CYTOKINE-BASED IMMUNE CELLS AND IMMUNOTHERAPEUTIC USE THEREOF

Abstract

The present invention relates to cytokine-based immune cells and an immunotherapeutic use thereof, and an immune cell according to an aspect is designed in such a form that a cytokine is linked to the surface of the cell via a linker, and thus the cytokine continuously stimulates the immune cell, thereby inducing proliferation and activation, and the cytokine does not affect surrounding cells, whereby side effects can be minimized.

Claims

1. Genetically engineered immune cells comprising: a transmembrane domain; a cytokine; and a peptide linker for linking the cytokine to the transmembrane domain.

2. The immune cells of claim 1, wherein the transmembrane domain is a transmembrane domain of receptor tyrosine kinases (RTKs).

3. The immune cells of claim 2, wherein the transmembrane domain of receptor tyrosine kinases may be a transmembrane domain of any one receptor selected from the group consisting of an epidermal growth factor receptor, an insulin receptor, a platelet-derived growth factor receptor, a vascular endothelial growth factor receptor, a fibroblast growth factor receptor, a cholecystokinin (CCK) receptor, a neurotrophic factor (NGF) receptor, a hepatocyte growth factor (HGF) receptor, an ephrin (Eph) receptor, an angiopoietin receptor, and a related to receptor tyrosine kinase (RTK) receptor.

4. The immune cells of claim 1, wherein the cytokine is any one selected from the group consisting of the bone morphogenetic protein (BMP) family, the chemokine ligand (CCL) family, the CKLF-like MARVEL transmembrane domain-containing member (CMTM) family, the C-X-C motif ligand (CXCL) family, the growth/differentiation factor (GDF) family, growth hormones, the interferon (IFN) family, the interleukin (IL) family, the tumor necrosis factor superfamily (TNFSF), glycophosphatidylinositol (GPI), secreted Ly-6/uPAR-related protein 1 (SLUPR-1), secreted Ly-6/uPAR-related protein 2 (SLUPR-2) and a combination thereof.

5. The immune cells of claim 4, wherein the interleukin is IL2, IL7, IL12, IL15, or IL21.

6. The immune cells of claim 4, wherein the IFN family is IFN-α, IFN-β, IFN-γ, IFN-ε, IFN-κ or IFN-ω.

7. The immune cells of claim 4, wherein the TNFSF is TNF, CD40L (TNFSF5), CD70 (TNFSF7; CD27L), EDA, FASL (TNFSF6), LTA (TNFSF1), LTB (TNFSF3), TNFSF4 (OX40L), TNFSF8 (CD153), TNFSF9 (4-1BBL), TNFSF10 (TRAIL), TNFSF11 (RANKL), TNFSF12 (TWEAK), TNFSF13, TNFSF13B, TNFSF14, TNFSF15, or TNFSF18.

8. The immune cells of claim 1, wherein the peptide linker is a flexible linker, and comprises 1 to 400 amino acid residues.

9. The immune cells of claim 1, wherein the peptide linker is (GGGGS).sub.n (SEQ ID NO: 1), (SGGGG).sub.n (SEQ ID NO: 2), (SRSSG).sub.n(SEQ ID NO: 3), (SGSSC).sub.n (SEQ ID NO: 4), (GKSSGSGSESKS).sub.n (SEQ ID NO: 5), (RPPPPC).sub.n (SEQ ID NO: 6), (SSPPPPC).sub.n (SEQ ID NO: 7), (GSTSGSGKSSEGKG).sub.n (SEQ ID NO: 8), (GSTSGSGKSSEGSGSTKG).sub.n (SEQ ID NO: 9), (GSTSGSGKPGSGEGSTKG).sub.n (SEQ ID NO: 10), or (EGKSSGSGSESKEF).sub.n (SEQ ID NO: 11), and n is an integer from 1 to 20.

10. The immune cells of claim 1, wherein the immune cells are transformed with a vector comprising a polynucleotide encoding a fusion protein comprising: a transmembrane domain; a cytokine; and a peptide linker for linking the cytokine to the transmembrane domain.

11. The immune cells of claim 10, wherein the vector is derived from a virus is selected from the group consisting of a lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, and vaccinia virus.

12. The immune cells of claim 1, wherein the cytokine binds to a cytokine receptor present on the surface of the immune cell to continuously stimulate the cell.

13. The immune cells of claim 1, wherein the immune cells are any one selected from the group consisting of macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils.

14. The immune cells of claim 1, wherein the immune cells are NK-92 cells.

15. (canceled)

16. A method for preventing or treating a cancer, the method comprising administering the the immune cells of claim 1 or a cell population thereof to an individual in need thereof.

17. A method for preventing or treating an infectious disease, the method comprising administering the immune cells of claim 1 or a cell population thereof to an individual in need thereof.

Description

DESCRIPTION OF DRAWINGS

[0071] FIG. 1 is a schematic view of a natural killer cell according to an exemplary embodiment.

[0072] FIG. 2 is a cleavage diagram of a lentiviral vector for the production of natural killer cells according to an exemplary embodiment.

[0073] FIG. 3 is a graph showing the proliferative ability of natural killer cells according to an exemplary embodiment.

[0074] FIG. 4 is a graph showing the viability of natural killer cells according to an exemplary embodiment.

MODES OF THE INVENTION

[0075] Hereinafter, the present invention will be described in more detail through Examples. However, these Examples are provided only for exemplarily describing the present invention, and the scope of the present invention is not limited by these Examples.

Example 1. Manufacture of Natural Killer Cells in which Cytokine is Linked to Linker

[0076] To manufacture natural killer cells in which a cytokine is linked to a linker, a vector including a transmembrane domain, a linker, and a polynucleotide encoding the cytokine was constructed.

[0077] Specifically, PCR primers were designed such that each cDNA could be introduced into a pair of Sfil restriction enzyme recognition sites compatible with each end of a PCR amplified insert in a lentiviral vector pLV2-EF1a-MTA. After an Sfil recognition region was digested, a lentiviral cytokine plasmid for each cytokine was constructed by individually ligating the insert into the Sfil-digested lentiviral vector. The insert was designed so as to include a structure of [cytokine-flexible linker-transmembrane domain], and a specific structure of the plasmid is as illustrated in FIG. 2.

[0078] Next, HEK-293FT cells were co-transfected with the above-manufactured lentiviral plasmid together with pCMVD8.9 and pVSVg viral packaging vectors at a ratio of 1:1:1. After the cells were incubated overnight, a DNA lipid complex was removed, and then a fresh medium was added to the cells. 48 hours later, an upper layer liquid containing the virus was collected, and the collected supernatant was filtered using a 0.22-μm polyether sulfone membrane filter unit (Millipore). The lentiviral particles obtained from the procedure were added to NK-92 cells (KCTC, Korean Collection for Type Cultures) in a growth medium containing 8 μg/mL of polybrene, and could be infected using a centrifugal force at a speed of 1200×g for 90 minutes. After 24 hours under the conditions of 37° C. and 5% CO.sub.2, the cells were incubated for 2 days or more after replacing the medium with a new medium. The medium was purchased from Thermo Fisher Scientific (Waltham, USA) and used.

[0079] The types of cytokines manufactured by the above method are shown in the following Table 1.

TABLE-US-00001 TABLE 1 Entrez NCBI. NCBI. Antibiotic Vector Sequence Gene Gene GeneID GeneSymbol NO Resistance Name Primer List Symbol List List 1 Amp pCR4- M13 (−21), 57152 SLURP1 [Homo 57152 SLURP1 TOPO M13 reverse, sapiens] T7, T3 2 Kan pCR- M13(−21), 651 BMP3 [Homo 651 BMP3 BluntII- M13 reverse, sapiens] TOPO T7, sp6 3 Kan pCR- M13(−21), 3586 IL10 [Homo 3586 IL10 BluntII- M13 reverse, sapiens] TOPO T7, sp6 4 Cam pDNR-LIB −21M13 3627 CXCL10 [Homo 3627 CXCL10 sapiens] 5 Cam pOTB7 −21M13, M13 2821 GPI [Homo 2821 GPI reverse, sp6, sapiens] T7 6 Cam pDNR-LIB −21M13 6356 CCL11 [Homo 6356 CCL11 sapiens] 7 Amp pCR4- M13 (−21), 9966 TNFSF15 9966 TNFSF15 TOPO M13 reverse, [Homo T7, T3 sapiens] 8 Amp pCR4- M13 (−21), 6346 CCL1 [Homo 6346 CCL1 TOPO M13 reverse, sapiens] T7, T3 9 Amp pPCR- M13(−21), 56477 CCL28 [Homo 56477 CCL28 Script Amp M13 reverse, sapiens] SK(+) T7, T3 10 Amp pDNR- M13(−21), T7 3558 IL2 [Homo 3558 IL2 Dual sapiens] 11 Amp pCMV- sp6, T7, −21M13, M13 6358 CCL14 [Homo 6358 CCL14 SPORT6 reverse sapiens] 12 Amp pINCY −21M13, M13 3589 IL11 [Homo 3589 IL11 reverse, T7, sapiens] sp6 13 Amp pCMV- sp6, T7, −21M13, M13 6351 CCL4 [Homo 6351 CCL4 SPORT6 reverse sapiens] 14 Amp pCMV- sp6, T7, −21M13, M13 6347 CCL2 [Homo 6347 CCL2 SPORT6 reverse sapiens] 15 Amp pCMV- sp6, T7, −21M13, M13 6367 CCL22 [Homo 6367 CCL22 SPORT6 reverse sapiens] 16 Amp pCMV- sp6, T7, −21M13, M13 6352 CCL5 [Homo 6352 CCL5 SPORT6 reverse sapiens] 17 Amp pDrive T7, sp6 6368 CCL23 [Homo 6368 CCL23 sapiens]

[0080] As a result, as illustrated in FIG. 1, natural killer cells were manufactured in which a cytokine was linked to a plasma membrane of the natural killer cells via a linker by a transmembrane domain. The proliferation and activation of cells may be induced by binding the cytokine to a cytokine receptor present on the surface of natural killer cells to continuously stimulate the natural killer cells.

Experimental Example 1. Analysis of Proliferative Ability and Viability of Natural Killer Cells Linked with Cytokine by Linker

[0081] In order to analyze the proliferative ability and viability of natural killer cells designed to be linked with the cytokine IL-2 of Example 1 by a linker, the proportion of living cells after being cultured for a certain period of time without supplying the cytokine was confirmed.

[0082] Specifically, natural killer cells in which interleukin-2 was linked to the plasma membrane via a linker through lentiviral infection were isolated using a flow cytometry device, and these cells were cultured in a medium obtained by adding 0.2 mM myo-inositol, 0.1 mM 2-mercarptoethanol, 0.02 mM folic acid, 12.5% horse serum, and 12.5% FBS to an MEM alpha medium and including 100 U/mL interleukin-2 for a week. Thereafter, NK-92 cells to which the cytokine was not attached and NK-92 cells to which interleukin-2 was attached were used as a control and an experimental group, respectively, and cultured for a certain period of time after changing the medium to a medium in which only interleukin-2 was excluded from the same medium composition, and then the number of living cells was confirmed by staining with Trypan blue 1:1. The medium was purchased from Thermo Fisher Scientific (Waltham, USA) and materials to be added were purchased from Sigma Aldrich (St. Louis, USA) and BD (Franklin Lakes, USA), and used.

[0083] The proliferative ability and viability of natural killer cells were analyzed by calculating cells stained blue with Trypan blue as dead cells and calculating unstained cells as living cells, and the results are illustrated in FIGS. 3 and 4, respectively.

[0084] FIG. 3 is a graph showing the proliferative ability of natural killer cells according to an exemplary embodiment.

[0085] FIG. 4 is a graph showing the viability of natural killer cells according to an exemplary embodiment.

[0086] As illustrated in FIGS. 3 and 4, it was confirmed that the natural killer cells according to a specific example increased their proliferation by 2-fold or more in an environment where external cytokines were absent compared to the natural killer cells of the control which was not genetically manipulated, and it was also confirmed that the cell viability also increased by about 1.6-fold or more.

[0087] These results mean that the cytokine linked to the surface of the natural killer cells according to a specific example is proliferated and activated without a separate supply of cytokine by stimulating itself without affecting other cells.