NOVEL TARGET FOR ANTI-CANCER AND IMMUNE-ENHANCING

Abstract

The present disclosure provides a pharmaceutical composition for treating or preventing cancer, comprising inhibitors of KIRREL3, CNTN4 and/or CD351. In addition, the present disclosure provides a pharmaceutical composition for immune-enhancing, comprising inhibitors of KIRREL3, CNTN4 and/or CD351. Furthermore, the present disclosure provides a method of screening of anti-cancer agent using KIRREL3, CNTN4 and/or CD351, and a method of providing information necessary for analysis of cancer prognosis using KIRREL3, CNTN4 and/or CD351.

Claims

1. A method of treating or preventing cancer, comprising administering one or more inhibitors of KIRREL3, CNTN4 and CD351 to a subject in need thereof.

2. The method according to claim 1, wherein the inhibitor is an antisense nucleic acid, a siRNA, a shRNA, a miRNA or a ribozyme that binds in a complementary manner to a DNA or a mRNA of one or more of KIRREL3, CNTN4 and CD351 gene.

3. The method according to claim 1, wherein the inhibitor is a compound, a peptide, a peptide mimetic, a fusion protein, an antibody or an aptamer that binds specifically to one or more of KIRREL3, CNTN4 and CD351 protein.

4. The method according to claim 1, wherein the cancer is stomach cancer, lung cancer, liver cancer, colorectal cancer, colon cancer, small intestinal cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenosis, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, renal cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer, leukemia, lymphoma, or fibroadenoma.

5. The method according to claim 1, wherein the inhibitor suppresses a function of cancer cells evading T cells.

6. A method of immune-enhancing, comprising administering one or more inhibitors of KIRREL3, CNTN4 and CD351 to a subject in need thereof.

7. The method according to claim 6, wherein the inhibitor inhibits expression or activity of one or more of KIRREL3, CNTN4 and CD351 in the subject to increase a level of T cell-mediated immune response.

8. The method according to claim 6, wherein the subject is in need of prevention, treatment or improvement of diseases related to immunodeficiency, lower immune function, immune system damage, or immunocompromising.

9. A method of screening an anti-cancer agent comprising: (a) treating a cancer cell with a candidate anti-cancer agent; and (b) measuring expression or activity of one or more of KIRREL3, CNTN4 and CD351 in the cancer cell.

10. The method according to claim 9, wherein the step (b) is conducted by determining a level of expression of a mRNA or a protein of one or more of KIRREL3, CNTN4 and CD351 or a level of suppression of T cell activity by one or more of KIRREL3, CNTN4 and CD351.

11. The method according to claim 9, further comprising: (c) determining the candidate anti-cancer agent to be the anti-cancer agent if a group treated with the candidate anti-cancer agent shows a significantly lower level of expression of mRNA of one or more of KIRREL3, CNTN4 and CD351 or protein of one or more of KIRREL3, CNTN4 and CD351; or a significantly lower level of suppression of T cell activity by one or more of KIRREL3, CNTN4 and CD351 compared to a group not treated with the candidate anti-cancer agent.

12. A method of providing information necessary for analysis of cancer prognosis comprising: measuring expression or activity of one or more of KIRREL3, CNTN4 and CD351 in cells or tissues isolated from a subject.

13. The method according to claim 12, wherein the expression or activity of one or more of KIRREL3, CNTN4 and CD351 is measured by determining a level of expression of a mRNA or a protein of one or more of KIRREL3, CNTN4 and CD351 or a level of suppression of T cell activity by one or more of KIRREL3, CNTN4 and CD351.

Description

BRIEF DESCRIPTION OF FIGURES

[0065] FIG. 1 shows the proliferation (%) of CD4+ T cells suppressed by KIRREL3.

[0066] FIG. 2 shows the proliferation (%) of CD8+ T cells suppressed by KIRREL3.

[0067] FIG. 3 shows the proliferation (%) of CD4+ T cells suppressed by CNTN4.

[0068] FIG. 4 shows the proliferation (%) of CD8+ T cells suppressed by CNTN4.

[0069] FIG. 5 shows the proliferation (%) of CD4+ T cells suppressed by CD351.

[0070] FIG. 6 shows the proliferation (%) of CD8+ T cells suppressed by CD351.

[0071] FIGS. 7A, 7B, 7C and 7D show the cytotoxicity (%) of PBMC when lung cancer cell line A549 and PBMC were treated with KIRREL3 inhibitors.

[0072] FIGS. 8A, 8B, 8C and 8D show the cytotoxicity (%) of PBMC when colon cancer cell line HCT-116 and PBMC were treated with KIRREL3 inhibitors.

[0073] FIGS. 9A, 9B, 9C and 9D show the cytotoxicity (%) of PBMC when breast cancer cell line MDA-MB-231 and PBMC were treated with KIRREL3 inhibitors.

[0074] FIGS. 10A, 10B, 10C and 10D show the cytotoxicity (%) of PBMC when gastric cancer cell line MKN-74 and PBMC were treated with KIRREL3 inhibitors.

[0075] FIGS. 11A, 11B, 11C and 11D show the cytotoxicity (%) of PBMC when leukemia cell line U937 and PBMC were treated with KIRREL3 inhibitors.

[0076] FIGS. 12A, 12B, 12C and 12D show the cytotoxicity (%) of PBMC when lung cancer cell line A549 and PBMC were treated with CNTN4 inhibitors.

[0077] FIGS. 13A, 13B, 13C and 13D show the cytotoxicity (%) of PBMC when colon cancer cell line HCT-116 and PBMC were treated with CNTN4 inhibitors.

[0078] FIGS. 14A, 14B, 14C and 14D show the cytotoxicity (%) of PBMC when breast cancer cell line MDA-MB-231 and PBMC were treated with CNTN4 inhibitors.

[0079] FIGS. 15A, 15B, 15C and 15D show the cytotoxicity (%) of PBMC when gastric cancer cell line MKN-74 and PBMC were treated with CNTN4 inhibitors.

[0080] FIGS. 16A, 16B, 16C and 16D show the cytotoxicity (%) of PBMC when leukemia cell line U937 and PBMC were treated with CNTN4 inhibitors.

[0081] FIGS. 17A, 17B, 17C and 17D show the cytotoxicity (%) of PBMC when lung cancer cell line A549 and PBMC were treated with CD351 inhibitors.

[0082] FIGS. 18A, 18B, 18C and 18D show the cytotoxicity (%) of PBMC when colon cancer cell line HCT-116 and PBMC were treated with CD351 inhibitors.

[0083] FIGS. 19A, 19B, 19C and 19D show the cytotoxicity (%) of PBMC when breast cancer cell line MDA-MB-231 and PBMC were treated with CD351 inhibitors.

[0084] FIGS. 20A, 20B, 20C and 20D show the cytotoxicity (%) of PBMC when gastric cancer cell line MKN-74 and PBMC were treated with CD351 inhibitors.

[0085] FIGS. 21A, 21B, 21C and 21D show the cytotoxicity (%) of PBMC when leukemia cell line U937 and PBMC were treated with CD351 inhibitors.

[0086] FIG. 22 shows the tumor size in mouse treated with KIRREL3 inhibitors.

[0087] FIG. 23 shows the tumor size in mouse treated with CNTN4 inhibitors.

[0088] FIGS. 24A, 24B and 24C show the tumor size in mouse treated with CD351 inhibitors.

EXAMPLES

[0089] In the following, exemplary embodiments of the inventive concept will be explained in further detail with reference to examples. However, the following examples are meant to exemplify the present invention, and the scope of the invention is not restricted by these examples.

Example 1. Inhibition of the Proliferation and Activity of T Cells

[0090] This example is to confirm whether KIRREL3, CNTN4 and CD351 suppress the proliferation and activity of the T cell, and ensures that cancer cells evade the T cell-mediated immune system.

1.1. Preparation of CD4+ Cells and CD8+ T Cells

[0091] Human blood was placed in a 10 ml tube coated with EDTA (or heparin) and mixed with PBS at a ratio of 1:1. Ficoll-Paque PLUS was placed in a 50 ml tube, and then the blood sample was added. After centrifugation, human PBMCs (peripheral blood mononuclear cells) were collected. The resultant was centrifuged, and the supernatant was removed. Then, RBC lysis (1) was added, pipetted, and stored on ice for 3 minutes. After that, 50 ml of 10% FBS RPMI1640 was added, and the mixture was centrifuged to remove the supernatant. Then, FACS buffer was added, and the supernatant was removed by centrifugation. Subsequently, 50 ml of MACS buffer (PBS containing 0.5% bovine serum albumin and 2 mM EDTA) was added, the number of cells was counted, and the supernatant was completely removed after centrifugation.

[0092] CD4+ T cells and CD8+ T cells were resuspended using 40 l of MACS buffer based on the number of 110.sup.7 cells in a 50 ml tube. 10 l of anti-CD4 and anti-CD8 biotin antibodies were added to the tube respectively, and then stored in the refrigerator for 5 minutes. Subsequently, 30 l of MACS buffer based on the number of 110.sup.7 cells was added to the resultant, and 20 l of anti-biotin microbeads were added and mixed. Then, CD4+ T cells and CD8+ T cells were separated using LS column, and were counted.

[0093] The prepared CD4+ T cells and CD8+ T cells were mixed with 1 l of CFSE (carboxyfluorescein succinimidyl ester) based on the number of 210.sup.6 cells, and stored at 37 C. for 3 minutes. Then, FBS was added into tubes containing the CD4+ T cells and CD8+ T cells respectively, and stored on ice 10 minutes. Thereafter, the supernatant was removed by centrifugation. The resultant was added with 30 ml of FACS buffer, pipetted, and centrifuged to remove the supernatant. Then, the resultant was mixed with 10 ml of 10% FBS RPMI1640, and the number of cells was counted.

1.2. Measurement of T Cell Activity

1.2.1. Inhibition of T Cell Activity by KIRREL3

[0094] The recombinant human IgG1 Fc protein (Cat. No. 110-HG) and the recombinant human PD-L1/B7-H1 Fc chimera protein (Cat. No. 156-B7) were purchased from R&D systems. The recombinant human KIRREL3 His Tag protein (Cat. No. 4910-K3) was purchased from R&D systems.

[0095] 7.5 g/ml or 10 g/ml of each protein was mixed with 2.5 g/ml of anti-CD3 antibody (BioLegend, Cat. No. 317325) in PBS, respectively. The resultant mixture was coated on 96-well plates at 4 C., and the wells were washed three times with PBS.

[0096] The CD4+ T cells and CD8+ T cells prepared in the Example 1.1 were added to each well of the 96-well plate at the number of 210.sup.6 cells in an amount of 200 l, and then incubated.

[0097] CD4+ T cells and CD8+ T cells were activated by anti-CD3 antibody for 72 hours. The proliferation of CD4+ T cells and CD8+ T cells can be confirmed by the degree of CFSE fluorescent cell staining, and was analyzed by flow cytometry using FACSDiVa software (BD Biosciences).

1.2.2. Inhibition of T-Cell Activity by CNTN4

[0098] The recombinant human IgG1 Fc protein (Cat. No. 110-HG) and the recombinant human PD-L1/B7-H1 Fc chimera protein (Cat. No. 156-B7) were purchased from R&D systems. The recombinant human CNTN4 His Tag protein (Cat. No. 2205-CN) was purchased from R&D systems.

[0099] 7.5 g/ml or 10 g/ml of each protein was mixed with 2.5 g/ml of anti-CD3 antibody (BioLegend, Cat. No. 317325) in PBS, respectively. The resultant mixture was coated on 96-well plates at 4 C., and the wells were washed three times with PBS.

[0100] The CD4+ T cells and CD8+ T cells prepared in the Example 1.1 were added to each well of the 96-well plate at the number of 210.sup.6 cells in an amount of 200 l, and then incubated.

[0101] CD4+ T cells and CD8+ T cells were activated by anti-CD3 antibody for 72 hours. The proliferation of CD4+ T cells and CD8+ T cells can be confirmed by the degree of CFSE fluorescent cell staining, and was analyzed by flow cytometry using FACSDiVa software (BD Biosciences).

1.2.3. Inhibition of T-Cell Activity by CD351

[0102] The recombinant human IgG1 Fc protein (Cat. No. 110-HG) and the recombinant human PD-L1/B7-H1 Fc chimera protein (Cat. No. 156-B7) were purchased from R&D systems. The recombinant human CD351 His Tag protein (Cat. No. 9278-FC) was purchased from R&D systems.

[0103] 10 g/ml of each protein was mixed with 1.0 g/ml, 2.0 g/ml, 4.0 g/ml, or 6.0 g/ml of anti-CD3 antibody (BioLegend, Cat. No. 317325) in PBS, respectively. The resultant mixture was coated on 96-well plates at 4 C., and the wells were washed three times with PBS.

[0104] The CD4+ T cells and CD8+ T cells prepared in the Example 1.1 were added to each well of the 96-well plate at the number of 210.sup.6 cells in an amount of 200 l, and then incubated.

[0105] CD4+ T cells and CD8+ T cells were activated by anti-CD3 antibody for 72 hours. The proliferation of CD4+ T cells and CD8+ T cells can be confirmed by the degree of CFSE fluorescent cell staining, and was analyzed by flow cytometry using FACSDiVa software (BD Biosciences).

1.3. Results

1.3.1. Inhibition of T-Cell Activity by KIRREL3

[0106] FIG. 1 and FIG. 2 show the percent proliferation (%) of CD4+ T cells and CD8+ T cells, respectively.

[0107] The control group treated with PD-L1 inhibited the proliferation of both CD4+ T cells and CD8+ T cells compared to the control group treated with IgG1. The PD-L1 binds to PD-1, a protein on the surface of T cells, and inhibits the proliferation of T cells. Accordingly, it results in suppressing the function of T cells attacking and killing cancer cells.

[0108] The group treated with KIRREL3 remarkably inhibited the proliferation of both CD4+ T cells and CD8+ T cells compared to the control group treated with IgG1. And also, the group treated with KIRREL3 inhibited the proliferation of both CD4+ T cells and CD8+ T cells similarly to the control group treated with PD-L1.

[0109] It means that if KIRREL3 is neutralized by blocking or knockdown, the T cell proliferation inhibition of KIRREL3 can be suppressed. Accordingly, the cancer treatment can be effectively achieved.

1.3.2. Inhibition of T-Cell Activity by CNTN4

[0110] FIG. 3 and FIG. 4 show the percent proliferation (%) of CD4+ T cells and CD8+ T cells, respectively.

[0111] The control group treated with PD-L1 inhibited the proliferation of both CD4+ T cells and CD8+ T cells compared to the control group treated with IgG1.

[0112] The group treated with CNTN4 remarkably inhibited the proliferation of both CD4+ T cells and CD8+ T cells compared to the control group treated with IgG1. And also, the group treated with CNTN4 inhibited the proliferation of both CD4+ T cells and CD8+ T cells similarly to the control group treated with PD-L1.

[0113] It means that if CNTN4 is neutralized by blocking or knockdown, the T cell proliferation inhibition of CNTN4 can be suppressed. Accordingly, the cancer treatment can be effectively achieved.

1.3.3. Inhibition of T-Cell Activity by CD351

[0114] FIG. 5 and FIG. 6 show the percent proliferation (%) of CD4+ T cells and CD8+ T cells, respectively.

[0115] The control group treated with PD-L1 significantly inhibited the proliferation of CD4+ T cells compared to the control group treated with IgG1, whereas it did not show a significant inhibition on the proliferation of CD8+ T cells compared to the control group treated with IgG1.

[0116] The group treated with CD351 remarkably inhibited the proliferation of both CD4+ T cells and CD8+ T cells compared to the control group treated with IgG1 as well as the control group treated with PD-L1.

[0117] It means that CD351 is neutralized by blocking or knockdown, the T cell proliferation inhibition of CD351 can be suppressed. Accordingly, the cancer treatment can be effectively achieved.

Example 2. PBMC Cytotoxic Function Assay

[0118] This example is to confirm whether the cytotoxic ability of PBMC against cancer cells is increased when KIRREL3, CNTN4 or CD351 is neutralized using inhibitors of KIRREL3, CNTN4 or CD351.

2.1. Preparation of PBMC

[0119] Human blood was placed in a 10 ml tube coated with EDTA (or heparin) and mixed with PBS at a ratio of 1:1. Ficoll-Paque PLUS was placed in a 50 ml tube, and then the blood sample was added. After centrifugation, human PBMCs were collected. The resultant was centrifuged, and the supernatant was removed. Then, RBC lysis (1) was added, pipetted, and stored on ice for 3 minutes. After that, 50 ml of 10% FBS RPMI1640 was added, and the mixture was centrifuged to remove the supernatant. Then, FACS buffer was added, and the supernatant was removed by centrifugation. Subsequently, 50 ml of MACS buffer (PBS containing 0.5% bovine serum albumin and 2 mM EDTA) was added, the number of cells was counted, and the supernatant was completely removed after centrifugation.

[0120] 96-well plates were coated with 1.0 g/ml of anti-CD3 antibody (BioLegend, Cat. No. 317325) in PBS at 4 C., and the wells were washed three times with PBS. The PBMC prepared in the above was mixed with 10% FBS RPMI1640, and was added to each well of the 96-well plate at the number of 610.sup.5 cells in an amount of 100 l. The PBMC was activated by anti-CD3 antibody for 72 hours.

2.2. Preparation of Cancer Cells

[0121] Lung cancer cell line A549, colon cancer cell line HCT-116, breast cancer cell line MDA-MB-231, gastric cancer cell line MKN-74, and leukemia cell line U937 were respectively mixed with 1 l of CFSE (carboxyfluorescein succinimidyl ester), and then stored at 37 C. for 3 minutes. Subsequently, FBS was added into tubes containing cancer cells and stored on ice for 10 minutes. Thereafter, the supernatant was removed by centrifugation. The resultant was added with 30 ml of FACS buffer, pipetted, and centrifuged to remove the supernatant. Then, 10% FBS RPMI1640 was added, pipetted, and centrifuged to remove the supernatant. Thereafter, the resultant was mixed with 10 ml of 10% FBS RPMI1640, and the number of cells was counted.

[0122] Each PBMC-containing well of the 96-well plate prepared in the Example 2.1 was added with the cancer cells at the number of 310.sup.4 cells in an amount of 100 l.

2.3. Measurement of Cytotoxicity of PBMC Against Cancer Cells

[0123] The mixtures of PBMCs and cancer cells were prepared in the Example 2.2. These mixtures were incubated for 24 hours with 10 g/mL of anti-human KIRREL3 antibody, anti-human CNTN4 antibody or anti-human CD351 antibody, or 50 nM of KIRREL3 siRNA, CNTN4 siRNA or CD351 siRNA.

[0124] Table 1 below provides the non-treated control group and Groups 1 to 4 using four neutralizing antibodies for blocking KIRREL3, and Table 2 below provides the non-treated control group and Groups 5 to 7 using three siRNAs for knockdown of KIRREL3.

TABLE-US-00001 TABLE 1 human KIRREL3 neutralizing antibody Control group Not treated Group 1 anti-human KIRREL3 antibody (R&D, AF4910) Group 2 anti-human KIRREL3 antibody (Bioss, bs-11864R) Group 3 anti-human KIRREL3 antibody (genetex, GTX32140) Group 4 anti-human KIRREL3 antibody (LSbio, LS-C336219)

TABLE-US-00002 TABLE2 humanKIRREL3siRNA Control Nottreated group Group5 Sense(5-CUCUCAAGUUACCCACAGUtt-3) (SEQIDNO:1) Antisense(5-ACUGUGGGUAACUUGAGAGtt-3) (SEQIDNO:2) Group6 Sense(5-GGAGAGGUGUACAGGACCAtt-3) (SEQIDNO:3) Antisense(5-UGGUCCUGUACACCUCUCCtt-3) (SEQIDNO:4) Group7 Sense(5-UCUCAAGUUACCCACAGUAtt-3) (SEQIDNO:5) Antisense(5-UACUGUGGGUAACUUGAGAtt-3) (SEQIDNO:6)

[0125] Table 3 below provides the non-treated control group and Groups 1 to 5 using five neutralizing antibodies for blocking CNTN4, and Table 4 below provides the non-treated control group and Groups 6 to 8 using three siRNAs for knockdown of CNTN4.

TABLE-US-00003 TABLE 3 human CNTN4 neutralizing antibody Control group Not treated Group 1 anti-human CNTN4 antibody (R&D, MAB2205) Group 2 anti-human CNTN4 antibody (abcam, ab137107) Group 3 anti-human CNTN4 antibody (abcam, ab131285) Group 4 anti-human CNTN4 antibody (LSbio, LS-C119876) Group 5 anti-human CNTN4 antibody (Abnova, PAB27653)

TABLE-US-00004 TABLE4 humanCNTN4siRNA Control Nottreated group Group6 Sense(5-CAGUAUCUUUGCCAGAAGUtt-3) (SEQIDNO:7) Antisense(5-ACUUCUGGCAAAGAUACUGtt-3) (SEQIDNO:8) Group7 Sense(5-GAUAAUGAGUCGGAAGUAAtt-3) (SEQIDNO:9) Antisense(5-UUACUUCCGACUCAUUAUCtt-3) (SEQIDNO:10) Group8 Sense(5-GUGACAAUAGACGAAAUCAtt-3) (SEQIDNO:11) Antisense(5-UGAUUUCGUCUAUUGUCACtt-3) (SEQIDNO:12)

[0126] Table 5 below provides the non-treated control group and Groups 1 to 3 using three neutralizing antibodies for blocking CD351, and Table 6 below provides the non-treated control group and Groups 4 to 6 using three siRNAs for knockdown of CD351.

TABLE-US-00005 TABLE 5 human CD351 neutralizing antibody Control group Not treated Group 1 anti-human CD351 antibody (Biolegend, 13730) Group 2 anti-human CD351 antibody (Creative diagnostics, CABT-BL4657) Group 3 anti-human CD351 antibody (Biobyt, orb183662)

TABLE-US-00006 TABLE6 humanCD351siRNA Control Nottreated group Group4 Sense(5-GAGAGAUGAACUGCUCAGUtt-3) (SEQIDNO:13) Antisense(5-ACUGAGCAGUUCAUCUCUCtt-3) (SEQIDNO:14) Group5 Sense(5-GAGAACUUCCAACUCAGUAtt-3) (SEQIDNO:15) Antisense(5-UACUGAGUUGGAAGUUCUCtt-3) (SEQIDNO:16) Group6 Sense(5-AGAGAACUUCCAACUCAGUtt-3) (SEQIDNO:17) Antisense(5-ACUGAGUUGGAAGUUCUCUtt-3) (SEQIDNO:18)

[0127] After twenty-four hours from incubating the mixtures of PBMCs and cancer cells with antibody or siRNA, cells were stained with 7-aminoactinomycin D (7-AAD; BD Pharmingen, San Diego, Calif., USA) to detect lysed cells. The cytotoxicity of PBMC against cancer cells was analyzed by determining FL-1 (CFSE) and FL-3 (7-AAD) staining using a FACSDiVa software (BD Biosciences).

2.4. Results

[0128] For the lung cancer cell line A549, FIGS. 7A, 7B, 7C and 7D show the results treated with KIRREL3 neutralizing antibody or siRNA, FIGS. 12A, 12B, 12C and 12D show the results treated with CNTN4 neutralizing antibody or siRNA, and FIGS. 17A, 17B, 17C and 17D show the results treated with CD351 neutralizing antibody or siRNA.

[0129] When the lung cancer cell line A549 and PBMC were treated with KIRREL3 neutralizing antibody, CNTN4 neutralizing antibody or CD351 neutralizing antibody, the cytotoxicity against lung cancer cell was significantly increased compared to the non-treated control group even though there is more or less degree of difference depending on the type of antibody. Further, the cytotoxicity against lung cancer cell was also significantly increased when it was treated with KIRREL3 siRNA, CNTN4 siRNA or CD351 siRNA.

[0130] Using KIRREL3 neutralizing antibody or siRNA, the results on the colon cancer cell line HCT-116 are shown in FIGS. 8A, 8B, 8C and 8D, the result on the breast cancer cell line MDA-MB-231 are shown in FIGS. 9A, 9B, 9C and 9D, the results on the gastric cancer cell line MKN-74 are shown in FIGS. 10A, 10B, 10C and 10D, and the results on the leukemia cell line U937 are shown in FIGS. 11A, 11B, 11C and 11D.

[0131] In addition, using CNTN4 neutralizing antibody or siRNA, the results on the colon cancer cell line HCT-116 are shown in FIGS. 13A, 13B, 13C and 13D, the results on the breast cancer cell line MDA-MB-231 are shown in FIGS. 14A, 14B, 14C and 14D, the results on the gastric cancer cell line MKN-74 are shown in FIGS. 15A, 15B, 15C and 15D, and the results on the leukemia cell line U937 are shown in FIGS. 16A, 16B, 16C and 16D.

[0132] In addition, using CD351 neutralizing antibody or siRNA, the results on the colon cancer cell line HCT-116 are shown in FIGS. 18A, 18B, 18C and 18D, the results on the breast cancer cell line MDA-MB-231 are shown in FIGS. 19A, 19B, 19C and 19D, the results on the gastric cancer cell line MKN-74 are shown in FIGS. 20A, 20B, 20C and 20D, and the results on the leukemia cell line U937 are shown in FIGS. 21A, 21B, 21C and 21D.

[0133] As shown in FIGS. 8A to 11D, 13A to 16D, and 18A to 21D, when one or more of KIRREL3, CNTN4 and CD351 were neutralized by antibodies or siRNAs, the results of increasing the cytotoxicity of PBMC were also observed in colon cancer, breast cancer, gastric cancer and leukemia.

Example 3. Tumor-Mouse Model Experiment

[0134] This example is to confirm whether the growth of tumor in mouse is suppressed when KIRREL3, CNTN4 or CD351 is neutralized using inhibitors of KIRREL3, CNTN4 or CD351.

3.1. Establishment of Tumor-Mouse Model

[0135] MC-38 cell line derived from C57bL6 colon adenocarcinoma cells was resuspended in 50 l PBS at the number of 210.sup.5 cells, and was subcutaneously injected into the flanks of 6-week-old female C57bL6 mice.

[0136] Table 7 below provides the non-treated control group and Group 8 using a siRNA for knockdown of KIRREL3.

TABLE-US-00007 TABLE7 mouseKIRREL3siRNA Control Nottreated group Group8 Sense(5-GUAAAGGAGAGGUCAUCAA-3) (SEQIDNO:19) Antisense(5-UUGAUGACCUCUCCUUUAC-3) (SEQIDNO:20)

[0137] Table 8 below provides the non-treated control group and Group 9 using a siRNA for knockdown of CNTN4.

TABLE-US-00008 TABLE8 mouseCNTN4siRNA Control Nottreated group Group9 Sense(5-GUGUAGACAAACUCUCUGU-3) (SEQIDNO:21) Antisense(5-ACAGAGAGUUUGUCUACAC-3) (SEQIDNO:22)

[0138] Table 9 below provides the non-treated control group and Groups 7, 8 and 9 using three siRNAs for knockdown of CD351.

TABLE-US-00009 TABLE9 mouseCD351siRNA Control Nottreated group Group7 Sense(5-GUCCAUCCAACACCACCUA-3) (SEQIDNO:23) Antisense(5-UAGGUGGUGUUGGAUGGAC-3) (SEQIDNO:24) Group8 Sense(5-CUGAUGAGGGAAAGAACUU-3) (SEQIDNO:25) Antisense(5-AAGUUCUUUCCCUCAUCAG-3) (SEQIDNO:26) Group9 Sense(5-CAGCUAAGCCCAGUGAACA-3) (SEQlDNO:27) Antisense(5-UGUUCACUGGGCUUAGCUG-3) (SEQIDNO:28)

[0139] In all Groups, the siRNA targeting mouse KIRREL3, mouse CNTN4 or mouse CD351 was injected into the tumor of mice three times at the interval of 5 days from the 11th day after injecting MC-38 cells. Specifically, 10 g siRNA and 7.5 l oligofectamine (Invitrogen) in PBS were mixed according to manufacturer's instruction, and then injected into the tumor tissue induced in mice at a dose of 0.5 mg/kg.

3.2. Results

[0140] FIG. 22 provides the result on the size of tumor in mice of the non-treated control group and Group 8 wherein KIRREL3 was knocked down. FIG. 23 provides the result on the size of tumor in mice of the non-treated control group and Group 9 wherein CNTN4 was knocked down. FIGS. 24A, 24B and 24C provide the results on the size of tumor in mice of the non-treated control group and Groups 7 to 9 wherein CD351 was knocked down.

[0141] In the non-treated control group, the tumor continued to grow after it occurred. Compared to the non-treated control group, the growth rate of tumor in mouse was remarkably inhibited in Groups whrein KIRREL3, CNTN4 or CD351 was knocked down. It means that when one or more of KIRREL3, CNTN4 and CD351 are blocked or knocked down to inhibit its activity or expression, the development of cancer is delayed or stopped and the occurrence of cancer is inhibited. Accordingly, one or more inhibitors of KIRREL3, CNTN4 and CD351 can be efficiently used to prevent cancer.

[0142] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. Such equivalents are intended to be encompassed by the following claims.