WO2022169291A1 - Modified killer cells, preparation method therefor, and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to modified killer cells, a preparation method therefor, and a pharmaceutical use thereof. More specifically, in the present invention, killer cells genetically modified to express a chemokine receptor, a cytokine, or a co-stimulatory molecule are used to promote immune response regulation and migration to tumor sites in hosts to alter the initial tumor microenvironments, whereby an effective anticancer therapeutic effect can be provided.

Description

Modified killer cells, methods for their preparation and pharmaceutical uses thereof

The present invention relates to a killer cell genetically modified to express a chemokine receptor, a cytokine, or a costimulatory molecule, a method for preparing the same, and a pharmaceutical use thereof.

Adoptive-cell therapy using killer cells is one of the promising therapeutic strategies for malignancy, but its implementation in clinical practice remains challenging. For successful tumor removal of adoptive-cell therapy, it is important that the killer cells migrate to the tumor site in vivo after adoptive transfer, and that the adoptive-transferred killer cells proliferate and survive. Recent studies have shown that immunotherapy is important to enhance the host immune response to tumorigenesis. This strategy can be overcome through co-administration of immunomodulatory cytokines such as IL-2 and IL-12 and anti-tumor cytokines such as IL-21, IL-23 and IL-27.

The tumor microenvironment is generated by the tumor, and immunosuppressive cells such as regulatory T cells (Treg) and myeloid suppressor cell (MDSC) are densely packed, so various immune cells are mobilized to the tumor site, but their antitumor function mainly responds to tumor-derived signals. is down-regulated.

IL-12 plays a central role in regulating both innate and adaptive immune responses, and by itself can induce potent anticancer effects.

Some chemokine ligands are expressed in tumors and are known to play important roles in tumor progression, such as promotion of tumor formation, maintenance of tumor cell growth, and induction of angiogenesis. Chemokine receptors are involved in the circulation, homing, maintenance and activation of immune cells, resulting in extensive infiltration of lymphocytes, thereby changing the tumor microenvironment.

Therefore, the present inventors pursued cytokines, which are T cell-stimulating factors that can stimulate the growth and function of killer cells, co-stimulatory molecules involved in T-cell immune response activation, proliferation and survival, and chemokine ligands expressed in tumors. The present invention was completed by developing a killer cell genetically modified with a chemokine receptor and using it to investigate the anticancer therapeutic effect accompanied by movement to the tumor site.

It is an object of the present invention to provide a killer cell genetically modified to express a chemokine receptor, cytokine or costimulatory molecule, a method for preparing the same, and a pharmaceutical use thereof.

In order to achieve the above object, the present invention provides a modified killer cell expressing at least one of a chemokine receptor (C-X-C chemokine receptor) and a cytokine.

The present invention also provides a method for producing a modified killer cell in vitro comprising introducing at least one of a chemokine receptor and a cytokine into the killer cell.

The present invention also provides an anticancer composition comprising the modified killer cells.

The present invention also provides the use of said modified killer cell for use in the manufacture of a medicament for the treatment of cancer.

The present invention also provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the modified killer cells.

The killer cells genetically modified with the chemokine receptor, cytokine or costimulatory molecule of the present invention track the chemokine ligand expressed in the tumor and move more efficiently to the tumor site. Adoptive therapy of genetically modified killer cells of the present invention enables effective tumor treatment by changing the initial tumor microenvironment by regulating the host's immune response and promoting migration of the killer cells to the tumor site.

1 shows the results of flow cytometry analysis of in vitro activated Pmel-1 T cells transduced with recombinant retroviruses encoding GFP, mouse CD70, and mouse CD70-OX40L (a) and therapeutic treatment of transgenic Pmel-1 CD8 T cells. Antitumor effects (b, c and d) are shown.

2 is an ELISA analysis result (a) and genes measuring the cytokine secretion of in vitro activated Pmel-1 T cells transduced with a recombinant retrovirus encoding mouse IL-2, IL-12 and IL-21 genes. The therapeutic antitumor effect (b, c, d and e) of metastatic Pmel-1 CD8 T cells is shown.

3 is a flow cytometric analysis result of ex vivo activated Pmel-1 T cells transduced with recombinant retroviruses encoding GFP and mouse CD70, IL-2-CD70, IL-12-CD70, IL-21-CD70 (a). ), ELISA analysis results (b, c, d, and e) measuring cytokine secretion in the culture supernatant of each group, and the therapeutic antitumor effect (f) of transgenic Pmel-1 CD8 T cells.

Figure 4 shows the results of flow cytometry analysis of in vitro activated Pmel-1 T cells transduced with recombinant retrovirus encoding GFP and mouse chemokine receptor CXCR3 (a) and the therapeutic antitumor effect of transgenic Pmel-1 CD8 T cells. (b, c, d and e) are shown.

Figure 5 shows the flow cytometry results of ex vivo activated Pmel-1 T cells transduced with recombinant retrovirus encoding GFP, mouse cytokines IL-2, IL-12, IL-2 and chemokine receptor CXCR3 (a); ELISA analysis results (b, c, d, and e) measuring the cytokine secretion of the corresponding culture solution of each group, and the therapeutic antitumor effect (f) of the transgenic Pmel-1 CD8 T cells are shown.

Figure 6 shows the results of flow cytometry analysis of in vitro activated Pmel-1 T cells transduced with recombinant retroviruses encoding GFP, mouse cytokine and chemokine receptor (a), cytokine secretion of the corresponding culture solution of each group. Results of ELISA analysis (b, c, d, and e), therapeutic antitumor effect of transgenic Pmel-1 CD8 T cells (f), and EliSpot results of measuring IFNγ secretion of transgenic Pmel-1 CD8 T cells (g) is shown.

Figure 7 shows the results of flow cytometry analysis of in vitro activated Pmel-1 T cells transduced with recombinant retrovirus encoding GFP, mouse cytokine and chemokine receptor (a), cytokine secretion of the corresponding culture solution of each group. The results of the measured ELISA analysis (b) and the therapeutic antitumor effect (c, d, e and f) of the transgenic Pmel-1 CD8 T cells are shown.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to modified killer cells expressing one or more of a C-X-C chemokine receptor and a cytokine.

The modified killer cell of the present invention is characterized in that it is a killer cell genetically modified to express a chemokine receptor, cytokine or co-stimulatory molecule. More specifically, genetically modified killer cells transduced with chemokine receptor and cytokine-encoding nucleic acids, either alone or in combination thereof, are transduced with costimulatory molecules or non-adoptive animal models. It is characterized in that it exhibits an excellent anti-tumor effect by inhibiting tumor formation compared to In particular, cancer cell-specific chemokine receptors track chemokine ligands expressed in tumors, enabling more efficient migration of genetically modified killer cells to the tumor site. In addition, the modified killer cells of the present invention change the initial tumor microenvironment by regulating the host's immune response and promoting migration to the tumor site, thereby enabling effective tumor treatment.

The chemokine receptor may be CXCR1, CXCR2, CXCR3, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR2, CCR4, CCR5, CCR7, CCR10, or the like. More specifically, it may be CXCR2, CXCR3 or CXCR5.

The cytokine may be IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, and the like.

The modified killer cells may further express costimulatory molecules in addition to chemokine receptors or cytokines.

As the co-stimulatory molecule, CD70, CD134L (OX40L), CD80, CD86, CD54, CD154 (CD40L) or CD137L (4-1BBL) may be used alone or in two or more types.

As used herein, the term "costimulatory molecule" refers to a substance that participates in the interaction between a receptor-ligand pair expressed on the surface of an antigen-presenting cell and a T cell. Two or more signals are required for T cells, one signal conferring specificity and produced by the interaction of the MHC/peptide complex with the TCR/CD3 complex, the second signal being non-antigen specific and a “costimulatory” signal. say This signal is known to be an activity provided by bone marrow-derived helper cells such as macrophages and dendritic cells. Co-stimulatory molecules mediate the necessary co-stimulatory signals under normal physiological conditions to perform complete activation of γδ T cells. In the present invention, the costimulatory molecule may be human or mouse CD70, CD134L (OX40L), CD80, CD86, CD54, CD154 (CD40L) or CD137L (4-1BBL).

The killer cells are tumor-infiltrated lymphocytes, ex vivo antigen-specific T cells, natural killer cells, natural killer T cells. , gamma delta T cells, or chimeric antigen receptor (CAR) or T cell receptor (TCR)-equipped T cells may be used.

The chimeric antigen receptor or T cell receptor-equipped T cell is a CD4 + T cell; CD8+ T cells; or recombinant tumor-infiltrated lymphocytes, recombinant natural killer cells, recombinant natural killer T cells, or recombinant gamma delta T cells into which a chimeric antigen receptor or T cell receptor has been introduced ( gammadelta T cells) and the like.

The modified killer cell can be prepared by introducing a nucleic acid encoding a chemokine receptor and/or cytokine into the killer cell using a known transformation technique. According to one embodiment, it can be prepared by introducing a nucleic acid encoding CXCR3 and/or IL-2, IL-12 or IL-21 into Pmel-1 T cells.

The nucleic acid is used in the broadest sense, and includes single-stranded (ss) DNA, double-stranded (ds) DNA, cDNA, (-)-RNA, (+)-RNA, dsRNA, and the like. Preferably, it is double-stranded DNA.

The present invention also relates to a method for producing a modified killer cell in vitro comprising the step of introducing at least one of a chemokine receptor and a cytokine into the killer cell.

The method may further comprise transducing the killer cell with a co-stimulatory molecule.

The introduction may be to introduce a vector into which a nucleic acid encoding a chemokine receptor, cytokine, costimulatory molecule, etc. is inserted into the killer cell using a known transformation technique.

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector capable of ligating additional DNA segments into the viral genome. Some vectors are capable of self-replication within a host cell when introduced into the host cell (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell and replicated along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are usually in the form of plasmids, since plasmids are the most commonly used type of vector, so "plasmid" and "vector" can be used interchangeably. However, the present invention provides other methods, such as viral vectors (e.g., adenoviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors) that serve equivalent functions. forms of expression vectors are also included. Preferably, retroviral vectors can be used.

The retrovirus is an RNA virus having a different life cycle from the lytic virus. Retroviruses in this respect are infectious entities that replicate through DNA intermediates. When a retrovirus infects a cell, the genome is converted to DNA formation by reverse transcriptase. The DNA copy serves as a template for the production of a new RNA genome and a virally encoded protein essential for the assembly of infectious viral particles. There are many retroviruses, such as murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma Virus (FuSV), Moloney Rodent Leukemia Virus (Mo-MLV), FBR Murine Osteosarcoma Virus (FBR MSV), Moloney Rodent Sarcoma Virus (Mo-MSV), Abelsol Murine Leukemia Virus (A-MLV), avian bone marrow Cytovirus-29 (MC29) and all other retroviral families, including avian erythrocytosis virus (AEV) and lentiviruses. A detailed list of retroviruses is described in Coffin et al (“Retroviruses” 1997 Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).

Transformation includes any method of introducing a nucleic acid into an organism, cell, tissue or organ, and as known in the art, can be performed by selecting an appropriate standard technique according to the type of killer cell. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation using silicon carbide fibers, agrobacterium mediated transformation, PEG, dextran sulfate, lipoproteins. Pectamine, and the like, but are not limited thereto.

According to one embodiment of the present invention, chemokine receptors such as CXCR2, CXCR3, CXCR5, cytokines such as IL-2, IL-12, IL-21, CD70, OX40L, CD80, CD83, CD54, CD32, 4-1BBL Each cDNA was prepared for the gene of a costimulatory molecule, such as, and inserted into each retroviral vector, and the retroviral vector was transduced into Pmel-1 T cells.

The present invention also relates to an anticancer composition comprising the modified killer cells.

The present invention also provides the use of said modified killer cell for use in the manufacture of a medicament for the treatment of cancer.

In general, the treatment of melanoma has been advanced by a wide range of immunotherapies, including immune checkpoint inhibitors and adoptive cell therapy, nevertheless, the therapeutic effect has been limited to a small fraction of patients. In addition, adoptive cell therapy, in which tumor-infiltrating lymphocytes obtained from tumor sites are expanded in vitro, by targeting a wide range of tumor antigens, including patient-specific neoantigens, has achieved great success in the treatment of melanoma, but in vitro expanded tumor-infiltrating lymphocytes Only a small fraction migrates to the tumor site after administration.

On the other hand, the modified killer cells of the present invention move more efficiently to the tumor site by tracking the chemokine ligand expressed in the tumor due to the chemokine receptor. Adoptive therapy of genetically modified killer cells of the present invention enables effective tumor treatment by changing the initial tumor microenvironment by regulating the host's immune response and promoting migration of the killer cells to the tumor site. According to one embodiment of the present invention, the antitumor effect of Pmel-1 T cells transduced with CXCR3 and IL-12 genes showed a therapeutic advantage compared to mice without costimulatory molecule (CD70) or adoptive transfer. Among them, the killer cells (IL-12-CXCR3) to which cytokines and chemokine receptors were simultaneously transferred had substantially higher antitumor effects in adoptively transferred mice.

Therefore, the anticancer composition of the present invention may be effective in treating melanoma.

In addition, the anticancer composition of the present invention, in addition to melanoma, lung cancer, stomach cancer, colon cancer, breast cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, perianal cancer, fallopian tube cancer Carcinoma, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph adenocarcinoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney cancer, ureter cancer, renal pelvic cancer, blood cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma or pituitary adenoma have.

The composition of the present invention may further include a pharmaceutically acceptable excipient or diluent in addition to the carrier.

In addition, the "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art to permit rapid, sustained or delayed release of the active ingredient upon administration to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.

The composition of the present invention may be administered by various routes, for example, oral, parenteral, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intrathecal administration, It can also be administered using implanted devices for sustained release or continuous or repeated release. The number of administration may be administered once a day or divided into several times within a desired range, and the administration period is not particularly limited.

The dosage of the composition of the present invention to a patient depends on many factors including the patient's height, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently. . Typically, the modified killer cells may be administered before and after 10 ⁹ to 10 ¹⁰ cells per m ² of body surface area at one time administration. Therefore, it is appropriate to administer about 2×10 ¹⁰ cells based on an average adult (about 60 kg), but the dosage may vary depending on the patient's various conditions and the type and amount of co-administered drugs as described above. have. Accordingly, the pharmaceutically active modified killer cells of the present invention may be administered in an amount of 10 ⁶ to 10 ¹⁰ cells/kg (body weight), and administrations below or above the exemplary range are also particularly administered in consideration of the above factors. If the dosing regimen is continuous infusion, it should be in the range of 10 ³ to 10 ⁹ cell units per kg body weight per minute.

The present invention also provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the modified killer cells.

As used herein, "therapeutically effective amount" means an amount that significantly inhibits the death of cancer cells or at least the growth of cancer tissues.

The subject may include humans, dogs, chickens, pigs, cattle, sheep, guinea pigs, monkeys, and the like.

Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

<Example 1> Preparation and Experimental Method of Genetically Modified Killer Cells

(mouse, cell line)

Six-week-old female C57BL/6 (B6) mice were purchased from Orient Bio (Seongnam, Kyunggi) and bred in the Catholic University of Korea Animal Laboratory under aseptic conditions. All procedures for animal research include guidelines and policies for rodent experiments provided by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (Approval number: CUMS-2019-0169-01) College of Medicine, and the management and management of laboratory animals. It was carried out according to the guide for use, the Laboratory Animal Welfare Act.

B16F10SK (B16) melanoma cells were purchased from the American Type Culture Collection (Manassas, VA) and Plat E retroviral packaging cells were purchased from Cell Biolabs (San Diego, CA), and all cell lines were cultured according to the manufacturer's recommendations.

(Production of Recombinant Retrovirus)

Mouse CD70 (NCBI Nucleotide ID: NM_011617.2 / Cat no: MG51129-G), CXCR3 (NCBI Nucleotide ID: NM_009910.3 / Cat no: MG50842-G), IL-2 (NCBI Nucleotide ID: NM_008366.2 / Cat no: MG51061-G), IL-12B(NCBI Nucleotide ID: NM_008352.2 / Cat no: MG51004-G), IL-12A(NCBI Nucleotide ID: NM_001159424.1 / Cat no: MG51308-G), IL-21 The gene for (NCBI Nucleotide ID: NM_021782.2 / Cat no: MG50137-M) was purchased from SinoBiological in the form inserted into the pMD18-T Simple Vector. The primer sets for PCR amplification of each gene are as follows:

1) CD70:

Forward primer: 5'-gctcacttacag gcggccgc gccacc atgccggaggaaggtcgccc-3' (SEQ ID NO: 1);

Reverse primer: 5'-ggtaagatgctc gaattc tcaagggcatatccactgaa-3' (SEQ ID NO: 2)

2) IL-2:

Forward primer: 5'-gctcacttacag gcggccgc gccacc atgtacagcatgcagctcgcat-3' (SEQ ID NO: 3);

Reverse primer: 5'-ggtaagatgctc gaattc ttattgagggcttgttgaga-3' (SEQ ID NO: 4)

3) IL12p35 T2A IL12p40:

1 ^st PCR, Forward primer: 5'-gctcacttacag gcggccgc gccacc atgtgtcaatcacgctacct-3' (SEQ ID NO: 5);

Overlapping Reverse primer: 5'-cacgtcaccgcatgttagaagacttcctctgccctcagcggccgcggcgga-3' (SEQ ID NO: 6);

Overlapping Forward primer: 5'-ctaacatgcggtgacgtggaggagaatcccggccct tccggaatgtgtcct-3' (SEQ ID NO: 7);

Reverse primer: 5'-ggtaagatgctc gaattc ctaggatcggaccctgcagg-3' (SEQ ID NO: 8);

2 ^nd PCR, Forward primer: 5'-gctcacttacag gcggccgc gccacc atgtgtcctcagaag-3' (SEQ ID NO: 9);

Reverse primer: 5'-ggtaagatgctc gaattc ctaggatcggaccctgcagg-3' (SEQ ID NO: 10)

4) IL-21:

Forward primer: 5'-gctcacttacag gcggccgc gccacc atggagaggacccttgtctg-3' (SEQ ID NO: 11);

Reverse primer: 5'-ggtaagatgctc gaattc ctaggagagatgctgatgaa-3' (SEQ ID NO: 12)

PCR was performed using TOYOBO cat no. KOD FX 1110 kit was used, 2x PCR buffer for KOD FX 12.5 μl, 2mM dNTPs 5 μl, 10pmol/μl Forward Primer 1.0 μl, 10pmol/μl Reverse Primer 1.0 μl, Template DNA (Plasmid DNA) 1 μl (50ng), PCR 25 μl of PCR mixture of 4 μl of grade water and 0.5 μl of KOD FX taq polymerase (1.0U/μl) was pre-denatured (94℃, 2min) 1 cycle, denatured (98℃, 10 sec)-annealed (58℃, 30 sec)-extension (68° C., 30 sec) was amplified under the conditions of 35 cycles and a final extension step (68° C., 5 min).

The PCR product was cloned into the retroviral vector pMP71 with Not I and EcoR I sites and sequenced to identify Taq polymerase errors. For generation of recombinant retroviruses, 5×10 ⁶ Plat-E cells were seeded in 100-mm culture plates coated with 5 μg/mL of poly-L-Lysine (Sigma, St. Louis, MO). After 20 h, 12 μg of cloned pMP71 plasmid and retroviral packaging plasmid (6.3 μg, pCL-Eco) were simultaneously transfected into Plat-E cells using lipofectamin 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. did it Two days later, the recombinant retrovirus was harvested, titrated with 293T cells, and used for transduction experiments.

(Transduction of Recombinant Retrovirus into Activated Killer Cells In Vitro)

killing Cells were obtained from the spleen of Pmel-1 mice. For in vitro pre-activation, anti-CD3ε (BioXcell, 145-2C11) 1 μg/mL, anti-CD28 (BioXcell, 37.51) 2 μg/mL in the presence of 100 IU/mL human interleukin 2 (hIL-2) CD8 T cells were co-cultured at 2.5×10 ⁵ cells per well in 96-well plates coated with . After 6 hours, the supernatant was discarded and recombinant retroviruses encoding costimulatory ligands, cytokines and chemokine receptors were individually added (Mulltiplicity of infection = 0.5). Cells were centrifuged at 2,500 rpm for 1 hour at 25° C. in the presence of 8 μg/mL polybrene (Sigma), anti-CD3ε (BioXcell, 145-2C11) 0.5 μg/mL, anti-CD28 (BioXcell, 37.51) 1 μg/mL, and replaced with a culture medium containing 100 IU/mL of human interleukin 2 (hIL-2). After 16 hours (overnight), the obtained genetically modified killer cells were used for adoptive transfer.

(Confirmation of function of gene-modified killer cells)

After the gene was introduced into the killer cells in the above manner, they were cultured for 3 days. To obtain a culture supernatant, IL-2, IL-12 and IL-21 cytokine secretion was measured using an ELISA kit (R&D Systems).

(Evaluation of therapeutic antitumor effect)

To evaluate the therapeutic effect in vivo, 1×10 ⁵ B16 cells were subcutaneously inoculated into the posterior flank of mice, and on day 7, genetically engineered 2×10 ⁶ killer cells were adoptively transferred once or three times at 3-day intervals. . Mice not adoptively transferred (No vax) were included as controls. Tumor growth was monitored by time course of in vivo bioluminescence imaging in individual mice, tumor size was measured in individual mice every 3-4 days using calipers, two opposite diameters were measured in individual mice, and tumor size was measured with a tumor area in ^mm2 . presented Mice were euthanized when the tumor area reached 400 mm ² . Results are expressed as mean tumor size (mm ² ) ± standard deviation for all treatment groups at various time points until the end of the experiment.

(Immunity evaluation)

To measure the antigen-specific CD8 T cell response, splenocytes were incubated with 1 μg/mL GolgiPlug (BD Bioscience, San Diego, CA) at 37°C. After 6 hours, intracellular IFNγdp eogo cells were stained using fluorescent binding antibodies to MHCII, CD8a and IFNγ. To evaluate the immune capacity of adoptively transferred mice, IFNγ-EliSpot (Millipore) was performed by obtaining CD8 T cells from the mouse spleen.

(Confirmation of adoptive transfer cells that have migrated to the tumor site/Confirmation of migration in vivo)

Seven days after the last adoptive transfer, mice in each group were sacrificed, the spleen and tumor sites were incised, and the tissues were separated using the gentleMACS Dissociator (Miltenyi) and filtered through a 70-㎛ nylon mesh. The proportion of adoptive metastasis cells in infiltrating T cells in the spleen and tumor sites was analyzed using a fluorescence-binding antibody against the adoptive metastasis cell marker CD90.1.

<Example 2> Expression and antitumor effect of costimulatory molecules metastasized to activated killer cells in vitro

1 shows the results of transduction of recombinant retroviruses encoding GFP, mouse CD70, and mouse CD70-OX40L (Pmel-GFP, Pmel-CD70, Pmel-CD70-OX40L, respectively) into in vitro activated Pmel-1 T cells. As shown, Figure 1a is the result of analyzing the expression of transduced Pmel-1 CD8 T cells by flow cytometry after obtaining cells from each group on the 3rd day after transduction, and Figures 1b-d are gene transfer Pmel-1 CD8 To show the therapeutic antitumor effect of T cells, B6 mice (3 per group) were subcutaneously inoculated with 1×10 ⁵ B16 cells on day 0 and 2×10 ⁶ transgenic Pmel-1 CD8 T cells were injected on day 7 received. Non-vaccinated mice (No vax) were included as controls. Tumor growth was monitored by time course of in vivo bioluminescence imaging in individual mice (left panel). Tumor size was measured in individual mice and tumor size was presented as tumor area in mm ² (right panel).

As a result of the experiment, the in vitro transduction efficiency of retroviruses showed that the expression level on CD8 T cells was subsequently increased (Fig. 1a), and all modified CD8 T cells were viable after transduction (not shown). . As a result of evaluating the therapeutic efficacy of adoptive transfer of transduced Pmel-1 T cells (based on Pmel-GFP, CD70, CD70-OX40L) against B16 melanoma established at 7 days (Fig. 1b-d), CD70 and CD70- The antitumor effect of Pmel-T cells transfected with the OX40L gene had a negligible therapeutic effect compared to mice that were not Pmel-GFP or adoptive transfer.

<Example 3> Anti-tumor effect of cytokines metastasized to activated killer cells in vitro

Figure 2 shows the result of transduction of the recombinant retrovirus encoding mouse IL-2, IL-12 and IL-21 genes into the in vitro activated Pmel-1 T cells, Figure 2a is Pmel-1 CD8 T cells On the 3rd day after transduction of the gene, the culture supernatant of each group was obtained and cytokine secretion was measured by ELISA, and FIGS. 2b-e show the therapeutic antitumor effect of the transgenic Pmel-1 CD8 T cells. B6 mice (3 per group) were subcutaneously inoculated with 1×10 ⁵ B16 cells on day 0 and received 2×10 ⁶ transgenic Pmel-1 CD8 T cells on day 7. Non-vaccinated mice (No vax) were included as controls. Tumor growth was monitored by time course of in vivo bioluminescence imaging in individual mice (left panel). Tumor size was measured in individual mice and tumor size was presented as tumor area in mm ² (right panel).

As a result of the experiment, it was shown that the introduced cytokine functioned normally on Pmel-1 T cells transduced with a retrovirus encoding a cytokine gene (Fig. 2a), and that all modified CD8 T cells were viable after transduction (Fig. not shown). As a result of evaluating the therapeutic efficacy of adoptive transfer against B16 melanoma established by transduced Pmel-1 T cells (based on Pmel-GFP, IL-2, IL-12, IL-21) for 7 days (Fig. 2b-e) ), IL-2, IL-12 and IL-21 gene-transduced Pmel-1 T cells showed therapeutic advantages compared to Pmel-GFP or non-adoptive mice, among which Pmel- IL-12 had a substantially higher antitumor effect in adoptive transfer mice.

<Example 4> Anti-tumor effect of costimulatory molecule-CD70 and cytokine-expressing killer cells

3 shows GFP and mouse CD70, IL-2-CD70, IL-12-CD70, and IL-21-CD70 (Pmel-GFP, Pmel-CD70, Pmel-IL-2, respectively) in activated Pmel-1 T cells in vitro. -CD70, Pmel-IL-12-CD70, Pmel-IL-21-CD70) shows the results of transduction with a recombinant retrovirus encoding the transduced, Figure 3a is the expression of the transduced Pmel-1 CD8 T cells. Cells from each group were obtained on the 3rd day after introduction and expression efficiency by flow cytometry, and FIGS. 3b-e are the results of measuring cytokine secretion by ELISA by obtaining the culture supernatant of each group, and FIG. 3f is the gene transfer Pmel-1 To show the therapeutic antitumor effect of CD8 T cells, B6 mice (3 per group) were subcutaneously inoculated with 1×10 ⁵ B16 cells on day 0 and 2×10 ⁶ transgenic Pmel-1 CD8 T cells on day 7 received. Non-vaccinated mice (No vax) were included as controls. Tumor growth was monitored by time course of in vivo bioluminescence imaging in individual mice (left panel). Tumor size was measured in individual mice and tumor size was presented as tumor area in mm ² (right panel).

As a result of the experiment, the in vitro transduction efficiency of retroviruses was subsequently increased in the expression level on CD8 T cells ( FIG. 3A ), and the transduced cytokines functioned normally ( FIGS. 3B-E ). All modified CD8 T cells were shown to be viable after transduction (not shown). Transduced Pmel-1 T cells (based on Pmel-GFP, Pmel-CD70, Pmel-IL-2-CD70, Pmel-IL-12-CD70, Pmel-IL-21-CD70) established B16 melanoma at 7 days As a result of evaluating the therapeutic efficacy of adoptive transfer against (Fig. 3f), the antitumor effect of Pmel-T cells transduced with IL-2-CD70, IL-12-CD70 and IL-21-CD70 genes was determined by Pmel-GFP. , showed a therapeutic advantage compared to mice that were not Pmel-CD70 or adoptively transferred, among which Pmel-IL-12-CD70 had a substantially higher antitumor effect in mice with adoptive transfer.

<Example 5> Chemokine receptor (CXCR3)-expressing killer cells antitumor effect

Figure 4 shows the result of transduction of in vitro activated Pmel-1 T cells with a recombinant retrovirus encoding GFP and mouse chemokine receptor CXCR3 (Pmel-GFP, Pmel-CXCR3, respectively), Figure 4a is the transduced The expression of Pmel-1 CD8 T cells is the result of obtaining cells from each group on the 3rd day after transduction and analyzing them by flow cytometry. As shown, B6 mice (3 per group) were subcutaneously inoculated with 1×10 ⁵ B16 cells on day 0 and received 2×10 ⁶ transgenic Pmel-1 CD8 T cells on day 7. Non-vaccinated mice (No vax) were included as controls. Tumor growth was monitored by time course of in vivo bioluminescence imaging in individual mice (left panel). Tumor size was measured in individual mice and tumor size was presented as tumor area in mm ² (right panel).

As a result of the experiment, the in vitro transduction efficiency of retroviruses showed that the expression level on CD8 T cells was subsequently increased (Fig. 4a), and all modified CD8 T cells were viable after transduction (not shown). . As a result of evaluating the therapeutic efficacy of adoptive transfer of transduced Pmel-1 T cells (Pmel-GFP, Pmel-CXCR3) against B16 melanoma established on day 7 (Fig. 4b-e), the chemokine receptor CXCR3 gene was transduced The antitumor effect of Pmel-1 T cells was substantially higher than that of Pmel-GFP or adoptive non-transferred mice.

<Example 6> Chemokine receptor CXCR3 and cytokine-expressing killer cells antitumor effect

5 shows GFP, mouse cytokines IL-2, IL-12, IL-2 and chemokine receptor CXCR3 (Pmel-GFP, Pmel-IL-2-CXCR3, Pmel-IL, respectively) in activated Pmel-1 T cells in vitro. -12-CXCR3, Pmel-IL-21-CXCR3) shows the transduction results with a recombinant retrovirus encoding, FIG. 5a shows the expression of transduced Pmel-1 CD8 T cells at 3 days after transduction. Cells of a group were obtained and expression efficiency by flow cytometry, and FIGS. 5b-e are the results of obtaining the culture supernatant of each group and measuring the cytokine secretion by ELISA, and FIG. 5f is the therapeutic effect of transgenic Pmel-1 CD8 T cells. To show antitumor effect, B6 mice (3 per group) were subcutaneously inoculated with 1×10 ⁵ B16 cells on day 0 and received 2×10 ⁶ transgenic Pmel-1 CD8 T cells on day 7. Non-vaccinated mice (No vax) were included as controls. Tumor growth was monitored by time course of in vivo bioluminescence imaging in individual mice (left panel). Tumor size was measured in individual mice and tumor size was presented as tumor area in mm ² (right panel).

As a result of the experiment, the in vitro transduction efficiency of retroviruses was subsequently increased in the expression level on CD8 T cells ( FIG. 5A ), and the transduced cytokines functioned normally ( FIGS. 5B-E ). All modified CD8 T cells were shown to be viable after transduction (not shown). Transduced Pmel-1 T cells (Pmel-GFP, Pmel-IL-2-CXCR3, Pmel-IL-12-CXCR3, Pmel-IL-21-CXCR3) were transferred to established B16 melanoma at 7 days of adoptive transfer. As a result of evaluating the therapeutic efficacy (Fig. 5f), the anti-tumor effect of Pmel-T cells transduced with the chemokine receptor CXCR3 gene showed a therapeutic advantage in the early stage of tumor growth compared to mice without Pmel-GFP or adoptive transfer. Among them, Pmel-IL-12-CXCR3 had a substantially higher antitumor effect in adoptively transferred mice.

<Example 7> Chemokine receptor CXCR3 and cytokine-expressing killer cells antitumor effect and tumor site migration

Fig. 6 shows the result of transduction of activated Pmel-1 T cells in vitro with recombinant retroviruses encoding GFP, mouse cytokine and chemokine receptors, and Fig. 6a is the expression of transduced Pmel-1 CD8 T cells. Cells from each group were obtained on the 3rd day after transduction, and the expression efficiency and the culture supernatant of each group were obtained by flow cytometry, and cytokine secretion was measured by ELISA, FIGS. 6b-e are the gene transfer Pmel-1 CD8 To show the therapeutic antitumor effect of T cells, B6 mice (3 per group) were subcutaneously inoculated with 1×10 ⁵ B16 cells on day 0 and 2×10 ⁶ transgenic Pmel-1 CD8 T cells once on day 7 or 3 times. Non-vaccinated mice (No vax) were included as controls. Tumor growth was monitored by time course of in vivo bioluminescence imaging in individual mice (left panel). Tumor size was measured in individual mice and tumor size was presented as tumor area in mm ² (right panel). 6f shows that the transgenic Pmel-1 CD8 T cells migrated to the tumor site by sacrificing mice of each group on day 15 after B16 cell inoculation were confirmed by flow cytometry. FIG. 6g shows that the CD8 T cells were isolated from the spleen of mice on the 35th day after inoculation of B16 cells, and the amount of IFNγ secretion to the B16 cells was confirmed through EliSpot.

As a result of the experiment, the in vitro transduction efficiency of retroviruses was subsequently increased in the expression level on CD8 T cells, and the transduced cytokines functioned normally ( FIG. 6A ). Results of evaluating the therapeutic efficacy of adoptive transfer of transduced Pmel-1 T cells (Pmel-GFP, Pmel-CXCR3, Pmel-IL-12, Pmel-IL-12-CXCR3) against B16 melanoma established on day 7 (Fig. 6b-e), the antitumor effect of Pmel-1 T cells transduced with the chemokine receptor CXCR3 and cytokine IL-12 showed a therapeutic advantage in the early stage of tumor growth compared to mice receiving 3 doses of Pmel-GFP. Among them, it was confirmed that adoptively transferred cells migrated to the tumor site in mice receiving Pmel-CXCR3 and Pmel-IL-12-CXCR3 transduced with the CXCR3 gene.

<Example 8> Chemokine receptor CXCR3 and cytokine-expressing anti-tumor effect according to the number of administration of killer cells

7 shows the results of transduction of activated Pmel-1 T cells in vitro with recombinant retroviruses encoding GFP, mouse cytokine and chemokine receptors, and FIG. 7a shows the expression of transduced Pmel-1 CD8 T cells. Cells from each group were obtained on the third day after transduction, and the expression efficiency of each group was obtained by flow cytometry, and FIG. 7b is the result of obtaining the culture supernatant of each group and measuring the cytokine secretion by ELISA, FIGS. 7c-f are the gene transfer Pmel -1 To show the therapeutic antitumor effect of CD8 T cells, B6 mice (3 per group) were subcutaneously inoculated with 1×10 ⁵ B16 cells on day 0 and 2×10 ⁶ gene transfer Pmel at 3 day intervals from the 7th day. -1 CD8 T cells were administered 3 times. Non-vaccinated mice (No vax) were included as controls. Tumor growth was monitored by time course of in vivo bioluminescence imaging in individual mice (left panel). Tumor size was measured in individual mice and tumor size was presented as tumor area in mm ² (right panel).

As a result of the experiment, the in vitro transduction efficiency of retroviruses was subsequently increased in the expression level on CD8 T cells, and the transduced cytokines functioned normally ( FIGS. 7A and 7B ). Treatment of mice receiving 3 doses of adoptive transfer for 7-day established B16 melanoma with transduced Pmel-1 T cells (Pmel-GFP, Pmel-CXCR3, Pmel-IL-12, Pmel-IL-12-CXCR3) As a result of evaluating the efficacy (Fig. 7c-f), the antitumor effect of Pmel-T cells transduced with the chemokine receptor CXCR3 was early in tumor growth compared to mice receiving Pmel-IL-12 and Pmel-IL-12-CXCR3. showed similar therapeutic benefits.

The present invention can be used in adoptive immunotherapy for the prevention or treatment of tumors.

Claims (14)

1. A modified killer cell that expresses one or more of a C-X-C chemokine receptor and a cytokine.
2. According to claim 1,

   The chemokine receptor is at least one selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR2, CCR4, CCR5, CCR7 and CCR10, a modified killer cell.
3. According to claim 1,

   The cytokine is at least one selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-21, modified killer cells.
4. According to claim 1,

   wherein the modified killer cell further expresses a costimulatory molecule.
5. 5. The method of claim 4,

   The costimulatory molecule is at least one selected from the group consisting of CD70, CD134L (OX40L), CD80, CD86, CD54, CD154 (CD40L) and CD137L (4-1BBL), a modified killer cell.
6. According to claim 1,

   Killer cells include tumor-infiltrated lymphocytes, ex vivo antigen-specific T cells, natural killer cells, natural killer T cells, A modified killer cell, at least one selected from the group consisting of gamma delta T cells and T cells with a chimeric antigen receptor (CAR) or T cell receptor (TCR).
7. 7. The method of claim 6,

   T cells equipped with chimeric antigen receptors or T cell receptors include CD4 + T cells, CD8 + T cells, recombinant tumor-infiltrated lymphocytes, recombinant natural killer cells, recombinant natural killer T cells ( At least one modified killer cell selected from the group consisting of natural killer T cells and recombinant gamma delta T cells.
8. A method for producing a modified killer cell in vitro comprising the step of introducing one or more of a chemokine receptor and a cytokine into the killer cell.
9. 9. The method of claim 8,

   The method further comprises the step of introducing a co-stimulatory molecule into the killer cell, the method for producing a modified killer cell in vitro.
10. An anticancer composition comprising the modified killer cell according to claim 1 .
11. 11. The method of claim 10,

    Cancers include lung cancer, stomach cancer, colon cancer, breast cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, perianal cancer, fallopian tube carcinoma, endometrial cancer, uterus Cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, Renal cancer, ureter cancer, renal pelvic cancer, blood cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma or pituitary adenoma, anticancer composition.
12. A method for treating cancer, comprising administering a therapeutically effective amount of the modified killer cell according to claim 1 to a subject in need thereof.
13. 13. The method of claim 12,

    Cancers include lung cancer, stomach cancer, colon cancer, breast cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, perianal cancer, fallopian tube carcinoma, endometrial cancer, uterus Cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, Renal cancer, ureter cancer, renal pelvic cancer, blood cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brain stem glioma or pituitary adenoma, a method of treating cancer.
14. Use of a modified killer cell according to claim 1 for the manufacture of a medicament for the treatment of cancer.

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