Mesenchymal stem cells in which NQO1 and CXCL10-fc are overexpressed, and use thereof

Abstract

The present invention relates to mesenchymal stem cells in which NAD(P)H quinone oxidoreductase 1 (NQO1) and a C-X-C motif chemokine ligand 10 (CXCL10)-Fc fusion protein are overexpressed, a preparation method therefor, and a use thereof, the CXCL10-Fc fusion protein comprising CXCL10 linked to an Fc region derived from immunoglobulin (Ig). According to the present invention, mesenchymal stem cells, in which adaptability to a stress environment represented by a tumor microenvironment is improved through the overexpression of NQO1 and infiltration of CD8 T cells into solid tumors is promoted through the overexpression of a CXCL10-Fc fusion protein, can be provided, and thus the mesenchymal stem cells can be effectively used as a cell therapeutic agent and the like effective for treating solid tumors.

Description

Mesenchymal stem cells overexpressing NQO1 and CXCL10-FC and their uses

[0001] The present invention relates to mesenchymal stem cells overexpressing NQO1 and CXCL10-Fc and the use thereof.

[0002] Traditional anticancer treatments such as surgical procedures, radiation therapy, and chemotherapy using cytotoxic drugs have clear limitations, and various studies including targeted therapies, immunotherapies, and gene therapies are being conducted to overcome these limitations. Among these, immunotherapies utilize the patient's own immune system to enhance tumor specificity and alleviate side effects, and this is a field that has been actively researched following the FDA approval of immune checkpoint inhibitors (ICIs) and CAR-T cell therapies.

[0003] However, immune checkpoint inhibitors have a limitation in that their response rates are low due to the numerical shortage of immune cells infiltrating the tumor; similarly, CAR-T cell therapies also suffer from low response rates because the infiltration of CAR-T cells into the tumor itself is difficult (Mongol GP, Proc Natl Acad Sci USA, 2017; Schietinger A., ​​Immunity, 2016; Cappell KM, Nat Rev Clin Oncol, 2013). Therefore, recent clinical studies are actively investigating combination therapies of immune checkpoint inhibitors and CAR-T cell therapies, as well as combination therapies that add small molecules such as cytokines, to improve the applicability and response rates in solid tumors. However, most studies focus on enhancing the function of the CAR-T cells themselves, and a fundamental solution to the problem of their inability to infiltrate into solid tumors has not been achieved.

[0004] Chemokines have been identified as a factor capable of resolving the problem of immune cell infiltration. Chemokines are molecules involved in the in vivo migration of not only immune cells but also various cell populations, operating on the principle of chemotaxis induced by concentration gradients. Chemokines are classified into various types based on their molecular structure, and among them, CXCL9, CXCL10, CXCL11, CCL5, and CX3CL1 are chemokines involved in the chemotaxis of CD8 T cells, a cell population that plays a key role in tumor immunity. Of these, CXCL9 and CXCL10 are known to play a significant role in direct migration to the affected site.

[0005] CXCL10 is a ligand for CXCR3 and is known to recruit macrophages, dendritic cells, and NK cells in addition to T cells. In patients with high CXCL10 expression, progression-free survival and overall survival were longer than in patients with low CXCL10 expression, and CXCL10 expression is also high in patients who responded to immune checkpoint inhibitors.

[0006] However, a therapeutic effect cannot be expected from the simple injection of CXCL10 alone. Injection methods that are likely to be released systemically due to the characteristic that chemotaxis is induced by a concentration gradient are likely insufficient to form a concentration gradient for the induction of CD8 T cells, and tumor-specific delivery is essential, especially considering that a correlation between blood concentration and poor prognosis has been reported for CXCL10, particularly in liver cancer.

[0007] Various methods have been devised for the delivery of tumor-specific substances. Among these, cell-based delivery systems are gaining prominence alongside the growth of the advanced biotechnology market. A wide variety of cells are used in the development of such cell therapies, with mesenchymal stem cells being actively utilized.

[0008] Mesenchymal stem cells (MSCs) are stem cells derived from the mesoderm, found in various tissues such as bone, cartilage, and adipose tissue, and possess multipotential differentiation. There are existing studies utilizing mesenchymal stem cells to deliver various small molecules specifically to tumors; these studies commonly cite the natural toxicity of MSCs against solid tumors, ease of genetic processing, and ease of maintenance in an in vitro environment as key advantages.

[0009] Accordingly, the inventors of the present invention have made efforts to develop a tumor-specific chemokine delivery system to infiltrate CD8 T cells into solid tumors. As a result, they confirmed that the ability to adapt to a stress environment, represented by the tumor microenvironment, is improved through the overexpression of NQO1 (NAD(P)H Quinone Oxidoreductase 1), and that the infiltration of CD8 T cells into solid tumors is promoted through the overexpression of a CXCL10-Fc fusion protein in which an Fc region derived from CXCL10 (CXC motif chemokine ligand 10) is linked to an immunoglobulin (Ig)-derived Fc region. Furthermore, they confirmed that using mesenchymal stem cells with improved performance through this process can enhance the therapeutic effect of adoptive cell transfer therapy through stable tumor-specific CXCL10 delivery, thereby completing the present invention.

[0010] The object of the present invention is to provide mesenchymal stem cells that overexpress NQO1 (NAD(P)H Quinone Oxidoreductase 1); and a CXCL10-Fc fusion protein in which an Fc region derived from immunoglobulin (Ig) is linked to CXCL10 (CXC motif chemokine ligand 10).

[0011] Another object of the present invention is to provide a method for producing mesenchymal stem cells comprising the step of transfecting a recombinant expression vector comprising NQO1 (NAD(P)H Quinone Oxidoreductase 1); and a polynucleotide encoding a CXCL10-Fc fusion protein in which an Fc region derived from immunoglobulin (Ig) is linked to CXCL10 (CXC motif chemokine ligand 10).

[0012] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of solid tumors comprising, as an active ingredient, one or more selected from the group consisting of the mesenchymal stem cells, cells differentiated from the mesenchymal stem cells, and components derived from the mesenchymal stem cells.

[0013] To solve the above problem, the present invention provides a mesenchymal stem cell overexpressing NQO1 (NAD(P)H Quinone Oxidoreductase 1); and a CXCL10-Fc fusion protein in which an Fc region derived from immunoglobulin (Ig) is linked to CXCL10 (CXC motif chemokine ligand 10).

[0014] According to the present invention, the expression of the NQO1 and the CXCL10-Fc fusion protein may be increased through genetic engineering.

[0015] At this time, it may be obtained by transfecting a recombinant expression vector containing a polynucleotide encoding the NQO1 and the CXCL10-Fc fusion protein.

[0016] In addition, the recombinant expression vector may be selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vacciniavirus vectors, herpesvirus vectors, lentivirus vectors, and avifoxvirus vectors.

[0017] At this time, the polynucleotide encoding the above NQO1 may include a base sequence represented by SEQ ID NO. 1.

[0018] In addition, the polynucleotide encoding the CXCL10-Fc fusion protein may include the base sequence represented by SEQ ID NO. 2.

[0019] According to the present invention, the mesenchymal stem cells may be one or more selected from the group consisting of bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, embryonic stem cell-derived mesenchymal stem cells, and induced pluripotent stem cell-derived mesenchymal stem cells.

[0020] According to the present invention, the ability to adapt to a stressful environment may be improved.

[0021] At this time, the stress environment may be one or more selected from the group consisting of a hypoxic environment, a nutrient-deficient environment, a hypoxic environment and a nutrient-deficient environment, an oxidative stress environment, an endoplasmic reticulum stress environment, and a mitochondrial stress environment.

[0022]

[0023] The present invention also provides a method for producing mesenchymal stem cells comprising the step of transfecting a recombinant expression vector comprising NQO1 (NAD(P)H Quinone Oxidoreductase 1); and a polynucleotide encoding a CXCL10-Fc fusion protein in which an Fc region derived from immunoglobulin (Ig) is linked to CXCL10 (CXC motif chemokine ligand 10).

[0024] According to the present invention, the polynucleotide encoding NQO1 may include a base sequence represented by SEQ ID NO. 1.

[0025] According to the present invention, the polynucleotide encoding the CXCL10-Fc fusion protein may include the base sequence represented by SEQ ID NO. 2.

[0026]

[0027] The present invention also provides a pharmaceutical composition for the prevention or treatment of solid tumors comprising, as an active ingredient, one or more selected from the group consisting of the mesenchymal stem cells, cells differentiated from the mesenchymal stem cells, and components derived from the mesenchymal stem cells.

[0028] According to the present invention, the solid tumor may be selected from the group consisting of glioblastoma, glioblastoma, meningioma, neuroblastoma, oral cancer, laryngeal cancer, pharyngeal cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, osteosarcoma, and sarcoma.

[0029]

[0030] The features and advantages of the present invention will become more apparent from the following detailed description based on the accompanying drawings.

[0031] Prior to this, terms and words used in this specification and claims shall not be interpreted in their ordinary and dictionary meanings, but must be interpreted in a meaning and concept consistent with the technical spirit of the invention, based on the principle that the inventor may appropriately define the concept of the terms to best describe his invention.

[0032] According to the present invention, mesenchymal stem cells capable of improving the ability to adapt to a stress environment represented by the tumor microenvironment through the overexpression of NQO1 and promoting the infiltration of CD8 T cells into solid tumors through the overexpression of CXCL10-Fc fusion protein can be provided, and said mesenchymal stem cells can be usefully utilized as a cell therapy agent effective for the treatment of solid tumors.

[0033] Figure 1 shows the results of analyzing the progression of apoptosis using Annexin V and PI when MSCs overexpressing NQO1 (NMSC) and normal MSCs were cultured for 48 hours under a stress environment. n=3.

[0034] Figure 2 shows the results of analyzing the extent of CFSE+ MSC detection within the tumor after 3 days, following CFSE labeling of NQO1-overexpressing MSCs (NMSCs) and normal MSCs, and subsequent injection of MC38 via subcuntaneous injection into the tumor formed therefrom via peritumor injection. n=3.

[0035] Figure 3 shows the results of analyzing whether CFSE+ MSCs are detected in each tissue after 3 days, following CFSE labeling of NQO1-overexpressing MSCs (NMSCs) and normal MSCs, and subsequent injection of the MC38 cell line via subcuntaneous injection into tumors formed via peritumor injection. n=3.

[0036] Figure 4 shows the results of measuring the amount of CXCL10-Fc fusion protein expressed by ELISA when MSCs engineered to overexpress NQO1 and CXCL10-Fc fusion protein (IP10Fc-NMSC) and MSCs engineered to overexpress CXCL10-Fc fusion protein (IP10Fc-MSC) were cultured for 48 hours under a stress environment, respectively. n=3.

[0037] Figure 5 shows the results of confirming, via transwell, the recruitment performance of activated mouse T cells using αCD3 antibody, αCD28 antibody, and recombinant human IL-2 for 48 hours, using a conditioned medium formed by culturing MSCs (IP10Fc-NMSC) engineered to overexpress NQO1 and CXCL10-Fc fusion proteins and normal MSCs in a hypoxic environment for 48 hours. n=3.

[0038] Figures 6 and 7 show the results of measuring the concentration of IP10-Fc in serum and the relative amount of IP10-Fc in each tissue by ELISA after 3 days, following tumor formation by subcutaneous injection of MC38, injection of MSCs engineered to overexpress NQO1 and CXCL10-Fc fusion proteins (IP10Fc-NMSC), normal MSCs, and purified IP10-Fc secreted from IP10Fc-NMSC via peritumor injection and intratumor injection, respectively. n=4.

[0039] Figures 8 and 9 show the results of analyzing the number of endogenous T cells and CFSE+ T cells in each tissue by flow cytometry after 3 days had passed since injecting MC38 via subcuntaneous injection to form a tumor, injecting MSCs (IP10Fc-NMSC) engineered to overexpress NQO1 and CXCL10-Fc fusion proteins, normal MSCs, and IP10-Fc proteins secreted from IP10Fc-NMSC via peritumor injection and intratumor injection, respectively, and then injecting mouse T cells activated with αCD3 antibody, αCD28 antibody, and recombinant human IL-2 via retro-orbital injection labeled with CFSE for 48 hours.

[0040] Figure 10 shows the results of tracking the growth of a tumor after injecting B16-OVA via subcuntaneous injection, injecting MSCs engineered to overexpress NQO1 and CXCL10-Fc fusion proteins (IP10Fc-NMSC) and normal MSCs via peritumor injection, and then injecting OT-I T cells.

[0041] Figure 11 shows the results of tracking changes in survival rates of each population after forming tumors by injecting B16-OVA via subcuntaneous injection, injecting MSCs engineered to overexpress NQO1 and CXCL10-Fc fusion proteins (IP10Fc-NMSC) and normal MSCs via peritumor injection, and then injecting OT-I T cells.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0043]

[0044] The present invention provides mesenchymal stem cells that overexpress NQO1 (NAD(P)H Quinone Oxidoreductase 1); and a CXCL10-Fc fusion protein in which an Fc region derived from immunoglobulin (Ig) is linked to CXCL10 (CXC motif chemokine ligand 10).

[0045] In this specification, the term "Mesenchymal Stem Cells (MSCs)" may refer to stem cells present in cartilage, bone tissue, adipose tissue, bone marrow stroma, etc., which are differentiated from the mesoderm formed by the division of a fertilized egg, and may include mesenchymal stem cells of mammals, such as animals including humans. Mesenchymal stem cells retain stemness and self-renewal ability and possess the ability to differentiate into various cells including chondrocytes, osteoblasts, muscle cells, and adipocytes, and may be extracted from bone marrow, adipose tissue, umbilical cord blood, synovial membrane, trabecular bone, muscle, infrapatellar fat pad, etc. Mesenchymal stem cells are cells capable of allotransplantation and xenotransplantation because they have immunomodulatory abilities that inhibit the activation and proliferation of T lymphocytes and B lymphocytes, inhibit the activation of natural killer cells (NK cells), and regulate the functions of dendritic cells and macrophages.

[0046] In this specification, the terms “Fc region (fragment crystallizable region),” “Fc domain,” “Fc portion,” or similar terms refer to a part of the antibody heavy chain constant region and include a hinge region and CH2 and CH3 fragments of the constant region. Referring to the EU numbering definition of a human IgG1 antibody, the Fc fragment is the amino acid sequence from positions 216 to 447 in the antibody constant region. The Fc region may be an antibody Fc region in the form of a hybrid mixture of the Fc regions of IgG1, IgG2, IgG3, IgG4, or IgD, or two or more isotypes. In one embodiment, the Fc region may be of human IgG1 origin. Additionally, the Fc region may be a form having a LALAPG mutation.

[0047] In this specification, the term “antibody” may mean an intact immunoglobulin of any isotype, an antigen-binding fragment capable of competing with an intact antibody for binding to a target antigen, or a combination thereof. For example, it may include mouse, chimeric, humanized, fully human antibodies, antigen-binding fragments thereof, or a combination thereof. An antibody may be a type of antigen-binding protein itself. An antibody generally comprises at least two full-length heavy chains and two full-length light chains, but in some cases, an antibody may comprise only heavy chains. The antibody may include a monospecific antibody that specifically binds to one target, and a multispecific antibody that specifically binds to multiple targets (e.g., a bispecific antibody and a trispecific antibody).

[0048] In this specification, the term “heavy chain” may include a full-length heavy chain and fragments thereof having a variable region sequence sufficient to provide binding specificity to an antigen or epitope. The full-length heavy chain may include a variable region domain and three constant region domains CH1, CH2, and CH3. The variable region (VH) domain is located at the amino terminus of the heavy chain polypeptide, the constant region (CH) domain is located at the carboxy terminus, and CH3 may be located closest to the carboxy terminus. The heavy chain may include isotypes of IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0049] The above antibody may be selected from all subtypes of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.). Preferably, the above antibody may be selected from IgG. The IgG-type antibody may be an IgG1, IgG2, IgG3, or IgG4 subtype, such as an IgG1 or IgG2 subtype. The IgG-type antibody comprises two heavy chains and two light chains, each heavy chain and light chain being joined via a disulfide bond to form two heavy-light chain structures (dimers), and the two formed heavy-light chains may be connected via a disulfide bond at the Fc site of the heavy chain. The IgG-type antibody described above may be a single-target antibody targeting a single antigen by including antigen-binding sites for the same antigen on both heavy-to-light chain structures, or a dual-specific antibody targeting two antigens by including antigen-binding sites for different antigens on both heavy-to-light chain structures.

[0050] In this specification, the term "hinge region" refers to a region included in the heavy chain of an antibody, existing between the CH1 and CH2 regions, and functioning to provide flexibility to the antigen-binding site within the antibody.

[0051] In this specification, the term "fusion protein" refers to a new protein formed by combining two or more different proteins or homologous proteins. Two or more heterologous proteins are combined in part and part, part and whole, or whole and whole. "CXCL10-Fc fusion protein" may refer to a protein formed by combining CXCL10 and immunoglobulin Fc protein in part and part, part and whole, or whole and whole. In one embodiment, the fusion protein may be in the form where CXCL10 is combined with the N-terminus of the Fc region.

[0052] According to the present invention, the mesenchymal stem cells may be genetically engineered to increase the expression of the NQO1 and CXCL10-Fc fusion proteins.

[0053] In this specification, the terms "genetic engineering" or "genetically engineered" may refer to the act of introducing one or more genetic modifications into a cell.

[0054] The above increase in expression or overexpression may mean that the expression of the same type of protein has a higher level compared to the expression of the intrinsic protein in a given genetically unmanipulated parent cell (e.g., wild type).

[0055]

[0056] Mesenchymal stem cells according to the present invention may be obtained by transfecting a recombinant expression vector comprising a polynucleotide encoding the NQO1 and the CXCL10-Fc fusion protein.

[0057] The most common method for overexpressing the NQO1 and CXCL10-Fc fusion proteins in stem cells is to artificially or experimentally introduce polynucleotides encoding the NQO1 and CXCL10-Fc fusion proteins into the stem cells to increase the copy number of the NQO1 and CXCL10-Fc genes. Injecting exogenous polynucleotides, which are not originally present in the cell, into the cell is called transfection, and the phenomenon in which the genetic traits of the cell change as a result is called transformation. The process of injecting the exogenous polynucleotides into the cell via a virus or a virus-derived vector is called transduction. In this specification, the terms "transfection," "transfection," and "transduction" are used interchangeably to refer to the introduction of exogenous polynucleotides into a cell to result in the cell acquiring genetic traits different from the normal type, or the process thereof.

[0058] In this specification, “polynucleotide” or “nucleic acid” refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) in a single- or double-stranded form. Unless otherwise limited, it also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

[0059] In this specification, "recombinant expression vector" refers to a genetic construct that includes an essential regulatory element operably linked to enable the expression of a polynucleotide (gene) insert, and is capable of expressing a target protein or target nucleic acid (RNA) in a suitable host cell. "Operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding the target protein or RNA are functionally linked to perform a general function. That is, it means that the nucleic acid sequence encoding the protein or RNA is linked in a manner that enables gene expression through the expression regulatory sequence; for example, a promoter and a nucleic acid sequence encoding the protein or RNA must be operably linked to influence the expression of the coding nucleic acid sequence. Operatory linkage with the recombinant vector may be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage may be performed using enzymes, etc., generally known in the art.

[0060] The recombinant expression vector of the present invention is not particularly limited in type as long as it is a vector commonly used in the field of cloning, and examples include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Preferably, a vector derived from a virus may be used. The viral vector of the present invention may be selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vacciniavirus vectors, herpesvirus vectors, lentivirus vectors, and avifoxvirus vectors.

[0061] The expression vector comprising the nucleic acid according to the present invention is a method known in the art, e.g., but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection (DEAE

[0062] It can be introduced into stem cells by dextran-mediated transfection, polybrene-mediated transfection, electroporation, a gene gun, and known methods for introducing nucleic acids into cells.

[0063] NQO1 according to the present invention may include the amino acid sequence of SEQ ID NO. 3, and preferably may consist of the amino acid sequence of SEQ ID NO. 3, but is not limited thereto, and variants of said amino acid sequence are included within the scope of the present invention. That is, the polypeptide consisting of SEQ ID NO. 3 of the present invention is a concept that includes functional equivalents of the polypeptide constituting it, for example, variants in which some amino acid sequences of the polypeptide have been modified by deletion, substitution, or insertion, but which can perform the same function as said polypeptide. Specifically, the polypeptide encoding said NQO1 may include amino acid sequences having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with any one of the amino acid sequences indicated by SEQ ID NO. 3. For example, it includes polypeptides having sequence homology of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. The “% of sequence homology” for the polypeptide is determined by comparing two optimally arranged sequences with a comparison region, where a portion of the polypeptide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal arrangement of the two sequences. In addition, NQO1 according to the present invention may be encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO. 1, preferably by a polynucleotide composed of the nucleic acid sequence of SEQ ID NO. 1, but is not limited thereto, and variants of said polynucleotide are included within the scope of the present invention.In the present invention, a mouse NQO1 sequence synthesized from MSC cDNA was used as the DNA sequence for NQO1 overexpression.

[0064] The CXCL10-Fc fusion protein according to the present invention may include the amino acid sequence of SEQ ID NO. 4 or be encoded by a polypeptide composed of the amino acid sequence of SEQ ID NO. 4, but is not limited thereto. That is, the CXCL10-Fc fusion protein may include an amino acid sequence having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with respect to the amino acid sequence represented by SEQ ID NO. 4. Additionally, the CXCL10-Fc fusion protein according to the present invention may be encoded by a polynucleotide including the nucleic acid sequence of SEQ ID NO. 2, and preferably by a polynucleotide composed of the nucleic acid sequence of SEQ ID NO. 2, but is not limited thereto, and a variant of the polynucleotide is included within the scope of the present invention. In the present invention, as DNA sequences for expressing a mouse CXCL10-human Fc fusion protein (IP10-Fc) using an EF1α promoter, a synthetic DNA sequence designed to match the amino acid sequence of a human Fc protein and a mouse CXCL10 sequence synthesized from the cDNA of an MSC were used. At this time, CXCL10 may be derived from cDNA derived from mRNA expressed by mesenchymal stem cells (MSCs), and the Fc region may be in a form that has increased translation efficiency through codon optimization.

[0065] According to the present invention, the mesenchymal stem cells may be one or more selected from the group consisting of bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, embryonic stem cell-derived mesenchymal stem cells, and induced pluripotent stem cell-derived mesenchymal stem cells, but are not limited thereto.

[0066] According to the present invention, the mesenchymal stem cells may have improved adaptability to a stressful environment.

[0067] At this time, the stress environment may be one or more selected from the group consisting of a hypoxic environment, a nutrient-deficient environment, a hypoxic environment and a nutrient-deficient environment, an oxidative stress environment, an endoplasmic reticulum stress environment, and a mitochondrial stress environment.

[0068]

[0069] The present invention also provides a method for producing mesenchymal stem cells comprising the step of transfecting a recombinant expression vector comprising NQO1 (NAD(P)H Quinone Oxidoreductase 1); and a polynucleotide encoding a CXCL10-Fc fusion protein in which an Fc region derived from immunoglobulin (Ig) is linked to CXCL10 (CXC motif chemokine ligand 10).

[0070] As the mesenchymal stem cells to which the manufacturing method of the present invention is applied have been described above, explanations regarding overlapping content will be omitted or described only briefly.

[0071] According to the present invention, the polynucleotide encoding NQO1 may include a base sequence represented by SEQ ID NO. 1.

[0072] According to the present invention, the polynucleotide encoding the CXCL10-Fc fusion protein may include the base sequence represented by SEQ ID NO. 2.

[0073]

[0074] The present invention also provides a pharmaceutical composition for the prevention or treatment of solid tumors comprising, as an active ingredient, one or more selected from the group consisting of the mesenchymal stem cells, cells differentiated from the mesenchymal stem cells, and components derived from the mesenchymal stem cells.

[0075] That is, the composition according to the present invention is a component derived from mesenchymal stem cells overexpressing NQO1 and CXCL10-Fc fusion proteins, and may include, without limitation, any component capable of exhibiting the characteristics or functions of said stem cells. Specifically, in the present invention, the concept of a component derived from said stem cells includes not only the mesenchymal stem cells overexpressing NQO1 and CXCL10-Fc fusion proteins themselves, but also their cultures, lysates, and extracts. In this specification, the term "culture" includes the culture medium itself obtained by culturing NQO1 and CXCL10-Fc fusion protein overexpressing stem cells according to the present invention in a suitable liquid medium, the filtrate obtained by removing NQO1 and CXCL10-Fc fusion protein overexpressing stem cells by filtering or centrifuging the culture medium (filtrate or supernatant after centrifugation), and the cell lysate obtained by sonicating the culture medium or treating the culture medium with a lysozyme, and preferably refers to the supernatant after centrifugation, but is not limited thereto. In addition, the culture medium may include both the concentrated culture medium and the dried culture medium.

[0076] More specifically, the components derived from stem cells in the present invention include carbohydrates, lipids, proteins (including peptides), glycoproteins, oligonucleotides, vitamins, and other metabolites produced from mesenchymal stem cells overexpressing NQO1 and CXCL10-Fc fusion proteins according to the present invention, as well as extracellular vesicles. In the present invention, "extracellular vesicle" refers to a membrane structure with a size of tens to hundreds of nanometers (preferably about 20 to 300 nm) composed of a double phospholipid membrane identical to the structure of a cell membrane (however, the particle size of exosomes may vary depending on the type of stem cell to be isolated, the isolation method, and the measurement method). Extracellular vesicles contain various sugars, proteins, miRNAs, mRNAs, DNA, etc. produced by the cell as cargo, and these cargoes are specific to the cell type. As such, since extracellular vesicles are secreted from cells containing biologically active substances produced by the cells, extracellular vesicles share the characteristics of the corresponding cells and thus can exert the biological activity of the cells themselves. In the present invention, "extracellular vesicle" encompasses exosomes and microvesicles, membrane vesicles, ectosomes, shedding vesicles, microparticles, or equivalents thereof.

[0077] The present invention also provides a cell therapeutic agent containing, as an active ingredient, mesenchymal stem cells overexpressing NQO1 and CXCL10-Fc fusion proteins or cells differentiated from said stem cells.

[0078] In this specification, the term "cell therapy" refers to a drug (under U.S. FDA regulations) used for therapeutic, diagnostic, and preventive purposes, consisting of cells and tissues produced by isolation, culture, and special processing from humans. It refers to a drug used for therapeutic, diagnostic, and preventive purposes through a series of actions, such as proliferating and / or selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or altering the biological characteristics of the cells by other means. Cell therapy products are broadly classified into somatic cell therapy products and stem cell therapy products depending on the degree of cell differentiation, and this invention relates particularly to stem cell therapy products.

[0079] In the present invention, the solid tumor may be selected from the group consisting of glioblastoma, glioblastoma, meningioma, neuroblastoma, oral cancer, laryngeal cancer, pharyngeal cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, osteosarcoma, and sarcoma.

[0080] In this specification, the term “composition” is considered to include not only a product containing a specific component, but also any product made directly or indirectly by the combination of a specific component.

[0081] The term “treatment” as used in this invention refers to any act of improving or beneficially altering the symptoms of a solid tumor by administering the pharmaceutical composition of this invention.

[0082] The above treatment for solid tumors may be applied to any mammal in which solid tumors may develop, and examples include humans and primates, as well as livestock such as cattle, pigs, sheep, horses, dogs, and cats without limitation, but preferably humans.

[0083] In the present invention, "administration" means introducing a specific substance to a patient by any appropriate method, and the route of administration of the compositions may be any general route as long as the drug can reach the target tissue. This may include, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, intranasal administration, intrapulmonary administration, rectal administration, etc.

[0084] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, wherein the term "pharmaceutically effective amount" in the present invention means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level may be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention used, and other factors well known in the medical field.

[0085] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered as a single or multiple doses. It is important to consider all of the above factors and administer an amount that obtains maximum effect with a minimum amount without side effects.

[0086] The dosage of the pharmaceutical composition of the present invention may be determined by a person skilled in the art by taking into consideration the purpose of use, the degree of toxicity of the disease, the age, weight, gender, medical history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered to mammals, including humans, at a dose of 0.0001 to 100 mg / kg per day, more preferably 0.1 to 1 mg / kg. The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once to three times a day or administered several times by dividing the dose.

[0087] The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for the treatment or prevention of solid tumors, further comprising a suitable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions, wherein the carrier may include a non-naturally occurring carrier.

[0088] Specifically, the above pharmaceutical composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, each according to conventional methods.

[0089] In the present invention, carriers, excipients, and diluents that may be included in the pharmaceutical composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0090] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0091] Liquid formulations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0092] The pharmaceutical composition of the present invention may be in the form of a sterile injectable formulation as a sterile injectable aqueous or oily suspension. This suspension may be formulated according to techniques known in the art using a suitable dispersant or wetting agent (e.g., Tween 80) and a suspending agent. The sterile injectable formulation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Vehicles and solvents that may be used permissibly include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile nonvolatile oils are typically used as solvents or suspension media. For this purpose, any nonvolatile oil with low irritation, including synthetic mono or diglycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in injectable formulations, as are pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially their polyoxyethylated forms.

[0093] The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration. These compositions may be prepared by mixing the compounds of the present invention with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperature. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0094] Parenteral administration of the pharmaceutical composition according to the present invention is particularly useful when the intended treatment involves a site or organ that is easily accessible by topical application. When applied topically to the skin, the pharmaceutical composition should be formulated as a suitable ointment containing an active ingredient suspended or dissolved in a carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petroleum jelly, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying waxes, and water. Alternatively, the pharmaceutical composition may be formulated as a suitable lotion or cream containing an active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The pharmaceutical composition of the present invention may also be applied topically to the lower intestinal tract by a rectal suppository or by a suitable enema. A locally applied transdermal patch is also included in the present invention.

[0095] The pharmaceutical composition of the present invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the field of pharmaceuticals and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and / or other solubilizing agents or dispersing agents known in the art.

[0096] The content of the above-mentioned preparation included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 0.0001 to 50 weight%, more preferably 0.01 to 10 weight% based on the total weight of the final composition.

[0097]

[0098] The present invention also provides a method for preventing or treating solid tumors, comprising the step of administering mesenchymal stem cells overexpressing NQO1 and CXCL10-Fc fusion proteins to a patient other than a human.

[0099] In the method for preventing or treating cancer according to the present invention, the mesenchymal stem cells and cancer overexpressing the NQO1 and CXCL10-Fc fusion proteins are the same as the concepts used in the pharmaceutical composition, so the description is replaced by the description thereof.

[0100] The method for the prevention or treatment of solid tumors according to the present invention comprises administering mesenchymal stem cells overexpressing the NQO1 and CXCL10-Fc fusion proteins to an individual at a therapeutically effective amount. It is preferable to apply a specific therapeutically effective amount for a specific individual differently depending on various factors and similar factors well known in the pharmaceutical field, including the type and degree of the response to be achieved, the specific composition including whether other agents are used in some cases, the individual's age, body weight, general health status, gender and diet, the time of administration, the route of administration and secretion rate of the composition, the duration of treatment, and drugs used together or simultaneously with the specific composition.

[0101] Therefore, it is preferable to determine the effective amount of mesenchymal stem cells overexpressing NQO1 and CXCL10-Fc fusion proteins suitable for the purpose of the present invention by taking into account the aforementioned matters.

[0102] The above patient is applicable to any mammal, and said mammal includes not only humans and primates, but also livestock such as cattle, pigs, sheep, horses, dogs, and cats.

[0103] [Example]

[0104] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples. Accordingly, the actual scope of the present invention is defined by the appended claims and their equivalents.

[0105]

[0106] Experimental method

[0107] Experimental materials

[0108] MSCs were produced by introducing the SV40 large T antigen into Bone Marrow-Derived MSCs (BM-MSCs) collected from C57BL6 mice using a lentiviral vector system. Specifically, bone marrow was collected from the femurs of 4–6 week old female C57BL6 mice and MSCs were obtained by subculturing the bone marrow in a culture dish at least three times. The SV40 large T antigen was introduced into these MSCs using the retroviral vector SSR #69. These MSCs expressed Sca-1, CD105, and CD44. Fetal bovine serum (FBS, cat no. S001-01) and antibiotics (Penicillin-Streptomycin (100X), cat no. LS202-02) for culturing these MSCs were purchased from Wellgene (Gyeongsan-si, Gyeongsangbuk-do, South Korea), respectively. The recombinant mouse CXCL10-human Fc (CXCL10-Fc) fusion protein was obtained by purifying it in a conditioning medium based on a plasmid introduced into the 293-F cell line via transfection using the lipofectamin 3000 transfection kit (cat no. L3000-008, invitrogen). Specifically, a plasmid containing the sequence encoding the recombinant mouse CXCL10-human Fc (CXCL10-Fc) fusion protein was expressed in the 293-F cell line to induce the recombinant mouse CXCL10-human Fc fusion protein to be secreted into the conditioning medium. From this conditioning medium, the recombinant mouse CXCL10-human Fc fusion protein was isolated using affinity chromatography with protein A. It was then separated from the chromatography column using 0.1M glycine buffer set to pH 3.0 and concentrated through an Amicon filter. The mouse NQO1 cDNA clone (MG57522-CY) was purchased and obtained from Sino Biological.Antibodies used in flow cytometry, Western blot, and enzyme immunoassay were obtained from Biolegend (San Diego, CA, USA), BD Biosciences (Franklin Lakes, NJ, USA), and ThermoFisher Scientific (Waltham, MA, USA). Annexin V / PI apoptosis detection was performed by BD Pharmaingen. TM The FITC Annexin V Apoptosis Detection Kit I (cat no. 556547, BD Biosciences) was used. Anti-CD3 antibody (cat no. 553057), anti-CD28 antibody (cat no. 102102), and human recombinant IL2 (cat no. 200-02-50UG) for T cell activation in an in vitro environment were obtained from Peprotech, BD Biosciences (Cranbury, NJ, USA), respectively.

[0109]

[0110] Preparation and culture of NMSC, IP10Fc-MSC, and IP10Fc-NMSC

[0111] MSCs (NMSC) overexpressing NQO1, IP10Fc-MSCs overexpressing CXCL10-Fc fusion protein, and IP10Fc-NMSCs overexpressing NQO1 and CXCL10-Fc fusion protein were prepared by constructing recombinant lentiviruses for the expression of NQO1, CXCL10-Fc fusion protein, and NQO1 and CXCL10-Fc fusion protein, respectively. To prepare the recombinant lentiviruses, HEK293FT cell lines (Invitrogen) were cultured and maintained in Dulbecco's Modified Eagle's Medium (DMEM; cat no. LM001-01, Wellgene) containing 10% FBS, penicillin, and a 1% antibiotic solution containing streptomycin. Lipofectamin 3000 transfection kit (cat no. L3000-008, Invitrogen) was used for lentivirus transfection.

[0112] Specifically, NQO1, CXCL10-Fc fusion protein, or NQO1 and CXCL10-Fc fusion protein expression vectors were prepared by inserting the sequences of SEQ NO. 1, SEQ NO. 2, or SEQ NO. 1 and SEQ NO. 2, respectively, between the NheI and EcoRI restriction enzyme sites of the FUW-EF1α-IRES-G418 plasmid or FUW-EF1α-IRES-Puro plasmid. 4 µg of each expression vector and 1.3 µg of each pRSV-Rev, pMDLg / pRRE, and pMD2.G packaging vector were combined with 10 µl of p3000 and 100 µl of Opti-mem TMI was added to Reduced Serum Medium (cat no. 31985062, Gibco). Separately, 10 µl of lipofectamine 3000 was added to 100 µl of Opti-mem, and the two solutions were mixed and reacted at room temperature for 20 minutes. This complex solution was added dropwise to the prepared HEK293FT cells and mixed uniformly. After culturing for 16 hours, the solution was replaced with 9 mL of fresh culture medium and the reaction was continued for an additional 32 hours.

[0113] After 32 hours, the supernatant was collected and stored at 4°C, and 9 mL of fresh culture medium was added to the cells. After 24 hours, the supernatant was collected, all supernatants were combined, and cell residues were removed by filtration through a 0.45 µm filter. Then, Lenti-X concentrator (cat no. 631232, Takara) was added in an amount equal to 1 / 3 of the total supernatant volume and treated for at least 16 hours. The treated supernatant was centrifuged at 1500 g for 45 minutes to obtain a lentivirus pellet, which was resuspended in 800 µL of PBS and stored at -80°C until use.

[0114] For the preparation of cell lines via transduction, 100 µl of a recombinant lentivirus vector suspension for expression was used. MSCs were cultured in a 6-well plate for at least 16 hours to allow attachment, and the medium was replaced with 2 ml of fresh medium. Two hours after the medium was replaced, an additional 1.9 ml of medium was added, and 1,5-Dimethyl-1,5-diazaundecamethyl-ene polymethobromide (cat no. TR-1003, Merck) was added to a concentration of 8 µg / ml. A cell line without the recombinant lentivirus vector suspension for expression was prepared in the same manner. Both cell lines were centrifuged at 1000g for 1 hour to precipitate virus particles, and the cells were cultured for 16 hours. After 16 hours, the medium was replaced with fresh medium. After 24 hours, transduced cells were selected based on the antibiotic resistance gene expressed in the introduced vector. This selection process was performed by treating with antibiotics for more than 7 days, and to ensure certainty in the selection process, antibiotics were treated for an additional 3 days after confirming through a microscope that all cell lines without the recombinant lentivirus vector suspension for expression had died.

[0115] The NMSC, IP10Fc-MSC, and IP10Fc-NMSC prepared in this way were each cultured in Dulbecco's Modified Eagle's Medium (DMEM; cat no. LM001-01, Wellgene) containing 10% FBS and a 1% antibiotic solution containing penicillin and streptomycin. Subsequently, they were plated in culture dishes of 35, 60, or 100 mm diameter in an incubator maintained at 37°C and 5% CO2.

[0116] A hypoxic culture chamber (LabMaster, Anseong-si, Gyeonggi-do, South Korea) was used to establish the stress environment. The establishment of the hypoxic environment followed the supplier's instructions. Serum-free medium was used to establish the serum deprivation environment.

[0117]

[0118] Preparation of samples for measuring CXCL10-Fc amounts by tissue type

[0119] Tissues were collected from the group administered IP10Fc-NMSC via peritumor injection or the group administered recombinant mouse CXCL10-human Fc fusion protein (IP10-Fc) via intratumoral injection. The collected tissues were homogenized using a homogenizer (MP Biosciences, Santa Ana, CA, USA). The supernatant of the homogenized tissue fluid was separated by centrifugation and analyzed using enzyme immunoassay.

[0120]

[0121] Preparation of samples for measuring T-cell numbers by tissue

[0122] Tissues were collected from the group administered IP10Fc-NMSC via peritumor injection or the group administered IP10-Fc via intratumoral injection. The collected tissues were converted into single-cell suspensions using a strainer (Corning, NY, USA) and DPBS containing 2% FBS and 0.5 mM EDTA. The tissue-specific single-cell suspensions were stained with antibodies conjugated with appropriate fluorescent dyes or labeled via CFSE, and then analyzed using a Novocyte Advanteon flow cytometer (Agilent, Santa Clara, CA, USA).

[0123]

[0124] Flow cytometry analysis

[0125] The prepared samples were stained using antibodies conjugated with appropriate fluorescent dyes. For staining, DPBS containing 2% FBS and 0.5 mM EDTA was used as the buffer, and the antibodies used are as follows (Table 1).

[0126] TargetFluorescent DyeManufacturerCD3BV785BiolegendCD4BV605BiolegendCD8BV421BiolegendCD44BV421BiolegendCD45PerCPBiolegendCD105PEBD BiosciencesSca-1APCBD BiosciencesViabilityeFlour506ThermoFisherScientific

[0127] Enzyme Immunoassay (ELISA)

[0128] The prepared samples were treated into 96-well plates coated with antibodies specific to human Fc. Prior to sample treatment, blocking was performed using 1% BSA for 1 hour. After sample treatment, the plates were incubated for 1 hour and 30 minutes, followed by three washes with DPBS containing 0.05% Tween20. Subsequently, the peroxidase-bound antibodies specific to human Fc were applied, and after 1 hour of incubation, the plates were washed three times with DPBS containing 0.05% Tween20. After washing was complete, TMB Substrate Solution (cat no. N301, ThermoFisher Scientific) was added, and the reaction was allowed to proceed by incubation at room temperature. When the reaction proceeded properly, the reaction was stopped using 3M HCl, and the amount of CXCL10-Fc fusion protein contained in the samples was measured by analyzing the absorbance. For the analysis, a serially diluted 1000 ng / mL human IgG solution was used as the standard solution.

[0129]

[0130] Transwell Migration Assay

[0131] A Transwell migration assay was performed using a Transwell 24-well plate with 5 µm pores (cat no. CLS3421, Corning). In the bottom chamber, 600 µl each of serum-free medium, serum-free medium supplemented with IP10-Fc at a concentration of 1 µg / ml, IP10Fc-NMSC, and MSC conditioning medium were added. The IP10Fc-NMSC and MSC conditioning media were obtained by culturing each cell type in a hypoxic environment for 48 hours. In the upper chamber, 0.8 x 10⁶ T cells activated for two days 6 200 µl of the suspension resuspended at a concentration of 1 / mL was added. After incubating for 6 hours following T cell treatment, the upper chamber was removed, and the number of T cells that fell into the bottom chamber was counted. To count the T cells, the bottom chamber was washed twice with PBS; the wash solution and culture medium were collected in one place and centrifuged at 500g for 5 minutes to obtain a T cell pellet. The T cell pellet was resuspended in PBS and incubated in Trypan Blue solution (cat no. T8154-100mL, Sigma-Aldrich) to stain dead cells. The total number of unstained cells was counted using a hemocytometer (cat no. HSU-0650010, Marienfeld superior).

[0132]

[0133] Annexin V / PI cell apoptosis detection

[0134] To evaluate changes in the pattern of apoptosis in MSCs under stress conditions due to NQO1 overexpression, Annexin V and PI double staining assays were performed. The concentrations of Annexin V and PI used followed the supplier's instructions. Cells aspirated in the same manner as in 1-8 were resuspended in commercially available binding buffer, and Annexin V-FITC and PI were added according to the supplier's instructions, respectively, followed by incubation at room temperature for 15 minutes. Subsequently, apoptosis was measured using a flow cytometer. Based on the measurement results, Annexin V-negative / PI-negative cells were classified as viable cells, Annexin V-positive / PI-negative cells as early-dead cells, Annexin V-negative / PI-positive cells as necrotic cells, and Annexin V-positive / PI-positive cells as late-dead cells.

[0135]

[0136] T-cell and MSC markers via CFSE

[0137] CellTrace to label T cells and MSCs TM The CFSE Cell Proliferation Kit (cat no. C34570, Invitrogen) was used. For CFSE labeling, 2 x 10 T cell suspensions were prepared using PBS containing 0.1% FBS. 7 At a concentration of / mL, the MSC suspension is 2x10 6 It was prepared at a concentration of 1 / mL. CFSE was added to the suspension to achieve a final concentration of 1.5 μM, and the reaction was incubated at room temperature for 8 minutes, after which the reaction was stopped using preheated FBS. After stopping the reaction, the cells were cultured in a water bath set at 37°C for 10 minutes and centrifuged at 500g for 5 minutes. The resulting pellet was washed twice with 2% FBS. The degree of labeling on the washed CFSE-labeled cells was confirmed using a flow cytometer.

[0138]

[0139] Tumor model

[0140] C57BL / 6NCrlOri mice from Orient Bio (Seongnam-si, Gyeonggi-do, South Korea) were used to construct the tumor models. The cell lines used were MC38, B16-OVA, and B16. To construct each tumor model, 1.0 x 10⁶ were administered via subcutaneous injection to 4-6 week old mice with dehaired flanks. 7 50 µl of a cancer cell line suspension prepared at a concentration of 1 ml was injected. When the injected cancer cells engrafted and formed a tumor, changes in size and survival rate were tracked.

[0141]

[0142] Adoptive cell delivery therapy in tumor models

[0143] In the constructed tumor model, the tumor size is 100 mm 3 Adoptive cell transfer was performed when [the condition was met]. T cells for adoptive cell transfer were activated by culturing them for 48 hours in T cell culture medium containing 2 μg / ml of anti-CD28 and 5 ng / ml of human recombinant IL-2 in culture dishes coated with anti-CD3 antibody. The T cell culture medium used was RPMI1640 liquid medium containing 55 μM β-Mercaptoethanol (cat no. 21985023, Gibco), 10% FBS, 1% sodium pyruvate (cat no. 11360070, Gibco), 1% Non-Essential Amino Acids Solution (cat no. 11140050, Gibco), 1% Penicillin-Streptomycin, and 20 mM HEPES (cat no. BB001-01, Wellgene). For cell delivery, an intravenous injection method was used with a 30G disposable sterile injection needle (cat no. K07415721, Seongsim Medical).

[0144]

[0145] Experimental results

[0146] Effect of NQO1 on the improvement of MSC adaptive capacity in stressful environments

[0147] To confirm the effect of NQO1 (NAD(P)H Quinone Oxidoreductase 1) on improving the adaptability of MSCs under various stress conditions, normal MSCs and MSCs overexpressing NQO1 (NMSCs) were cultured for 6 hours in various environments to evaluate apoptosis. The experimental conditions were hypoxic, serum-free, and hypoxic + serum-free environments. Annexin V / PI double staining analysis was performed on cells cultured for 6 hours under each condition. As a result, unlike normal MSCs, the proportion of cells undergoing apoptosis was significantly reduced in MSCs overexpressing NQO1 (NMSCs) (Figure 1). Specifically, this difference was particularly pronounced in the serum-free environment, with the proportion of cells undergoing apoptosis decreasing by 21.53% (P= 0.0001) in the serum-free environment and by 31.77% (P< 0.0001) in the hypoxic + serum-free environment. In addition, MSCs (NMSCs) overexpressing NQO1 showed a 59.45% (P= 0.0308) improved survival performance when delivered into the tumor compared to normal MSCs without overexpression (Fig. 2). Meanwhile, MSCs inherently possess a targeting property for tumors, and this property was equally expressed in MSCs (NMSCs) overexpressing NQO1 (Fig. 3).

[0148] In conclusion, the present invention confirmed that the adaptive ability within a tumor can be improved by reducing MSC apoptosis in various stress environments through the overexpression of NQO1 (Figs. 1-3).

[0149]

[0150] T cell recruiting effects following the secretion of NQO1 and CXCL10-Fc fusion proteins

[0151] To verify the production capacity of CXCL10-Fc fusion protein by MSCs overexpressing NQO1 and CXCL10-Fc fusion protein (IP10Fc-NMSC), normal MSCs and MSCs overexpressing NQO1 (NMSCs) were cultured in various environments for 48 hours, and the amount of CXCL10-Fc fusion protein (rmIP10Fc) was measured. As a result, the amount of CXCL10-Fc fusion protein (rmIP10Fc) was significantly higher in MSCs overexpressing NQO1 than in normal MSCs (Fig. 4). Specifically, increases in expression of 21.66% (P< 0.0001), 12.42% (P= 0.0003), 79.87% (P= 0.0112), and 36.13% (P= 0.0059) were confirmed in normal, hypoxic, serum-free, and hypoxic+serum-free environments, respectively. A transwell migration assay was performed to confirm the recruit effect of T cells. After culturing for 6 hours under each condition, the number of cells recruited into the bottom chamber was measured using a hemocytometer, and it was confirmed that the number of T cells significantly increased in the conditioned medium formed by IP10Fc-NMSC (Fig. 5). Specifically, the number of T cells increased by 92.96% (P= 0.0006) compared to the serum-free medium environment and by 33.33% (P= 0.0409) compared to the conditioned medium formed by MSCs; this level was not significantly different from the results in serum-free medium supplemented with 1 μg / mL of purified recombinant mouse CXCL10-human Fc fusion protein. When this IP10Fc-NMSC was introduced into the tumor, the extent of leaching of the delivered CXCL10-Fc fusion protein (rmIP10Fc) into the serum and other organs was significantly reduced (Figs. 6-7). Specifically, the concentration in the serum decreased from 2.70 ± 0.35 μg / mL when administered directly via intratumoral injection to 0.67 ± 0.17 μg / mL (P= 0.0057).In addition, while no significant change in the amount of fusion protein per unit mass was observed in the tumor, it was confirmed that it decreased by 86.77% (P= 0.0023), 97.81% (P= 0.0016), 85.23% (P= 0.0016), 95.55% (P= 0.0022), and 99.99% (P= 0.0079) in the lung, liver, heart, kidney, and spleen, respectively. Based on this, the tissue infiltration assay results also confirmed that the T cell induction performance by IP10Fc-NMSC was present in the B16 tumor (Figs. 8-9). Specifically, unlike in the lung and peritumoral lymph nodes, the number of endogenous T cells and adoptive T cells per unit mass in the tumor increased by 61.94% (P= 0.1075) and 85.06% (P= 0.038), respectively, compared to when standard MSCs were injected. Likewise, compared to when the fusion protein was directly administered via intratumoral injection, the number increased by 75.00% (P= 0.0366) and 89.51% (P= 0.0395), respectively. When these numbers were normalized to the number of T cells per unit mass in the lung, a normal organ, the number increased by 66.04% (P= 0.0182) and 84.50% (P= 0.006), respectively, compared to when general MSCs were injected, and by 87.69% (P= 0.0034) and 90.57% (P= 0.0058), respectively, compared to when the fusion protein was directly administered via intratumoral injection.

[0152]

[0153] Enhancement of the therapeutic effect of adoptive cell delivery therapy using MSCs overexpressing NQO1 and CXCL10-Fc fusion proteins

[0154] To determine whether the therapeutic effect of adoptive cell delivery therapy could be enhanced by the T cell recruiting performance of MSCs (IP10Fc-NMSC) overexpressing NQO1 and CXCL10-Fc fusion proteins, IP10Fc-NMSC was injected via peritumor injection into mice injected with a B16-OVA tumor the day before adoptive cell delivery therapy, and changes in tumor growth curves and survival rates were tracked. As a result, significant differences were observed in the tumor growth curve (P= 0.0037) and survival rate curve (P= 0.0466) (Figs. 10-11).

[0155] In conclusion, it was confirmed that using mesenchymal stem cells according to the present invention can induce efficient infiltration of T cells into a tumor through the overexpression of CXCL10-Fc fusion protein, thereby enabling the treatment of solid tumors (Figs. 6-11).

[0156]

[0157] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0158] According to the present invention, mesenchymal stem cells capable of enhancing adaptability to stressful environments and promoting the infiltration of CD8 T cells into solid tumors can be provided, and thus can be usefully utilized in the field of cell therapy effective for treating solid tumors.

Claims

1. NQO1 (NAD(P)H Quinone Oxidoreductase 1); and Mesenchymal stem cells overexpressing a CXCL10-Fc fusion protein in which CXCL10 (CXC motif chemokine ligand 10) and an immunoglobulin (Ig)-derived Fc region are linked.

2. In Paragraph 1, Mesenchymal stem cells characterized by being genetically engineered to increase the expression of the above NQO1 and the above CXCL10-Fc fusion protein.

3. In Paragraph 2, Mesenchymal stem cells characterized by being obtained by transfecting a recombinant expression vector comprising a polynucleotide encoding the above NQO1 and the above CXCL10-Fc fusion protein.

4. In Paragraph 3, Mesenchymal stem cells characterized by the above-mentioned recombinant expression vector being selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vacciniavirus vectors, herpesvirus vectors, lentivirus vectors, and avifoxvirus vectors.

5. In Paragraph 3, A mesenchymal stem cell characterized in that the polynucleotide encoding the above NQO1 comprises a base sequence represented by SEQ ID NO.

1.

6. In Paragraph 3, A mesenchymal stem cell characterized in that the polynucleotide encoding the above CXCL10-Fc fusion protein comprises the nucleotide sequence represented by SEQ ID NO.

2.

7. In Paragraph 1, The mesenchymal stem cells are characterized by being one or more selected from the group consisting of bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, embryonic stem cell-derived mesenchymal stem cells, and induced pluripotent stem cell-derived mesenchymal stem cells.

8. In Paragraph 1, Mesenchymal stem cells characterized by improved ability to adapt to stressful environments.

9. In Paragraph 8, A mesenchymal stem cell characterized in that the above stress environment is one or more selected from the group consisting of a hypoxic environment, a nutrient-deficient environment, a hypoxic environment and a nutrient-deficient environment, an oxidative stress environment, an endoplasmic reticulum stress environment, and a mitochondrial stress environment.

10. A method for producing mesenchymal stem cells comprising the step of transfecting a recombinant expression vector comprising NQO1 (NAD(P)H Quinone Oxidoreductase 1); and a polynucleotide encoding a CXCL10-Fc fusion protein in which an Fc region derived from immunoglobulin (Ig) is linked to CXCL10 (CXC motif chemokine ligand 10).

11. In Paragraph 10, A method for producing mesenchymal stem cells characterized in that the polynucleotide encoding the above NQO1 comprises a base sequence represented by SEQ ID NO.

1.

12. In Paragraph 10, A method for producing mesenchymal stem cells, characterized in that the polynucleotide encoding the above CXCL10-Fc fusion protein comprises the nucleotide sequence represented by SEQ ID NO.

2.

13. A pharmaceutical composition for the prevention or treatment of solid tumors comprising, as an active ingredient, one or more selected from the group consisting of mesenchymal stem cells according to claim 1, cells differentiated from said mesenchymal stem cells, and components derived from said mesenchymal stem cells.

14. In Paragraph 13, A pharmaceutical composition for the prevention or treatment of solid tumors, characterized in that the above-mentioned solid tumor is selected from the group consisting of glioblastoma, glioblastoma, meningioma, neuroblastoma, oral cancer, laryngeal cancer, pharyngeal cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, osteosarcoma, and sarcoma.

Images