NK cell-activating fusion protein, NK cell, and pharmaceutical composition including same

Abstract

A fusion protein for cancer treatment and a use thereof is disclosed. The fusion protein for preventing or treating cancer of the present invention includes a fusion polypeptide including: an antibody or fragment thereof binding to a tumor-associated antigen; a linker; and a NK cell-inducing protein of CXCL16, wherein a co-administration of the fusion polypeptide along with the NK cells, an immunocyte therapeutic agent, greatly increases an influx of the NK cells into cancer expressing a certain antigen, thereby having a remarkable effect on preventing or treating cancer.

Claims

1. A fusion polypeptide, consisting of: an antibody or fragment thereof that binds to a tumor-associated antigen; a linker comprising a furin cleavage site; and a natural killer (NK) cell-inducing protein of CXCL16, wherein the CXCL16 consists of the amino acid sequence of SEQ ID NO: 17.

2. The fusion polypeptide, according to claim 1, wherein the tumor-associated antigen is at least one selected from the group consisting of mesothelin, PD-L1 (programmed death-ligand 1), Her2 (human EGFR-related 2), CD19, MUC1, EGFR, VEGFR, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, 4-1BB, 5T4, AGS-5 and AGS-16.

3. The fusion polypeptide, according to claim 1, wherein the antibody is a single-chain Fv fragment (scFv).

4. The fusion polypeptide, according to claim 1, wherein the furin cleavage site comprises the amino acid sequence of SEQ ID NO: 15.

5. A nucleic acid, coding the fusion polypeptide according to claim 1.

6. An expression vector, comprising the nucleic acid coding the fusion polypeptide according to claim 5.

7. A host cell, comprising the expression vector according to claim 6.

8. The host cell, according to claim 7, wherein the host cell is one selected from the group consisting of COS, CHO, HeLa and myeloma cell lines.

9. A pharmaceutical composition for preventing or treating a cancer, comprising a fusion polypeptide, wherein the fusion polypeptide consists of: an antibody or fragment thereof that binds to a tumor-associated antigen; a linker comprising a furin cleavage site; and a NK cell-inducing protein of CXCL16, wherein the CXCL16 consists of the amino acid sequence of SEQ ID NO: 17.

10. The pharmaceutical composition for preventing or treating cancer, according to claim 9, wherein the tumor-associated antigen is at least one selected from the group consisting of mesothelin, PD-L1, Her2, CD19, MUC1, EGFR, VEGFR, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, 4-1BB, 5T4, AGS-5 and AGS-16.

11. The pharmaceutical composition for preventing or treating a cancer, according to claim 9, wherein the cancer is one selected from the group consisting of pancreatic cancer, breast cancer, prostate cancer, gastric cancer, liver cancer or lung cancer.

12. A pharmaceutical composition for preventing or treating cancer, comprising a fusion polypeptide, wherein the fusion polypeptide consists of: an antibody or fragment thereof that binds to a tumor-associated antigen; a linker comprising a furin cleavage site; and a NK cell-inducing protein of CXCL16, and Natural killer cells, wherein the CXCL16 consists of the amino acid sequence of SEQ ID NO: 17.

13. A method of preventing or treating a cancer comprising administering to a subject an effective amount of fusion polypeptide, wherein the fusion polypeptide consists of: an antibody or fragment thereof that binds to a tumor-associated antigen; a linker comprising a furin cleavage site; and a NK cell-inducing protein of CXCL16, wherein the CXCL16 consists of the amino acid sequence of SEQ ID NO: 17.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph of showing results of identifying a degree of migration of expanded natural killer (NK) cells according to a chemokine type.

(2) FIG. 2 is a schematic diagram of showing an expression vector for preparing a fusion polypeptide according to the present invention.

(3) FIG. 3 is a graph of showing results of identifying that the fusion polypeptide prepared according to the present invention recognizes and binds to mesothelin present on a surface of a pancreatic cancer cell line by means of a mesothelin-recognizing site.

(4) FIG. 4 is a graph of showing results of identifying that the fusion polypeptide prepared according to the present invention recognizes and binds to PD-L1 present on a surface of a pancreatic cancer cell line by means of a PD-L1-recognizing site.

(5) FIG. 5 is a graph of showing results of identifying that the fusion polypeptide prepared according to the present invention recognizes and binds to Her2 present on a surface of a pancreatic cancer cell line by means of a Her2-recognizing site.

(6) FIG. 6 is a graph of showing results of identifying that the fusion polypeptide prepared according to the present invention binds to a pancreatic cancer cell line to release CXCL16.

(7) FIG. 7 is a graph of showing results of identifying that a migration ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to mesothelin.

(8) FIG. 8 is a graph of showing results of identifying that an influx of NK cells is increased according to treatment of a Panc-1 cell line with the fusion polypeptide of the present invention, comprising an antibody binding to PD-L1.

(9) FIG. 9 is a graph of showing results of identifying that the influx of NK cells is increased according to treatment of an HT-29 cell line with the fusion polypeptide of the present invention, comprising an antibody binding to PD-L1.

(10) FIG. 10 is a graph of showing results of identifying that the influx of NK cells is increased according to treatment of a Panc-1 cell line with the fusion polypeptide of the present invention, comprising an antibody binding to Her2.

(11) FIG. 11 is a graph of showing results of identifying that the influx of NK cells is increased according to treatment of an MCF7 cell line with the fusion polypeptide of the present invention, comprising an antibody binding to Her2.

(12) FIG. 12 is a graph of showing results of identifying that a migration ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to CD19.

(13) FIG. 13 is a graph of showing results of identifying that the migration ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to MUC-1.

(14) FIG. 14 is a graph of showing results of identifying that the migration ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to EGFR.

(15) FIG. 15 is a graph of showing results of identifying that the migration ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to VEGFR.

(16) FIG. 16 is a graph of showing results of identifying that an invasion ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to mesothelin.

(17) FIG. 17 is a graph of showing results of identifying that the invasion ability of NK cells is increased according to treatment of a Panc-1 cell line with the fusion polypeptide of the present invention, comprising an antibody binding to PD-L1.

(18) FIG. 18 is a graph of showing results of identifying that the invasion ability of NK cells is increased according to treatment of an HT-29 cell line with the fusion polypeptide of the present invention, comprising an antibody binding to PD-L1.

(19) FIG. 19 is a graph of showing results of identifying that the invasion ability of NK cells is increased according to treatment of a Panc-1 cell line with the fusion polypeptide of the present invention, comprising an antibody binding to Her2.

(20) FIG. 20 is a graph of showing results of identifying that the invasion ability of NK cells is increased according to treatment of an MCF7 cell line with the fusion polypeptide of the present invention, comprising an antibody binding to Her2.

(21) FIG. 21 is a graph of showing results of identifying that the invasion ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to CD19.

(22) FIG. 22 is a graph of showing results of identifying that the invasion ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to MUC-1.

(23) FIG. 23 is a graph of showing results of identifying that the invasion ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to EGFR.

(24) FIG. 24 is a graph of showing results of identifying that the invasion ability of NK cells is increased according to treatment of various cancer cell lines with the fusion polypeptide of the present invention, comprising an antibody binding to VEGFR.

(25) FIG. 25 is a graph of showing results of identifying that the invasion ability of NK cells is increased by means of a fusion polypeptide (Her2 scFv NRP-body) prepared to recognize Her2 according to the present invention.

(26) FIG. 26 is a graph of showing the induction of NK cells into a cancer tissue according to an administration of the fusion polypeptide prepared according to the present invention as well as the NK cells. FIG. 26A is a diagram showing that an influx of natural killer cells into cancer tissues is greatly increased by NRP-body. FIG. 26B is a diagram showing a total number of influx cells by the addition of NRP-body.

(27) FIG. 27 is a graph of showing results of identifying a therapeutic effect by administering mesothelin scFv fusion polypeptide prepared according to the present invention into an animal model with transplanted pancreatic cancer along with the NK cells. FIG. 27A is a diagram showing results of identifying the tumor growth inhibitory effect after administering a fusion polypeptide prepared in Example 2 together with natural killer cells, and FIG. 27B is a diagram showing results of identifying an increase in migration of NK cells in tumor tissues with a fluorescence Image program.

(28) FIG. 28 is a graph of showing results of identifying a therapeutic effect by administering PD-L1 scFv fusion polypeptide prepared according to the present invention into an animal model with transplanted pancreatic cancer along with the NK cells. FIG. 28A is a diagram showing results of identifying an increase in migration of NK cells in tumor tissues with a fluorescence Image program after administering a fusion polypeptide prepared in Example 2 together with natural killer cells, and FIG. 28B is a diagram showing results of identifying the tumor growth inhibitory effect.

(29) FIG. 29 is a graph of showing results of identifying a therapeutic effect by administering Her2 scFv fusion polypeptide prepared according to the present invention into an animal model with transplanted pancreatic cancer along with the NK cells. FIG. 29A is a diagram showing results of identifying an increase in migration of NK cells in tumor tissues with a fluorescence Image program after administering a fusion polypeptide prepared in Example 2 together with natural killer cells, and FIG. 29B is a diagram showing results of identifying the tumor growth inhibitory effect.

(30) FIG. 30 is a graph of showing a change in distribution of NK cells upon treatment of the NK cells with CXCL16 and IL-2 for a short period of time. FIG. 30A is a diagram showing results of identifying a change in distribution of cells from CD56.sup.dim to CD56.sup.bright by means of CXCL16 treatment according to an elapse of time, and FIG. 30B is a graph showing quantification of CD56.sup.bright cells (%) over time.

(31) FIG. 31 is a graph of showing a change in distribution of NK cells upon treatment of the NK cells with CXCL16 and IL-2 for a long period of time. FIG. 31A is a diagram showing results of identifying a change in distribution of NK cells upon treatment with IL-2 and IL-2+CXCL16, and FIG. 31B is a graph showing quantification of CD56.sup.bright cells (%) over time in the IL-2 and CXCL16 treated groups.

(32) FIG. 32 is a graph of showing results of identifying an increase in cell deaths by means of CD56.sup.brightCD16.sup.+ NK cells, which are distributed upon treatment of the NK cells with the fusion polypeptide prepared according to the present invention.

MODE FOR INVENTION

(33) Hereinafter, the present invention will be described in detail through preferred Examples for better understanding of the present invention. However, the following Examples are provided only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.

<Example 1> Identification of Migration of Expanded Natural Killer Cells by Means of Chemokine

(34) In order to identify a degree of migration of expanded natural killer (NK) cells according to a chemokine type, the expanded NK cells were collected and centrifuged at 1,500 rpm. Then, supernatant was removed therefrom and washed with PBS, after which the number of cells was counted. As a chemokine, CXCL9, CXCL10, CXCL11 and CXCL16 were divided by 10 nM onto a bottom layer of a Boyden chamber plate, and the expanded NK cells were divided by 2×10.sup.5 cells onto an upper layer of the Boyden chamber plate. After that, the resulting cells were cultured in a CO.sub.2 incubator at 37° C. for two hours, after which the bottom layer was collected therefrom and centrifuged at 1,500 rpm. Then, a PBS washing was performed, after which a CD56-PE staining was carried out at 4° C. for 30 minutes and washed with PBS. For an FAC analysis, Count Bright Absolute Counting Beads (Invitrogen) were divided by 50 ul thereto, and the FACS analysis was performed.

(35) The results thereof were shown in FIG. 1.

(36) As identified in FIG. 1, it was identified that CXCL16 shows a remarkable effect on the migration of the expanded NK cells compared to other chemokine types.

<Example 2> Preparation and Purification of a Fusion Polypeptide [NK Cell Recruitment Protein (NRP)-Body]

(37) Prepared was a recombinant vector, to which the followings were bound: a scFv sequence for recognizing a cancer-targeting antigen; a furin sequence for serving as a linker; and CXCL16 (NK cell Recruitment Protein; NRP) for inducing an influx of NK cells at the highest efficiency.

(38) A structure of the particular recombinant vector, to which the scFv sequence for recognizing mesothelin as a target antigen was bound, was shown in FIG. 2.

(39) A pcDNA3.1 vector was decomposed with a Sfi1 enzyme for two hours and purified to prepare a vector for ligation. To prepare mesothelin scFv, an amplification was performed through a PCR based on a primer sequence as shown in a following table 1 to obtain a mesothelin scFv base sequence of SEQ ID NO: 2, after which the vector, an insertion sample and T4 ligase were mixed together, and cultured at 25° C. for two hours to perform a ligation between the vector and the insertion. A resulting product was inserted into a Sfi1 enzyme site of the pcDNA3.1 vector.

(40) TABLE-US-00001 TABLE 1 Primer sequence for preparing mesothelin scFv Sequence Mesothelin scFv 5′-GGCCCAGCCGGCCATGCAGGTACAACTGCA Forward primer GCAG-3′ (SEQ ID NO: 20) Mesothelin scFv 5′-GGCCCTTGGTGGAGGCACTCGAGACGGTGA Reverse primer CCAGGGTTC-3′ (SEQ ID NO: 21)

(41) To prepare PD-L1 scFv, an amplification was performed through the PCR based on a primer sequence as shown in a following table 2 to obtain a PD-L1 scFv base sequence comprising a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, after which the ligation between the vector and the insertion was performed by means of the same method as the method for preparing the said vector, to which mesothelin scFv was bound, such that a resulting product was inserted into the Sfi1 enzyme site of the pcDNA3.1 vector.

(42) TABLE-US-00002 TABLE 2 Primer sequence for preparing PD-L1 scFv Sequence PD-L1 scFv 5′-GGCCCAGCCGGCCATGCAGGTCCAACTTGTG Forward primer CAGTC-3′ (SEQ ID NO: 22) PD-L1 scFv 5′-GGCCCTTGGTGGACCAAGCTGGAGATCAAA-  Reverse primer 3′ (SEQ ID NO: 23)

(43) To prepare Her2 scFv, the amplification was performed through the PCR based on a primer sequence as shown in a following table 3 to obtain a Her2 scFv base sequence comprising a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 11, after which the ligation between the vector and the insertion was performed by means of the same method as the method for preparing the said vector, to which mesothelin scFv was bound, such that a resulting product was inserted into the Sfi1 enzyme site of the pcDNA3.1 vector.

(44) TABLE-US-00003 TABLE 3 Primer sequence for preparing Her2 scFv Sequence Her2 scFv 5′-GGCCCAGCCGGCCATGGAGGTTCAGCTGGT Forward primer GGA-3′ (SEQ ID NO: 24) Her2 scFv 5′-GGCCCTTGGTACCAAGGTGGAGATCAAA- Reverse primer 3′ (SEQ ID NO: 25)

(45) Also, to prepare CD19, MUC-1, EFGR and VEGFR scFv, a synthesis was performed on a base sequence for scFv (CD19 scFv comprising a heavy chain of SEQ ID NO: 29 and a light chain of SEQ ID NO: 31; MUC-1 scFv comprising a heavy chain of SEQ ID NO: 33 and a light chain of SEQ ID NO: 35; EGFR scFv comprising a heavy chain of SEQ ID NO: 37 and a light chain of SEQ ID NO: 39; and VEGFR scFv comprising a heavy chain of SEQ ID NO: 41 and a light chain of SEQ ID NO: 43) based on an amino acid sequence of each scFv, after which the ligation between the vector and the insertion was performed by means of the same method as the method for preparing the said vector, to which mesothelin scFv was bound, such that a resulting product was inserted into the Sfi1 enzyme site of the pcDNA3.1 vector.

(46) CXCL16 and a furin cleavage site were amplified through the PCR based on a primer sequence as shown in a following table 4, and a Not1 enzyme site behind immunoglobulin present in the vector was used. The vector, into which scFv for recognizing a target antigen was inserted, was decomposed with a Not1 enzyme for two hours, and purified, after which the vector, the insertion, i.e. a CXCL16 sample, and a ligase enzyme were mixed together and cultured at 25° C. for two hours to perform the ligation between the vector and the insertion.

(47) TABLE-US-00004 TABLE 4 Primer sequence for preparing CXCL16  and the furin cleavage site Sequence CXCL16, Furin 5′-CACACTGGCGGCCGCACGGGTGAAGCGGAAC cleavage site GAGGGCAG-3′ (SEQ ID NO: 26) Forward primer CXCL16, Furin 5′-AATCTCGAGCGGCCGCCTAAGGAAGTAAATG cleavage site CTTCTGGTG-3′ (SEQ ID NO: 27) Reverse primer

(48) Fusion polypeptide (NRP-body) was mass-produced by transfecting a CHO (Chinese hamster ovary) cell, from which furin was removed, with the prepared expression vector. The CHO cell transfected with the said expression vector was cultured in a 150 mm plate, then cultured in a roller bottle incubator for 72 hours, and then collected therefrom. A collected culture fluid was centrifuged, after which only supernatant thereof was purified by using a protein A-agarose column of an AKTA protein purification system (GE Healthcare Life Sciences), such that the fusion polypeptide was produced.

<Example 3> Identification of Binding of the Fusion Polypeptide to an Antigen

(49) Mesothelin-recognizing fusion polypeptide (mesothelin scFv NRP-body) (0.1-2 μg/ml) prepared in Example 2 above was divided into 2×10.sup.5 Panc-1 cells, which are pancreatic cancer cell lines, and cultured at 4° C. for 20 minutes. After that, the cells were collected therefrom and washed with PBS, after which FC antibodies (1 μg/ml), to which FITC was bound, were divided thereto and cultured at 4° C. for 20 minutes. After that, the cells were collected therefrom again, then washed with PBS, and then analyzed by means of an FACS.

(50) The results thereof were shown in FIG. 3.

(51) As identified in FIG. 3, it was identified that mesothelin present on a surface of the pancreatic cancer cell line is recognized through a mesothelin-recognizing site of the fusion polypeptide prepared in Example 2 above, such that the fusion polypeptide is bound to the cell surface.

(52) Also, in order to identify that the fusion polypeptide of the present invention is bound to a surface of a target cell line, even if antigen-recognizing sites are different, the FACS analysis was performed even on the PD-L1 scFv fusion polypeptide and Here scFv fusion polypeptide, which were prepared in Example 2 above, under the same condition as the experiment on antigen-binding of the said mesothelin scFv fusion polypeptide, wherein the results thereof were shown in FIG. 4 (PD-L1 scFv NRP-body) and FIG. 5 (Her2 scFv NRP-body).

(53) As identified in FIGS. 4 and 5, it was identified that the PD-L1 scFv NRP-body and the Her2 scFv NRP-body recognize PD-L1 or Her2 present on a surface of a pancreatic cancer cell line respectively through an antigen-recognizing site, and the fusion polypeptide is specifically bound to the cell surface, and thus identified for the fusion polypeptide of the present invention that the antibody specifically binding to a target antigen may be differently applied depending on a target tumor-associated antigen.

<Example 4> Identification of Characteristics of CXCL16 Release

(54) Through a human CXCL16 ELISA, it was identified if a furin cleavage site of the fusion polypeptide (NRP-body) prepared in Example 2 above is cleaved by means of the furin of a cancer cell line and CXCL16 is released.

(55) The CXCL16 ELISA was performed according to a method of Human CXCL16 ELISA kit (# DCX160) of an R&D system. For an ELISA analysis, the mesothelin scFv fusion polypeptide (mesothelin scFv NRP-body) was divided in an amount of 0.5 μg/mL and 50 μl/well into a 96-well plate for ELISA (R&D) and left alone at room temperature for two hours, such that a resulting absorbed one was used for that analysis. The said plate was washed, after which a peroxidase label was added thereto in an amount of 200 μl/well as a secondary antibody, and left alone at room temperature for two hours. The said plate was washed with Tween-PBS, after which an ABTS substrate solution was added thereto to carry out color development, such that an absorbance was measured at OD 415 nm by using a plate reader.

(56) The results thereof were shown in FIG. 6.

(57) As identified in FIG. 6, it was identified that the fusion polypeptide (NRP-body) is bound to mesothelin of a pancreatic cancer cell line, i.e. Panc-1, after which a furin cleavage site of the fusion polypeptide is cleaved by means of furin of the cancer cell, such that CXCL16 is released.

<Example 5> Identification of an Increase in Migration Ability (Influx) of NK Cells by Means of CXCL16 Released from the Fusion Polypeptide

(58) A Boyden chamber system was used to identify if the fusion polypeptide prepared in Example 2 above recognizes and binds to cancer expressing a target antigen, after which CXCL16, a protein for inducing an influx of NK cells, is released to increase an influx of the NK cells.

(59) HPDE, Panc-1 (ATCC, Cat.CRL-1469), HCT116 (ATCC, Cat.CCL-247), MCF7 (ATCC, Cat.HTB-22) and HT-29 (ATCC, Cat.HTB-38) cell lines were divided by 2×10.sup.5 onto a bottom layer of a Boyden Chamber assay plate (Fisher Scientific, #07-200-155), and cultured in a CO.sub.2 incubator at 37° C. for two hours. The mesothelin scFv-fusion polypeptide was divided in an amount of 1 μg/ml into each cell line above, and cultured in the CO.sub.2 incubator at 37° C. for four hours. The NK cells were labeled with CFSE (BioLegend, # RUO 423801), then divided by 2×10.sup.5 onto an upper layer, and then cultured in the CO.sub.2 incubator at 37° C. for four hours. After that, the cells were collected from the bottom layer, and a distribution of CFSE-labeled NK cells was identified through the FACS.

(60) The results thereof were shown in FIG. 7.

(61) As identified in FIG. 7, it was identified that a migration ability of human expanded NK cells is increased by means of CXCL16 released from the mesothelin scFv fusion polypeptide, and further identified that a degree of increased influx of the NK cells varies depending on a type of cancer cell line.

(62) Also, the PD-L1 scFv-fusion polypeptide and the Her2 scFv-fusion polypeptide prepared in Example 2 above were divided into Panc-1, HT-29 or MCF7 cell lines, and thus identified that an influx of the NK cells is increased through the Boyden chamber system under the same condition as in the experiment on the said mesothelin scFv-fusion polypeptide, wherein the results thereof were shown in FIG. 8 (PD-L1 scFv NRP-body for Panc-1), FIG. 9 (PD-L1 NRP-body for HT-29), FIG. 10 (Her2 NRP-body for Panc-1) and FIG. 11 (Her2 scFv NRP-body for MCF7).

(63) As identified in FIGS. 8 to 11, it was identified that the migration ability of the human expanded NK cells is increased by means of CXCL16 released from each fusion polypeptide just like the mesothelin scFv-fusion polypeptide.

(64) Also, the CD-19, MUC-1, EGFR and VEGFR scFv-fusion polypeptides prepared in Example 2 above were divided into HPDE, K562 (ATCC, Cat.CCL-243), HCT116 (ATCC, Cat.CCL-247), Panc-1 (ATCC, Cat.CRL-1469) or MCF7 (ATCC, Cat.HTB-22) cell lines, and thus identified whether an influx of the NK cells is increased or not under the same condition as in the experiment on the said mesothelin scFv-fusion polypeptide through the Boyden chamber system, wherein the results thereof were shown in FIG. 12 (CD19 scFv NRP-body), FIG. 13 (MUC-1 scFv NRP-body), FIG. 14 (EGFR scFv NRP-body) and FIG. 15 (VEGFR scFv NRP-body), respectively.

(65) As identified in FIGS. 12 to 15, it was identified that the migration ability of the human expanded NK cells is increased by means of CXCL16 released from the fusion polypeptide, and further identified that a degree of increased influx of the NK cells varies depending on a type of cancer cell line.

<Example 6> Identification of an Invasion Ability of NK Cells by CXCL1.6 Released from the Fusion Polypeptide

(66) An invasion assay was used to identify if each fusion polypeptide prepared in Example 2 above recognizes and binds to a target antigen expressed on a cancer cell, after which CXCL16, a protein for inducing an influx of NK cells, is released to increase an invasion ability of the NK cells into cancer cells.

(67) Particularly, HPDE, Panc-1, HCT116, MCF7, HT-29 and K562 cell lines were divided by 2×10.sup.5 onto a bottom layer of the Boyden Chamber assay plate (Fisher Scientific, #07-200-155), and cultured in a CO.sub.2 incubator at 37° C. for two hours, after which the fusion polypeptide prepared in Example 2 was divided in an amount of 1 μg/ml into each cell line above. The upper layer was treated with matrigel (BD, #354234), after which the NK cells were divided by 2×10.sup.5 thereto, and cultured in the CO.sub.2 incubator at 37° C. for 48 hours. After that, the upper layer was collected therefrom and stained with crystal violet for one hour, after which a picture was randomly taken from three portions of the upper layer, such that the invasion ability of the NK cells was measured by means of an image J program.

(68) The results of each fusion polypeptide were shown in FIGS. 16 to 24, respectively.

(69) As identified in FIGS. 16 to 24, it was identified that the invasiveness of human expanded NK cells is increased by means of CXCL16 released from the fusion polypeptide, and further identified that a degree of increased invasion ability of the NK cells varies depending on a type of cancer cell line.

<Example 7> Identification of an Induced Death of Cancer Cell Lines by Increasingly Introduced NK Cells

(70) It was identified about an efficacy of antibody-dependent cellular cytotoxicity (ADCC) on inducing a death of cancer cell lines by means of NK cells, which are increasingly introduced after a release of CXCL16 from the fusion polypeptide prepared in Example 2 above.

(71) Panc-1 cell lines were divided by 2×10.sup.5 into a 96-well plate, and cultured in a CO.sub.2 incubator at 37° C. for two hours. The target cells were treated with the mesothelin scFv-fusion polypeptide in an amount of 1 μg/ml, and cultured in the CO.sub.2 incubator at 37° C. for two hours. The NK cells were added thereto by 2×10.sup.5 to set a ratio of target cell and effector cell at 1:1, and cultured in the CO.sub.2 incubator at 37° C. for four hours. The cells were collected therefrom, then washed with PBS, then stained with Annexin V (1 μg/ml) and PI (1 μg/ml) for 30 minutes, and then analyzed with the FACS.

(72) The results thereof were shown in FIG. 25.

(73) As identified in FIG. 25, it was identified that the death of cancer cells is remarkably increased by means of the NK cells, which are increasingly introduced after a release of CXCL16.

<Example 8> Identification of a Therapeutic Efficacy of the Fusion Polypeptide in an Animal Model with Transplanted Cancer

(74) The fusion polypeptide prepared in Example 2 was injected into an animal model with transplanted cancer to identify an effect thereof in vivo.

(75) For an in vivo experiment, a six-week female NSG (NOD.Cg-PrkdcscidIl2rgtm1wjl/SzJ) mouse was used. The management of mice was performed under the authority of the Animal Care Committee of the Laboratory Animal Resource Center in the Korea Research Institute of Bioscience and Biotechnology. Panc-1 was injected into a mouse pancreas, after which a tumor was formed for two weeks, and mesothelin, PD-L1 or Her2 scFv fusion polypeptide (5 mg/kg) was intraperitoneally injected at an interval of five days.

(76) For an experiment on tumor growth, the NK cells were I.V. injected in an amount of 1×10.sup.7/mouse. For a tumor growth observation, a growth of Panc-1, which expresses luciferase, was observed by using an IVIS Living Image 3.0 program. For an experiment on the migration ability of the NK cells, the NK cells stained with DiR were intravenously injected in an amount of 1×10.sup.7/mouse and observed with the IVIS fluorescence Image program and FACS.

(77) The results thereof were shown in FIGS. 26 to 29. FIG. 26 shows an induction of the NK cells into a cancer tissue according to an administration of the fusion polypeptide prepared in Example 2 above as well as the NK cells, and FIGS. 27, 28 and 29 show results of identifying an therapeutic effect by administering mesothelin scFv NRP-body, PD-L1 scFv NRP-body and Her2 scFv NRP-body respectively along with the NK cells.

(78) As identified in A of FIG. 26, the influx of the NK cells into the cancer tissue was greatly increased by means of the NRP-body. As shown in B of FIG. 26, such agonistic effect occurred only with an addition of the NRP-body.

(79) Also, as identified in FIGS. 27 to 29, in case of administering the fusion polypeptide prepared in Example 2 along with the NK cells, the tumor growth was remarkably inhibited, and the migration of the NK cells into the tumor tissue was greatly increased.

(80) From the results above, it was identified that the fusion polypeptide of the present invention increases the influx of the NK cells, an immunocyte therapeutic agent, thereby showing a remarkable effect on cancer treatment.

<Example 9> Identification of a Characteristic Change of NK Cells According to CXCL16 Treatment

(81) To identify a characteristic change in the NK cells by means of CXCL16 released from the fusion polypeptide, the NK cells were treated with IL-2 and CXCL16, which promote a growth of the NK cells, at a concentration of 200 U and 100 nM respectively for 0, 1, 2, 8 or 16 hours, and a distribution of CD56.sup.dim and CD56.sup.bright was identified through the FACS, wherein the results thereof were shown in FIG. 30 and the cells in a square at the top right indicate CD56.sup.bright cells.

(82) As identified in FIG. 30, it was identified that a distribution of cells were changed from CD56.sup.dim to CD56.sup.bright by means of CXCL16 treatment according to an elapse of time.

(83) Also, the treatment with IL-2 and CXCL16 was simultaneously performed for a long period of time (14 days) in a similar way to the experimental method above, after which a change in CD56 expression was identified, wherein the results thereof were shown in FIG. 31.

(84) As identified in FIG. 31, a change into CD56.sup.bright cells was identified in an experimental group dosed with IL2 and CXCL16 together (IL-2+CXCL16 of FIG. 31A and CXCL16 of FIG. 31B).

(85) From the results above, it was identified that CXCL16 changes CD56.sup.dim into CD56.sup.bright having a large ADCC effect, thereby having an influence on characteristics of the NK cells.

<Example 10> Characteristic Change of NK Cells According to Treatment with the Fusion Polypeptide (NRP-Body)

(86) It was identified about a change in an efficacy of the ADCC, which induced a death of cancer cells according to a distribution of the NK cells changed by means of CXCL16.

(87) Panc-1 cell lines were divided by 2×10.sup.5 into a 96-well plate, and cultured in a CO.sub.2 incubator at 37° C. for two hours. The NK cells were added thereto by 2×10.sup.5 to set a ratio of target cell and effector cell at 1:1, and cultured in the CO.sub.2 incubator at 37° C. for four hours. The cells were collected therefrom, then washed with PBS, then stained with Annexin V (1 μg/ml) and PI (1 μg/ml) for 30 minutes, and then analyzed with the FACS.

(88) The results thereof were shown in FIG. 32.

(89) As identified in FIG. 32, it was identified that the death of cancer cells is increased by means of CD56.sup.bright CD16.sup.+ NK cells, which are increased by CXCL16 of the fusion polypeptide.