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

Abstract

The present invention addresses the problem of providing an anti-tumor immune response activator which inhibits an immune checkpoint function based on interaction between VSIG4 and Siglec-7, achieves an antibody having antitumor activity, and includes the antibody as an active ingredient. The present invention provides an anti-tumor immune response activator comprising an antibody that inhibits intermolecular interaction between VSIG4 and Siglec-7 or a functional fragment thereof.

Description

Anti-VSIG4 and anti-Siglec-7 inhibitory antibodies against the intermolecular interaction between VSIG4 and Siglec-7

[0001] The present invention relates to anti-VSIG4 antibodies and anti-Siglec-7 antibodies that inhibit the intermolecular interaction between VSIG4 and Siglec-7 and activate anti-tumor immune responses, as well as anti-tumor immune response activators containing these antibodies.

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

[0003] Recently, Bio-Techne 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 expressed primarily on monocytes, NK cells, and T cells. Therefore, the interaction between VSIG4 and Siglec-7 is thought to regulate immune checkpoints. Anti-VSIG4 and anti-Siglec-7 antibodies that inhibit this interaction are predicted to be effective as immune checkpoint inhibitors against tumors that highly express VSIG4, including glioma, ovarian cancer, multiple myeloma, gastric cancer, and pancreatic cancer. However, no antibodies have been identified that actually inhibit immune checkpoint function based on the interaction between VSIG4 and Siglec-7 and activate antitumor immune responses.

[0004] US10752689B2

[0005] In view of the above situation, the present invention aims to obtain an antibody that inhibits immune checkpoint function based on the interaction between VSIG4 and Siglec-7, has anti-tumor activity, and provides an anti-tumor immune response activator that contains this antibody as an active ingredient.

[0006] The present inventors have conducted extensive research to solve the above-mentioned problems. As a result, they have succeeded in obtaining anti-VSIG4 antibodies and anti-Siglec-7 antibodies that inhibit the intermolecular interaction between VSIG4 and Siglec-7, inhibit immune checkpoint function based on the interaction between VSIG4 and Siglec-7, and have anti-tumor activity. They have also found that such antibodies can be used as anti-tumor immune response activators. Based on these findings, the present invention has been completed. Specifically, the gist of the present invention relates to the following:

[0007] [1] An anti-tumor immune response activator comprising an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7. [2] The anti-tumor immune response activator according to [1], wherein the antibody or functional fragment thereof is an anti-VSIG4 antibody or a functional fragment thereof and / or an anti-Siglec-7 antibody or a functional fragment thereof. [3] The anti-tumor immune response activator according to [1] or [2], wherein the antibody or functional fragment thereof is an antibody or 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 another VSIG4.[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) an antibody or functional fragment thereof comprising a heavy-chain variable region comprising 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, and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a light-chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 10; (2) an antibody or functional fragment thereof comprising a heavy-chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having VSIG4-binding activity, 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 VSIG4-binding activity; (3) An antibody or functional fragment thereof comprising: a heavy chain variable region comprising 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, and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16; and a light chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and (4) an antibody or functional fragment thereof comprising: a heavy chain variable region consisting of (having) the amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence and having binding activity to Siglec-7; and a light chain variable region consisting of (having) the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence and having binding activity to Siglec-7. [5] The anti-tumor immune response activator according to any one of [1] to [4], for activating an NK cell immune response and / or an T cell immune response.[6] The anti-tumor immune response activator according to any one of [1] to [5], for enhancing the cytotoxicity of NK cells and / or enhancing the cytokine production of NK cells. [7] An anti-tumor pharmaceutical composition comprising the anti-tumor immune response activator according to any one of [1] to [6]. [8] An antibody or a functional fragment thereof, which 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 functional fragment thereof selected from the following (1) to (4): (1) an antibody or functional fragment thereof comprising a heavy chain variable region comprising 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, and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 10; (2) an antibody or functional fragment thereof comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence and having VSIG4-binding activity, 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 VSIG4-binding activity; (3) a CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, (4) an antibody or functional fragment thereof comprising a heavy chain variable region comprising CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO: 15 and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and (5) an antibody or functional fragment thereof comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence and having binding activity to Siglec-7, and 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 with said amino acid sequence and having binding activity to Siglec-7.

[0008] The present invention can also be embodied in the following aspects:

[10] A method for activating an anti-tumor immune response, comprising administering to a subject an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7; Use of an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for activating an anti-tumor immune response; An antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for use in activating an anti-tumor immune response; Use of an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for producing an anti-tumor immune response activator; or A method for producing an anti-tumor immune response activator, comprising formulating an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7.

[0009]

[11] A method for preventing or treating tumors (or cancer), comprising administering to a subject an antibody or functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7; use of an antibody or functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for the prevention or treatment of tumors (or cancer); an antibody or functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for use in the prevention or treatment of tumors (or cancer); use of an antibody or functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for the production of an antitumor pharmaceutical composition; or a method for producing an antitumor pharmaceutical composition, comprising formulating an antibody or functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7.

[0010]

[12] An immune checkpoint inhibitor comprising an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7; a method for inhibiting an immune checkpoint, comprising administering to a subject an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7; use of an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for immune checkpoint inhibition; an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for use in immune checkpoint inhibition; use of an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 for the production of an immune checkpoint inhibitor; or a method for producing an immune checkpoint inhibitor, comprising formulating an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7.

[0011] The present invention provides anti-VSIG4 antibodies and anti-Siglec-7 antibodies that inhibit the intermolecular interaction between VSIG4 and Siglec-7, inhibit the immune checkpoint function based on the interaction between VSIG4 and Siglec-7, and have antitumor activity. Antibodies that inhibit the intermolecular interaction between VSIG4 and Siglec-7 inhibit the immune checkpoint function between VSIG4 and Siglec-7, have antitumor activity, and are expected to be effective in treating tumors that express VSIG4.

[0012] Figure 1 shows the interaction of VSIG4 with Siglec family proteins. (A) Schematic diagram of the VSIG4-COMP protein. The C-terminus of the extracellular domain (ECD) of VSIG4 contains rat Cd4 domain 3+4, a rat COMP pentamerization motif, FLAG, and a 6xHis tag. (B) Binding assays were performed using ELISA with VSIG4-COMP protein and purified proteins of all human Siglec family molecules. The figures show the mean absorbance ± standard deviation of three independent experiments. Siglec-13 is not conserved in humans and is omitted. VSIG4 was shown to bind to Siglec-1, -4, and -7. (C) The purity of the VSIG4-COMP and Siglec-7-Fc proteins used in the binding analysis was demonstrated by silver staining (photo). (D) VSIG4-COMP protein bound to the surface of HEK293 + vector and + Siglec-7 cells was quantified by flow cytometry, suggesting that greater 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 greater Siglec-7 binds to VSIG4-expressing cells. Figure 2 shows the sialic acid dependence of the interaction between VSIG4 and Siglec-7. (A) Sialyl groups were removed from VSIG4-COMP protein using sialidase. Sialyl groups were confirmed using an anti-Siaα2-3 antibody (photo). (B) Binding of VSIG4-COMP protein and Siglec-7-Fc protein with or without sialidase treatment was evaluated using Alpha Technology. Sialyl group removal significantly reduced the binding signal. (C) Schematic diagram of candidate O-glycosylation sites in the extracellular domain of VSIG4. The seven predicted glycosylation sites are concentrated in two regions: one located between the two immunoglobulin-like loop structures IgV and IgC2 of VSIG4 (S139, S141, T144, T146, and T147) and the other located near the membrane (T241 and T245).SP: signal peptide; TM: transmembrane region. (D) Silver staining of VSIG4-COMP mutant proteins with mutations introduced at potential O-glycosylation sites in VSIG4 (photograph). (E) ELISA binding assay of VSIG4-COMP mutants with Siglec-7-Fc. Compared to wild-type (WT) VSIG4, the S141A, T144A, T147A, and T241A mutants exhibited equal or superior binding to Siglec-7. However, the S139, T146A, and T245A mutants exhibited lower binding to Siglec-7, with the T146A mutant exhibiting the lowest binding affinity. This suggests that sialic acid modification at the T146 residue is important for binding to Siglec-7. The graph shows relative values, with the absorbance of wild-type (WT) VSIG4 set at 1. 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 NK-92 cells do not. Siglec-7 overexpression was 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, VSIG4 expression was not detected in HEK293 and Jurkat cells, and VSIG4 overexpression was generated. 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.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 cocultured as effector cells with Jurkat + vector or + VSIG4 cells as target cells. Decreased cytotoxicity was observed only when NK-92 + Siglec-7 cells were cocultured with Jurkat + VSIG4 cells. (D) Cytotoxicity was measured when NK-92 + vector or + Siglec-7 cells were cocultured 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. Figure 5 shows that binding of VSIG4 to Siglec-7 reduces T cell receptor signaling activity. (A) Siglec-7 expression was not observed in Jurkat NFAT-Luc cells, and Siglec-7-expressing cells were generated. (B) Jurkat NFAT-Luc + vector or +Siglec-7 cells were plated on plates coated with anti-CD3 antibody and IgG or VSIG4-Fc protein, and T cell receptor signaling activity was detected by luciferase assay. VSIG4 protein reduced luciferase activity in Jurkat NFAT-Luc + Siglec-7 cells. 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 the known binding molecules C3b and Siglec-1, -4, and -7 was detected by ELISA, and the addition of anti-VSIG4 antibody was examined to determine whether the absorbance, indicating the interaction, decreased. The results showed that the 2C7 antibody neutralized the interaction of VSIG4 with Siglec-1, -4, and -7. However, it did not inhibit the interaction of VSIG4 with C3b, suggesting that the interaction occurs at a region distinct from that of the Siglec family.Furthermore, the 2F12 antibody was suggested to have neutralizing activity against the interaction between VSIG4 and Siglec-7. Figure 7 shows the neutralizing activity of the anti-Siglec-7 antibody (clone 4G7) against the interaction between Siglec-7 and its ligand. The interaction of VSIG4 with Siglec-7 was detected by ELISA to determine whether the addition of the anti-Siglec-7 antibody reduced the absorbance, indicating the interaction. The results showed that the 4G7 antibody neutralized the interaction between Siglec-7 and VSIG4. Figure 8 shows the results of narrowing down the epitope of the anti-VSIG4 antibodies (clones 2C7 and 2F12). (A) First, we prepared the HA-VSIG4 construct by replacing the endogenous signal peptide sequence (amino acids 1-19 in SEQ ID NO: 2) of the full-length VSIG4 amino acid sequence with a sequence consisting of an Igκ-leader signal peptide followed by an HA tag (a). Next, a series of deletion mutants (b, c, d) were constructed 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 mutants were transiently transfected into 293FT cells, and cell surface expression was detected by flow cytometry using APC-conjugated anti-HA antibody and FITC-conjugated anti-VSIG4 (2C7, 2F12) antibodies. The 2C7 antibody recognized full-length VSIG4 and VSIG4 lacking the first loop, but showed significantly reduced reactivity with VSIG4 lacking both the first loop and 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 mutants lacking amino acid residues 138-147 or two loops. This suggests that the majority of the epitope for the anti-VSIG4 antibody (clone 2C7, 2F12) is contained within the amino acid residues 138-147. Figure 9 shows the effects of anti-VSIG4 (2C7) and anti-Siglec-7 (4G7) antibodies as immune checkpoint inhibitors.(A) When KHYG-1 cells were cocultured as effector cells with 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), the addition of anti-Siglec-7 (4G7) neutralizing antibody restored IFN-γ secretion. (C) When NK-92 + Siglec-7 cells were cocultured as effector cells with Jurkat + vector or + VSIG4 cells as target cells, forced expression of VSIG4 reduced the cytotoxicity of NK-92 cells, but the addition of anti-VSIG4 (2C7) neutralizing antibody restored cytotoxicity. Figure 10 shows the amino acid sequences of the heavy and light chains of the anti-VSIG4 antibody (clone 2C7) used in each example. In the figure, the signal sequence is a signal peptide sequence inherent in the antibody molecule for extracellular secretion. Figure 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 inherent in the antibody molecule for extracellular secretion.

[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 "equal to or less than" or "less than," and the lower limit may be "equal to or greater than" or "more than."

[0014] The present invention is based on the immune checkpoint inhibitory activity of an antibody or functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7, and such an antibody or functional fragment thereof activates an anti-tumor immune response based on immune checkpoint inhibition. (Hereinafter, "antibodies or functional fragments thereof" may be collectively referred to as "antibodies, etc.") That is, one aspect of the present invention relates to an anti-tumor immune response activator based on immune checkpoint inhibition, comprising an antibody, etc. that inhibits the intermolecular interaction between VSIG4 and Siglec-7. A further 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 embodied as an immune checkpoint inhibitor comprising such an antibody, etc.

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

[0016] Siglec-7, which interacts with VSIG4, is an immunosuppressive receptor expressed in immune cells and functions as an "immune checkpoint molecule." Its ligand, VSIG4, is expressed in cancer 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 in the target 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 inhibit the function of VSIG4 protein as a ligand for immune checkpoint molecules. In another preferred embodiment, antibodies 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, such as primates (e.g., humans, chimpanzees, gorillas, etc.), rodents (e.g., mice, rats, guinea pigs, etc.), cows, horses, pigs, sheep, goats, llamas, camels, dogs, cats, rabbits, etc. Preferred are humans, mice, etc.

[0018] An example of the full-length amino acid sequence of human VSIG4 protein is shown in SEQ ID NO: 2 (NP_009199.1), and an example of the nucleotide sequence encoding it is shown in SEQ ID NO: 1 (NM_007268.3). The VSIG4 protein may be a human VSIG4 protein consisting of the amino acid sequence shown in SEQ ID NO: 2, a functional variant thereof, or a homolog thereof. 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. It preferably retains its function as a ligand for immune checkpoint molecules (i.e., the ability to bind to immune checkpoint molecules on immune cells and suppress immune responses mediated by immune checkpoint molecules). The amino acid sequences and gene nucleotide sequences of VSIG4 proteins derived from various organisms can be easily obtained from publicly known databases (e.g., GenBank).

[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 nucleotide sequence encoding it 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 variant thereof, or a homolog thereof. The Siglec-7 protein may consist of the amino acid sequence shown in SEQ ID NO: 3, 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. Preferably, the Siglec-7 protein retains its function as an immune checkpoint molecule (i.e., the ability to bind to a ligand of an immune checkpoint molecule and suppress an immune response on immune cells). The amino acid sequences and nucleotide sequences of the genes of Siglec-7 proteins from various organisms can be easily obtained from publicly known databases (e.g., GenBank).

[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 (for example, default, i.e., initial setting parameters).

[0021] In one embodiment, the anti-VSIG4 antibody may specifically bind to the extracellular region of the VSIG4 protein (particularly, a region containing an O-glycosylation site in the extracellular region of the VSIG4 protein). In one embodiment, the anti-VSIG4 antibody may recognize as an epitope a region contained in the region containing an O-glycosylation site in the extracellular region of the VSIG4 protein (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). In a preferred embodiment, the anti-VSIG4 antibody may recognize as an epitope a region contained in the region containing an O-glycosylation site in the extracellular region of the VSIG4 protein, i.e., a region from positions 138 to 245 (SEQ ID NO: 13) of the amino acid sequence shown in SEQ ID NO: 2, more preferably a region from positions 138 to 231 (e.g., a region consisting of a partial sequence of 6 amino acids or more), or a region corresponding to these regions in the amino acid sequence of another VSIG4 protein. In a more preferred embodiment, the epitope of the anti-VSIG4 antibody may be partially or entirely contained within the region from positions 138 to 147 in the amino acid sequence of SEQ ID NO: 2. Here, for example, a "region corresponding to the region contained in the amino acid sequence of VSIG4 set forth in SEQ ID NO: 13 in another VSIG4" refers to a region that can be identified as the region contained in the amino acid sequence of VSIG4 set forth in SEQ ID NO: 13. The amino acid sequence set forth in SEQ ID NO: 13 is derived from humans, but VSIG4 from other organisms (individuals, species, etc.) may contain portions with different amino acid sequences. That is, if another organism contains an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 13 (i.e., an amino acid sequence that is highly conserved among organisms, for example, an amino acid sequence that has 80% or more, preferably 90% or more, sequence identity to SEQ ID NO: 13), a "region corresponding to the region contained in the amino acid sequence of VSIG4 set forth in SEQ ID NO: 13 in another VSIG4" refers to, for example, such a highly conserved region, or a region that has been determined by a homology search or other method to correspond to the region contained in the amino acid sequence of VSIG4 set forth in SEQ ID NO: 13.

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

[0023] Antibodies of 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 the VSIG4 or Siglec-7 protein or a partial fragment thereof (for example, the above-mentioned epitope) as an antigen, chimeric and humanized antibodies produced using genetic recombination techniques, and human antibodies produced using human antibody-producing transgenic animals, etc. When the antibodies of the present invention are administered to humans as pharmaceuticals, humanized or human antibodies are preferred in terms of side effects.

[0024] The antibody of the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7 may be either a polyclonal or monoclonal antibody, preferably a monoclonal antibody, as long as it can specifically bind to the VSIG4 protein or the Siglec-7 protein. Furthermore, the antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7 may be a monospecific antibody that recognizes one 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 produce hybridomas, culturing the hybridomas or administering them to an animal to cause ascites tumorigenesis in the animal, and isolating and purifying the culture medium or ascites fluid. Antibody-producing cells that can be used include spleen cells, lymph nodes, and antibody-producing cells in peripheral blood, with spleen cells being particularly preferred.

[0026] A preferred embodiment of the antibody of the present invention that inhibits the intermolecular interaction between VSIG4 and SIGLEC7 is a chimeric antibody. Examples of "chimeric antibodies" include those whose variable regions are derived from immunoglobulins of non-human animals (e.g., mice, rats, hamsters, chickens) and whose constant regions are derived from human immunoglobulins. For example, chimeric antibodies can be produced by immunizing mice with an antigen, excising the antigen-binding variable region from the mouse monoclonal antibody gene, and then combining it with an antibody constant region derived from human bone marrow. Human immunoglobulin-derived constant regions have unique amino acid sequences depending on the isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, IgE, or IgY. The constant region of the recombinant chimeric antibody of the present invention may be the constant region of a human immunoglobulin belonging to any isotype. Preferably, it is a human IgG constant region. Expression vectors can be constructed using the gene encoding the chimeric antibody thus produced. Host cells are transformed with the expression vector to obtain transformed cells that produce chimeric antibodies, and the transformed cells are cultured to obtain the desired chimeric antibodies 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 is a humanized antibody. Here, a "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, has been grafted onto a human antibody gene (CDR grafting). Specifically, this refers to a humanized antibody in which some or all of its CDRs are derived from a monoclonal antibody of a non-human mammal (such as a mouse, rat, or hamster), the framework regions of its variable regions are derived from a human immunoglobulin, and its constant regions are derived from a human immunoglobulin.

[0028] For example, recombinant humanized antibodies derived from mouse monoclonal antibodies can be produced by genetic engineering. Specifically, DNAs for the mouse heavy chain CDRs and mouse light chain CDRs are isolated from a hybridoma producing the mouse monoclonal antibody, and the entire human heavy chain gene region except for the human heavy chain CDRs and the entire human light chain gene region except for the human light chain CDRs are isolated from human immunoglobulin genes.

[0029] The isolated human heavy chain gene grafted with 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 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. Host cells are transformed with the expression vector prepared in this way to obtain humanized antibody-producing transformants, and the desired humanized antibody is obtained from the culture supernatant by culturing the transformed cells.

[0030] Another preferred embodiment of the antibody of the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7 is a human antibody. A "human antibody" is an antibody in which all regions constituting the immunoglobulin, including the heavy chain variable region, heavy chain constant region, and light chain variable region, and light chain constant region, are derived from a gene encoding human immunoglobulin. This antibody 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 genetic locus of a non-human mammal, such as a mouse, can be immunized with an antigen in a manner similar to the method for producing polyclonal or monoclonal antibodies described above.

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

[0032] Antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 may be optionally modified, such as glycosylated, acetylated, formylated, amidated, phosphorylated, or PEGylated.

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

[0034] Specific examples of antibodies or functional fragments thereof that inhibit the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention include (a-1) the 2C7 antibody, an anti-VSIG4 neutralizing antibody, or a functional fragment thereof; (a-2) an anti-VSIG4 antibody or a functional fragment thereof that has a specific sequence as an epitope; and (b-1) the 4G7 antibody, an anti-Siglec-7 neutralizing antibody, or a functional fragment thereof. That is, one aspect of the present invention provides the following antibodies (hereinafter sometimes referred to as "antibodies of the present invention") or functional fragments thereof that inhibit the intermolecular interaction between VSIG4 and Siglec-7. The antibodies of the present invention can be produced using the methods described in the Examples.

[0035] (a-1) 2C7 antibody An antibody or functional fragment thereof comprising: a heavy chain variable region comprising 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, and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 7; and a light chain variable region comprising 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 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 included 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 An antibody or functional fragment thereof comprising: a heavy chain variable region comprising 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, and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16; and a light chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19.

[0038] As long as the antibodies of the present invention maintain their properties of binding 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 acids (e.g., 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 to 2) may be substituted, deleted, added, or inserted in the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibodies. While such substitutions, deletions, or additions may be introduced into the CDRs, they are preferably introduced into regions other than the CDRs. Furthermore, to maintain the properties of the present invention, the amino acid substitutions are preferably conservative substitutions. Here, conservative substitutions refer to the replacement of an amino acid residue with another chemically similar amino acid residue so as not to substantially alter the activity of the peptide. Examples include the replacement of a hydrophobic residue with another hydrophobic residue, or the replacement of a polar residue with another polar residue having the same charge. Examples of functionally similar amino acids that can be substituted include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine; polar (neutral) amino acids such as glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine; positively charged (basic) amino acids such as arginine, histidine, and lysine; and negatively charged (acidic) amino acids such as aspartic acid and glutamic acid. The amino acid sequence of the antibody of the present invention containing 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 percent identity of, for example, 90% or more (more preferably, 95%, 96%, 97%, 98%, or 99% or more) to the amino acid sequence before the modification, and an amino acid sequence in which the light chain variable region after the amino acid sequence modification has a percent identity of, for example, 90% or more (more preferably, 95%, 96%, 97%, 98%, or 99% or more) to the amino acid sequence before the modification.

[0039] Alternatively, the antibody used as the anti-tumor immune response activator may be an antibody that competes with the 2C7 antibody for binding to VSIG4 and inhibits the intermolecular interaction between VSIG4 and Siglec-7 upon binding to VSIG4. Alternatively, the antibody used as the anti-tumor immune response activator may be an antibody that competes with the 4G7 antibody for binding to Siglec-7 and inhibits the intermolecular interaction between VSIG4 and Siglec-7 upon binding to Siglec-7. Here, "compete" means that, when antibody-antigen binding is measured by surface plasmon resonance (SPR), for example, the presence of a competing antibody significantly reduces the binding between the antigen and the antibody.

[0040] <Anti-tumor immune response activators, immune checkpoint inhibitors> The above-mentioned antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 can cause immune checkpoint inhibition. Furthermore, by inhibiting VSIG4 and / or Siglec-7 function, such antibodies and the like cause immune checkpoint inhibition in immune cells, thereby relieving the suppression of anti-tumor immune responses (cancer immune responses) contributed by immune checkpoint molecules and their ligands, activating the anti-tumor immune responses, and enhancing effective anti-tumor immune responses (cancer immune responses). In other words, antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 can be used as anti-tumor immune response activators that cause immune checkpoint inhibition, relieving the suppression of anti-tumor immune responses (cancer immune responses) contributed by immune checkpoint molecules, activating the anti-tumor immune responses, and enhancing effective anti-tumor immune responses (cancer immune responses).

[0041] That is, one aspect of the present invention provides an anti-tumor immune response activator comprising an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7 (hereinafter, sometimes referred to as the "anti-tumor immune response activator of the present invention"). Another aspect of the present invention is an immune checkpoint inhibitor comprising an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7.

[0042] Here, one embodiment of cells in which immune checkpoint inhibition is induced is a cell expressing Siglec-7 protein on its cell surface. Examples of such cells include, but are not limited to, immune cells such as NK cells and T cells. More specifically, antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention can be used as immune checkpoint inhibitors and anti-tumor immune response activators, preferably for immune cells such as NK cells and T cells. Even more specifically, antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention can be used as immune checkpoint inhibitors and anti-tumor immune response activators that activate NK cell immune responses, preferably by enhancing NK cell cytotoxicity and / or NK cell cytokine production (or "NK cell cytotoxicity enhancers" or "NK cell cytokine production enhancers"). Furthermore, antibodies and the like that inhibit the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention can be used as immune checkpoint inhibitors and anti-tumor immune response activators that activate T cell immune responses.

[0043] The immune checkpoint inhibitor and anti-tumor immune response activator contain, as an active ingredient, an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7. The immune checkpoint inhibitor and anti-tumor immune response activator can contain a prophylactically or therapeutically effective amount of the antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7. The antibody or the like can be used alone or in combination of any two or more types.

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

[0045] The optional ingredients, dosage, administration method, target tumor, administration subject, etc. for formulation can also be applied to the immune checkpoint inhibitor and anti-tumor immune response activator as described below in the section <Anti-tumor pharmaceutical composition>. Furthermore, the immune checkpoint inhibitor and anti-tumor immune response activator can also be used as reagents, and the production, use, etc. of the reagents can be carried out based on conventional methods in the field of molecular biology.

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

[0047] <Anti-tumor pharmaceutical composition> The present invention provides an anti-tumor pharmaceutical composition that contains an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7, and whose mechanism of action is to activate (or induce) an anti-tumor immune response based on immune checkpoint inhibition. Such a pharmaceutical composition is useful for preventing or treating tumors (or cancer) based on immune checkpoint inhibition of immune cells such as NK cells and T cells.

[0048] Here, in the present invention, tumor (or cancer) prevention includes preventing tumors (or cancer) from occurring. Tumor (or cancer) prevention also includes preventing or suppressing further progression of tumors (or cancer) at various stages, such as the early stages of onset. Tumor (or cancer) prevention may also be preventing recurrence of tumors (or cancer). Tumor (or cancer) treatment means improving, alleviating, or preventing or suppressing progression of tumors (or cancer).

[0049] That is, one aspect of the present invention provides an anti-tumor pharmaceutical composition comprising an antibody or the like according to the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7. A further aspect of the present invention provides an anti-tumor pharmaceutical composition comprising an immune checkpoint inhibitor comprising an antibody or the like according to the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7. A further aspect of the present invention provides an anti-tumor pharmaceutical composition comprising an anti-tumor immune response activator of the present invention that comprises an antibody or the like according to the present invention that inhibits the intermolecular interaction between VSIG4 and Siglec-7. Hereinafter, these may be referred to as the "anti-tumor 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 cancers; gastrointestinal cancers such as small intestine cancer, colon cancer, gastrointestinal stromal tumor (GIST), gastrointestinal carcinoid, gastric cancer, and esophageal cancer; cancers that occur near the intestinal tract, such as liver cancer, gallbladder and biliary tract cancer, pancreatic cancer, pancreatic and gastrointestinal neuroendocrine tumors, Langerhans cell histiocytosis, renal cell carcinoma, renal pelvis and ureter cancer, and adrenal tumors; pelvic cancers such as bladder cancer, urethral cancer, prostate cancer, testicular tumors, penile cancer, uterine cancer, cervical cancer, uterine tumors, ovarian tumors, and female genital cancer; cancers that occur in the chest, such as lung cancer, thymic tumors, mesothelioma, and breast cancer; myeloid cancers such as hematopoietic tumors, leukemia, myeloproliferative disorders, and multiple myeloma; and osteosarcoma, soft tissue sarcoma, and malignant lymphoma that occur near the intestinal tract. Preferred examples of tumors (or cancers) that can be prevented or treated in the present invention include glioma, ovarian cancer, multiple myeloma, gastric cancer, pancreatic cancer, and the like, which highly express VSIG4.

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

[0052] The antitumor pharmaceutical composition of the present invention may consist solely of an antibody or the like, or may be formulated together with other ingredients according to conventional methods in the pharmaceutical field. The antitumor pharmaceutical composition of the present invention may be used in combination with an active antitumor ingredient other than the active ingredient, an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7, as long as the effect is not impaired. Furthermore, because the antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7 according to the present invention can activate an antitumor immune response, the antitumor pharmaceutical composition of the present invention can also be administered as an adjuvant for cancer treatment in combination with other antitumor measures.

[0053] When the antibody or other agent inhibiting the intermolecular interaction between VSIG4 and Siglec-7 of the present invention is used in combination with other antitumor active ingredients, it may be in the form of a combination drug containing the antibody or other agent and one or more other antitumor active ingredients in a single dosage form, or it may be in a form in which a unit dosage form of a formulation containing the antibody or other agent and a unit dosage form of a formulation containing one or more other antitumor active ingredients are combined and administered at the time of use. When separately prepared formulations containing the antibody or other agent and formulations containing one or more other antitumor active ingredients are administered in combination, the two can be mixed immediately before administration and administered as a single dosage unit, or they can be administered simultaneously or sequentially as separate dosage forms. The cancer treatment adjuvant can be administered as appropriate, for example, before, simultaneously with, or after the administration of the anticancer agent, based on the administration schedule of the commonly used anticancer agent.

[0054] The antitumor pharmaceutical composition of the present invention may contain, as an active ingredient, an antibody that inhibits the intermolecular interaction between VSIG4 and Siglec-7, and / or other components, as well as carriers, diluents, and excipients commonly used in the pharmaceutical field. For example, lactose, magnesium stearate, and the like are used as carriers and excipients for tablets. Aqueous solutions for injection include physiological saline, PBS, and isotonic solutions containing glucose and other adjuvants, which may be used in combination with appropriate solubilizers, such as alcohol, polyalcohols such as propylene glycol, and nonionic surfactants. Oily solutions include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The antitumor pharmaceutical composition of the present invention can be administered in various forms, including oral administration in the form of tablets, capsules, granules, powders, syrups, and the like, and parenteral administration in the form of injections, infusions, suppositories, and the like.

[0055] The dosage varies depending on symptoms, age, body weight, etc., but typically, for oral administration, the active ingredient for an adult is, for example, about 0.01 mg to 1000 mg per day, which can be administered once or in divided doses. 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, 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.

[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 antitumor pharmaceutical composition of the present invention can be administered to these subjects.

[0058] <Methods> The present invention also provides a method for inhibiting immune checkpoints, comprising administering to a subject (or patient) an immune checkpoint inhibitor or an antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7. The present invention also provides 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 to a subject the above-mentioned antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7, an immune checkpoint inhibitor, or an anti-tumor immune response activator. The present invention also provides a method for preventing or treating tumors (or cancers) based on immune checkpoint inhibition, comprising administering to a subject the above-mentioned antibody or the like that inhibits the intermolecular interaction between VSIG4 and Siglec-7, an immune checkpoint inhibitor, an anti-tumor immune response activator, or an anti-tumor pharmaceutical composition. In these methods, immune checkpoint inhibition is preferably directed against cells or the like that express VSIG4 protein on their cell surface and immune cells that express Siglec-7 protein on their cell surface, preferably cancer cells and NK cells, T cells, or the like.

[0059] The dosage, administration method, target tumor, administration subject, etc. in the above method can also be applied to the above method as described above in the section <Antitumor pharmaceutical composition>.

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

[0061] <Materials and Methods> (Cell Culture) KHYG-1 cells, a human NK cell line, 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 (BioWest, Nouaille, France), 100 units / mL penicillin, 100 μg / 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 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 an incubator at 37°C with 8% CO2, using a RemoteShake rotary shaker (Wakembee Tech Co., Ltd., Kyoto, Japan) with shaking at 125 rpm.

[0063] All cells except Expi293-F cells were cultured at 37°C in an incubator (Panasonic Corporation, Osaka, Japan) set at a CO2 concentration of 5%.

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

[0065]

[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, internally, the ccdB gene and chloramphenicol resistance gene was inserted into the SmaI site of the pHEK293 Ultra Expression Vector II (Takara Bio Inc., Shiga, Japan), a vector highly expressed in HEK293 cells. Furthermore, a human IgG2-Fc sequence (hereafter referred to as Fc) was inserted into the XhoI-SphI site to create the pHEK-Fc destination vector. Alternatively, a tag sequence consisting of rat Cd4 domains 3 + 4, rat COMP (cartilage oligomeric matrix protein) pentamerization domain, FLAG, and a tandem 6 × His sequence (hereafter referred to as the COMP tag) was inserted into the XhoI-SphI site to create the pHEK-COMP destination vector. The entry clone and destination vector were then recombined using LR clonase (Invitrogen) to create expression vectors bearing the Fc or COMP tag.

[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. Expression vectors for VSIG4 mutant proteins were constructed by PCR-amplified DNA sequences in which predicted O-glycosylation sites S139, S141, T144, T146, T147, T241, and T245 were substituted with alanine. These DNA sequences were then cloned into the ENTR / D-TOPO vector and recombined with the pHEK-COMP vector using LR clonase.

[0068] (Purification of Siglec family, VSIG4, and VSIG4 mutant proteins) Fc- or COMP-tagged proteins of Siglec-2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -14, and -15, VSIG4, and VSIG4 mutants were produced as follows: 30 μg of expression vector, 6 μg of pHEK293 Enhancer Vector (Takara Bio Inc.), and 36 μL of TransIT-PRO Reagent (Mirus Bio, Madison, WI, USA) were added to 3 mL of Opti-MEM and incubated at room temperature for 15 minutes. The transfection mix was then transferred to 3 × 10 cells suspended in 30 mL of Expi293 Expression Medium. 7Transfection was performed by adding 1000 Expi293-F cells to the cells. The cells were cultured at 125 rpm in a humidified incubator with 8% CO2 at 37°C for 4 days, allowing the protein to be secreted into the culture supernatant. The culture supernatant was passed through a 0.45 μm filter (Merck Millipore) to completely remove the cells. Protein purification was performed using MicroSpin Columns (GE Healthcare, Little Chalfont, UK). Fc-tagged proteins were incubated overnight at 4°C with Protein A Sepharose (GE Healthcare) washed twice with binding buffer (20 mM Na3PO4, pH 7.0). The eluate was then eluted with elution buffer (0.1 M glycine, pH 3.0) and neutralized with 1 M Tris-HCl (pH 9.0). COMP-tagged proteins were prepared by adding 1 / 10 the volume of 5M NaCl to the culture supernatant, followed by the addition of 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 incubating overnight at 4°C. The proteins were then eluted with elution buffer (20 mM Na3PO4, 500 mM NaCl, 500 mM imidazole, pH 7.4) and dialyzed overnight at 4°C against PBS using a Slide-A-Lyzer MINI Dialysis Device (Thermo Fisher Scientific). 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). Concentration was performed using Amicon Ultra Centrifugal Filters (10K, 0.5 mL) (Merck Millipore).Protein concentration was quantified by the Bradford method using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA).

[0069] Binding assay using ELISA. Binding assays between VSIG4-COMP protein and Siglec family proteins were performed 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 diluted to 20 nM in carbonate buffer. 100 μL of each was added to a Nunc MaxiSorp™ flat-bottom plate (Thermo Fisher Scientific) 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 blocked for 1 hour at room temperature. 100 μL of VSIG4-COMP protein diluted to 10 nM in blocking solution was added and incubated for 1 hour at room temperature. After washing three times with PBS-T, 100 μL of anti-FLAG M2 antibody-HRP (Sigma-Aldrich) diluted 1:10,000 was added to each well and incubated for an additional hour at room temperature in the dark. After washing four times with PBS-T, 100 μL of TMB Soluble Reagent (ScyTek, Logan, UT, USA) was added to each well and incubated for 30 minutes at room temperature in the dark. The color reaction was stopped by adding TMB Stop Buffer (ScyTek). 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. VSIG4-COMP protein and VSIG4-COMP protein containing 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 solution was added to a Nunc MaxiSorp flat-bottom plate in duplicate. The plate was then left to stand at 4°C overnight for immobilization.After washing twice with PBS-T, 300 μL of 1% BSA / PBS was added to each well and blocking was performed for 1 hour at room temperature. 100 μL of Siglec-7-Fc protein diluted to 50 nM in blocking solution was added and incubated for 1 hour at room temperature. 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 well and incubated for an additional hour at room temperature in the dark. After washing four times with PBS-T, 100 μL of TMB Soluble Reagent was added to each well and incubated for 30 minutes at room temperature in the dark. The color reaction was stopped by adding TMB Stop Buffer. The absorbance at 450 nm was measured using an EnSight Multimode Plate Reader.

[0070] (Protein Desialylation) 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. Sialic acid removal was confirmed by Western blotting 50 ng of protein using an anti-Siaα2-3 monoclonal antibody (HYB4, Fujifilm Wako Pure Chemical Corporation) to detect α2-3 sialic acid. The FLAG tag contained in the VSIG4-COMP protein was used as a loading control, and was detected with an anti-FLAG-M2 antibody (Sigma-Aldrich).

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

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

[0073] Siglec-7 was introduced into NK92 cells, and VSIG4 was introduced into Jurkat cells as follows. Virus solutions were prepared in the same manner using the respective pLenti7.3 / V5-DEST expression vectors and ViraPower Lentiviral Packaging Mix. The virus solutions were then added to a 24-well microbial culture plate (AS ONE Corporation, Osaka, Japan) pre-coated with RetroNectin (Takara Bio Inc., Shiga, Japan) and centrifuged at 2000 rpm for 3 hours at room temperature. After removing the supernatant, the plates were washed once with PBS and seeded with NK92 cells. The cells were then centrifuged at 1200 rpm for 5 minutes to allow contact with the RetroNectin-bound virus, and the plates were then cultured for 24 hours for infection. Stable cell lines were isolated using a FACSAria III (BD, Franklin Lakes, NJ, USA) to isolate GFP-positive and anti-Siglec-7 antibody-stained fractions. VSIG4 gene knockout was performed in P31 / FUJ cells using the lentiviral vector pLV-U6g-EPCG (Sigma), which expresses both a VSIG4 short guide RNA (HS0000100372) and Cas9 protein. After lentiviral infection, VSIG4 knockout cells were isolated by isolating GFP-positive but anti-VSIG4 antibody-negative fractions.

[0074] (Expression analysis using flow cytometry) 1.0 × 10 cells were cultured in HBSS (Gibco / Thermo Fisher Scientific) supplemented with 1% FBS to a final concentration. 6 The cells were adjusted to a density of 1.0 × 10 cells / mL, and then 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 to 1.0 × 10 cells / mL with HBSS + 1% FBS. 7The cells were prepared at a concentration of 1000 cells / mL, and propidium iodide (Fujifilm Wako Pure Chemical Corporation) was added to a final concentration of 1 μg / mL. The cells were 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 diluted to 1 × 10 with HBSS + 1% BSA (Fujifilm Wako Pure Chemical Industries, Ltd.). 6 The cells were prepared at a concentration of 0.5 μg per mL. 0.5 μg of purified VSIG4 or Siglec-7 protein, either Fc- or COMP-tagged, was incubated with FITC anti-human IgG Fc antibody or FITC anti-FLAG M2 antibody, depending on the protein tag, for 20 minutes at room temperature. Then, 100 μL of the cell preparation solution was added and incubated for an additional 30 minutes at room temperature. After washing once with HBSS + 1% FBS, the cells were passed through a 35 μm cell strainer and analyzed using a FACS Verse.

[0076] (VSIG4 and Siglec-7 binding assay using Alpha technology) Proteins were diluted in 0.2% BSA / PBS. VSIG4-COMP was diluted from 40 nM to 1.2 μM (final concentrations: 10 nM to 300 nM), and Siglec-7-Fc protein was prepared at 400 nM (final concentration: 100 nM). Five μL of each Fc- and COMP-tagged protein was added to a 384-well plate (PerkinElmer, Waltham, MA, USA) and incubated at room temperature for 60 minutes. Subsequently, 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, was added and incubated at room temperature for 60 minutes in the dark. The intermolecular binding was detected as emission at 520-620 nm when excited at a wavelength of 680 nm, and detection was performed using an EnSight Multimode Plate Reader (PerkinElmer).

[0077] (Western blotting) Cultured cells were washed with PBS and then lysed in 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) on ice for 30 minutes. The cells were then centrifuged at 12,000 rpm for 10 minutes in a centrifuge cooled to 4°C, and the supernatant was collected. 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, boiled at 95°C for 5 minutes, and used as a sample for Western blotting. SDS-PAGE gel electrophoresis was performed on a 10% acrylamide:bis separating gel with 10 μg of sample loaded per well in SDS-PAGE electrode buffer (25 mM Tris, 0.2 M glycine, 0.1% SDS) at a constant current of 20–40 mA. For blotting, a semi-dry method was used. The PVDF membrane (Merck Millipore) was presoaked in methanol and then in transfer buffer (50 mM Tris, 40 mM glycine, 20% methanol, 0.1% SDS). The membrane was then blotting-impacted for 60 minutes at a constant current of 110 mA in a Transblot SD cell (Bio-Rad). The PVDF membrane was then blocked in 3% skim milk / PBS-T (PBS-0.1% Tween 20). After washing once with PBS-T, the membrane was incubated overnight at 4°C with a 1:1000 dilution of primary antibody. After washing three times with PBS-T, HRP-labeled antibodies of each animal species were added as secondary antibodies at a dilution of 1:1000 in 0.5% skim milk solution and incubated at room temperature for 1 hour.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] (Production of anti-VSIG4 and anti-Siglec-7 monoclonal antibodies) Hybridoma production was outsourced to the Cell Engineering Institute (Osaka, Japan). Purified VSIG4-Fc and Siglec-7-Fc proteins were used as antigens, and B6D2F1 / Slc mice were immunized. Lymphocytes were collected from the iliac lymph nodes of immunized mice and fused with SP2 mouse myeloma cells to produce hybridomas. Hybridoma culture supernatants were screened for neutralizing activity to select clones. Hybridoma clones were cultured in Hybridoma-SFM medium (Thermo Fisher Scientific). After approximately two weeks of culture, the culture supernatant was collected and passed through a 0.45 μm filter to completely remove cells. Antibodies were purified using MicroSpin Columns, similar to the purification of Fc fusion proteins.

[0079] (Assessment 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. 50 μL of each solution was added to a Nunc MaxiSorp™ flat-bottom plate and allowed to stand overnight at 4°C for immobilization. After washing twice with PBS-T, 100 μL of 1% BSA / PBS was added to each plate for blocking at room temperature for 1 hour. After washing twice with PBS-T, a mixture of 50 μL of serially diluted antibody solution (0.01–10 μg / mL) and 50 μL of 100 nM VSIG4-COMP protein was added and incubated 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 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.

[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 added in 50 μL portions to Nunc MaxiSorp™ flat-bottom plates. The plates were then immobilized overnight at 4°C. After washing twice with PBS-T, 100 μL of 1% BSA / PBS was added to each plate for blocking at room temperature for 1 hour. After washing twice with PBS-T, a mixture of 50 μL of serially diluted antibody solution (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 anti-human IgG-HRP diluted 1:2000 was added to each plate 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 6 CFSE (Fujifilm Wako Pure Chemical Corporation) was added to the cells at 2 μg / mL, and the cells were then rotated and mixed at room temperature for 20 minutes in the dark. After washing twice with PBS, the cells were cultured at 2 × 10 cells / mL using assay medium containing RPMI-1640 and 1% FBS. 5The 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 the desired effector:target ratio (E:T ratio) using the assay medium. The E:T ratio was set at 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). A well containing target cells alone served as a control (% spontaneous cell death). The plate was centrifuged at 1000 rpm for 5 minutes and incubated at 37°C with 5% CO2 for 4 hours. The cells were harvested, centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and propidium iodide added to a final concentration of 50 μg / mL. The cells were then passed through a 35 μm cell strainer and analyzed using a 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 + 50 IU / mL human IL-2. 6 VSIG4- or vector-transfected HEK293 cells were harvested using 0.5 mM EDTA, washed with PBS, and then diluted to 2 × 10 5The cells were incubated at a concentration of 100 μL / mL. Antibodies were added to the cell suspension before administration and incubated at room temperature for 30 minutes. 100 μL of each cell suspension was added to a 96-well Ultra-low attachment round bottom plate and incubated at 37°C with 5% CO2 for 24 hours. The supernatant was then collected from each well. IFN-γ levels in the culture supernatant were quantified using a Human IFN-γ ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the protocol.

[0083] (Detection of cytokine secretion using antibody array) 36 cytokines were detected using the Proteome Profiler Human Cytokine Array Kit (R&D Systems). KHYG-1 cells were cultured at 1 × 10 in RPMI-1640 + 1% FBS + 50 IU / mL human IL-2. 6 VSIG4- or vector-transfected HEK293 cells were harvested using 0.5 mM EDTA, washed with PBS, and then diluted to 2 × 10 5 The cells were incubated at 37°C with 5% CO2 for 24 hours, and the supernatant was collected from each well. The reaction was performed according to the kit's recommended protocol. Cytokine secretion was detected using an Amersham Imager 680 (GE Healthcare) as a function of the HRP substrate. The amount of cytokine secretion was measured by quantifying the pixel density of the antibody spots against each cytokine using Image J. The amount of cytokine secretion from KHYG-1 cells co-cultured with HEK293 + vector cells was set at 1, and the amount of cytokine secretion from HEK293 + VSIG4 cells was expressed as a relative value.

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

[0085] (Method for 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) was replaced with an Igκ leader sequence followed by an HA tag, was cloned into the pHEK293 Ultra Expression Vector (Takara Bio Inc.). Subsequently, a series of deletion mutant expression vectors were constructed 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). These vectors were transiently transfected into 293FT cells using PEI MAX, and cell surface expression was detected by flow cytometry using an APC-labeled anti-HA antibody (Biolegend) and FITC-labeled anti-VSIG4 (2C7, 2F12) antibodies. FITC labeling of anti-VSIG4 (2C7, 2F12) antibodies was performed using the Fluorescein Labeling Kit - NH2 (Dojindo Laboratories) according to the recommended protocol.

[0086] Results: First, we investigated the interaction between VSIG4 and Siglec family molecules. Based on a previously established method for detecting interactions between IgSF molecules (Kasai et al., Cancer Sci, 2022; 113(5):1669-1678), VSIG4 protein was purified by tagging the C-terminus of the extracellular domain of VSIG4 with a tandem sequence consisting of rat Cd4 domain 3+4, rat COMP (cartilage oligomerize protein) pentamerization domain, FLAG, and 6xHis (hereafter referred to as the COMP tag) (Fig. 1A). Binding of the VSIG4-COMP protein to purified Siglec-1 to -15 proteins (except Siglec-13, which is not conserved in humans) was examined by ELISA, suggesting that VSIG4 binds to Siglec-1, -4, and -7 (Fig. 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 cell surface interaction between VSIG4 and Siglec-7, we constructed Siglec-7 and VSIG4 transfectants in 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 these cells by flow cytometry. Silver staining, which confirmed the size and purity of each protein, is shown in Figure 1C. The results showed that the binding of VSIG4-COMP was enhanced in the Siglec-7-expressing cells (Figure 1D), and that the binding of Siglec-7-Fc was enhanced in the VSIG4-expressing cells (Figure 1E), suggesting that VSIG4 and Siglec-7 interact with each other on a cell-by-cell basis.

[0088] The Siglec family is a receptor that recognizes sialic acid, which is modified at the terminal glycan of glycoproteins or glycolipids. Therefore, we predicted that Siglec-7 recognizes the sialic acid modification of VSIG4 when binding to VSIG4. To confirm this, we removed sialic acid from the VSIG4-COMP protein using sialidase (Fig. 2A), and then evaluated binding to Siglec-7-Fc protein using Alpha technology. As a result, removal of sialic acid significantly reduced the binding signal (Fig. 2B). This suggests that Siglec-7 recognizes sialic acid modified on the VSIG4 protein.

[0089] Protein glycosylation can be categorized into two types: N-glycosylation and O-glycosylation. While no predicted N-glycosylation sites were found in the extracellular domain of VSIG4, 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 loops of VSIG4, and T241 and T245 are located between the second loop and the transmembrane domain (Fig. 2C). To identify the glycosylation sites essential for interaction with Siglec-7, we generated VSIG4-COMP mutant proteins in which S or T at each candidate site was replaced by A (Fig. 2D). The interaction of these mutants with Siglec-7-Fc was examined by ELISA. Compared with wild-type VSIG4, the S141A, T144A, T147A, and T241A mutants exhibited equal or superior binding ability to Siglec-7, whereas the S139, T146A, and T245A mutants exhibited lower binding ability to Siglec-7, with the T146A mutant exhibiting the lowest binding ability (Fig. 2E). This suggests that the sialic acid modification at T146 is important for binding to Siglec-7.

[0090] Next, we investigated whether the interaction between VSIG4 and Siglec-7 is involved in immune checkpoint regulation using NK cell and T cell models. Siglec-7 is endogenously expressed in normal human NK cells and the human NK cell line KHYG-1. However, expression was not observed in the human NK cell line NK-92, and we generated Siglec-7-transfected lines (Figure 3A). VSIG4 is endogenously expressed in the human acute monocytic leukemia cell line P31 / FUJ, and we generated VSIG4 knockout lines using the CRISPR-Cas9 system. Furthermore, expression was not observed in HEK293 or Jurkat cells, and we generated VSIG4-transfected lines (Figure 3B).

[0091] NK cells exhibit cytotoxicity against target cells and secrete cytokines, including IFN-γ. Therefore, we investigated whether the interaction between VSIG4 and Siglec-7 inhibits cytotoxicity and cytokine secretion when NK cells are cocultured with target cells (HEK293 and Jurkat cells). First, KHYG-1 cells, which endogenously express Siglec-7, were cocultured with HEK293+vector and +VSIG4 cells for 24 hours, and IFN-γ secreted into the culture supernatant was measured by ELISA. IFN-γ secretion from KHYG-1 cells was significantly suppressed in coculture with the VSIG4-expressing cells (Fig. 4A). In addition, when the secretion of cytokines other than IFN-γ was examined using a cytokine antibody array, it was suggested that the secretion of CCL1 / I-309, MIP-1α / MIP-1β, CCL5 / RANTES, GM-CSF, IL-8, and IL-10 was suppressed in coculture with VSIG4-expressing cells (Fig. 4B).

[0092] Next, NK-92 cells were cocultured with Jurkat cells for 4 hours, and cytotoxicity was analyzed by detecting propidium iodide-positive dead cells using flow cytometry. No difference in cytotoxicity of NK-92+vector cells against Jurkat+vector and +VSIG4 cells was observed. However, when NK-92+Siglec-7 cells were used, cytotoxicity was significantly suppressed by coculture with a VSIG4-expressing cell line (Figure 4C). Furthermore, when NK-92+Siglec-7 cells were used to examine cytotoxicity against P31 / FUJ cells, which endogenously express VSIG4, coculture with a VSIG4-knockout cell line significantly enhanced cytotoxicity (Figure 4D). These findings suggest that NK cell activity is suppressed via the interaction between VSIG4 and Siglec-7.

[0093] Siglec-7 has been reported to be highly expressed in activated T cells, particularly memory CD8+ T cells (Fan et al., Cancer Sci, 2023 Feb;114(2):370-383). As a model for measuring T cell activity, we used Jurkat NFAT-Luc cells transfected with a plasmid expressing luciferase (Luc) downstream of the NFAT transcription factor binding domain. Upon T cell receptor signaling with anti-CD3 antibody, these cells express luciferase through activation of the NFAT transcription factor. Jurkat cells do not endogenously express Siglec-7, so we generated a Siglec-7 transfectant (Figure 5A). We then examined whether luciferase activity was suppressed by binding of the VSIG4-Fc protein to Siglec-7 upon T cell receptor stimulation with anti-CD3 antibody. The results showed that luciferase activity in Jurkat NFAT-Luc + Siglec-7 cells was reduced by the addition of VSIG4-Fc, whereas the activity remained unchanged in the vector-transfected cells (Fig. 5B), suggesting that T cell activation 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. Anti-VSIG4 antibodies (clone 2C7 and 2F12) and anti-Siglec-7 antibodies (clone 4G7) were obtained. The neutralizing activity of these antibodies was evaluated by ELISA. First, we examined the binding of VSIG4 to the known binding molecules complement C3b and Siglec-1, -4, and -7 by ELISA, and examined whether the addition of anti-VSIG4 antibody reduced the binding signal. The results showed that anti-VSIG4 antibody (clone 2C7) neutralized the binding of VSIG4 to Siglec-1, -4, and -7. However, it did not neutralize the binding of VSIG4 to complement C3b. Furthermore, it was suggested that the anti-VSIG4 antibody (clone 2F12) had neutralizing activity against the binding of VSIG4 to Siglec-7 (Fig. 6).

[0095] Next, we investigated the binding of Siglec-7 to VSIG4 by ELISA and whether the addition of anti-Siglec-7 antibody reduced the binding signal. The results showed that anti-Siglec-7 antibody (clone 4G7) neutralized the binding of Siglec-7 to VSIG4 (Fig. 7).

[0096] Furthermore, we analyzed the epitope of anti-VSIG4 antibodies (clone 2C7 and 2F12). To clarify which region of VSIG4 they recognize, we constructed an expression vector for HA-VSIG4, in which an HA tag was added to the N-terminus of VSIG4. We then generated a series of VSIG4 mutants by sequentially deleting the first loop (amino acid residues 20-137 in SEQ ID NO: 2), the region where O-glycosylation sites are concentrated (S139-T147) (amino acid residues 138-147), and the second loop (amino acid residues 148-231) (Fig. 8A). Anti-VSIG4 antibody (clone 2C7) bound to full-length VSIG4 and VSIG4 lacking the first loop, but showed little binding to the mutant lacking the amino acid residues 138-147 where O-glycosylation sites are concentrated, and no binding to the mutant lacking both loops (Fig. 8B). Clone 2F12 also bound to full-length VSIG4 and VSIG4 lacking the first loop, but not to mutants lacking the amino acid residues 138-147 or two loops, indicating that the majority of the epitopes of the 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 cocultures 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 treatment with anti-VSIG4 (2C7) antibody (Fig. 9A). Treatment with anti-Siglec-7 (4G7) antibody also restored IFN-γ secretion (Fig. 9B). Furthermore, in cocultures of NK-92 + Siglec-7 cells and Jurkat + VSIG4 cells, cytotoxicity, which was suppressed by the interaction between VSIG4 and Siglec-7, was restored by treatment with anti-VSIG4 (2C7) antibody (Fig. 9C). Therefore, 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 VSIG4-expressing tumors.

[0098] As described above, the present invention provides an anti-VSIG4 (clone 2C7) mouse monoclonal antibody and an anti-Siglec-7 mouse monoclonal antibody (clone 4G7) that have inhibitory activity against the molecular interaction between VSIG4 and Siglec-7. Furthermore, NK cell cytotoxicity and cytokine secretion were reduced through the VSIG4-Siglec-7 interaction, resulting in a decrease in activity. Administration of the anti-VSIG4 (2C7) antibody restored NK cell cytotoxicity and cytokine secretion. In a similar experiment, the anti-Siglec-7 (4G7) antibody also restored NK cell cytokine secretion. Furthermore, suppression of T cell activity via the VSIG4-Siglec-7 interaction was observed. These findings suggest that anti-VSIG4 (2C7) and anti-Siglec-7 (4G7) antibodies function as VSIG4-Siglec-7 immune checkpoint inhibitors, enhancing the antitumor activity of NK cells and / or T cells, and are expected to be therapeutically effective against VSIG4-expressing tumors.

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

[0100] <Explanation of sequences> SEQ ID NO: 1: DNA sequence of the full-length VSIG4 gene SEQ ID NO: 2: Amino acid sequence of the full-length VSIG4 protein SEQ ID NO: 3: DNA sequence of the full-length Siglec-7 gene SEQ ID NO: 4: Amino acid sequence of the 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 anti-Siglec-7 antibody 4G7

Claims

1. An anti-tumor immune response activator comprising an antibody or a functional fragment thereof that inhibits the intermolecular interaction between VSIG4 and Siglec-7.

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

3. The anti-tumor immune response activator described in claim 1, wherein the antibody or functional fragment thereof is an antibody or 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.

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) an antibody or functional fragment thereof comprising a heavy chain variable region comprising 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, and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO:10; (2) an antibody or functional fragment thereof comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:11 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to VSIG4, 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; (3) An antibody or a functional fragment thereof comprising: a heavy chain variable region comprising 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, and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and (4) an antibody or a functional fragment thereof comprising: a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to Siglec-7, and 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 with said amino acid sequence, and having binding activity to Siglec-7.

5. The anti-tumor immune response activator according to claim 1 for activating the immune response of NK cells and / or activating the immune response of T cells.

6. The anti-tumor immune response activator according to claim 1 for enhancing the cytotoxicity of NK cells and / or enhancing the cytokine production of 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 functional fragment thereof selected from the following (1) to (4): (1) an antibody or functional fragment thereof comprising a heavy chain variable region comprising 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, and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO:10; (2) an antibody or functional fragment thereof comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:11 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to VSIG4, 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; (3) a CDR-H1 consisting of the amino acid sequence set forth in SEQ ID NO:14, (4) an antibody or functional fragment thereof comprising a heavy chain variable region comprising CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO: 15 and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region comprising 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 CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and (5) an antibody or functional fragment thereof comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to Siglec-7, and 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 with said amino acid sequence, and having binding activity to Siglec-7.

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