TRANSFORMED IMMUNE CELLS INDUCING CHEMOTAXIS TOWARDS HETEROGENEOUS IMMUNE CELLS

Abstract

Immune cells expressing IL-7, CCL19, or a combination thereof, and a composition containing the immune cells, which is useful for preventing or treating cancer or infectious diseases are disclosed. A method for preventing or treating cancer or infectious diseases, which include administering a therapeutically effective amount of single type of immune cells, specifically natural killer cells, is also disclosed. Complementary immune responses between the patient's endogenous T cells and injected natural killer cells exhibit multifaceted and synergistic therapeutic effects. Co-administration of T cells and the immune cells other than T cells, specifically natural killer cells, also significantly improves therapeutic effects by allowing these heterogeneous cell populations to act in a lesion-concentrated manner.

Claims

1. An immune cell expressing a nucleic acid molecule encoding IL-7 (interleukin-7) or a functional portion thereof, a nucleic acid molecule encoding CCL19 (C—C Motif Chemokine Ligand 19) or a functional portion thereof, or a combination thereof.

2. The immune cell according to claim 1, wherein the immune cell is an immune cell other than a T cell.

3. The immune cell according to claim 2, wherein the immune cell other than T cell is a natural killer cell.

4. A method for preventing or treating cancer or infectious diseases comprising administering the immune cell according to claim 1 to a subject in need thereof.

5. A method for inducing proliferation or homing of a heterogeneous immune cell comprising introducing a nucleic acid molecule encoding IL-7 (interleukin-7) or a functional portion thereof, a nucleic acid molecule encoding CCL19 (C—C Motif Chemokine Ligand 19) or a functional portion thereof, or a combination thereof, into an immune cell.

6. The method according to claim 5, wherein the heterogeneous immune cell is a T cell or a dendritic cell.

7. The method according to claim 5, wherein the nucleic acid molecule encoding IL-7 (interleukin-7) or a functional portion thereof, the nucleic acid molecule encoding CCL19 (C—C Motif Chemokine Ligand 19), and/or a functional portion thereof, or a combination thereof are inserted together into single gene delivery system or each separately into two different gene delivery systems.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 represents a schematic diagram showing the structure of the pEF1-IRES empty vector used as a negative control vector.

[0048] FIG. 2 represents a schematic diagram showing the structure of the pEF1-IRES vector into which IL-7 and CCL19 genes are inserted.

[0049] FIG. 3 represents a schematic diagram showing the structure of the pEF1-IRES vector into which CCL19 and IL-7 genes are inserted.

[0050] Hereinafter, the present invention will be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

Example 1: Construction of IL-7 and CCL19 Expression Vectors

[0051] Expression vectors were constructed to prepare human NK cells expressing IL-7 and CCL19. A pEF1α-IRES bicistronic mammalian expression vector (Takara, CAT #631970) under the control of human elongation factor 1 alpha (EF1α) promoter was used as an expression vector.

[0052] The ORF sequences of IL-7 and/or CCL19 genes to be inserted into the vector were codon-optimized (SEQ ID NO:3 and SEQ ID NO:4, respectively) and synthesized. IL-7 or CCL19 was cloned into the multicloning site A (MCS A) of the pEF1α-IRES vector, and CCL19 or IL-7 was cloned into MCS B. Here, an internal ribosome entry site (IRES) was positioned between the two MCSs in the pEF1α-IRES so that the two genes could be co-expressed. The structure of the empty vector, the negative control, is shown in FIG. 1, the structure of the IL-7/CCL19 expression vector is shown in FIG. 2, and the structure of the CCL19/IL-7 expression vector is shown in FIG. 3, respectively.

Example 2: Confirmation of the Expression of IL-7 and CCL19

[0053] The NK92 cell line was purchased from ATCC (CAT #CRL-2407), and cultured in α-MEM (Alpha Minimum Essential Medium) containing 12.5% horse serum (Sigma, CAT #H1270), 12.5% fetal bovine serum (FBS, Gibco, CAT #10099141), and interleukin-2 (IL-2, Peprotech, CAT #200-02). In order to prepare a NK92 cell line expressing IL-7 and CCL19 (hereinafter, “transformed NK92 cell line”), electroporation was performed at a density of 1×10.sup.6 NK92 cells/0.1 mL.

[0054] The electroporation was performed by Neon™ Transfection System (ThermoFisher, CAT #MPK5000) was used under the following conditions: 1800-1850 v voltage, 10 ms pulse length, 1 time, and 10-20 μg DNA (negative control vector, IL-7/CCL19 expression vector, or CCL19/IL-7 expression vector prepared in Example 1). After culturing for 1 day in 2 mL of RPM11640 medium (Gibco #11875168) containing 10% FBS, the expression level of IL-7 or CCL19 was measured or a transwell assay were performed.

[0055] In order to measure the expression level of IL-7 and CCL19 in the transformed NK92 cell line, the culture medium was collected in a 6-well plate. After centrifugation at 800×g for 5 minutes, the supernatant was separated. 0.1 mL per well of the supernatant was appropriately diluted and used for IL-7 or CCL19 enzyme linked immunosorbent assay (ELISA; IL-7, Komabiotech, CAT #k0331215; CCL19, Abcam, CAT #ab100601) to measure the expression level of IL-7 or CCL19.

[0056] The ELISA for IL-7 and CCL19 was performed according to the standard testing methods of each manufacturer. The final values measured at a wavelength of 450 nm are shown in Table 1.

TABLE-US-00001 TABLE 1 Amount of IL-7 and CCL19 secretion in transformed cells Cell Gene IL-7 (pg/mL) CCL19 (pg/mL) NK92 Empty vector N.D 4.2 IL-7/CCL19 1,656 242 CCL19/IL-7 157 368 *N.D: Not Detected

[0057] The results of Table 1 indicate that the IL-7/CCL19 NK92 cell line and the CCL19/IL-7 NK92 cell line secreted IL-7 and CCL19, while IL-7 and CCL19 were not detected or were detected at insignificant levels in the negative control cells injected with the empty vector.

Example 3: Analysis of T Cell Chemotaxis and Proliferation Induced by IL-7 and CCL19 Expressing NK Cells

[0058] To confirm the effect of the secretion of IL-7 and CCL19 by NK cells on T cell chemotaxis, a 12-well transwell (Corning, CAT #CLS3421) was used. 5 μm polycarbonate was used as the chamber filter.

Comparison of IL-7/CCL19 NK92 Cells and Negative Controls Using HuT78 T Cell Line

[0059] 1×10.sup.7 cells of HuT78 (ATT, CAT #TIB-161), the T cells, were cultured for 24 hours in IMDM (Gibco, CAT #12440053) medium containing 1% PS without FBS. Thereafter, 100 μl of HuT78 cells at a density of 5×10.sup.6 cells/mL were dispensed into the upper chamber using the same medium as above, and the culture solution cultured for 2-3 days in the IL-7/CCL19 NK92 cell line prepared in Example 2 was dispensed in the lower chamber.

[0060] After culturing for 1 day in an incubator at 37° C. and 5% CO.sub.2, the upper chamber was removed and further cultured for 3 days. To measure the number of cells in the lower chamber, 400 μl of medium per well was centrifuged at 800×g for 5 minutes to remove the supernatant and obtain a concentrated sample. 10 μl of Trypan blue was mixed with 10 μl of the concentrated sample at a 1:1 ratio and measured with Countess™ II (Invitrogen, CAT #AMQAX1000). The results were converted into the total number of viable cells in 400 μl as shown in Table 2.

TABLE-US-00002 TABLE 2 Chemotaxis of the HuT78 T cell line by transformed NK cells Number of cells in the lower chamber Experimental Group (mean ± standard deviation) Negative control cells  3,836 ± 276 IL-7/CCL19 NK92 cells 11,989 ± 887

Comparison of IL-7/CCL19 NK92 Cells, CCL19/IL-7 NK92 Cells and Negative Control Using PBMC-Derived T Cells

[0061] To evaluate the chemotaxis of peripheral blood mononuclear cells (PBMC)-derived T cells, T cells were isolated from PBMC using the EasySep™ Human T Cell Isolation Kit (STEMCELL #17951). After diluting the T cells at a concentration of 2×10.sup.6 cells/mL with RPM11640 medium containing 2% FBS, 100 μl was dispensed into the upper chamber. In the lower chamber, the culture solution cultured for 1 day in IL-7/CCL19 and CCL19/IL-7 transformed NK92 cell lines prepared in Example 2 was dispensed.

[0062] After culturing for 1 day in an incubator at 37° C. and 5% CO.sub.2, the upper chamber was removed and further cultured for 3 days. To measure the number of cells in the lower chamber, 500 μl of medium per well was centrifuged at 800×g for 5 minutes to remove the supernatant and obtain a concentrated sample. 10 μl of Trypan blue was mixed with 10 μl of the concentrated sample at a 1:1 ratio and measured with Countess™ II. The results were converted into the total cell numbers as shown in Table 3.

TABLE-US-00003 TABLE 3 PBMC-derived T cell chemotaxis by transformed NK92 cell line Number of T cells migrating to lower chamber (mean ± standard deviation, T cell type Experimental group (n = 2) 10.sup.4 cells) PBMC-derived Negative control NK92 cells 1.89 ± 0.75 T cells IL-7/CCL19 NK92 cells 9.35 ± 0.53 CCL19/IL-7 NK92 cells 6.46 ± 0.16

[0063] As shown in Tables 2 and 3, the number of T cells that migrated to the lower chamber was significantly higher in the IL-7/CCL19 and CCL19/IL-7 NK92 cell line group compared to the negative control empty vector-introduced NK92 cell line. This indicates that the NK92 cell transformed with IL-7 and CCL19 induced chemotaxis of T cells and proliferation of migrated T cells.

Example 4: Measurement of Cancer Cell Killing and Secretion of the Related Factors by Transformed NK92 Cell Lines and T Cells Migrated Thereby

[0064] After HepG2 liver cancer cell line (ATCC #HB-8065) was diluted in RPM11640 medium containing 10% FBS to a concentration of 2×10.sup.5 cells/mL, 0.1 mL was dispensed into each well of a 96-well plate and cultured for 1 day. T cell migration was induced for 1 day by transferring 0.5 mL RPM11640 medium containing the transformed NK92 cell line cultured for 1 day by electroporation in the same manner as in Example 2 to the lower chamber of the transwell in the same manner as in Example 3. 2×10.sup.5 cells/0.1 mL of T cells isolated from PBMC were transferred to the upper chamber. Here, 1×10.sup.5 CD3/CD28 Dynabeads and 100U IL-2 were added to the lower chamber to activate the migrating T cells. After culturing for 1 day, the upper chamber was removed and transferred 0.1 mL per well to the 96-well plate in which the HepG2 liver cancer cell line was being cultured. After culturing for 3 more days and washing three times using PBS (phosphate buffer saline), the medium was replaced with 0.1 mL of 10% FBS-RPM11640 medium containing 1% of CCK-8 and further cultured for 2 hours. In order to evaluate the cell viability of the HepG2 liver cancer cell line, absorbance was measured at 450 nm and the degree of cell viability was calculated using [Equation 1], as shown in Table 4.

[00001]$\begin{array}{cc}\mathrm{HepG}2\mathrm{cell}\mathrm{line}\mathrm{viability}=100⨯\frac{\left(\mathrm{Test}\mathrm{group}\mathrm{absorbance}\mathrm{value}-\mathrm{RPMI}1640\mathrm{medium}\mathrm{absorbance}\mathrm{value}\right)}{\left(\mathrm{Normal}\mathrm{group}\mathrm{absorbance}\mathrm{value}-\mathrm{RPMI}1640\mathrm{medium}\mathrm{absorbance}\mathrm{value}\right)}& \left[\mathrm{Equation}1\right]\end{array}$

[0065] In addition, the secretion of granzyme-B (Grz-B), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) from activated T cells and activated NK cells was measured using the medium collected from the cell viability measurement above, via the enzyme-linked immunosorbent assay (ELISA; GrzB, abcam #ab235635; IFN-γ, komabiotech #K0331121; TNF-α, komabiotech #K0331131), of which the results are shown in Table 5. Each value in Table 4 and 5 was expressed as mean±standard deviation.

TABLE-US-00004 TABLE 4 Cytotoxicity for HepG2 liver cancer cell line Cancer cell line Experimental group (n = 3) Cell viability HepG2 Medium (Normal group) 100.0 ± 7.8  Negative control group (Empty vector NK92) 83.8 ± 2.1 IL-7/CCL19 NK92 cells 33.2 ± 3.3 CCL19/IL-7 NK92 cells 52.2 ± 3.5

TABLE-US-00005 TABLE 5 Secretion Grz-B, IFN-γ and TNF-α Cancer Experimental group Grz-B IFN-γ TNF-α cell line (n = 2) (pg/mL) (pg/mL) (pg/mL) HepG2 Medium (Normal group) .sup. 592 ± 179 N.D N.D Negative control 1,259 ± 81   8 ± 1 N.D (Empty vector NK92) IL-7/CCL19 NK92 cells 2,573 ± 450 320 ± 28 195 ± 37 CCL19/IL-7 NK92 cells 1,460 ± 466 71 ± 3  33 ± 10 *N.D: Not Detected

[0066] As shown in Table 4, significantly higher cell killing activity toward HepG2 was observed in the experimental group of IL-7/CCL19 and CCL19/IL-7 NK92 cell line compared to the empty vector-introduced NK92 cell as a negative control. As shown in Table 5, the factors related to the cytotoxicity for HepG2 cell line also showed higher secretion in the groups of IL-7/CCL19 and CCL19/IL-7 NK92 cell line compared to the negative control group. Accordingly, it was confirmed that both the IL-7/CCL19 NK cell line and the CCL19/IL-7 NK cell line may be applied as an effective anti-cancer treatment.

[0067] Having described specific embodiment of the present invention in detail above, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.