM_007268.3). The VSIG4 protein is a human VSIG4 protein consisting of the amino acid sequence shown in SEQ ID NO: 2, a functional mutant thereof, or a mutant thereof. The VSIG4 protein may be a homologue. The VSIG4 protein may consist of the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 70% or more, preferably 80% or more, for example 90% or more, 93% or more, 95% or more, 98% or more, 99% or more, or 99.5% or more percent identity to the amino acid sequence, and preferably retains a function as a ligand for immune checkpoint molecules (a function of binding to immune checkpoint molecules on immune cells and suppressing immune responses caused by immune checkpoint molecules). Amino acid sequences of VSIG4 proteins derived from various organisms The sequences and gene sequences can be easily obtained from public databases (GenBank, etc.). This can be done.

[0019] An example of the full-length amino acid sequence of human Siglec-7 protein is shown in SEQ ID NO: 4 (NP_055200.1), and an example of the base sequence encoding the same is shown in SEQ ID NO: 3 (NM_014385.4). The Siglec-7 protein may be a human Siglec-7 protein consisting of the amino acid sequence shown in SEQ ID NO: 3, a functional mutant thereof, or or a homolog thereof. The Siglec-7 protein may be the amino acid sequence represented by SEQ ID NO:3. The amino acid sequence may be an amino acid sequence having 70% or more, preferably 80% or more, for example 90% or more, 93% or more, 95% or more, 98% or more, 99% or more, or 99.5% or more percent identity to the amino acid sequence, and preferably has a function as an immune checkpoint molecule (a function of binding to a ligand of an immune checkpoint molecule and suppressing an immune response on an immune cell). The amino acid sequences and gene nucleotide sequences of Siglec-7 proteins derived from various organisms can be easily obtained from known databases (GenBank, etc.).

[0020] In this specification, "% identity" may be a value calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., default, i.e., initial setting parameters).

[0021] In one embodiment, the anti-VSIG4 antibody is an antibody that binds to the extracellular domain of the VSIG4 protein (particularly the It may be one that specifically binds to a region containing an O-glycotype modification site in the extracellular domain. In one embodiment, the anti-VSIG4 antibody recognizes an O-glycosylation site in the extracellular domain of the VSIG4 protein. A region contained in the region (e.g., a sequence of 6 amino acids or more, typically 6 to 20 amino acids or 6 to 10 amino acids, e.g., 6, 7, 8, 9, or 10 amino acids) is used as an epitope. It may be something you recognize. In a preferred embodiment, the anti-VSIG4 antibody inhibits the modification of O-glycosylation in the extracellular domain of the VSIG4 protein. The region containing the modification site is from position 138 to position 245 in the amino acid sequence shown in SEQ ID NO:2. (SEQ ID NO: 13), more preferably a region included in the region from position 138 to position 231 (for example, a region consisting of a partial sequence of 6 amino acids or more), or the amino acid sequence of another VSIG4 protein. The regions in the array that correspond to these regions may be recognized as epitopes. In a further preferred embodiment, the epitope of the anti-VSIG4 antibody may be part or all of the epitope contained within the region from positions 138 to 147 in the amino acid sequence shown in SEQ ID NO:2. Here, for example, the "region corresponding to the region contained in the amino acid sequence of SEQ ID NO: 13 of VSIG4 in another VSIG4" refers to a region corresponding to the region contained in the amino acid sequence of SEQ ID NO: 13 of VSIG4. The amino acid sequence of SEQ ID NO: 13 is a human-derived amino acid sequence, but the amino acid sequence in VSIG4 of other organisms (individuals, species, etc.) is different. Parts may be included. In other words, if an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 13 exists in another organism (i.e., an amino acid sequence that is highly conserved between organisms, for example, an amino acid sequence that has 80% or more, preferably 90% or more, sequence identity with SEQ ID NO: 13), a "region corresponding to the 'region contained in the amino acid sequence set forth in SEQ ID NO: 13 of VSIG4' in another VSIG4" refers to, for example, such a highly conserved region, or a region that has been determined by techniques such as homology search to correspond to the region contained in the amino acid sequence set forth in SEQ ID NO: 13 of VSIG4.

[0022] The antibody of the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7 is an immunoglobulin It may be of any class of molecule, for example IgG, IgE, IgM, IgA, IgD, or IgY, and further any subclass, for example IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.

[0023] Antibodies according to the present invention that inhibit the intermolecular interaction between VSIG4 and Siglec-7 include polyclonal and monoclonal antibodies obtained by immunizing a mammal such as a mouse with an antigen, such as a VSIG4 or Siglec-7 protein or a partial fragment thereof (for example, the above-mentioned epitopes), as well as chimeric and humanized antibodies produced using gene recombination technology, and human antibodies produced using human antibody-producing transgenic animals, etc. When the antibody according to the present invention is administered to humans as a pharmaceutical, humanized or human antibodies are desirable from the viewpoint of side effects.

[0024] The antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention may be either a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody, so long as it can specifically bind to the VSIG4 protein or the Siglec-7 protein. The antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7 may be a monospecific antibody that recognizes one type of antigen, or a multispecific antibody that recognizes multiple antigens, preferably a monospecific antibody.

[0025] Polyclonal antibodies can be prepared by isolating and purifying serum obtained from an animal immunized with an antigen (e.g., the above-mentioned epitope fragment). Monoclonal antibodies can be prepared by fusing antibody-producing cells obtained from an animal immunized with an antigen with myeloma cells to prepare hybridomas, culturing the hybridomas or administering the hybridomas to an animal to cause ascites cancer in the animal, and isolating and purifying the above-mentioned culture fluid or ascites. As antibody-producing cells, spleen cells, lymph nodes, and antibody-producing cells in peripheral blood can be used, and spleen cells are particularly preferred.

[0026] A preferred embodiment of the antibody that inhibits the intermolecular interaction between VSIG4 and SIGLEC7 according to the present invention is a chimeric antibody. An example of a "chimeric antibody" is a chimeric antibody whose variable region is derived from the immunoglobulin of a non-human animal (mouse, rat, hamster, chicken, etc.) and whose constant region is derived from human immunoglobulin. For example, a chimeric antibody can be produced by immunizing a mouse with an antigen, excising the variable region that binds to the antigen from the gene of the mouse monoclonal antibody, and combining it with the antibody constant region derived from human bone marrow. The constant region derived from immunoglobulin has a unique amino acid sequence depending on the isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, IgE, or IgY, and the constant region of the recombinant chimeric antibody of the present invention may be the constant region of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. An expression vector can be prepared using the gene of the chimeric antibody thus prepared. A host cell is transformed with the expression vector to obtain a chimeric antibody-producing transformed cell, and the transformed cell is cultured to obtain the desired chimeric antibody from the culture supernatant.

[0027] Another preferred embodiment of the antibody of the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7 An example of such a humanized antibody is a humanized antibody. Here, the term "humanized antibody" refers to an antibody in which only the DNA sequence of the antigen-binding site (CDR; complementarity determining region) of a non-human animal antibody such as a mouse is transplanted (CDR grafting) into a human antibody gene. Specifically, the term refers to a humanized antibody in which a part or all of the CDRs are CDRs derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework region of the variable region is a framework region of the variable region derived from human immunoglobulin, and the constant region is a constant region derived from human immunoglobulin.

[0028] For example, a recombinant humanized antibody derived from a mouse monoclonal antibody can be produced by genetic engineering, that is, by isolating the DNA of the mouse heavy chain CDR region and the DNA of the mouse light chain CDR region from a hybridoma producing a mouse monoclonal antibody, and isolating the entire region of the human heavy chain gene other than the human heavy chain CDR and the entire region of the human light chain gene other than the human light chain CDR from a human immunoglobulin gene.

[0029] The human heavy chain gene grafted with the isolated DNA of the mouse heavy chain CDR region is introduced into an appropriate expression vector so as to be expressible, and similarly, the human light chain gene grafted with the DNA of the mouse light chain CDR region is introduced into another appropriate expression vector so as to be expressible. Alternatively, the human heavy chain and light chain genes grafted with mouse CDRs can be introduced into the same expression vector so as to be expressible. A humanized antibody-producing transformed cell is obtained by transforming a host cell with the expression vector thus prepared, and the transformed cell is cultured to obtain the desired humanized antibody from the culture supernatant.

[0030] Another preferred embodiment of the antibody of the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7 An example of such an antibody is a human antibody. A "human antibody" is an antibody in which all regions, including the heavy chain variable region, heavy chain constant region, and light chain variable region, light chain constant region, constituting an immunoglobulin, are immunoglobulins derived from a gene encoding human immunoglobulin, and can be produced by introducing a human antibody gene into a mouse. Specifically, for example, a transgenic animal produced by incorporating at least a human immunoglobulin gene into the gene locus of a mammal other than a human, such as a mouse, can be produced by immunizing the animal with an antigen in the same manner as in the production method of a polyclonal antibody or monoclonal antibody described above.

[0031] The antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7 may be in any antibody form. For example, in addition to the above-mentioned chimeric antibodies, humanized antibodies, and human antibodies, the antibody may be a multispecific antibody (such as a bispecific antibody), a single-chain antibody, a minibody, a diabody, a triabody, etc. The antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7 may also be a whole antibody or a functional fragment of the antibody (which in the present invention is synonymous with the antigen-binding fragment of an antibody, for example, Fab, F(ab')2, Fab', Fv, scFv, sdFv, etc.).

[0032] Antibodies that inhibit the intermolecular interaction between VSIG4 and Siglec-7 inhibit glycosylation, acetylation, and , formylation, amidation, phosphorylation, or PEGylation. Good too.

[0033] The antibodies and the like obtained as described above can be isolated, purified, and recovered by known purification methods. Examples of the purification methods include affinity purification, reverse phase chromatography, reverse phase high performance liquid chromatography (RP-HPLC), ion exchange chromatography, and other chromatography methods. These include, but are not limited to, the purification method, gel filtration, column purification, polyacrylamide gel electrophoresis (PAGE), and the like, or any combination thereof.

[0034] The present invention relates to an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. Specific examples of the fragment include the following: (a-1) 2C7 antibody, which is an anti-VSIG4 neutralizing antibody, or a functional fragment thereof; (a-2) an anti-VSIG4 antibody having a specific sequence as an epitope, or a functional fragment thereof; (b-1) An example is the 4G7 antibody, which is an anti-Siglec-7 neutralizing antibody, or a functional fragment thereof. That is, in one aspect of the present invention, there is provided a method for inhibiting the intermolecular interaction between VSIG4 and Siglec-7, comprising: The present invention provides the above antibody (hereinafter sometimes referred to as the "antibody of the present invention") or a functional fragment thereof. The antibody of the present invention can be produced using the methods described in the Examples.

[0035] (a-1) 2C7 antibody CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO:5; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO:6; A heavy chain variable region comprising CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:7; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 8; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 9; An antibody or a functional fragment thereof comprising a light chain variable region and CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO:10.

[0036] (a-2) An antibody or a functional fragment thereof that recognizes as an epitope a region contained in the amino acid sequence of VSIG4 set forth in SEQ ID NO: 13 or a region corresponding to the same region in other VSIG4.

[0037] (b-1) 4G7 antibody CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO: 14; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO: 15; A heavy chain variable region comprising a CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 17; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 18; An antibody or a functional fragment thereof comprising: a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO:19.

[0038] In addition, as long as the antibody of the present invention maintains its properties of having binding activity to VSIG4 or Siglec-7 and activating anti-tumor immune responses by inhibiting the intermolecular interaction between VSIG4 and Siglec-7, one or several amino acid residues may be added to the amino acid sequence of each of the heavy chain variable region and the light chain variable region of the antibody. There may be substitution, deletion, addition or insertion of 1 to 20, 1 to 10, 1 to 5, 1 to 3 or 1 to 2 amino acids. Such substitution, deletion or addition may be introduced into the CDR, but is preferably introduced into a region other than the CDR. In addition, the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention. Here, conservative substitution means replacing an amino acid residue with another chemically similar amino acid residue so as not to substantially alter the activity of the peptide. For example, a hydrophobic residue may be replaced with another hydrophobic residue, a polar residue may be replaced with another polar residue having the same charge, etc. Examples of functionally similar amino acids that can be replaced in this way include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of positively charged (basic) amino acids include arginine, histidine, lysine, etc. Examples of negatively charged (acidic) amino acids include aspartic acid, glutamic acid, etc. The amino acid sequence of the antibody of the present invention, which contains substitutions, deletions, additions or insertions in the amino acid sequence, is, for example, an amino acid sequence in which the heavy chain variable region after the amino acid sequence modification has a % identity of, for example, 90% or more (more preferably, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence before modification, and an amino acid sequence in which the light chain variable region after the amino acid sequence modification has a % identity of, for example, 90% or more (more preferably, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence before modification.

[0039] Furthermore, the antibody used as the anti-tumor immune response activator may be an antibody that competes with the 2C7 antibody in binding to VSIG4 and inhibits the intermolecular interaction between VSIG4 and Siglec-7 by binding to VSIG4. In addition, the antibody used as the antitumor immune response activator has the same binding activity as 4G7 in binding to Siglec-7. This antibody competes with the antibody and binds to Siglec-7, inhibiting the intermolecular interaction between VSIG4 and Siglec-7. It's fine. Here, the term "compete" refers to, for example, the reaction between an antibody and an antigen as determined by surface plasmon resonance (SPR) method. This refers to the fact that, when the binding between an antigen and an antibody is measured, the binding between the antigen and the antibody is significantly reduced in the presence of a competing antibody.

[0040] <Anti-tumor immune response activators, immune checkpoint inhibitors> Antibodies that inhibit the above-mentioned intermolecular interaction between VSIG4 and Siglec-7 are immune checkpoint inhibitors. In addition, such antibodies, etc. can cause immune checkpoint inhibition in immune cells by inhibiting the functions of VSIG4 and / or Siglec-7, thereby releasing the suppression of anti-tumor immune response (cancer immune response) contributed by immune checkpoint molecules and their ligands, activating the anti-tumor immune response, and enhancing an effective anti-tumor immune response (cancer immune response). In other words, antibodies, etc. that inhibit the intermolecular interaction between VSIG4 and Siglec-7 can be used as anti-tumor immune response activators that cause immune checkpoint inhibition, release the suppression of anti-tumor immune response (cancer immune response) contributed by immune checkpoint molecules, activate the anti-tumor immune response, and enhance an effective anti-tumor immune response (cancer immune response).

[0041] That is, in one embodiment of the present invention, an antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7 or a functional fragment thereof (hereinafter, may be referred to as "the anti-tumor immune response activator of the present invention"). The present invention also relates to an antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7, or a compound that inhibits the intermolecular interaction between VSIG4 and Siglec-7, The present invention may also be embodied as an immune checkpoint inhibitor, including a target fragment thereof.

[0042] Here, one embodiment of the cells in which immune checkpoint inhibition is induced is a cell expressing Siglec-7 protein on the cell surface, and examples of such cells include NK cells, T cells, and the like. More specifically, the antibody or the like according to the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7 is an immune checkpoint inhibitor. and an antitumor immune response activator, preferably an immune cell such as a NK cell or a T cell. They can be used as checkpoint inhibitors and antitumor immune response activators. More specifically, the antibodies and the like according to the present invention that inhibit the intermolecular interaction between VSIG4 and Siglec-7 activate the immune response of NK cells, preferably enhance the cytotoxicity of NK cells and / or can be used as an immune checkpoint inhibitor and an antitumor immune response activator (or an "NK cell cytotoxicity enhancer" or an "NK cell cytokine production enhancer") that enhances the cytokine production of NK cells. Antibodies that block interactions between immune cells activate the immune response of T cells, an immune checkpoint It can be used as an inhibitor and activator of anti-tumor immune response.

[0043] Immune checkpoint inhibitors and antitumor immune response activators interact with VSIG4 and Siglec-7 It contains as an active ingredient an antibody that inhibits intermolecular interactions. Immune checkpoint inhibitors and antitumor immune response activators inhibit the intermolecular interaction between VSIG4 and Siglec-7. The antibody or the like may be contained in a prophylactically or therapeutically effective amount. Any combination of two or more of them may be used.

[0044] The immune checkpoint inhibitor and anti-tumor immune response activator may consist only of an antibody, etc., or may be formulated together with optional ingredients based on conventional methods in the formulation field.

[0045] In addition, the contents described in the section <Antitumor pharmaceutical composition> below regarding optional components, dosage, administration method, target tumor, administration target, etc. for formulation can also be applied to the immune checkpoint inhibitor and antitumor immune response activator. In addition, the immune checkpoint inhibitor and antitumor immune response activator can be used as a reagent, and the production, use, etc. of the reagent can be performed based on a conventional method in the field of molecular biology.

[0046] Immune checkpoints such as antibodies that inhibit the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention The immune checkpoint inhibitory effect (immune checkpoint inhibitory activity) and antitumor immune response activation ability can be evaluated using as an index an increase in the production of inflammatory cytokines (e.g., IFN-γ, etc.) (preferably a statistically significant increase), an increase in cytotoxicity (preferably a statistically significant increase), or an increase in the degree of T cell activation (preferably a statistically significant increase) in immune cells, such as NK cells, treated with an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7, compared to cells not treated with an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7. More specifically, the immune checkpoint inhibitory effect and antitumor immune response activation ability of an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7 can be evaluated by measuring the amount of cytokines in the culture medium in a co-culture of NK cells expressing Siglec-7 and cells expressing VSIG4, and comparing the amount with a control not treated with the antibody or the like, or by measuring the cytotoxicity and comparing the amount with a control not treated with the antibody or the like.

[0047] <Antitumor pharmaceutical composition> The present invention relates to an immune control comprising an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7. The present invention provides an antitumor pharmaceutical composition having an action mechanism of activating an antitumor immune response (or inducing an antitumor immune response) based on checkpoint inhibition. Such a pharmaceutical composition is capable of inhibiting NK cells, T cells, and the like. The present invention is useful for preventing or treating tumors (or preventing or treating cancer) based on the inhibition of immune checkpoints against immune cells such as cells.

[0048] Here, in the present invention, tumor (or cancer) prevention includes not causing tumors (or cancers). Tumor (or cancer) prevention also includes preventing or suppressing further progression of tumors (or cancers) at various stages, such as the early stage of onset. Tumor (or cancer) prevention may be prevention of recurrence of tumors (or cancers). Tumor (or cancer) treatment means improving, alleviating, and preventing or suppressing progression of tumors (or cancers).

[0049] That is, in one aspect of the present invention, the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention is The present invention provides an anti-tumor pharmaceutical composition comprising an inhibitory antibody and the like. In a further aspect of the present invention, a compound according to the present invention is provided which inhibits the intermolecular interaction between VSIG4 and Siglec-7. The present invention provides an anti-tumor pharmaceutical composition comprising an immune checkpoint inhibitor, such as an antibody that inhibits tumor growth factor receptors (GFR) and / or a tumor growth factor receptor (TGR). In a further aspect of the present invention, a compound according to the present invention is provided which inhibits the intermolecular interaction between VSIG4 and Siglec-7. The present invention also provides an anti-tumor pharmaceutical composition comprising the anti-tumor immune response activator of the present invention, which comprises an antibody that inhibits tumor growth. Hereinafter, these may be referred to as "the antitumor pharmaceutical composition of the present invention."

[0050] Tumors (or cancers) that can be prevented or treated by the present invention include, but are not limited to, brain tumors such as gliomas; head and neck cancer; digestive tract cancers such as small intestine cancer, colon cancer, gastrointestinal stromal tumor (GIST), digestive tract carcinoid, gastric cancer, and esophageal cancer; cancers occurring near the intestinal tract, such as liver cancer, gallbladder and biliary tract cancer, pancreatic cancer, pancreatic and digestive tract neuroendocrine tumors, and Langerhans cell histiocytosis, renal cell cancer, renal pelvis and ureter cancer, and adrenal tumors; pelvic cancers such as bladder cancer, urethral cancer, prostate cancer, testicular tumor, penile cancer, uterine cancer, cervical cancer, uterine tumor, ovarian tumor, and female genital cancer; cancers occurring in the chest, such as lung cancer, thymic tumor, mesothelioma, and breast cancer; myeloid system cancers such as hematopoietic tumors, leukemia, myeloproliferative disorders, and multiple myeloma; and osteosarcoma, soft tissue sarcoma, and malignant lymphoma occurring near the intestinal tract. The tumors (or cancers) that are the targets of prevention or treatment in the present invention are gliomas, ovarian cancers, and multiple myelomas that highly express VSIG4. Preferred examples of the cancer include myeloma, gastric cancer, and pancreatic cancer.

[0051] The antitumor pharmaceutical composition of the present invention includes an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7. The antitumor pharmaceutical composition of the present invention comprises as an active ingredient VSIG4 and Siglec-7. The composition may contain an antibody or the like that inhibits the intermolecular interaction in a prophylactically or therapeutically effective amount. The antibody or the like may be used alone or in combination of any two or more kinds. The amount of the antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7, which is the active ingredient in the antitumor pharmaceutical composition of the present invention, may be Although it generally differs depending on the dosage form, the amount of active ingredient can be set within the range of, for example, about 0.01 to 99.9% by mass so as to achieve a desired dosage.

[0052] The antitumor pharmaceutical composition of the present invention may consist of only the antibody, etc., or may be formulated together with other ingredients based on a conventional method in the pharmaceutical field. The antitumor pharmaceutical composition of the present invention may contain an antibody, etc. that inhibits the intermolecular interaction between VSIG4, which is the active ingredient, and Siglec-7, as long as the effect is not hindered. In addition, the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention can be used in combination with an antitumor active ingredient other than VSIG4. Since antibodies and the like that inhibit this interaction can activate anti-tumor immune responses, the anti-tumor pharmaceutical composition of the present invention can also be administered as an adjuvant for cancer treatment in combination with other anti-tumor measures.

[0053] The antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7 of the present invention is administered together with other antitumor active ingredients. When used in combination with other active antitumor ingredients, a single dosage form may contain both the antibody, etc. and one or more other active antitumor ingredients. It may be in the form of a combination drug containing two or more kinds, and may be a formulation containing an antibody, etc. in a unit dosage form. The composition may be administered in a unitary dosage form in combination with a preparation containing one or more other antitumor active ingredients at the time of use. When administering a combination of a formulation containing an antibody, etc., which has been separately prepared, and a formulation containing one or more other antitumor active ingredients, the two are mixed immediately before administration to form a single dosage unit. The adjuvant agent for cancer treatment can be administered before, at the same time, or after the administration of the anticancer drug, as appropriate, based on the administration schedule of the anticancer drug that is usually used.

[0054] The antitumor pharmaceutical composition of the present invention is characterized by the intermolecular interaction between the active ingredients VSIG4 and Siglec-7. In addition to the inhibitory antibody, etc., the composition may contain, as optional components, carriers, diluents, and excipients that are commonly used in the pharmaceutical field. For example, lactose, magnesium stearate, etc. are used as carriers and excipients for tablets. Physiological saline, PBS, glucose, and the like are used as aqueous solutions for injection. As the oily liquid, sesame oil, soybean oil, etc. are used, and as the solubilizing agent, benzyl benzoate, benzoyl benzoate, etc. are used. The antitumor pharmaceutical composition of the present invention can be administered in various forms, including oral administration using tablets, capsules, granules, powders, syrups, etc., and parenteral administration using injections, drips, suppositories, etc.

[0055] The dosage varies depending on symptoms, age, body weight, etc., but in general, for oral administration, the amount of active ingredient for an adult is, for example, about 0.01 mg to 1000 mg per day, which can be administered once or in several divided doses. In addition, for parenteral administration, for example, about 0.01 mg to 1000 mg per dose can be administered by subcutaneous injection, intramuscular injection, or intravenous injection.

[0056] The subject of prevention or treatment in the present invention may be any animal, but is preferably a mammal, for example, a primate such as a human, chimpanzee, or gorilla, a rodent such as a mouse, rat, or guinea pig, or a mammal such as a cow, horse, pig, sheep, goat, llama, camel, dog, cat, or rabbit.

[0057] The subjects for prevention or treatment in the present invention (preferably mammals, particularly humans) are subjects who have been diagnosed as having a high likelihood of developing a tumor or subjects who have developed a tumor, and the anti-tumor pharmaceutical composition of the present invention can be administered to these subjects.

[0058] <Method> The present invention also relates to an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7, also provides a method of inhibiting an immune checkpoint, comprising administering an immune checkpoint inhibitor to a subject (or patient). The present invention also relates to an immunological agent, such as an antibody that inhibits the above-mentioned intermolecular interaction between VSIG4 and Siglec-7. Also provided is a method for activating an anti-tumor immune response (or a method for inducing an anti-tumor immune response) based on immune checkpoint inhibition, comprising administering a checkpoint inhibitor or an anti-tumor immune response activator to a subject. The present invention also relates to an immunological agent, such as an antibody that inhibits the above-mentioned intermolecular interaction between VSIG4 and Siglec-7. Also provided is a method for preventing or treating tumors (or cancers) based on immune checkpoint inhibition, comprising administering a checkpoint inhibitor, an anti-tumor immune response activator, or an anti-tumor pharmaceutical composition to a subject. In these methods, immune checkpoint inhibition inhibits the expression of VSIG4 protein on the cell surface. It is preferable that the present invention is directed to cells expressing the Siglec-7 protein on the cell surface and immune cells expressing the Siglec-7 protein on the cell surface, for example, cancer cells and NK cells, T cells, etc. It is preferable that there is.

[0059] In addition, the contents explained in the above section <Antitumor pharmaceutical composition> regarding the dosage, administration method, target tumor, administration subject, etc. in the above method can also be applied to the above method. EXAMPLES

[0060] The present invention will be described in detail below with reference to examples. However, these are merely illustrative of the present invention and the scope of the present invention is not limited to these examples.

[0061] Materials and Methods (cell culture) Human NK cell line KHYG-1 cells were purchased from the JCRB Cell Bank (Osaka, Japan), and NK92 and NK92 MI cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). KHYG-1 cells were cultured in RPMI-1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 20% FBS (Bio West, Nouaille, France), 100 units / mL penicillin and 100 g / mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 100 IU / mL human IL-2 (PeproTech, Cranbury, NJ, USA). NK92 cells were cultured in Alpha MEM (Nacalai Tesque, Inc.) supplemented with 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA), 1.5 g / L sodium bicarbonate (Thermo Fisher Scientific), 0.2 mM inositol (Nacalai Tesque, Inc.), 0.02 mM folic acid (Nacalai Tesque, Inc.), 0.1 mM 2-mercaptoethanol (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan), 12.5% ​​FBS, 12.5% ​​horse serum (Thermo Fisher Scientific), and 100 IU / mL human IL-2. Human normal NK cells were purchased from Japan BioTherapy Institute (Tokyo, Japan) and cultured using AlyS505N-7 (Cell Science Institute, Sendai, Miyagi, Japan). The human acute monocytic leukemia cell line P31 / FUJ was purchased from the JCRB Cell Bank, the human acute T-cell leukemia cell line Jurkat was purchased from Riken Cell Bank (Ibaraki, Japan), and Jurkat NFAT-Luc cells were purchased from Invivogen (San Diego, CA, USA). They were cultured in RPMI-1640 supplemented with 10% FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0062] HEK293 cells and 293FT cells were purchased from ATCC and Thermo Fisher Scientific, respectively, and cultured in DMEM (Nacalai Tesque, Inc.) supplemented with 10% FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin. Expi293-F cells were purchased from Thermo Fisher Scientific and cultured in Expi293 Expression Medium (Thermo Fisher Scientific) supplemented with 50 units / mL penicillin and 50 μg / mL streptomycin. The cells were cultured in 125 mL Erlenmeyer flasks (Thermo Fisher Scientific) in a 37°C incubator with a CO2 concentration of 8%, using a rotary shaker RemoteShake (Wakembee Tech Co., Ltd., Kyoto, Japan) with shaking at 125 rpm.

[0063] All cells except Expi293-F cells were incubated in a 5% CO2 incubator (Panasonic). The cells were cultured at 37°C using a 30-mL PBS (Nic Corporation, Osaka, Japan).

[0064] (antibody) The primary antibodies used in flow cytometry and Western blotting in this study are listed in Table 1. Horseradish peroxidase (HRP)-conjugated secondary antibodies used in Western blotting were purchased from Merck Millipore (Burlington, MA, USA), anti-rat IgG-HRP, and anti-mouse IgG-HRP, respectively. Anti-human IgG-HRP was purchased from Invitrogen (Carlsbad, CA, USA). Mouse anti-Human Siglec-7 S7.7 (BioLegend, San Jose, CA) was used as a neutralizing antibody against Siglec-7. Diego, CA, USA) was used.

[0065] [Table 1]

[0066] (Construction of expression vectors for Siglec family, VSIG4 and VSIG4 mutant proteins) The extracellular domains of Siglec-2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -14, -15 and VSIG4 were cloned into the pENTR / D-TOPO vector (Thermo Fisher Scientific) to obtain entry clones. A Gateway cassette containing two attR recombination sequences and, inside the sequences, a ccdB gene and a chloramphenicol resistance gene was inserted into the SmaI site of pHEK293 Ultra Expression Vector II (Takara Bio Inc., Shiga, Japan), a vector that is highly expressed in HEK293 cells. Human IgG2-Fc sequence (hereafter abbreviated as Fc) was inserted into the XhoI-SphI site to prepare pHEK-Fc destination vector, or a tag sequence consisting of rat Cd4 domains 3 + 4, rat COMP (cartilage oligomeric matrix protein) pentamerization domain, FLAG, and 6 × His arranged in tandem (hereafter COMP tag) was inserted into the XhoI-SphI site to prepare pHEK-COMP destination vector. Recombination of the entry clone and destination vector using LR clonase (Invitrogen) was performed to prepare expression vectors with the Fc or COMP tag added.

[0067] Glycosylation sites in the extracellular domain of VSIG4 were predicted using NetOGlyc (http: / / www.cbs.dtu.dk / services / NetOGlyc / ), a program that predicts O-glycosylation sites using neural networks. The expression vector for VSIG4 mutant proteins was constructed by substituting alanine for the predicted O-glycosylation sites S139, S141, T144, T146, T147, T241, and T245. Each was amplified by PCR and cloned into the ENTR / D-TOPO vector, and then cloned into the pHEK-COMP vector and the LR clone. It was produced by recombination using enzymes.

[0068] (Siglec family, purification of VSIG4 and VSIG4 mutant proteins) Fc- or COMP-tagged proteins of Siglec-2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -14, -15, VSIG4, and VSIG4 mutants were produced as follows: 30 μg of expression vector, 6 μg of pHEK293 Enhancer Vector (Takara Bio Inc.) and 10 μg of β-lactamase (β-lactamase) were added to 3 mL of Opti-MEM. 36 μL of TransIT-PRO Reagent (Mirus Bio, Madison, WI, USA) was added and incubated at room temperature for 15 min. The transfection mix was then transfected with 3 × 10 7 Transfection was performed by adding 1000 μg of Expi293-F cells to the The cells were cultured at 125 rpm for 4 days in a humidified incubator at 37°C with 8% CO2, and the proteins were secreted into the culture supernatant. The culture supernatant was filtered through a 0.45 μm filter (Merck Millipore The cells were completely removed by passing the cells through a column (GE Healthcare, Little Chalfont, UK). Proteins were purified using MicroSpin Columns (GE Healthcare, Little Chalfont, UK). Fc-tagged proteins were prepared by reacting the culture supernatant with Protein A Sepharose (GE Healthcare) that had been washed twice with Binding buffer (20 mM Na3PO4, pH 7.0) at 4°C overnight, eluting the proteins with Elution buffer (0.1 M glycine, pH 3.0), and neutralizing the mixture with 1 M Tris-HCl (pH 9.0) to obtain purified proteins. COMP-tagged proteins were prepared by adding 1 / 10 volume of 5M NaCl to the culture supernatant, adding Ni Sepharose (GE Healthcare, Little Chalfont, UK) washed twice with binding buffer (20 mM Na3PO4, 500 mM NaCl, 20 mM imidazole, pH 7.4), and reacting overnight at 4°C. The proteins were eluted using elution buffer (20 mM Na3PO4, 500 mM NaCl, 500 mM imidazole, pH 7.4), and then dialyzing overnight at 4°C against PBS using a Slide-A-Lyzer MINI Dialysis Device (Thermo Fisher Scientific) to obtain purified proteins. The purity and molecular weight of each purified protein were confirmed by silver staining using a Silver Stain MS Kit (Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan), and when concentration was required, Amicon Ultra Centrifugal Filters (10 K, 0.5 mL) (Merck Millipore) were used. Protein concentrations were quantified by the Bradford method using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA).

[0069] (ELISA binding assay) The binding assay between VSIG4-COMP protein and Siglec family proteins was carried out as follows. Normal human IgG (Sigma-Aldrich), recombinant human Siglec-1 protein (R&D Systems), and Fc fusion proteins of Siglec-2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -14, and -15 purified from Expi293-F cells were dissolved in carbonate buffer at a concentration of 20 nM. After dilution, 100 μL of each solution was added to a Nunc MaxiSorp™ flat-bottom plate (Thermo Fisher Scientific) in duplicate and left to stand overnight at 4°C for immobilization. Wash twice with PBS-T. Afterwards, 300 μL of 1% BSA / PBS was added to each well and blocking was performed at room temperature for 1 hour. 100 μL of VSIG4-COMP protein diluted to 10 nM with the mixing solution was added and incubated at room temperature for 1 hour. The cells were washed three times with PBS-T and then 100 μl of anti-FLAG M2 antibody-HRP (Sigma-Aldrich) diluted 1:10,000 was added. After washing four times with PBS-T, the reaction mixture was incubated at room temperature for 1 hour in the dark. 100 μL of TMB Soluble Reagent (ScyTek, Logan, UT, USA) was added to each well and incubated at room temperature for 30 minutes in the dark, after which TMB Stop Buffer (ScyTek) was added to stop the color reaction. The absorbance at 450 nm was measured using an EnSight Multimode Plate Reader. The binding assay between VSIG4-COMP mutant proteins and Siglec-7-Fc protein was performed as follows. The VSIG4-COMP protein and the VSIG4-COMP protein with S139A, S141A, T144A, T146A, T147A, T241A, and T245A mutations were diluted in carbonate buffer to a concentration of 50 nM, and 100 μL of each was added to a Nunc MaxiSorp flat-bottom plate in duplicate and allowed to stand overnight at 4°C for immobilization. After washing twice with PBS-T, 300 μL of 1% BSA / PBS was added to each plate and blocking was performed at room temperature for 1 hour. 100 μL of Siglec-7-Fc protein diluted to 50 nM with blocking solution was added and reacted at room temperature for 1 hour. After washing three times with PBS-T, 100 μL of anti-human IgG antibody-HRP (Thermo Fisher Scientific) diluted 1:2000 was added to each plate and reacted at room temperature for 1 hour in the dark. After washing four times with PBS-T, 100 μL of TMB Soluble Reagent was added and incubated at room temperature for 30 minutes in the dark, and then TMB Stop Buffer was added to stop the color reaction. The absorbance at 450 nm was measured using an EnSight Multimode Plate Reader.

[0070] (Protein desialylation) The VSIG4-COMP protein was desialylated using α2-3,6,8 Neuraminidase (New England Biolabs, Ipswich, MA, USA). 2 μg of protein was reacted with Neuraminidase in GlycoBuffer (New England Biolabs) at 37°C for 1 hour, and then inactivated by heating at 65°C for 10 minutes. The removal of sialic acid was confirmed by Western blotting 50 ng of protein and detecting α2-3 sialic acid using anti-Siaα2-3 monoclonal antibody (HYB4, Fujifilm Wako Pure Chemical). The FLAG tag contained in the VSIG4-COMP protein was used as a loading control and detected with anti-FLAG-M2 antibody (Sigma-Aldrich).

[0071] (Construction of lentiviral vectors) VSIG4 is a sequence up to the stop codon to add the V5 tag, and Siglec-7 is a sequence up to the stop codon. The full-length sequence including Don was cloned into pENTR / D-TOPO vector to obtain an entry clone. The destination vector was pLenti6 / V5-DEST (Thermo Fisher Scientific) for blasticidin selection or pLenti7.3 / V5-DEST (Thermo Fisher Scientific) for GFP selection, and expression vectors were generated by recombination with the entry vector using LR clonase.

[0072] (Creation of stable gene expression cells) To introduce VSIG4 and Siglec-7 into HEK293 cells, we used the Lenti6-V5 / DEST expression vector. Vector and packaging vector ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific) was transfected into 293FT cells using PEI MAX (Polysciences, Warrington, PA, USA), and after 2 days of culture, the supernatant was passed through a 0.45 μm filter to obtain a virus solution. HEK293 cells were infected with the virus solution for 48 hours and selected with 10 μg / mL blasticidin to obtain a stable expression line.

[0073] Siglec-7 was introduced into NK92 cells and VSIG4 was introduced into Jurkat cells as follows. Each pLenti7.3 / V5-DEST expression vector and ViraPower Lentiviral Packaging Mix were A virus solution was obtained in the same manner as above using RetroNectin (Takara Bio Inc., Shiga, Japan). The mixture was added to a 24-well plate (As One Corporation, Osaka, Japan) coated with RetroNectin and centrifuged at 2000 rpm for 3 hours at room temperature. After removing the supernatant, the plate was washed once with PBS and seeded with NK92 cells. The cells were contacted with the virus bound to RetroNectin by centrifugation at 1200 rpm for 5 minutes and cultured for 24 hours for infection. Stable expression cells were obtained by isolating GFP-positive fractions and fractions that stained positive with anti-Siglec-7 antibody using a FACSAriaIII (BD, Franklin Lakes, NJ, USA). The VSIG4 gene was knocked out in P31 / FUJ cells using the lentiviral vector pLV-U6g-EPCG (Sigma), which expresses both a short guide RNA (HS0000100372) against VSIG4 and the Cas9 protein. After lentivirus infection, VSIG4 knockout cells were obtained by isolating GFP-positive fractions and fractions that stained negative with anti-VSIG4 antibody.

[0074] (Expression analysis using flow cytometry) 1.0 × 10 in HBSS (Gibco / Thermo Fisher Scientific) supplemented with 1% FBS. 6 The cells were adjusted to a density of 1.0 × 10 cells / mL, and APC anti-VSIG4 or APC anti-Siglec-7 was added and incubated at room temperature for 30 minutes in the dark. The cells were washed twice with PBS and then diluted with HBSS + 1% FBS at 1.0 × 10 7 The cells were then cultured at a final concentration of 1 μg / mL, and propidium iodide (Fujifilm Wako Pure Chemical Corporation) was added to a final concentration of 1 μg / mL. The cells were then passed through a 35 μm cell strainer (Corning, Corning, NY, USA) to obtain single cells, which were then analyzed using a FACS Verse (BD).

[0075] (Protein binding analysis using FACS) HEK293 cells transfected with VSIG4 or Siglec-7 were detached from the dish using 0.5 mM EDTA and cultured at 1 × 10 6 The cells were prepared at a concentration of 100 μL per mL. 0.5 μg of purified VSIG4 or Siglec-7 protein with Fc or COMP tags was incubated with FITC anti-human IgG Fc antibody or FITC anti-FLAG M2 antibody, respectively, at room temperature for 20 minutes, and then added to 100 μL of the cell preparation solution described above and incubated at room temperature for another 30 minutes. After washing once with HBSS + 1% FBS, the cells were passed through a 35 μm cell strainer and analyzed using FACS Verse.

[0076] (VSIG4 and Siglec-7 binding assay using Alpha technology) Proteins were diluted in 0.2% BSA / PBS, and VSIG4-COMP was diluted from 40 nM to 1.2 μM. (final concentration 10 nM - 300 nM), and Siglec-7-Fc protein was 400 nM (final concentration 100 nM). The Fc- and COMP-tagged proteins were added in an amount of 5 μL each to a 384-well plate (PerkinElmer, Waltham, MA, USA) and incubated at room temperature for 60 min. Next, 5 μL each of Anti-FLAG Alpha Donor Beads and Alpha Screen Protein A Acceptor Beads (PerkinElmer) diluted to 80 μg / mL in 0.2% BSA / PBS were added and incubated at room temperature for 60 min in the dark. Intermolecular binding was detected as emission at 520-620 nm when excited at a wavelength of 680 nm, and was detected using an EnSight Multimode Plate Reader (PerkinElmer).

[0077] (Western Blotting) After washing the cultured cells with PBS, Triton lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 200 μM AEBSF, 10 μM leupeptin, 1 μM pepstatin A, 1 μM DTT) was added and the cells were lysed on ice for 30 minutes. Then, the cells were lysed on a centrifuge cooled to 4°C. The supernatant was collected by centrifugation at 12,000 rpm for 10 min. Lysis buffer and 4 × SDS sample buffer (0.25 M Tris-HCl [pH 6.8], 40% glycerol, 8% SDS, 20% βME, 0.2% BPB) were added to the supernatant to adjust the concentration to 1 μg / μL, and the supernatant was boiled at 95°C for 5 min to prepare a sample for Western blotting. SDS-PAGE gel electrophoresis was performed using a 10% acrylamide: bis separating gel, with 10 μg of sample injected into each well, at a constant current of 20-40 mA, using SDS-PAGE electrode buffer (25 mM Tris, 0.2 M glycine, 0.1% SDS). Blotting was performed using the semi-dry method. The PVDF membrane (Merck Millipore) was soaked in methanol and then in transfer buffer (50 mM Tris, 40 mM Glycine, 20% methanol, 0.1% SDS) in that order, and then blotted for 60 minutes at a constant current of 110 mA using a Transblot SD cell (Bio-Rad). The PVDF membrane was then soaked in 3% skim milk / PBS-T (PBS-0.1% Tween 20) for blocking. After washing once with PBS-T, the primary antibody diluted 1:1000 was reacted overnight at 4°C. After washing three times with PBS-T, the HRP-labeled antibody of each animal species was added as the secondary antibody, also diluted 1:1000, and reacted for 1 hour at room temperature. A 0.5% skim milk solution was used as the diluent. After washing again three times with PBS-T, the sections were reacted with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and detected using an Amersham Imager 680 (GE Healthcare).

[0078] (Preparation of anti-VSIG4 and anti-Siglec-7 monoclonal antibodies) Hybridomas were produced by the Institute of Cell Engineering (Osaka, Japan). Purified VSIG4-Fc and Siglec-7-Fc proteins were used as antigens, and B6D2F1 / Slc mice were used as immunized animals. Lymphocytes were collected from the iliac lymph nodes of mice immunized with the antigen and transfected with SP2 mouse myeloma cells. The hybridomas were produced by fusion of the hybridomas. The hybridoma culture supernatant was screened for neutralizing activity and clones were selected. Hybridoma clones were cultured in Hybridoma-SFM medium (Thermo Fisher Scientific). The culture supernatant obtained after approximately 2 weeks of culture was collected and passed through a 0.45 μm filter to completely remove cells. The antibodies were purified using MicroSpin Columns in the same manner as for the purification of Fc fusion proteins.

[0079] (Evaluation of neutralizing activity of anti-VSIG4 antibodies using ELISA) Recombinant Human Siglec-1 protein, Complement C3b (Sigma-Aldrich), Siglec-4-Fc, and Siglec-7-Fc proteins were prepared in carbonate buffer at a concentration of 100 nM and then diluted with Nunc MaxiSorp. Add 50 μL to each of the ELISA kits and incubate overnight at 4°C to form a solid phase. After washing twice with PBS-T, 100 μL of 1% BSA / PBS was added to each well and blocking was performed at room temperature for 1 hour. After washing twice with PBS-T, a mixture of 50 μL of antibody solution serially diluted to 0.01 - 10 μg / mL and 50 μL of 100 nM VSIG4-COMP protein was added and blocking was performed at room temperature for 1 hour. After washing three times with PBS-T, 100 μL of anti-FLAG M2 antibody-HRP diluted 1:2000 was added to each well. After washing four times with PBS-T, 100 μL each of TMB Soluble Reagent was added and reacted at room temperature for 10 minutes in the dark. The reaction was stopped by adding 1 μL of each solution. The absorbance was measured.

[0080] (Assessment of neutralizing activity of anti-Siglec-7 antibodies using ELISA) VSIG4-COMP protein was prepared at a concentration of 100 nM in carbonate buffer and incubated with Nunc MaxiSorp (commercially available). Add 50 μL of each to a flat-bottom plate and leave it at 4°C overnight to immobilize. After washing twice with PBS-T, 100 μL of 1% BSA / PBS was added to each well and blocking was performed at room temperature for 1 hour. After washing twice with PBS-T, a mixture of 50 μL of antibody solution serially diluted to 0.01 - 10 μg / mL and 50 μL of 100 nM Siglec-7 protein was added and incubated at room temperature for 1 hour. After washing three times with PBS-T, 100 μL of 1:2000 diluted anti-human IgG-HRP was added to each well and incubated at room temperature for 1 hour in the dark. After washing four times with PBS-T, 100 μL of TMB Soluble Reagent was added to each well and incubated at room temperature for 10 minutes in the dark. The reaction was stopped by adding 100 μL of TMB Stop Buffer to each well. The absorbance at 450 nm was measured using an EnSight Multimode Plate Reader.

[0081] (Cytotoxicity Assay) Tumor cells used as targets for cytotoxicity were washed with PBS and then diluted to 1 × 10 6CFSE (FUJIFILM Wako Pure Chemical Corporation) was added to a final concentration of 2 μg / mL, and the cells were rotated and mixed at room temperature for 20 minutes in the dark. After washing twice with PBS, the cells were cultured at 2 × 10 5 The cells were prepared at a concentration of 100 μL / mL. When antibodies were administered, the antibodies were added after preparation and incubated at room temperature for 30 minutes. NK-92 cells, the effector used to measure cytotoxicity, were also prepared at a given effector:target ratio (E:T ratio) using the assay medium. The E:T ratio was set to 5:1 for Jurkat cells and 10:1 for P31 / FUJ cells. 100 μL of each cell suspension was added to a 96-well Ultra-low attachment round bottom plate (Corning), and a well containing only target cells was used as a control (% spontaneous cell death). The plate was centrifuged at 1000 rpm for 5 minutes and incubated at 37°C with a CO2 concentration of 5% for 4 hours. The cells were collected and centrifuged at 1000 rpm for 5 minutes, after which the supernatant was discarded, propidium iodide was added to a final concentration of 50 μg / mL, and the cells were passed through a 35 μm cell strainer before analysis using FACS Verse. Specific cell death of NK cells was calculated as (%Dead targets - %Spontaneous cell death) / (100 - %Spontaneous cell death) × 100.

[0082] (IFN-γ secretion assay) KHYG-1 cells were cultured at 1 × 10 in RPMI-1640 + 1% FBS + 50IU / mL human IL-2. 6 VSIG4 or vector-transfected HEK293 cells were harvested using 0.5 mM EDTA and washed with PBS. After that, 2 × 10 5 When administering antibodies, add them to the cell suspension in advance. The cells were incubated at room temperature for 30 minutes. Each cell suspension was then cultured in a 96-well Ultra-low attachment round. 100 μL of each solution was added to the bottom plate, and the plate was incubated at 37°C with 5% CO2 for 24 hours. The supernatant was then collected from each well. IFN-γ in the culture supernatant was quantified using a ELISA kit (Microbial Immunosorbent Assay Co., Ltd., MN, USA) according to the protocol.

[0083] (Detection of cytokine secretion using antibody arrays) 36 samples were analyzed using the Proteome Profiler Human Cytokine Array Kit (R&D Systems). KHYG-1 cells were cultured in RPMI-1640 + 1% FBS + 50IU / mL human IL-2. Using 1 × 10 6 VSIG4 or vector-transfected HEK293 cells were incubated in 0.5 mM EDTA After washing with PBS, 2 x 10 5 100 μL of each cell suspension was added to a 96-well Ultra-low attachment round bottom plate and incubated in a CO2 atmosphere at 5% After 24 hours of incubation at 37°C, the supernatant was collected from each well. The reaction was carried out according to the protocol, and cytokine secretion was detected as reactivity to the HRP substrate using an Amersham Imager 680 (GE Healthcare). The amount of cytokine secretion was quantified using Image J by quantifying the pixel density of the antibody spot for each cytokine, and the amount of cytokine secretion from KHYG-1 cells when co-cultured with HEK293 + vector cells was set to 1, and the amount of secretion when co-cultured with HEK293 + VSIG4 cells was expressed as a relative value.

[0084] (Luciferase assay of T cell activity) Jurkat NFAT-Luc cells were transfected with Siglec-7 expression vector or control vector. The cells were activated using anti-human CD3ε antibody (BioLegend). The anti-human CD3 antibody was diluted with PBS to a final concentration of 2 μg / mL, and VSIG4-Fc or normal human IgG was diluted to a final concentration of 5 μg / mL. 100 μL of the diluted protein solution was added to a 96-well plate (TPP) and incubated at 4°C overnight. The next day, the protein dilution solution was removed, and the plate was washed twice with PBS. Jurkat-Lucia NFAT cells transfected with Siglec-7 expression vector and control vector were then added at 1.0 × 10 6 After 24 hours of incubation, 20 μL of the cell suspension was taken and mixed with 50 μL of QUANTI-Luc (InvivoGen) for reaction. Luciferase luminescence was immediately measured using a GloMax 20 / 20 Luminometer (Promega, Madison, WI, USA). The reading time was 1 second.

[0085] (Determining the epitope of anti-VSIG4 antibodies using HA-VSIG4 deletion mutants) HA-VSIG4, in which the endogenous signal peptide sequence (amino acids 1-19 in SEQ ID NO:2) of the full-length amino acid sequence of VSIG4 was replaced with an amino acid sequence consisting of an Igκ leader sequence followed by an HA tag, was cloned into pHEK293 Ultra Expression Vector (Takara Bio Inc.). Next, the first loop (amino acid residues 20-137), the amino acid sequence with five potential O-glycosylation sites (amino acid residues 138-147), the second loop (amino acid residues 148-231), We constructed expression vectors for a series of deletion mutants in which the amino acid residues were successively deleted. The cells were transiently transfected with PEI MAX, and expression on the cell surface was detected by flow cytometry using APC-labeled anti-HA antibody (Biolegend) and FITC-labeled anti-VSIG4 (2C7, 2F12) antibody. FITC labeling of anti-VSIG4 (2C7, 2F12) antibody was performed using the Fluorescein Labeling Kit - NH2 (Dojindo Laboratories) according to the recommended protocol.

[0086] <Result> First, we investigated the interaction between VSIG4 and the Siglec family of molecules. Based on a method for detecting molecular interactions (Kasai et al, Cancer Sci, 2022; 113(5):1669-1678), VSIG4 protein was purified by adding a tag consisting of rat Cd4 domain 3+4, rat COMP (cartilage oligomerize protein) pentamerization domain, FLAG, and 6×His linked in tandem (hereafter referred to as the COMP tag) to the C-terminus of the extracellular domain of VSIG4 (Figure 1A). Binding of the VSIG4-COMP protein to purified proteins of Siglec-1 to -15 (however, Siglec-13 is not conserved in humans) was examined by ELISA, suggesting that VSIG4 binds to Siglec-1, -4, and -7 (Figure 1B). Siglec-7 contains an intracellular inhibitory motif ITIM (immunoreceptor tyrosine-based inhibitory motif), whereas Siglec-1 and Siglec-4 do not. Therefore, only Siglec-7 is thought to be an inhibitory receptor involved in the control of immune checkpoints.

[0087] To investigate the interaction between VSIG4 and Siglec-7 on the cell surface, we created Siglec-7 and VSIG4-transfected HEK293 cells and detected the binding of VSIG4-COMP and Siglec-7-Fc (human IgG2 Fc domain fused to the C-terminus of the extracellular domain of Siglec-7) proteins to the cells by flow cytometry. Figure 1C shows silver staining to confirm the size and purity of each protein. As a result, the binding of VSIG4-COMP was enhanced in the Siglec-7-expressing cells (Figure 1D), and the binding of Siglec-7-Fc was enhanced in the VSIG4-expressing cells (Figure 1E), suggesting that VSIG4 and Siglec-7 actually interact with each other on the cell surface.

[0088] The Siglec family is a receptor that recognizes sialic acid, which is modified at the terminus of the glycan of glycoproteins or glycolipids. Therefore, it was predicted that Siglec-7 recognizes the sialic acid modification of VSIG4 when binding to VSIG4. To confirm this, sialic acid was removed from the VSIG4-COMP protein using sialidase (Fig. 2A), and the Siglec-7-Fc protein was The binding of sialic acid was evaluated using Alpha technology. The expression of sialic acid modified on the VSIG4 protein was significantly decreased (Fig. 2B).

[0089] There are two types of glycosylation in proteins: N-type and O-type. The extracellular domain of VSIG4 has N-type While no predicted glycosylation sites were found, seven O-glycosylation sites were predicted by the NetOGlyc 4.0 program. Among these, S139, S141, T144, T146, and T147 are located between the two immunoglobulin-like loop structures of VSIG4, and T241 and T245 are located between the second loop and the transmembrane domain. VSIG4-COMP mutant proteins were constructed in which S or T at each of the candidate glycosylation sites was replaced by A (Fig. 2D). The interaction of these mutant proteins with Siglec-7-Fc was examined by ELISA. Compared with wild-type VSIG4, the S141A, T144A, T147A, and T241A mutants had equal or higher binding ability to Siglec-7, whereas the S139, T146A, and T245A mutants had lower binding ability to Siglec-7, with the T146A mutant having the lowest binding ability (Fig. 2E). These results suggest that sialic acid modification at the T146 residue is important for the binding to Siglec-7.

[0090] We next investigated whether the interaction between VSIG4 and Siglec-7 is involved in the regulation of immune checkpoints. We investigated whether Siglec-7 is involved in the regulation of NK cell proliferation and T cell proliferation in normal human NK cells and T cell models. VSIG4 is endogenously expressed in the human NK cell line KHYG-1. However, no expression was observed in the human NK cell line NK-92, and a Siglec-7-introduced line was generated (Fig. 3A). VSIG4 is endogenously expressed in the human acute monocytic leukemia cell line P31 / FUJ, and a VSIG4 knockout line was generated using the CRISPR-Cas9 system. No expression was observed in HEK293 or Jurkat cells, and a VSIG4-introduced line was generated (Fig. 3B).

[0091] NK cells have the property of showing cytotoxicity against target cells and secreting cytokines including IFN-γ. Therefore, NK cells were co-cultured with target cells (HEK293 and Jurkat cells) to investigate the mechanism of action of NK cells. The interaction between VSIG4 and Siglec-7 mediates cytotoxicity and cytokine secretion in the presence of VSIG4. First, KHYG-1 cells, which endogenously express Siglec-7, were co-cultured with HEK293+vector and +VSIG4 cells for 24 hours, and IFN-γ secreted into the culture supernatant was measured by ELISA. As a result, IFN-γ secretion from KHYG-1 cells was significantly suppressed in co-culture with the VSIG4-expressing line (Fig. 4A). In addition, when secretion of cytokines other than IFN-γ was examined using a cytokine antibody array, the secretion of CCL1 / I-309 was significantly suppressed in co-culture with the VSIG4-expressing line. It has been suggested that the secretion of MIP-1α / MIP-1β, CCL5 / RANTES, GM-CSF, IL-8, and IL-10 is suppressed. was achieved (Figure 4B).

[0092] Next, NK-92 cells and Jurkat cells were co-cultured for 4 hours, and cytotoxicity was analyzed by detecting propidium iodide-positive dead cells using flow cytometry. As a result, no difference was observed in the cytotoxicity of NK-92+vector cells against Jurkat+vector and +VSIG4 cells, but when NK-92+Siglec-7 cells were used, the cytotoxicity was significantly suppressed by co-culture with a VSIG4-expressing cell line (Figure 4C). In addition, when the cytotoxicity of NK-92 cells against P31 / FUJ cells, which endogenously express VSIG4, was examined, the cytotoxicity was significantly enhanced by co-culture with a VSIG4 knockout cell line when NK-92+Siglec-7 cells were used (Figure 4D). These facts indicate that the activity of NK cells is suppressed via the interaction between VSIG4 and Siglec-7.

[0093] It has been reported that Siglec-7 is highly expressed in activated T cells, especially memory CD8+ T cells. (Fan et al, Cancer Sci, 2023 Feb;114(2):370-383) As a model for measuring T cell activity, a plasmid expressing luciferase (Luc) downstream of the transcription factor NFAT binding domain was used. We used Jurkat NFAT-Luc cells transfected with the NFAT-Luc gene. These cells were then cloned using anti-CD3 antibody to identify T cell receptors. When somatic signals are input, the transcription factor NFAT is activated and luciferase is expressed. Jurkat cells do not endogenously express Siglec-7, so we generated a Siglec-7 transfectant (Fig. 5A). We examined whether luciferase activity was suppressed by binding of VSIG4-Fc protein to Siglec-7 when T cell receptor was stimulated with anti-CD3 antibody. As a result, luciferase activity was reduced in Jurkat NFAT-Luc + Siglec-7 cells by addition of VSIG4-Fc, whereas no change in activity was observed in vector transfectant (Fig. 5B). These results suggest that T cell activity is suppressed via the interaction between VSIG4 and Siglec-7.

[0094] To obtain anti-VSIG4 and anti-Siglec-7 antibodies that inhibit the interaction between VSIG4 and Siglec-7, we immunized mice with VSIG4-Fc and Siglec-7-Fc, respectively, to generate monoclonal antibodies. As a result, anti-VSIG4 antibodies (clone 2C7, 2F12) and anti-Siglec-7 antibodies (clone 4G7) were obtained. The neutralizing activity of these antibodies was evaluated by ELISA. First, the binding of VSIG4 to the known binding molecules, complement C3b and Siglec-1, -4, and -7, was detected by ELISA, and it was examined whether the addition of anti-VSIG4 antibody reduced the binding signal. The results showed that anti-VSIG4 antibody (clone 2C7) had neutralizing activity against the binding of VSIG4 to Siglec-1, -4, and -7. On the other hand, it did not show neutralizing activity against the binding of VSIG4 to complement C3b. It was also suggested that anti-VSIG4 antibody (clone 2F12) had neutralizing activity against the binding of VSIG4 to Siglec-7 (Figure 6).

[0095] Next, we investigated whether the binding of Siglec-7 to VSIG4 was reduced by the addition of anti-Siglec-7 antibody, and found that anti-Siglec-7 antibody (clone 4G7) had neutralizing activity against the binding of Siglec-7 to VSIG4. It was shown that (Figure 7).

[0096] Furthermore, we analyzed the epitopes of anti-VSIG4 antibodies (clones 2C7 and 2F12). To clarify whether VSIG4 recognizes this region, we constructed an expression vector for HA-VSIG4 in which an HA tag was added to the N-terminus of VSIG4, and created a series of mutants in which the first loop (amino acid residues at positions 20-137 in SEQ ID NO: 2), the region where O-glycosylation sites from S139 to T147 are concentrated (amino acid residues at positions 138-147), and the second loop (amino acid residues at positions 148-231) of VSIG4 were sequentially deleted (Fig. 8A). An anti-VSIG4 antibody (clone 2C7) bound to full-length VSIG4 and VSIG4 lacking the first loop, but hardly bound to a mutant lacking the amino acid residues at positions 138-147 where O-glycosylation sites are concentrated, and did not bind at all to a mutant lacking both loops (Fig. 8B). Clone 2F12 also bound to full-length VSIG4 and VSIG4 lacking the first loop, but did not bind to mutants lacking amino acid residues 138-147 or two loops, indicating that most of the epitopes of anti-VSIG4 antibodies (clones 2C7 and 2F12) are contained within the amino acid residues 138-147.

[0097] Finally, we examined the immune checkpoint inhibitory activity of anti-VSIG4 and anti-Siglec-7 antibodies, which inhibit the interaction between VSIG4 and Siglec-7. In coculture of KHYG-1 cells and HEK293 + VSIG4 cells, IFN-γ secretion, which was reduced by the interaction between VSIG4 and Siglec-7, was restored by administration of anti-VSIG4 (2C7) antibody (Fig. 9A), and administration of anti-Siglec-7 (4G7) antibody also reduced IFN-γ secretion. We observed recovery of VSIG4 secretion (Figure 9B). Furthermore, in co-culture of NK-92 + Siglec-7 cells and Jurkat + VSIG4 cells, the cytotoxicity suppressed by the interaction between VSIG4 and Siglec-7 was restored by administration of anti-VSIG4 (2C7) antibody (Figure 9C). Thus, it was suggested that anti-VSIG4 (2C7) antibody and anti-Siglec-7 (4G7) antibody have immune checkpoint inhibitory activity and could be used as therapeutic antibodies against tumors expressing VSIG4. [Industrial Applicability]

[0098] As described above, the present invention provides a compound having inhibitory activity against the intermolecular interaction between VSIG4 and Siglec-7. The present inventors provided anti-VSIG4 (clone 2C7) mouse monoclonal antibody and anti-Siglec-7 mouse monoclonal antibody (clone 4G7) having the following structures: In addition, the cytotoxicity and cytokine secretion of NK cells were reduced through the interaction between VSIG4 and Siglec-7, and a decrease in activity was observed. However, administration of anti-VSIG4 (2C7) antibody restored the cytotoxicity and cytokine secretion of NK cells. In a similar experiment, anti-Siglec-7 (4G7) antibody In addition, recovery of NK cell cytokine secretion was also observed. In addition, suppression of T cell activity was observed through VSIG4-Siglec-7 interaction. These results suggest that anti-VSIG4 (2C7) and anti-Siglec-7 (4G7) antibodies bind to the VSIG4-Siglec-7 domain. function as immune checkpoint inhibitors and inhibit the antitumor activity of NK cells and / or T cells It has been suggested that this signaling pathway may be involved in the treatment of VSIG4-expressing tumors, and this may be an effective treatment for tumors that express VSIG4.

[0099] VSIG4 expression correlates with poor prognosis in multiple myeloma, glioma, ovarian cancer, and gastric cancer. It has been reported that the antibody inhibits the intermolecular interaction between VSIG4 and Siglec-7, inhibits the immune checkpoint interaction between VSIG4 and Siglec-7, and has antitumor activity. Antibodies such as the antibody of the present invention are expected to be put to practical use as immune checkpoint inhibitors targeting these cancer types. In particular, there is a high demand for therapeutic drugs for glioma and ovarian cancer, which are intractable cancers.

[0100] <Array description> SEQ ID NO: 1: DNA sequence of the full-length VSIG4 gene SEQ ID NO:2: Amino acid sequence of full-length VSIG4 protein SEQ ID NO: 3: DNA sequence of full-length Siglec-7 gene SEQ ID NO: 4: Amino acid sequence of full-length Siglec-7 protein SEQ ID NO: 5: Amino acid sequence of CDR-H1 of anti-VSIG4 antibody 2C7 SEQ ID NO: 6: Amino acid sequence of CDR-H2 of anti-VSIG4 antibody 2C7 SEQ ID NO: 7: Amino acid sequence of CDR-H3 of anti-VSIG4 antibody 2C7 SEQ ID NO: 8: Amino acid sequence of CDR-L1 of anti-VSIG4 antibody 2C7 SEQ ID NO: 9: Amino acid sequence of CDR-L2 of anti-VSIG4 antibody 2C7 SEQ ID NO: 10: Amino acid sequence of CDR-L3 of anti-VSIG4 antibody 2C7 SEQ ID NO: 11: Amino acid sequence of the heavy chain variable region of anti-VSIG4 antibody 2C7 SEQ ID NO: 12: Amino acid sequence of the light chain variable region of anti-VSIG4 antibody 2C7 SEQ ID NO: 13: Amino acid sequence of the region containing the epitope of anti-VSIG4 antibody 2C7 SEQ ID NO: 14: Amino acid sequence of CDR-H1 of anti-Siglec-7 antibody 4G7 SEQ ID NO: 15: Amino acid sequence of CDR-H2 of anti-Siglec-7 antibody 4G7 SEQ ID NO: 16: Amino acid sequence of CDR-H3 of anti-Siglec-7 antibody 4G7 SEQ ID NO: 17: Amino acid sequence of CDR-L1 of anti-Siglec-7 antibody 4G7 SEQ ID NO: 18: Amino acid sequence of CDR-L2 of anti-Siglec-7 antibody 4G7 SEQ ID NO: 19: Amino acid sequence of CDR-L3 of anti-Siglec-7 antibody 4G7 SEQ ID NO: 20: Amino acid sequence of the heavy chain variable region of anti-Siglec-7 antibody 4G7 SEQ ID NO: 21: Amino acid sequence of the light chain variable region of the anti-Siglec-7 antibody 4G7

Claims

1. The present invention relates to an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. Tumor immune response activator.

2. The antibody or functional fragment thereof is an anti-VSIG4 antibody or a functional fragment thereof and / or The anti-tumor immune response activator according to claim 1, which is an anti-Siglec-7 antibody or a functional fragment thereof.

3. The antibody or functional fragment thereof is selected from the group consisting of the amino acid sequence of SEQ ID NO: 13 of VSIG4. Antibodies that recognize the epitope in the region or in the region corresponding to the same region in other VSIG4. The anti-tumor immune response activator according to claim 1 , which is a polypeptide of the present invention or a functional fragment thereof.

4. The anti-tumor immune response activator according to claim 1, wherein the antibody or functional fragment thereof is selected from the following (1) to (4): (1) CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO:5; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO:6; A heavy chain variable region comprising CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:7; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 8; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 9; and a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 10, or a functional fragment thereof; (2) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 or a heavy chain variable region having 90% identical to said amino acid sequence; A heavy chain variable region having an amino acid sequence having the above sequence identity and having binding activity to VSIG4. Area and A light chain variable region having the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to VSIG4. an antibody or a functional fragment thereof comprising (3) CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO: 14; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO: 15; A heavy chain variable region comprising a CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 17; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 18; and a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and (4) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 or a heavy chain variable region having 90% identical to said amino acid sequence; a heavy chain variable region having an amino acid sequence having the above sequence identity and having binding activity to Siglec-7; An antibody or a functional fragment thereof comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and having binding activity to Siglec-7.

5. The present invention relates to a method for activating an immune response of NK cells and / or an immune response of T cells. The anti-tumor immune response activator according to claim 1.

6. The anti-tumor immune response activator according to claim 1 for enhancing the cytotoxicity of NK cells and / or enhancing cytokine production by NK cells.

7. An antitumor pharmaceutical composition comprising the antitumor immune response activator according to claim 1.

8. An antibody or a functional fragment thereof that recognizes as an epitope a region contained in the amino acid sequence of VSIG4 set forth in SEQ ID NO: 13 or a region corresponding to the same region in other VSIG4.

9. An antibody or a functional fragment thereof selected from the following (1) to (4): (1) CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO:5; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO:6; A heavy chain variable region comprising CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:7; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 8; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 9; and a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 10, or a functional fragment thereof; (2) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 or a heavy chain variable region having 90% identical to said amino acid sequence; A heavy chain variable region having an amino acid sequence having the above sequence identity and having binding activity to VSIG4. Area and A light chain variable region having the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to VSIG4. an antibody or a functional fragment thereof comprising (3) CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO: 14; CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO: 15; A heavy chain variable region comprising a CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16; CDR-L1 consisting of the amino acid sequence set forth in SEQ ID NO: 17; CDR-L2 consisting of the amino acid sequence set forth in SEQ ID NO: 18; and a light chain variable region comprising CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and (4) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 or a heavy chain variable region having 90% identical to said amino acid sequence; a heavy chain variable region having an amino acid sequence having the above sequence identity and having binding activity to Siglec-7; An antibody or a functional fragment thereof comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and having binding activity to Siglec-7.

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