KR20230077650A - Natural killer cells-specific chimeric antigen receptor and the use thereof - Google Patents

Abstract

The present invention relates to a natural killer cell-specific chimeric antigen receptor (NK-CAR), and the like, wherein an NK-CAR comprising a specific combination of natural killer cell-specific activation receptor-derived domains effectively activates natural killer cells to target cells. It was completed by confirming that it could enhance the killing ability. Specifically, the inventors prepared three natural killer cell-specific CARs composed of combinations of specific domains, and natural killer cells expressing the NK-CAR produced cytokines and granzyme B in response to target cells. It was confirmed that the activation of AKT and ERK was further enhanced, and degranulation was promoted to exert a more excellent apoptotic activity. That is, since the NK-CAR according to the present invention can enhance the apoptotic activity by strongly inducing the activation of natural killer cells, it can be used as an immunotherapy for the treatment of various diseases including cancer. In particular, CAR-based anti-cancer treatment using natural killer cells does not cause cytokine release syndrome or neurotoxicity, and since autologous and allogeneic natural killer cells can be used, it has the advantage of having fewer side effects than CAR-T. Therefore, natural killer cells expressing NK-CAR according to the present invention are expected to be used as cell therapy agents with maximized disease treatment effects while reducing side effects.

Description

Natural killer cell-specific chimeric antigen receptor and the use thereof {Natural killer cells-specific chimeric antigen receptor and the use thereof}

The present invention relates to natural killer cell-specific chimeric antigen receptors, natural killer cells expressing the same, and uses thereof.

Cancer is one of the diseases that account for the largest share of the causes of death of modern people. It is a disease caused by changes in normal cells due to gene mutations, etc. refers to something Cancer is particularly characterized by "uncontrolled cell growth", and by this abnormal cell growth, a cell mass called a tumor is formed and invades surrounding tissues and, in severe cases, may metastasize to other organs of the body. . Cancer is an intractable chronic disease that in many cases cannot be fundamentally cured even when treated with surgery, radiation, and drug therapy, causing pain to patients and ultimately leading to death. In particular, in recent years, the incidence of cancer in the world is increasing by more than 5% every year due to the increase in the elderly population and environmental degradation. It is estimated that 10,000 people will die of cancer.

Cancer drug treatment, that is, anticancer drugs, are generally cytotoxic compounds that treat cancer by attacking and killing cancer cells, but show high side effects because they damage normal cells as well as cancer cells. Therefore, targeted anticancer drugs have been developed to reduce side effects. However, in the case of these targeted anticancer drugs, although side effects could be lowered, they showed a limitation in that resistance developed with a high probability. Therefore, interest in immuno-cancer agents that reduce problems due to toxicity and resistance by using the body's immune system is rapidly increasing. As an example of such an immune anti-cancer agent, an immune checkpoint inhibitor has been developed that specifically binds to PD-L1 on the surface of cancer cells, inhibits the binding of T cells to PD-1, activates T cells, and makes them attack cancer cells. However, even in the case of these immune checkpoint inhibitors, since the types of cancer exhibiting effects are not diverse, there is an urgent need to develop new immune checkpoint inhibitors that exhibit the same therapeutic effect in various cancers.

Recently, studies on anti-cancer immune cell therapy in which cancer-attacking immune cells are directly injected into the body to induce cancer cell death have been actively conducted. Natural killer cells (NK cells), one of the representative immune cells used in anticancer immune cell therapy, are a type of cytotoxic lymphocyte responsible for the innate immune response. Morphologically, they have large granules in the cytoplasm, It accounts for 5 to 20% of blood lymphocytes. Natural killer cells have various immunoreceptors on their surface, so they can distinguish between cancer cells and normal cells, so they have the advantage of being able to immediately detect and remove cancer cells. In particular, natural killer cells not only inhibit the occurrence, proliferation, and metastasis of cancer cells and virus-infected cells, but also can effectively remove cancer stem cells, so they can prevent the recurrence of cancer caused by cancer stem cells and are considered effective anti-cancer immune cells. . In addition, various clinical studies have shown that when natural killer cells isolated from relatives or normal people are introduced into patients, the immune rejection reaction is extremely low compared to other immune cells, and thus the possibility of using them as cell therapy agents is attracting attention.

On the other hand, chimeric antigen receptors (CARs) are artificial receptors designed to deliver antigen specificity to immune cells, and they include antigen-specific components, transmembrane components selected to activate immune cells and provide specific immunity. , and intracellular components. Recently, cancer immunotherapy using cells into which a gene encoding such a chimeric antigen receptor has been introduced has been attempted. The most representative immunotherapy is a method of treating cancer by collecting T cells from a patient, introducing and amplifying a gene encoding a chimeric antigen receptor, and translocating the gene back to the patient, that is, therapy using CAR-T cells. am. However, despite having excellent anticancer effects, CAR-T cells have side effects such as cytokine release syndrome or neurotoxicity, and there is a limitation in that autologous T cells must be used to prevent immune rejection. Furthermore, there are problems in that the manufacturing process of autologous CAR-T is very complicated, the manufacturing cost is considerable, and there are still problems of side effects on immunity. In order to overcome this, research on an allogenic off-the-shelf CAR-T is being conducted, but a CAR-T capable of solving the above problems has not yet been developed.

Therefore, in recent years, natural killer cell-based treatment "CAR-NK", which has less risk of immune side effects compared to CAR-T and is easy to manufacture allogeneic cell therapy products, is attracting attention. Compared to other immune cells, natural killer cells have a wider range of cancer cell recognition, and can produce allogeneic cells without separate genetic manipulation. In addition, there is an advantage in that allogeneic cells can be used. In addition, unlike T cells, NK cells are more efficient in that they can be provided off-the-shelf and do not need to be customized for individual patients. It is expected that it will be applicable to -T-resistant carcinoma.

In other words, CAR-NK therapeutics using NK cells can be mass-produced, which can reduce the cost of treatment, and is considered an alternative treatment that can reduce solid cancer treatment and cytokine side effects due to low immunogenicity. However, studies on chimeric antigen receptors to date have been mostly based on T cell components, and it is difficult to apply them to NK cells. Therefore, there is a need to develop new chimeric antigen receptors tailored to the characteristics of NK cells.

Republic of Korea Patent Registration No. 10-1755431

The present invention was made to solve the above problems, and a chimeric antigen receptor comprising a specific combination of natural killer cell-specific activating receptor domains effectively activates natural killer cells to promote target cell (e.g., cancer cell) killing activity It is done by confirming that it can be done.

Accordingly, an object of the present invention is to provide a natural killer cell-specific chimeric antigen receptor.

Another object of the present invention is to provide natural killer cells expressing the chimeric antigen receptor.

Another object of the present invention is to provide a nucleic acid molecule encoding the chimeric antigen receptor and/or an expression vector containing the same.

Another object of the present invention is to provide a cell transformed with the expression vector.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, containing natural killer cells expressing the chimeric antigen receptor as an active ingredient.

Another object of the present invention is to provide a cell therapy agent for preventing or treating cancer, comprising natural killer cells expressing the chimeric antigen receptor as an active ingredient.

Another object of the present invention is (S1) transforming the expression vector into isolated cells; and

(S2) To provide a method for producing a natural killer cell-specific chimeric antigen receptor, comprising culturing the transformed cell obtained in step (S1).

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

The present invention provides a natural killer cell-specific chimeric antigen receptor, comprising: (i) an antigen-binding domain; (ii) a CD8 or CD28 hinge domain; (iii) DAP10 intracellular signaling domain; (iv) a 2B4 intracellular signaling domain; and (v) a CD3z intracellular signaling domain, wherein the chimeric antigen receptor is expressed in natural killer cells.

In one embodiment of the present invention, the antigen-binding domain may be a tumor antigen-specific antibody or an antigen-binding fragment thereof, but is not limited thereto.

In another embodiment of the present invention, the chimeric antigen receptor may satisfy one or more of the following characteristics, but is not limited thereto:

(a) the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 2;

(b) the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 3;

(c) the DAP10 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7;

(d) the 2B4 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 8; and

(e) the CD3z intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 9.

In another embodiment of the present invention, the chimeric antigen receptor may further include a CD28 transmembrane domain, but is not limited thereto.

In another embodiment of the present invention, the CD28 transmembrane domain may include the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

In another embodiment of the present invention, the chimeric antigen receptor may further include at least one selected from the group consisting of a DAP10 extracellular domain and a DAP10 transmembrane domain, but is not limited thereto.

In another embodiment of the present invention, the chimeric antigen receptor may satisfy one or more of the following characteristics, but is not limited thereto:

(f) the DAP10 extracellular domain comprises the amino acid sequence of SEQ ID NO: 5; and

(g) the DAP10 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6.

In another embodiment of the present invention, the chimeric antigen receptor may further include a signal peptide, but is not limited thereto.

In another embodiment of the present invention, the signal peptide may be a CD8 signal peptide, but is not limited thereto.

In another embodiment of the present invention, the tumor antigen is CD19, TAG72, IL13Rα2 (Interleukin 13 receptor alpha-2 subunit), CD52, CD33, CD20, TSLPR, CD22, CD30, GD3, CD171, NCAM (Neural cell adhesion molecule ), FBP (Folate binding protein), Le(Y) (Lewis-Y antigen), PSCA (Prostate stem cell antigen), PSMA (Prostate-specific antigen membrane), CEA (Carcinoembryonic antigen), HER2 (Human epidermal growth factor receptor) 2),　Mesothelin,　CD44v6 (Hyaluronate receptor variant 6), B7-H3, Glypican-3, ROR1 (receptor tyrosine kinase like orphan receptor 1), Survivin, FOLR1 (folate receptor 1), WT1 (Wilm's tumor 1), VEGFR2 ( It may be one or more selected from the group consisting of vascular endothelial growth factor 2), EGFR, and KRAS, but is not limited thereto.

In another embodiment of the present invention, the antigen-binding fragment may be selected from the group consisting of scFv, (scFv) ₂ , Fab, Fab' and F(ab') ₂ , but is not limited thereto.

In another embodiment of the present invention, the tumor antigen-specific antibody or antigen-binding fragment thereof may include the following heavy chain variable region and light chain variable region, but is not limited thereto:

(i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28; and

(ii) a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

In another embodiment of the present invention, the tumor antigen-specific antibody or antigen-binding fragment thereof may include the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

In addition, the present invention provides natural killer cells expressing the natural killer cell-specific chimeric antigen receptor according to the present invention. That is, the present invention provides a CAR-NK cell containing (expressing) the chimeric antigen receptor.

In one embodiment of the present invention, the natural killer cells may satisfy one or more characteristics selected from the group consisting of, but are not limited thereto:

(a) increased secretion of one or more selected from the group consisting of MIP-1α, granzyme B, and INF-γ upon antigen recognition;

(b) an increase in one or more activities selected from the group consisting of AKT signaling and ERK signaling upon antigen recognition; and

(c) Increased level of degranulation upon antigen recognition.

In addition, the present invention provides a nucleic acid molecule encoding a natural killer cell-specific chimeric antigen receptor according to the present invention.

In addition, the present invention provides an expression vector containing the nucleic acid molecule.

In addition, the present invention provides an isolated cell into which the expression vector is introduced.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising natural killer cells expressing natural killer cell-specific chimeric antigen receptors according to the present invention as an active ingredient. Preferably, the antigen-binding domain of the chimeric antigen receptor is a tumor antigen-specific antibody or antigen-binding fragment thereof.

In addition, the present invention provides a cell therapy agent for preventing or treating cancer, comprising natural killer cells expressing a natural killer cell-specific chimeric antigen receptor according to the present invention as an active ingredient.

In addition, the present invention provides a method for preventing or treating cancer, comprising administering natural killer cells expressing the natural killer cell-specific chimeric antigen receptor to a subject in need thereof.

In addition, the present invention provides the natural killer cell-specific chimeric antigen receptor, a nucleic acid molecule encoding the chimeric antigen receptor, an expression vector containing the nucleic acid molecule, and the introduction of the expression vector for the prevention or treatment of cancer. Provided are the uses of the cells, and/or natural killer cells expressing the chimeric antigen receptor.

In addition, the present invention provides the natural killer cell-specific chimeric antigen receptor, a nucleic acid molecule encoding the chimeric antigen receptor, an expression vector containing the nucleic acid molecule, and the expression vector for the production of a drug for cancer treatment. The use of the introduced cells and/or natural killer cells expressing the chimeric antigen receptor is provided. The drug may be a cell therapy agent for cancer treatment.

In one embodiment of the present invention, the cancer is colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharynx cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer , tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, hematological cancer, squamous cell carcinoma, adenocarcinoma of the lung, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine It may be at least one selected from the group consisting of adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, gastrointestinal cancer, glioblastoma, ovarian cancer, endometrial cancer, salivary gland cancer, vulvar cancer, and head and neck cancer, but is not limited thereto. .

The present invention also includes (S1) introducing the expression vector into an isolated cell; and (S2) culturing the cells into which the expression vector has been introduced.

The present invention also provides a method for producing natural killer cells expressing a natural killer cell-specific chimeric antigen receptor, comprising introducing the expression vector into the isolated natural killer cells.

The present invention relates to a natural killer cell-specific chimeric antigen receptor (NK-CAR), and the like, wherein an NK-CAR comprising a specific combination of natural killer cell-specific activation receptor-derived domains effectively activates natural killer cells to target cells. It was completed by confirming that it could enhance the killing ability. Specifically, the inventors prepared three natural killer cell-specific CARs composed of combinations of specific domains, and natural killer cells expressing the NK-CAR produced cytokines and granzyme B in response to target cells. It was confirmed that the activation of AKT and ERK was further enhanced, and degranulation was promoted to exert a more excellent apoptotic activity. That is, since the NK-CAR according to the present invention can enhance the apoptotic activity by strongly inducing the activation of natural killer cells, it can be used as an immunotherapy for the treatment of various diseases including cancer. In particular, CAR-based anti-cancer treatment using natural killer cells does not cause cytokine release syndrome or neurotoxicity, and since autologous and allogeneic natural killer cells can be used, it has the advantage of having fewer side effects than CAR-T. Therefore, natural killer cells expressing NK-CAR according to the present invention are expected to be used as cell therapy agents with maximized disease treatment effects while reducing side effects.

1 shows the structures of chimeric antigen receptors NK-CAR1, NK-CAR2, and NK-CAR3 according to the present invention, and a control 2 ^nd generation CAR.
Figure 2a shows the result of confirming the ratio of GFP and NK-CAR expressing cells 3 days after infection of NKL cells with the NK-CAR retrovirus according to the present invention by flow cytometry.
Figure 2b shows the result of confirming the ratio of GFP and NK-CAR expressing cells among NKL cells by isolating and culturing GFP-expressing cells 3 days after retrovirus infection by flow cytometry.
Figure 2c shows the result of confirming the ratio of GFP and NK-CAR expressing cells 3 days after infection of NK-92 cells with the NK-CAR retrovirus according to the present invention by flow cytometry.
FIG. 2D shows the results obtained by isolating and culturing GFP-expressing cells from NK-92 cells 3 days after retrovirus infection, and then confirming the ratio of GFP- and NK-CAR-expressing cells among them by flow cytometry.
Figure 3a shows the results of measuring the apoptotic activity of an empty vector, a control 2 ^nd CAR, or NK-CAR expressing NKL cells according to the present invention on a CD19 positive REH cell line by Europium assay (EV: empty vector, hereinafter the same) .
Figures 3b and 3c show the apoptotic activity of NK92 cells expressing an empty vector, a control 2 ^nd CAR, or an NK-CAR according to the present invention against CD19 positive REH cell line (Fig. 3b) and RAMOS cell line (Fig. 3c) Europium assay Indicates the result of measurement with .
4a and 4b show MPI-1α (FIG. 4a) and granzyme secreted from NKL cells after co-culture of an empty vector, a control 2 ^nd CAR, and CAR-NK expressing NKL cells according to the present invention with a CD19 positive REH cell line, respectively. B (Fig. 4b) shows the results of measuring the level by ELISA.
Figure 5a shows the result of confirming the degree of activation of AKT and ERK signals by Western blot after stimulating NKL cells or NK-CAR expressing NKL cells with CD19-positive REH cells for 2 or 5 minutes.
Figure 5b shows the result of confirming the activation of AKT and ERK signals by Western blot after stimulating NKL cells or NK-CAR expressing NKL cells with recombinant human CD19 Fc chimera for 2 or 5 minutes.
Figure 6 shows the results of measuring the degree of degranulation of NK92 cells after co-culture of NK92 cells expressing an empty vector, a control 2 ^nd CAR, or an NK-CAR according to the present invention with CD19-positive cells (REH or Ramos). indicate
Figure 7 shows the ratio of Interferon-γ-positive NK92 cells after co-culture of NK92 cells expressing an empty vector, a control 2 ^nd CAR, or an NK-CAR according to the present invention with CD19-positive cells (REH or Ramos). show the result.
8 shows an example of a preferred amino acid sequence of NK-CAR1.
9 shows an example of a preferred amino acid sequence of NK-CAR2.
10 shows an example of a preferred amino acid sequence of NK-CAR3.

The present invention relates to a natural killer cell (NK cell)-specific chimeric antigen receptor (CAR), etc., wherein a chimeric antigen receptor containing a specific combination of natural killer cell-specific activated receptor domains is It was completed after confirming that it is effective in activating natural killer cells and can induce cancer cell specific death.

Specifically, in one embodiment of the present invention, a retroviral vector of three NK-CAR genes and a T cell-based CAR (control) gene according to the present invention was constructed (Example 1), and a retrovirus into which the vector was introduced was used to express chimeric antigen receptors in natural killer cells (NKL cells and NK92 cells) (Example 2).

In another embodiment of the present invention, as a result of evaluating the cancer cell killing activity of NK-CAR expressing NKL cells and NK92 cells, both NK-CAR expressing NKL cells and NK92 cells kill target cells (cancer cells) more effectively than the control group. confirmed (Example 3).

In another embodiment of the present invention, as a result of evaluating the cytokine secretion ability of NK-CAR-expressing NKL cells, when NK-CAR-expressing NKL cells were stimulated with target cells (cancer cells), MIP-1α and granzyme B levels were decreased compared to the control group. It was confirmed that the amount of secretion was further increased (Example 4).

In another embodiment of the present invention, as a result of evaluating the degree of AKT and ERK signal activation according to target antigen recognition of NK-CAR-expressing NKL cells, NK-CAR-expressing NKL cells were stimulated with target antigen protein or cancer cells expressing target antigen When compared to the control group, it was confirmed that the phosphorylation levels of AKT and ERK were further increased (Example 5).

In another example of the present invention, it was confirmed that NK-CAR-expressing NK92 cells showed a higher degree of degranulation according to target antigen recognition than the control group (Example 6).

In another embodiment of the present invention, it was confirmed that NK-CAR-expressing NK92 cells produced a higher degree of cytokine production according to target antigen recognition than the control group (Example 7).

That is, the natural killer cell-specific chimeric antigen receptor according to the present invention can more effectively activate natural killer cells in response to a target and enhance their apoptotic activity, thereby preventing and treating various diseases including cancer. It is expected to be used as a new immunotherapy method.

Hereinafter, the present invention will be specifically described.

The present invention provides a natural killer cell-specific chimeric antigen receptor characterized in that it is expressed in natural killer cells.

In the present invention, "chimeric antigen receptor (CAR)" refers to synthetic receptors capable of targeting a specific antigen. The CAR according to the present invention has the structure of a receptor including an antigen-binding domain, a transmembrane domain, and a cytoplasmic domain, preferably a hinge region ( hinge region).

In the present invention, "antigen-binding domain" refers to a protein or polypeptide domain capable of specifically recognizing and binding a target antigen. In the present invention, "antigen" refers to a polypeptide, compound, or other substance that can specifically bind to humoral immune mediators such as antibodies or cellular immune mediators such as T cell receptors. do.

The antigen according to the present invention is not limited to a specific type, and those skilled in the art can appropriately select an appropriate antigen and an antigen-binding domain capable of specifically binding to the antigen according to the purpose. For example, in the case of preventing or treating an infectious disease caused by a bacterium or virus, an antigen-binding domain specific to the bacterium or virus-specific protein may be selected.

Preferably, the antigens are tumor antigens. The tumor antigens include both tumor-specific antigens that are expressed only in cancer cells as well as tumor-associated antigens that are expressed in normal cells but are expressed at a particularly high frequency or are more active in cancer cells. do. For example, the tumor antigen may be a surface protein expressed only in cancer cells or at a higher frequency in cancer cells. The tumor antigen is not limited to specific types, but preferably, CD19, TAG72, IL13Rα2 (Interleukin 13 receptor alpha-2 subunit), CD52, CD33, CD20, TSLPR, CD22, CD30, GD3, CD171, NCAM (Neural cell adhesion molecule), FBP (Folate binding protein), Le(Y) (Lewis-Y antigen), PSCA (Prostate stem cell antigen), PSMA (Prostate-specific membrane antigen), CEA (Carcinoembryonic antigen), HER2 (Human epidermal growth factor receptor 2),　Mesothelin,　CD44v6 (Hyaluronate receptor variant 6), B7-H3, Glypican-3, ROR1 (receptor tyrosine kinase like orphan receptor 1), Survivin, FOLR1 (folate receptor 1), WT1 (Wilm's tumor 1) , VEGFR2 (Vascular endothelial growth factor 2), EGFR (Epidermal growth factor receptor), and KRAS.

In the present invention, the "hinge region" refers to a region that is located between the antigen-binding domain and the transmembrane domain and serves as a flexible linker, and is also referred to as a "spacer". The hinge region allows the antigen-binding domain to be properly positioned when the antigen-binding domain binds to an antigen to form a stable binding with the antigen. For example, the hinge region has the purpose of extending an antigen binding domain from the cell membrane of a cell expressing the CAR.

In the present invention, "transmembrane domain (TM)" means any polypeptide or oligopeptide that functions to connect extracellular and intracellular signaling domains across cell membranes. The transmembrane domain can penetrate the cell membrane so that the antigen-binding domain of the chimeric antigen receptor is located on the cell surface (outside the cell) and the intracellular signaling domain is located inside the cell. That is, the transmembrane domain serves as a support for the chimeric antigen receptor and at the same time connects the antigen binding domain (or hinge domain) and the intracellular signaling domain.

In the present invention, "intracellular signaling domain (cytoplasmic domain or cytoplasmic signaling domain, CYP)" means a polypeptide or oligopeptide inside the cell membrane. The intracellular signaling domain serves to receive the signal transmitted by the antigen-binding domain and transmit the signal into the cell expressing the CAR. The signal is transmitted inside the cell and causes activation or inhibition of a biological process. The intracellular signaling domain is not particularly limited to that type, as long as it transmits a signal capable of inducing activation of NK cells when the antigen-binding domain binds to an antigen. In the present invention, the intracellular signaling domain may include one or more intracellular co-stimulatory domains. The co-stimulatory domain is located in the extracellular or intracellular portion of the CAR and serves to transmit signals to cells expressing the CAR. That is, the co-stimulatory domain contributes to inducing a sufficient response (activation) of NK cells according to the binding of the target antigen. The co-stimulatory domain is, for example, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and / or may be selected from B7-H3 derived domains, but is not limited thereto.

Each domain of the chimeric antigen receptors according to the present invention may optionally be linked by a short oligopeptide or polypeptide linker. The linker is not particularly limited in its length or type, as long as it can induce NK cell activation through the intracellular domain when the target antigen binds to the extracellular antigen-binding domain, and linkers known in the art can be applied without limitation. can

Each domain of the chimeric antigen receptor according to the present invention may include each of the above domains as well as a modified form of each of the above domains. In this case, the modification may be performed by substituting, deleting, or adding one or more amino acids in the amino acid sequence of the wild-type antibody and domain without modifying the functions of the antibody and domain. Typically, the substitution is an alanine, or can be made by conservative amino acid substitution that does not affect the charge, polarity or hydrophobicity of the entire protein.

The type of immune cells (T cells, NK cells, NKT cells, macrophages, etc.) in which the chimeric antigen receptor according to the present invention can be expressed is not limited. Preferably, the CAR of the present invention is a natural killer cell-specific CAR and is characterized in that it is expressed in natural killer cells. Preferably, the CAR according to the present invention is characterized in that it is located in the cell membrane of natural killer cells. That is, the CAR according to the present invention is specialized for activating the function of natural killer cells, and when combined with a target antigen, activates a signaling pathway that activates the target cell death function of natural killer cells, resulting in natural killer cells induces to specifically kill target cells. The chimeric antigen receptor according to the present invention can be expressed in allogeneic cells as well as autologous cells of the subject to be administered.

The hinge domain of the chimeric antigen receptor according to the present invention may be a domain derived from CD8, CD28, IgG1, IgG4, and/or KIR (killer immunoglobulin-like receptor), but is not limited thereto, and commonly used in the art. Hinge domains can be applied without limitation.

Preferably, in the present invention, the hinge domain may be a CD8 (preferably, CD8α) hinge domain. Preferably, the CD8 hinge domain may include the amino acid sequence of SEQ ID NO: 2, more preferably may consist of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto, and variants of the amino acid sequence are within the scope of the present invention. included within That is, the CD8 hinge domain has 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 2, respectively. It may contain an amino acid sequence.

Alternatively, the hinge domain of the chimeric antigen receptor in the present invention may be a CD28 hinge domain. Preferably, the CD28 hinge domain includes the amino acid sequence of SEQ ID NO: 3 or may be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto. That is, the CD28 hinge domain has 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 3, respectively. It may contain an amino acid sequence.

The intracellular signaling domain of the chimeric antigen receptor according to the present invention may be a DAP10, 2B4, and/or CD3z-derived domain, but is not limited thereto, and can transmit a signal that activates NK cells by stimulating a target antigen. Anything can be applied without limitation.

In the present invention, the DAP10 intracellular signaling domain may include the amino acid sequence of SEQ ID NO: 7 or be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, but is not limited thereto. That is, the DAP10 intracellular signaling domain is 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more of the amino acid sequence represented by SEQ ID NO: 7. It may contain homologous amino acid sequences.

In addition, in the present invention, the 2B4 intracellular signaling domain may include the amino acid sequence of SEQ ID NO: 8 or be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8, but is not limited thereto. That is, the 2B4 intracellular signaling domain is 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more of the amino acid sequence represented by SEQ ID NO: 8. It may contain homologous amino acid sequences.

In the present invention, the CD3z (CD3ζ) intracellular signaling domain may include the amino acid sequence of SEQ ID NO: 9 or be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 9, but is not limited thereto. That is, the CD3z intracellular signaling domain is 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more of the amino acid sequence represented by SEQ ID NO: 9, respectively. It may contain homologous amino acid sequences.

Preferably, the chimeric antigen receptor according to the present invention may further include a CD28 transmembrane domain. In addition, the CD28 transmembrane domain may include the amino acid sequence of SEQ ID NO: 4 or be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. That is, the CD28 transmembrane domain has 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 4, respectively. The branch may include an amino acid sequence.

Preferably, the chimeric antigen receptor according to the present invention may further include a DAP10 extracellular domain. In addition, the DAP10 extracellular domain may include the amino acid sequence of SEQ ID NO: 5 or be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5, but is not limited thereto. That is, the DAP10 extracellular domain has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the amino acid sequence represented by SEQ ID NO: 5. The branch may include an amino acid sequence.

Preferably, the chimeric antigen receptor according to the present invention may further include a DAP10 transmembrane domain. In addition, the DAP10 transmembrane domain may include the amino acid sequence of SEQ ID NO: 6 or be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, but is not limited thereto. That is, the DAP10 transmembrane domain has 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 6, respectively. The branch may include an amino acid sequence.

Preferably, the chimeric antigen receptor according to the present invention may further include a signal peptide. In the present invention, the "signal peptide" is an amino acid sequence located at the N-terminus of a protein, and serves to induce the newly synthesized protein to move to a specific location such as the endoplasmic reticulum (ER). In the present invention, the signal peptide may be derived from a molecule selected from, for example, CD8 (CD8α), GM-CSF receptor α, Ig-κ, and IgG1 heavy chain, but is not limited thereto. Preferably, in the present invention, the signal peptide may be a CD8 signal peptide. In addition, the CD8 signal peptide may include the amino acid sequence of SEQ ID NO: 10 or be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10, but is not limited thereto. That is, the DAP10 transmembrane domain has 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 10, respectively. The branch may include an amino acid sequence. The signal peptide according to the present invention is preferably located at the N-terminus of a recombinant protein (CAR) and is used to transfer the protein to the cell surface. Alternatively, the entire sequence may be truncated so that no portion of the signal peptide sequence is present.

Preferably, the antigen-binding domain is the antigen-specific antibody or fragment thereof (preferably, an antigen-binding fragment of the antibody). In one embodiment of the present invention, the antigen-binding domain is an antigen-binding domain that specifically binds to a tumor antigen, and is a tumor antigen-specific antibody or antigen-binding fragment thereof.

In the present invention, "antibody" means an immunoglobulin molecule immunologically reactive with a specific antigen (epitope), and includes polyclonal antibodies, monoclonal antibodies, and functional fragments thereof. (fragments) are included. The term may also include forms produced by genetic engineering, such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies). The antibody includes a heavy chain and/or light chain variable region (VH, heavy chain variable region; VL, light chain variable region). The variable region includes, as a primary structure, a portion forming an antigen-binding site of an antibody molecule, and the antibody of the present invention may be composed of a partial fragment including the variable region.

The term "epitope" refers to a specific three-dimensional molecular structure within an antigen molecule to which an antibody specifically binds.

In the present invention, "fragments" of an antibody means (functional) fragments that retain the antigen-binding function of an antibody, and are preferably antigen-binding fragments of the antibody. The fragment is used to include scFv, (scFv) ₂ , Fab, Fab' and F(ab') ₂ as well as nanobody fragments and the like. The definitions of these fragments are well known in the art. Preferably, the antigen-binding fragment is a scFv.

Among antibody fragments, Fab has a structure having variable regions of light and heavy chains, constant region of light chain, and first constant region (CH1) of heavy chain, and has one "antigen" binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Double-chain Fv (two-chain Fv) is a heavy chain variable region and a light chain variable region are connected by a non-covalent bond. Single-chain Fv (scFv) generally has a dimer-like structure like double-chain Fv because the heavy chain variable region and light chain variable region are covalently linked through a peptide linker or directly linked at the C-terminus. can achieve

More specifically, a "single-chain Fv" or "scFv" antibody fragment refers to a protein in which the light and heavy chain variable regions of an antibody are connected by a linker consisting of a peptide sequence of 15 or less amino acids connected. These domains are present in a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain and the VL domain to allow the scFv to form a desired structure for antigen binding. As used herein, an “Fv” fragment is an antibody fragment that contains the complete antibody recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, virtually covalent association, for example a scFv.

An antibody exhibits antigenic specificity according to changes in the sequence of the variable region. The variable region of the antigen binding site is divided into a framework region (FR) with low variability and a complementarity determining region (CDR) with high variability, and both heavy and light chains are divided into CDR1, 2, and 3. It has two CDR regions and four FR regions. The complementarity determining region is a region that imparts binding specificity to an antigen among variable regions of an antibody. The CDRs of each chain are typically named CDR1, CDR2, CDR3 sequentially starting from the N-terminus, and also identified by the chain in which the particular CDR is located.

In one embodiment of the present invention, the tumor antigen-specific antibody or antigen-binding fragment thereof according to the present invention may include the following heavy chain variable region and / or light chain variable region:

(i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and/or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28; and

(ii) a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and/or a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

In the present invention, the CDRs may include biological equivalents thereof. That is, each CDR may include an amino acid sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to the amino acid sequence indicated above. .

In another embodiment of the present invention, the tumor antigen-specific antibody or antigen-binding fragment thereof according to the present invention may contain the amino acid sequence of SEQ ID NO: 1 or be encoded by a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, but Not limited. That is, the antibody or fragment thereof has 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 1, respectively. The branch may include an amino acid sequence. Most preferably, the tumor antigen-specific antibody or antigen-binding fragment thereof may be an anti-CD19 antibody or a scFv fragment thereof.

The natural killer cell-specific chimeric antigen receptor according to the present invention may include a combination of an antigen binding domain, a CD8 domain, a CD28 domain, a DAP10 domain, a 2B4 domain, and/or a CD3z domain. More specifically, the natural killer cell-specific chimeric antigen receptor according to the present invention comprises an antigen binding domain, a CD8 or CD28 hinge domain, a DAP10 intracellular signaling domain, a 2B4 intracellular signaling domain, and a CD3z intracellular signaling domain. can include Preferably, the chimeric antigen receptor may further include a CD28 transmembrane domain. It may further include at least one selected from the group consisting of DAP10 extracellular domain and DAP10 transmembrane domain.

In a preferred embodiment of the present invention, the natural killer cell-specific chimeric antigen receptor comprises a combination of an antigen binding domain, a CD8 domain, a DAP10 domain, a 2B4 domain, and a CD3z domain; comprises a combination of an antigen binding domain, a CD28 domain, a DAP10 domain, a 2B4 domain, and CD3z; A combination of an antigen binding domain, a CD8 domain, a CD28 domain, a DAP10 domain, a 2B4 domain, and a CD3z domain.

In a more preferred embodiment of the present invention, the natural killer cell-specific chimeric antigen receptor comprises antigen binding domain, CD8 hinge domain, DAP10 extracellular domain, DAP10 transmembrane domain, DAP10 intracellular signaling domain, 2B4 intracellular signaling domain, and CD3z intracellular signaling domain (NK-CAR1). In another embodiment of the present invention, the natural killer cell-specific chimeric antigen receptor is an antigen binding domain, CD28 hinge domain, DAP10 extracellular domain, DAP10 transmembrane domain, DAP10 intracellular signaling domain, 2B4 intracellular signaling domain , and a CD3z intracellular signaling domain (NK-CAR2). In another embodiment of the present invention, the natural killer cell-specific chimeric antigen receptor comprises an antigen binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a DAP10 intracellular signaling domain, a 2B4 intracellular signaling domain, and a CD3z intracellular Signaling domain may be sequentially included (NK-CAR3).

The chimeric antigen receptor according to the present invention may include the aforementioned polypeptide domains as well as biological equivalents thereof. For example, additional changes may be made to the amino acid sequence of each domain in order to further improve the antigen recognition ability and/or intracellular signal transduction ability of the chimeric antigen receptor.

Also, preferably, the natural killer cell-specific chimeric antigen receptor according to the present invention is 70% or more, more preferably 80% or more, even more preferably 90% or more of the amino acid sequence represented by any one of SEQ ID NOs: 23 to 25. It may include an amino acid sequence having a sequence identity of 95% or more, most preferably 95% or more.

A polypeptide (or nucleic acid molecule) represented by a specific sequence herein may include the corresponding sequence as well as its biological equivalent. That is, considering a mutation having a biologically equivalent activity of a polypeptide (nucleic acid molecule), the polypeptide (or nucleic acid molecule) of one aspect is also interpreted to include a sequence showing substantial identity with the sequence described in SEQ ID NO. Specifically, a polypeptide (nucleic acid molecule) comprising an amino acid sequence (nucleotide sequence) represented by a specific sequence number is not limited to the amino acid sequence (nucleotide sequence), and variants of the amino acid sequence (nucleotide sequence) are within the scope of the present invention. included within A polypeptide molecule (nucleic acid molecule) composed of an amino acid sequence (nucleotide sequence) represented by a specific sequence number of the present invention is a functional equivalent of a polypeptide molecule (nucleic acid molecule) constituting it, for example, a polypeptide molecule (nucleic acid molecule) A concept that includes variants in which some amino acid sequences (nucleotide sequences) have been modified by deletion, substitution, or insertion, but which are functionally identical to the corresponding polypeptides (nucleic acid molecules). am. Specifically, the polypeptide (nucleic acid molecule) disclosed in the present invention is 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more of the amino acid sequence represented by a specific sequence number. It may contain an amino acid sequence (nucleotide sequence) having the above sequence homology. For example, 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%, 100% sequence homology It includes a polypeptide (nucleic acid molecule) with The "percentage of sequence homology" for a polypeptide (nucleic acid molecule) is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polypeptide sequence (nucleotide sequence) in the comparison region corresponds to the optimal alignment of the two sequences. It may contain additions or deletions (i.e., gaps) relative to the reference sequence (which does not contain additions or deletions).

In addition, the present invention provides a cell expressing a natural killer cell-specific chimeric antigen receptor according to the present invention. The cells may be in vitro or in vivo cells, and may be autologous cells and/or allogeneic cells (i.e., allogeneic cells) of an individual that requires the cells, or a heterogeneous cell thereof. (or derived from) xenogenic cells. Preferably, the cells may be prepared from autologous cells and/or syngeneic cells of an individual in need thereof. In a preferred embodiment of the present invention, the cell expresses the chimeric antigen receptor in its cell membrane. The cells may be immune cells such as T cells, NK cells, NKT cells, and macrophages. Preferably, the cells are natural killer cells. That is, the present invention provides CAR-NK cells expressing the chimeric antigen receptor according to the present invention. Since NK cells have the most immediate and powerful killing ability among immune cells, the CAR-NK cells of the present invention can be a powerful immunotherapy in the treatment of diseases such as cancer.

In the present invention, "natural killer cells (NK cells)" are a type of cytotoxic lymphocytes derived from bone marrow. Natural killer cells account for 5 to 20% of all lymphocytes and are responsible for innate immunity. Although there is no major histocompatibility complex or antibody on the surface, natural killer cells provide an immediate immune response against virus-infected cells, cancer cells, and other transformed cells. do. The natural killer cells include natural killer cells isolated from individuals as well as natural killer cells cultured or modified therefrom, and commercially available natural killer cell lines may also be used. That is, the natural killer cell of the present invention is not limited to a specific type, and it is sufficient as long as it has the same or similar molecular characteristics and biological activity to natural killer cells. In one embodiment, the natural killer cells may be selected from, for example, HANK1, NKL, NK92, NK-YS, YT, NOI-90, and NK101, but this is only an example, but is not limited thereto. The natural killer cells expressing the chimeric antigen receptor according to the present invention may be CAR-NK cells (Chimeric antigen receptor natural killer cells).

Natural killer cells expressing the chimeric antigen receptor according to the present invention, when recognizing a target through the receptor, are activated more quickly or at a higher level than natural killer cells that do not express the chimeric antigen receptor. It is characterized in that it can more quickly or effectively kill target cells.

For example, natural killer cells expressing the chimeric antigen receptor according to the present invention may be characterized by increased cytokine production and/or secretion upon recognizing a target antigen. That is, natural killer cells expressing the NK-CAR of the present invention may produce and/or secrete cytokines at a higher degree than natural killer cells that do not express the NK-CAR upon target recognition. Preferably, the cytokine is secreted from activated natural killer cells and induces the death of target cells. Preferably, the cytokine is a cytokine capable of inducing an anticancer effect by inhibiting the growth, proliferation, migration, and/or metastasis of cancer cells. The cytokine is not limited to specific types, but may preferably be MIP-1α (Macrophage inflammatory protein-1α) and/or Interferon-γ (interferon-γ, IFN-γ). . However, those skilled in the art will recognize that, in addition to the above-mentioned cytokines, any cytokines related to the apoptosis mechanism of natural killer cells may be included.

Natural killer cells expressing the chimeric antigen receptor according to the present invention may be characterized by increased production and/or secretion of granzyme when recognizing a target antigen. That is, natural killer cells expressing the NK-CAR of the present invention may produce and/or secrete granzymes at a higher degree than natural killer cells that do not express the NK-CAR upon target recognition. The granzymes include both granzymes A and B.

In addition, natural killer cells according to the present invention may be characterized in that the activation of natural killer cells and/or the activity of signal transduction pathways related to target cell killing ability increase upon recognizing the target antigen. Preferably, the signal transduction pathway is a signal transduction pathway related to the anticancer activity of natural killer cells (ie, a signal transduction pathway for killing or inhibiting the growth of cancer cells). That is, natural killer cells expressing the NK-CAR of the present invention may have higher activity of the signaling pathway than natural killer cells that do not express the NK-CAR upon target recognition. Representative examples of the signaling pathway include the AKT signaling pathway and the ERK signaling pathway. That is, when the natural killer cell expressing the chimeric antigen receptor according to the present invention recognizes the target antigen, the phosphorylation of AKT and/or ERK in the cell increases and the AKT and/or ERK signaling pathway can be activated. there is.

In addition, natural killer cells expressing the chimeric antigen receptor according to the present invention may be characterized in that the degree (level) of degranulation increases upon recognizing the target antigen. That is, natural killer cells expressing the NK-CAR of the present invention can undergo degranulation more actively than natural killer cells that do not express the NK-CAR upon target recognition. As proof of this, the natural killer cells expressing the chimeric antigen receptor according to the present invention can increase the level of CD107a more than natural killer cells that do not express the chimeric antigen receptor when recognizing the target.

In addition, the present invention provides a nucleic acid molecule (ie, polynucleotide) encoding a natural killer cell-specific chimeric antigen receptor according to the present invention.

As used herein, the term "polynucleotide" refers to an oligomer or polymer comprising two or more linked nucleotides or nucleotide derivatives bonded to each other, usually by phosphodiester bonds, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). indicate Polynucleotides may also contain, for example, nucleotide analogs, or "backbone" linkages other than phosphate diester linkages, such as phosphate triester linkages, phosphoramidate linkages, phosphorothioate linkages, thioester linkages, or peptide linkages (peptide linkages). nucleic acids), including DNA and RNA derivatives. Polynucleotides include single-stranded and/or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as analogs of either RNA or DNA.

That is, the nucleic acid molecule according to the present invention includes genes encoding domains constituting the chimeric antigen receptor according to the present invention. Each of the above genes may be directly linked to each other's N or C terminus, or may be linked through a linker sequence. Preferably, the nucleic acid molecule according to the present invention may include one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 11 to 22. As with the polypeptides described above, polynucleotides according to the present invention include functional equivalents thereof. Therefore, the nucleic acid molecule according to the present invention contains at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 22. may include a nucleotide sequence having 95% or more sequence homology.

Specifically, the region encoding the CD8 hinge domain in the nucleic acid molecule according to the present invention may include the nucleotide sequence represented by SEQ ID NO: 12 or 13. In addition, the region encoding the CD28 hinge domain may include the nucleotide sequence represented by SEQ ID NO: 14. In addition, the region encoding the CD28 transmembrane domain may include the nucleotide sequence represented by SEQ ID NO: 15. In addition, regions encoding the DAP10 extracellular domain, transmembrane domain, and intracellular signaling domain may include the nucleotide sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. In addition, the 2B4 intracellular signaling domain may include the nucleotide sequence represented by SEQ ID NO: 19. In addition, the CD3z intracellular signaling domain may include the nucleotide sequence represented by SEQ ID NO: 20.

A person skilled in the art can select an appropriate antigen binding domain-encoding gene according to the purpose. For example, when the natural killer cell-specific chimeric antigen receptor is for anticancer treatment, those skilled in the art can utilize a gene encoding a tumor antigen-specific antibody or fragment thereof known in the art, or a newly created gene sequence can be used. In one embodiment of the present invention, the nucleic acid molecule of the natural killer cell-specific chimeric antigen receptor may include a polynucleotide encoding a CD-19 specific antibody or a fragment thereof. Preferably, the polynucleotide or fragment thereof may include the nucleotide sequence of SEQ ID NO: 11.

In addition, the nucleic acid molecule according to the present invention may include a region encoding the CD8 signal peptide (preferably, including the nucleotide sequence of SEQ ID NO: 21).

The present invention also provides an expression vector comprising the nucleic acid molecule according to the present invention. That is, the present invention provides a recombinant vector comprising the nucleic acid molecule according to the present invention.

In the present invention, "recombinant vector" refers to a vector capable of expressing a peptide or protein encoded by a heterologous nucleic acid inserted into the vector, preferably a target protein (in the present invention, NK A vector prepared to express a specific CAR). The "vector" refers to any medium for the introduction and / or transfer of a base into a host cell in vitro, in vivo or in vivo, and a replicating unit capable of binding another DNA fragment to cause replication of the bound fragment ( replicon), and "replication unit" refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosomes, viruses, etc.)

Vectors according to the present invention may be linear DNA, plasmid DNA or recombinant viral vectors, but are not limited thereto. The recombinant viral vectors include, for example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, lentiviral vectors, retroviral vectors and adeno-associated viral vectors. Preferably, the vector is a retroviral vector. The present inventors used the pMXs-IRES-GFP retroviral vector in a specific example.

The recombinant vector of the present invention preferably includes a promoter, which is a transcription initiation factor to which RNA polymerase binds, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and termination of transcription and translation. It may include a regulatory sequence, a terminator, and the like, more preferably a polyhistidine tag (an amino acid motif composed of at least 5 or more histidine residues), a signal peptide gene, and an endoplasmic reticulum retention signal peptide ), a cloning site, and the like, and may further include a tag gene, a marker gene for selection such as an antibiotic resistance gene for selecting transformants, and the like. In the recombinant vector, the polynucleotide sequence of each gene is operably linked to a promoter. As used herein, the term "operably linked" refers to a functional linkage between a nucleotide expression control sequence, such as a promoter sequence, and another nucleotide sequence, whereby the control sequence is involved in the transcription of the other nucleotide sequence. and/or regulate detoxification.

The recombinant vector may be constructed using a prokaryotic or eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (eg, pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) ), a ribosome binding site for initiation of translation and a transcription/translation termination sequence. In the case of using a eukaryotic cell as a host, the origin of replication at which the vector operates in the eukaryotic cell may include, but is not limited to, the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, and the BBV origin of replication. it is not going to be In addition, promoters derived from the genome of mammalian cells (eg, metallotionine promoter) or promoters derived from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and the tk promoter of HSV) can be used, and usually have a polyadenylation sequence as a transcription termination sequence.

Examples of the gene for the tag include Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag (polyhistidine tag), Myc tag, S tag, SBP tag, IgG-Fc tag, and CTB. tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, Xpress tag, etc. can be included. Preferably, the vector according to the present invention may contain a myc tag. More preferably, the myc tag may include the nucleotide sequence of SEQ ID NO: 22.

In the present invention, the nucleic acid or the vector may be transfected or transfected into a virus-producing cell, that is, a packaging cell line. A variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for "transfection" or "transfection", such as electrophoresis, calcium phosphate precipitation; DEAE-dextran transfection or lipofection or the like can be used. The virus containing the gene of interest (a natural killer cell-specific chimeric antigen receptor-encoding nucleic acid molecule) according to the present invention can proliferate in the packaging cell line and be released extracellularly, and the virus can The NK-CAR may be transduced into cells, such as NK cells, in which the final expression is desired. The nucleic acid of the virus "transduced" into the cell is used to produce a target protein (natural killer cell-specific chimeric antigen receptor) with or without integration into the genome of the cell.

The present invention can provide isolated cells into which the expression vector according to the present invention has been introduced (transformation, transfection, transfection, etc.). The cell herein refers to a cell for proliferating (amplifying) the expression vector, not a cell that ultimately expresses a natural killer cell-specific chimeric antigen receptor. That is, the cell refers to a host cell directly transduced/transformed/transfected with the aforementioned nucleic acid molecule or expression vector. For example, when the expression vector is a viral vector, the cell may be a packaging cell for producing a virus containing the viral vector. The selection of a suitable host is believed to be obvious to one skilled in the art from the teachings herein.

In addition, the present invention comprises the steps of (S1) introducing the expression vector according to the present invention into a cell; and (S2) culturing cells into which the expression vector has been introduced.

The step (S1) is a step of amplifying the expression vector of the present invention using a host cell or introducing the expression vector into a host cell to express the vector. That is, by introducing the expression vector into an appropriate host cell (transformation, transfection, transfection, etc.), the expression vector is replicated inside the cell, or a protein or the like is induced to be expressed from the expression vector (viral vector When used), a virus containing the expression vector may be induced to be produced. The step (S1) may be appropriately performed by a person skilled in the art according to the type of vector and cell used. For example, when the expression vector is a viral vector, the expression vector may be introduced into a host cell by transfection.

The step (S2) refers to culturing the cells so that the target gene introduced into the cells is sufficiently amplified or expressed in the cells. The culture, after the expression vector is introduced into the host cell, is performed for a period sufficient for the expression vector to express the target protein in the host cell (or, for a period sufficient for the expression vector to replicate or containing the target gene). for a period of time sufficient to produce viruses). More preferably, the culturing may be performed for a period of time sufficient to allow the protein to be secreted into the culture medium in which the host cell is cultured (or, for a period sufficient to release the virus containing the target gene to the outside of the cell). .

The method includes the steps of obtaining (or amplified) natural killer cell chimeric antigen receptors generated (or amplified) from the cells after the step (S2), an expression vector containing a nucleic acid molecule encoding the same, or a virus containing the expression vector. may further include. The acquisition method is appropriately selected and controlled in consideration of the characteristics of the chimeric antigen receptor, expression vector, or virus produced in the host cell, the characteristics of the host cell, the expression method, or the targeting of the chimeric antigen receptor/expression vector/virus. can If necessary, in order to release and recover chimeric antigen receptors/expression vectors/viruses present in specific organelles or cytoplasm of cells to the outside of cells, cells can be lysed within a range that does not affect the functional structure of antibodies or their antigen-binding fragments. may be In addition, the virus containing the expression vector can be recovered by a method such as obtaining a medium in which host cells are cultured and centrifuging to remove impurities.

The obtained chimeric antigen receptor/expression vector/virus may further undergo a process of further removing impurities and concentrating through methods such as chromatography, filtration through a filter, etc., and dialysis. Separation or purification of the obtained chimeric antigen receptor can be carried out by separation and purification methods commonly used for proteins, such as chromatography. The chromatography may include, for example, affinity chromatography including a protein A column, protein G column, or protein L column, ion exchange chromatography, or hydrophobic chromatography. In addition to the above chromatography, the chimeric antigen receptor may be separated and purified by further combining filtration, ultrafiltration, salting out, dialysis, and the like. Isolation or purification of the virus can be achieved by filtering the medium through a porous filter to remove impurities and concentrating.

In addition, the present invention provides a natural killer cell-specific chimeric antigen receptor, comprising the step of introducing into natural killer cells an expression vector containing a nucleic acid molecule encoding the natural killer cell-specific chimeric antigen receptor according to the present invention Provided is a method for producing natural killer cells expressing the present invention. That is, the present invention provides a method for producing a CAR-NK cell comprising a chimeric antigen receptor according to the present invention. The expression vector may be an expression vector amplified through the method for preparing a natural killer cell-specific chimeric antigen receptor described above. The expression vector itself may be introduced into natural killer cells, or may be introduced in a form included in a virus. A person skilled in the art can select an appropriate introduction method depending on the type of expression vector and natural killer cell. For example, when the expression vector is a viral vector, the vector may be introduced into cells by infecting natural killer cells with a virus containing the expression vector (transduction). The nucleic acid of the virus transduced into the cell may be inserted into the genome of the natural killer cell or expressed without being inserted, and the natural killer cell-specific chimeric antigen receptor of the present invention may be expressed therefrom.

In addition, the present invention provides natural killer cells expressing the natural killer cell-specific chimeric antigen receptor according to the present invention; an expression vector containing a nucleic acid molecule encoding the natural killer cell-specific chimeric antigen receptor; And / or provides a pharmaceutical composition for preventing or treating cancer, comprising cells containing the expression vector as an active ingredient. Here, the antigen-binding domain of the natural killer cell-specific chimeric antigen receptor is characterized in that it is an antigen-binding domain that specifically binds to a tumor antigen. The natural killer cells may be autologous cells or allogeneic cells of an individual to whom the composition is administered.

In the present invention, "cancer" is used synonymously with "tumor" and refers to a condition characterized by typically uncontrolled cell growth or proliferation. Cancers that can be prevented or treated using the chimeric antigen receptor according to the present invention include solid tumors and non-solid tumors (eg, hematological cancer, etc.). In addition, in the present invention, cancer types include carcinoma, blastoma, and sarcoma, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcoma, carcinoma, and melanoma, but are not limited thereto .

The specific type of cancer of the present invention is not limited, but colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharynx cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer , tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, hematological cancer, squamous cell carcinoma, adenocarcinoma of the lung, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, gastrointestinal cancer, glioblastoma, ovarian cancer, endometrial cancer, salivary gland cancer, vulvar cancer, head and neck cancer, and the like. Preferably, the cancer according to the present invention is a cancer expressing a target antigen recognized by the chimeric antigen receptor of the present invention. That is, the cancer expresses a tumor antigen that can be recognized by the antigen-binding domain of the chimeric antigen receptor of the present invention. For example, when the CAR according to the present invention contains an anti-CD19 scFv as a cancer cell-specific antigen-binding domain, the cancer is preferably a cancer in which CD19 is overexpressed or has a higher activity in cancer cells than in normal cells. .

In the present invention, "hematologic cancer" refers to cancer arising from components of blood (leukocytes, red blood cells, platelets, etc.), bone marrow producing blood, and lymphatic system constituting the immune system (lymphocytes, lymph nodes, lymphatic vessels, etc.). Examples include leukemia, malignant lymphoma, and multiple myeloma. More specific examples of blood cancer include Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia, Chronic Myelogenous Leukemia, Multiple Myeloma, and Lymphoma etc.

Preferably, the cancer is lymphoma, B cell lymphoma, acute lymphoma, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, MALT lymphoma, marginal zone It may be selected from marginal zone lymphoma, peripheral T cell lymphoma, anaplastic large cell lymphoma, lymphoblastic lymphoma, Hodgkin's lymphoma, and the like.

The content of natural killer cells expressing the NK-CAR, the expression vector, and/or cells containing the expression vector in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc. For example, it may be 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.

The pharmaceutical compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods. , tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta agents, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external agents are creams, gels, patches, sprays, ointments, and warning agents. , lotion, liniment, pasta, or cataplasma may have formulations such as the like.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin Binders such as hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch arc, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, Hydroxypropyl Methyl Cellulose, Corn Starch, Agar Powder, Methyl Cellulose, Bentonite, Hydroxypropyl Starch, Sodium Carboxymethyl Cellulose, Sodium Alginate, Calcium Carboxymethyl Cellulose, Calcium Citrate, Sodium Lauryl Sulfate, Silicic Anhydride, 1-Hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid; may be used.

Additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like may be used.

In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.

Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

Suspension agents according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Agents may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.

The suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolen (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Buytyrum Tego-G -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hyde Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), testosterone triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.

In the present invention, "subject" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. of mammals.

In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.

In the present invention, "prevention" refers to any action that suppresses or delays the onset of a desired disease, and "treatment" means that a desired disease and its associated metabolic abnormalities are improved or treated by administration of the pharmaceutical composition according to the present invention. Any action that is beneficially altered, and "improvement" means any action that reduces a parameter related to a desired disease, for example, the severity of a symptom, by administration of the composition according to the present invention.

In addition, the present invention provides natural killer cells expressing the natural killer cell-specific chimeric antigen receptor according to the present invention; an expression vector containing a nucleic acid molecule encoding the natural killer cell-specific chimeric antigen receptor; And / or providing a kit for preventing or treating cancer, including the expression vector. The kit according to the present invention is not limited in specific form as long as it is for the prevention or treatment of cancer, and the production, storage, introduction and expression of the chimeric antigen receptor according to the present invention into natural killer cells, administration of the natural killer cells, etc. It may include without limitation any components and devices for.

In addition, the present invention provides a cell therapy agent for preventing or treating cancer, comprising natural killer cells expressing a natural killer cell-specific chimeric antigen receptor according to the present invention as an active ingredient.

In the present invention, "cellular therapeutic agents" are cells or tissues prepared from humans through isolation, culture, and special manipulation and used for the purpose of diagnosis, prevention, or treatment of a specific disease. The FDA defines cell therapy as a drug. Specifically, cell therapy products are manufactured through a series of processes in which living autologous, allogeneic, or xenogeneic cells are propagated and selected in vitro, or by changing the biological properties of cells by other methods, in order to restore the functions of cells or tissues. , it is used for diagnosis, prevention, and treatment of diseases.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Preparation of anti-CD19-CAR expression vector

Based on the NK cell-specific signaling domain, three anti-CD19-DAP10-2B4-CD3ζ CAR artificial synthetic genes were prepared according to the present invention (below (1) to (3)), and anti-CD19 as a control An artificial synthetic gene for -CD28-CD3ζ CAR was also prepared ((4) below).

The type and composition of each chimeric antigen receptor is as follows:

(1) myc-tag, scFv of anti-CD19 monoclonal antibody binding to CD19, CD8 hinge domain, DAP10 extracellular domain, DAP10 transmembrane domain, DAP10 intracellular signaling domain, 2B4 intracellular signaling domain, and a one-molecule chimeric protein consisting of the CD3ζ (also referred to as CD3z) intracellular signaling domain (termed "NK-CAR1");

(2) myc-tag, scFv of anti-CD19 monoclonal antibody binding to CD19, CD28 hinge domain, DAP10 extracellular domain, DAP10 transmembrane domain, DAP10 intracellular signaling domain, 2B4 intracellular signaling domain, and One molecule of chimeric protein consisting of the CD3ζ intracellular signaling domain (named "NK-CAR2");

(3) myc-tag, scFv of anti-CD19 monoclonal antibody binding to CD19, CD8 hinge domain, CD28 transmembrane domain, DAP10 intracellular signaling domain, 2B4 intracellular signaling domain, and CD3ζ intracellular signaling One molecule of chimeric protein consisting of domains (named “NK-CAR3”); and

(4) One molecule chimera consisting of myc-tag, scFv of anti-CD19 monoclonal antibody binding to CD19, CD8 hinge domain, CD28 transmembrane domain, CD28 intracellular signaling domain, and CD3ζ intracellular signaling domain Protein (T cell derived CAR; termed "2 ^nd CAR" or "2 ^nd generation CAR").

pMXs-CAR-IRES-GFP retroviral vector 3 expressing NK-CAR according to the present invention by cloning a nucleic acid fragment containing each of the artificial genes into a pMXs-IRES-GFP retroviral vector digested with EcoR1-Xho1 One retroviral vector expressing species and control T-CARs was constructed. That is, the vector encodes the chimeric antigen receptor according to the present invention together with the fluorescent protein GFP. The structures of NK-CAR1, NK-CAR2, NK-CAR3, and the control 2 ^nd CAR according to the present invention are shown in FIG. 1 .

Example 2. Construction of NK-CAR retroviral solution

E. coli DH5α was transformed with the four types of pMXs-CAR-IRES-GFP retroviral vectors prepared in Example 1, respectively, to obtain transformants. The plasmid DNA cloned in the transformant was purified using the HiSpeed Plasmid Midi Kit (Qiagen, #12643), and prepared as DNA for transfection. The DNA for transfection was subjected to the following manipulations.

Using X-tremeGENE9 (Roche, #6365787001) according to the manufacturer's instructions, the DNA for transfection was transfected into Plat A cells, a retroviral packaging cell line. The medium was replaced 24 hours after the transfection, and 24 hours after the medium was changed, the supernatant containing the amphotropic retrovirus was obtained and filtered through a 0.45 μm filter (Nalgene, #725-2545). Using this supernatant, 10 μg/mL polybrene (Sigma, #H9268) was used according to the manufacturer's instructions to inject amphotropic virus into natural killer cell lines, NKL and NK92 cell lines, at 700 G, 30 minutes, and 32 ° C. Spinfection was performed twice.

Cells expressing GFP among the cells on day 3 of virus infection were separated using BD FACSAria ^TM II Cell Sorter (BD Biosciences) and cultured. Cells 3 days after isolation were stained with Alexa ^Fluor® 647-Myc-Tag (9B11) Mouse mAb (Cell Signaling, #2233S) and analyzed using BD Accuri ^™ C6 (BD Biosciences). GFP-positive cells mean cells infected with the retrovirus prepared in this example, and Alexa Fluor ^® 647-positive cells mean cells expressing the NK-CAR according to the present invention.

First, the ratio of Alexa Fluor ^® 647-positive cells among GFP-positive cells (virus-infected NKL cells) and the ratio of NKL cells expressing NK-CAR were measured. As shown in Fig. 2a, about 7% of the NKL cells were infected with the CAR-retrovirus, and about 85% of them expressed the NK-CAR according to the present invention. About 12% of NKL cells were infected with the ^2nd CAR virus, and about 95% of NKL cells expressing the ^2nd CAR were identified.

Subsequently, NKL cells expressing GFP among NKL cells 3 days after virus infection were isolated and cultured with BD FACSAria ^TM II Cell Sorter (BD Biosciences), and to confirm their GFP and NK-CAR expression, as described above Similarly, it was stained with Alexa Fluor ^® 647-Myc-Tag (9B11) Mouse mAb and analyzed with BD Accuri ^TM C6 (BD Biosciences). As a result, GFP was expressed in more than 99% of the cells, indicating that only virus-infected NKL cells were isolated and cultured (FIG. 2b). As a result of observing the ratio of NK-CAR expressing cells to GFP positive cells, the ratio of NK-CAR1 expressing cells was 70%, the ratio of NK-CAR2 expressing cells was 85%, and the ratio of NK-CAR3 expressing cells was 96% confirmed (Fig. 2b). The above result means that structural differences of NK-CARs affect protein expression.

In addition, the ratio of cells positive for Alexa Fluor ^® 647 among GFP-positive cells (NK92 cells infected with the virus) and the ratio of NK92 cells expressing NK-CAR were measured. As shown in Figure 2c, about 22 to 26% of NK92 cells were infected with NK-CAR virus, and NK92 cells expressing NK-CAR were about 32 to 85% of them. About 35% of the NK92 cells were infected with the ^2nd CAR virus, and about 94% of the NK92 cells were expressing the ^2nd CAR.

Subsequently, NK 92 cells expressing GFP among NK92 cells 3 days after virus infection were isolated and cultured with BD FACSAria ^TM II Cell Sorter (BD Biosciences), and to confirm their GFP and NK-CAR expression, the above-described cells were cultured. Stained with Alexa ^Fluor® 647-Myc-Tag (9B11) Mouse mAb as described above and analyzed with BD Accuri ^™ C6 (BD Biosciences). As a result, GFP was expressed in more than 99% of the cells, indicating that only virus-infected NK 92 cells were isolated and cultured (FIG. 2d). As a result of observing the ratio of NK-CAR-expressing cells to GFP-positive cells, it was confirmed that the ratio of NK-CAR2-expressing cells was 63.7% and the ratio of NK-CAR3-expressing cells was 96.6% (FIG. 2d). This result, like the previous result, means that the structural difference of NK-CAR affects protein expression.

Example 3. Evaluation of antigen-specific apoptosis activity of NK-CAR expressing NK cells

In this example, the apoptotic activity of NK cells introduced with the NK-CAR prepared in the above example was evaluated. First, in order to evaluate the apoptotic activity of NKL cells introduced with NK-CAR, CD19-positive cell line REH on day 2 of culture was used as a target cell, and NK-CAR expressing NKL cells on day 3 of culture rested for 24 hours, and As a control, NKL into which pMXs-IRES-GFP (empty vector) was introduced was used as an effector cell. In order to rest the effector cells, the cells on the second day of culture were washed with serum free RPMI1640, suspended in 10 ml of rested RPMI1640 (+5% FBS, 0.5% pen/strep, 0.5% sodium pyruvate) medium and cultured for 24 hours. . In addition, in order to evaluate the apoptotic activity of NK92 cells introduced with NK-CAR, CD19-positive cell lines REH and Ramos on day 2 of culture were used as target cells, and NK-CAR expressing NK92 cells on day 2 of culture without resting, And as a control, NK92 into which pMXs-IRES-GFP (empty vector) was introduced was used as an effector cell.

The apoptotic activity of NK-CAR-expressing NKL and NK92 cells was measured by Europium assay, respectively. First, the target cells were suspended in rested IMDM (+5% FBS, 0.5% pen/strep) to a concentration of 1.0×10 ⁶ cells/400 μl, and then 40 μM BATDA (Perkin Elmer, #C136-100) was added to 5.0% CO. It was kept warm for 30 minutes in a 37°C, CO ₂ incubator equilibrated with ₂ gas. Then, wash with rested IMDM containing 1 mM sulfinpyrazone (Sigma, #S9509) (rested IMDM containing 1 mM sulfinpyrazone is named assay media), and suspend in assay media so that the concentration is 5.0×10 ⁴ cells/1 ml. 100 μl was added. In addition, the effector cells are suspended in assay media, and the Effector to Target (E:T) ratio is 20:1, 10:1, or 5:1 in the case of NK-CAR expressing NKL cells; For NK-CAR expressing NK92 cells, 100 μl was added at a ratio of 4:1, 2:1, or 1:1. Instead of effector cells, medium was prepared as a spontaneous control and 100 μl of 2% Triton X-100 was added as a maximum lysis control.

After preparing the cells and each control group, the 96-well plate was insulated in a 37°C CO ₂ incubator equilibrated with 5.0% CO ₂ gas. NK-CAR-expressing NKL cells were kept warm for 2 hours, and NK-CAR-expressing NK92 cells were kept warm for 30 minutes.

Subsequently, 20 μl of the supernatant was reacted with 20% europium solution (Perkin Elmer, #C135-100) containing 0.3 M acetic acid for 5 minutes, and then the fluorescence intensity was measured with a Victor X4 multilabel plate reader (Perkin Elmer) to kill the cells. Activity (%) was calculated. The formula for cell killing activity (%) is as follows: Cell killing activity (%) = 100 × (measured value of each well-measured value of Spontaneous control) / (measured value of Maximum lysis control-measured value of Spontaneous control) .

Europium assay results are shown in Figures 3a to 3c. First, Figure 3a shows the apoptotic activity of NK-CAR introduced NKL cells. EV (Empty vector) refers to cells infected with the virus made by the pMXs-IRES-GFP vector, and 2 ^nd CAR and NK-CAR mean NKL cells infected with the virus made by the pMXs-CAR-IRES-GFP vector. . As shown in the figure, when the effector to target ratio is 20: 1, the EV group did not kill REH, a CD19-positive cell line, and the ^2nd CAR killed about 60% of REH cells, and the NK-CAR killed about 70 to 80 % of REH cells were killed. In addition, even when the effector to target ratio was 10:1 or 5:1, the REH killing activity of NKL cells expressing the NK-CAR according to the present invention was higher than that of control cells expressing 2 ^nd CAR.

Figures 3b and 3c show the killing activity of NK-CAR-introduced NK92 cells against REH cells (Figure 3b) and RAMOS cells (Figure 3c), respectively. EV (Empty vector) means cells infected with the virus made by the pMXs-IRES-GFP vector, and ^2nd CAR and NK-CAR mean NK92 cells infected with the virus made by the pMXs-CAR-IRES-GFP vector. . As shown in the figure, the apoptotic activity of the NK-CAR expressing cells of the present invention was significantly higher than that of the 2 ^nd CAR expressing cells in both REH cells and RAMOS cells, and these results were the same at all Effector to Target ratios.

The above results show that as NK-CAR was introduced into NKL and NK92 cells, NKL and NK92 cells acquired the ability to recognize CD19 and kill target cells specifically for CD19. In addition, it is shown that the NK-CAR3 of the present invention prepared based on a specific NK activating receptor combination (NKG2D+2B4) is particularly effective in activating NK cells. In particular, cells expressing NK-CAR3 showed more excellent apoptosis activity than other NK-CAR expressing cells, which shows that NK-CAR3 among NK-CARs according to the present invention is particularly effective in activating NK cells.

Example 4. Confirmation of cytokine secretion ability of NK-CAR expressing NK cells

Macrophage inflammatory protein-1α (also called MIP-1α; CCL3) is a chemotactic cytokine known as a chamokine and is secreted from activated natural killer cells. Granzyme B is a serine protease most commonly found in the granules of natural killer cells and cytotoxic T cells, and perforin-mediated target cell death by these cells (perforin-dependent target cell death). It is an important factor in inducing death) mechanism and is secreted together with perforin. That is, since MIP-1α and granzyme B are measures of the degree of activation of natural killer cells, in order to confirm that the NK-CAR according to the present invention effectively induces the apoptosis function of NKL cells, NK-CAR expressing cells MIP-1α and granzyme B levels were measured.

CD19-positive cell line REH on day 2 of culture was used as a target cell, NKL cells expressing NK-CAR on day 3 of culture rested for 24 hours, and ^2nd CAR transduced NKL cells as a control, pMXs-IRES-GFP (empty vector; EV ) Transduced NKL cells, and simple NKL cells (NKL cells without vector introduction) were used as effector cells. To rest the effector cells, the cells on the second day of culture were washed with serum free RPMI1640, suspended in 10 ml of rested RPMI1640 (+5% FBS, 0.5% pen/strep, 0.5% sodium pyruvate), and cultured for 24 hours.

^{96 _} It was aliquoted into well plates.

They were co-cultured for 8 hours in a 37°C CO ₂ incubator equilibrated with 5.0% CO ₂ gas. Thereafter, the cell culture medium was centrifuged to obtain a supernatant, and Human CCL3/MIP-1alpha DuoSet ELISA kit (R&D Systems, #DY270) and Human Granzyme B DuoSet ELISA kit (R&D Systems, #DY2906) were used according to the manufacturer's instructions. was used to confirm the secretion of MIP-1α and Granzyme B in the supernatant.

As a result, as shown in FIGS. 4a and 4b , MIP-1α and granzyme B secreted by activated NK cells were produced and secreted only by cells expressing CAR. In addition, it was found that the amount of cytokine secretion of NKL cells expressing the NK-CAR according to the present invention was significantly higher than that of NKL cells expressing the ^2nd CAR. The above results show that the NK-CAR based on a specific receptor combination according to the present invention effectively activates the target cell killing function of natural killer cells and promotes cytokine secretion.

Example 5. Confirmation of activation of AKT and ERK signaling pathways in NK-CAR expressing NK cells

Natural killer cells that recognize the target antigen activate AKT and ERK signals to exert antitumor effects. Therefore, it was confirmed whether the NK-CAR-expressing natural killer cells according to the present invention effectively recognize the target antigen (CD19) and activate specific AKT and ERK signals. To this end, CD19-positive cell line REH or recombinant human CD19 Fc chimera were used as stimulants to stimulate NK-CAR-introduced NKL cells and control NKL cells on day 3 of culture resting for 24 hours. , and confirmed changes in AKT and ERK signals. To rest the cells, the cells on the second day of culture were washed with serum free RPMI1640, suspended in 10 ml of rested RPMI1640 (+5% FBS, 0.5% pen/strep, 0.5% sodium pyruvate), and cultured for 24 hours.

The process of obtaining a cell lysate by stimulating NKL cells with REH cells is as follows. CD19 positive cell line REH cells, anti-CD19-DAP10-2B4 CAR (a structure without CD3ζ intracellular signaling domain in NK-CAR2) introduced NKL cells, and control NKL cells were 5.0×10 ⁶ cells/100 μl/sample. After each suspension in rested RPMI (+5% FBS, 0.5% pen/strep, 0.5% sodium pyruvate), it was cooled at 4℃ for 10 minutes. Then, REH cells and CAR transduced NKL cells; After mixing REH cells and control NKL cells, respectively, centrifugation was performed and the pellets were cooled at 4°C for 10 minutes. Thereafter, stimulation was performed in a 37°C water bath for 2 and 5 minutes, and after the stimulation, the stimulation was terminated by cooling in ice for 5 minutes. After washing with DPBS, lysis buffer was added, vortexed, and cell lysis was performed on ice. Cell lysates were then obtained by centrifugation.

The process of obtaining a cell lysate by stimulating NKL cells with a recombinant human CD19 Fc chimera is as follows. Protein G (Invitrogen, #1003D) 4.0×10 ⁷ beads/sample was washed with wash buffer (1X PBS + 0.01% Tween-20 + 1% FBS). Here, 4 μg/sample of recombinant human CD19 Fc chimera (R&D, #9269-CD) was added, and rotation incubation was performed at 4°C for 1 hour. Then, it was washed with wash buffer and suspended in rested RPMI (+5% FBS, 0.5% pen/strep, 0.5% sodium pyruvate) to form 4.0×10 ⁷ beads/100 μl/sample, and recombinant human CD19 Fc chimera A coated Protein G beads solution was prepared. Anti-CD19-DAP10-2B4 CAR (a structure without CD3ζ intracellular signaling domain in NK-CAR2) introduced NKL cells and control NKL cells at 2.0×10 ⁶ cells/sample were centrifuged to form a pellet and refrigerated for a minute. It was resuspended in beads solution, centrifuged to make pellets, and cooled at 4℃ for 10 minutes. Thereafter, stimulation was performed in a 37°C water bath for 2 and 5 minutes, and after the stimulation, the stimulation was terminated by cooling in ice for 5 minutes. Then, the cells were washed with DPBS, vortexed in lysis buffer, and then cell lysed on ice. Cell lysates were then obtained by centrifugation.

The cell lysate obtained by the above method was subjected to SDS-PAGE using an 8% acrylamide gel, and the protein was transferred to a PVDF membrane (immobilon, #IPVH00010) by a semi-dry transfer method. After blocking with 5% skim milk, antigen-antibody reaction was performed. Primary antibodies were treated at 4°C for 16 hours, and the list of primary antibodies used is as follows: Phospho-Akt (cell signaling, #CS9271), AKT (cell signaling, #CS9272), Phospho-p44/42 Erk1/ 2 (cell signaling, #CS9101), Erk1/2 (cell signaling, #CS4695). The secondary antibody (mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology, #SC2357)) was treated at room temperature for 1 hour. Then, proteins were detected with ImageQuant LAS 4000 (Fujifilm) using SuperSignal ^TM WestPico PLUS Chemiluminescent Substrate (Thermo Scientific, #34580).

The results of stimulating the NK-CAR (a structure without the CD3ζ intracellular signaling domain in NK-CAR2)-expressing cell lines with CD19-positive REH cells are shown in FIG. 5a. When the same amount of AKT and ERK were loaded, the level of phosphorylated AKT (pS476-pAKT) compared to total AKT increased significantly when NK-CAR expressing cells were stimulated with REH for 5 minutes, and the level of phosphorylated ERK compared to total ERK also increased. - It was found to be markedly increased in CAR-expressing cells. On the other hand, control NKL cells that do not express NK-CAR showed no significant difference in the phosphorylation levels of AKT and ERK even when stimulated with REH. The results of stimulation of a cell line expressing NK-CAR (a construct lacking the CD3ζ intracellular signaling domain in NK-CAR2) with the recombinant human CD19 Fc chimera are shown in FIG. 5B . Similar to the above results, AKT and ERK signals were not activated in NKL cells that did not express NK-CAR even when stimulated with CD19, but phosphorylation of AKT and ERK in NKL cells expressing NK-CAR was stimulated with CD19 for 5 minutes. markedly increased.

The above results show that natural killer cells expressing the NK-CAR according to the present invention can effectively recognize the target antigen, CD19, and specifically activate AKT and ERK signals.

Example 6. Confirmation of cytotoxic degranulation of NK-CAR expressing NK cells

Degranualation refers to a cellular process that releases antimicrobial, cytotoxic substances and the like from secretory vesicles called granules. Degranulation is observed in granulocytes (neutrophils, basophils, and eosinophils) or immune cells such as mast cells, NK cells, and T cells, and is mainly aimed at attacking invading microorganisms. In particular, activated NK cells induce apoptosis by releasing substances including perforin and granzyme on the surface of target cells through degranulation. The granular inner surface of NK cells is coated with CD107a. After degranulation, CD107a is exposed on the surface of cytotoxic lymphocytes and thus protects the outer membrane of lymphocytes from perforin-induced damage. Degranulation assays of NK cells based on externalization of CD107a allow direct detection of activation of NK cells in response to antigen. Therefore, in this Example, the activation of NK cells by the NK-CAR was verified by measuring the degree of degranulation of NK cells into which the NK-CAR of the present invention was introduced.

Specifically, to evaluate the cytotoxic degranulation of CAR-introduced NK92 cells, CD19-positive cell lines REH and Ramos cells on day 2 of culture were used as target cells, NK-CAR-introduced NK92 cells on day 2 of culture, and 2 ^nd CAR-transduced NK92 cells were used. In addition, pMXs-IRES-GFP transduced NK92 cells were used as effector cells.

Target cells are 1.0×10 ⁵ cells/50 μl/sample, effector cells are 1.0×10 ⁵ cells/100 μl/sample, anti-CD107a/LAMP1-PE (clone H4A3, BD Bioscience, #555801) is 2 μl/50 Suspended in IMDM (+10% FBS, 1% pen/strep) to μl/sample and dispensed into 96 well plates to make a final volume of 200 μl, which was equilibrated with 5.0% CO ₂ gas at 37°C and CO ₂ Co-cultured for 2 hours in an incubator. Then, the cell pellet was suspended in DPBS (+1% FBS) by centrifugation and anti-CD3-PerCP (clone SK7, BD Bioscience, #347344), anti-CD56-APC (clone NCAM16.2, BD Bioscience, #341025) ), and anti-CD107a/LAMP1-PE (clone H4A3, BD Bioscience, #555801), respectively, and labeled for 35 minutes in a dark room at 4°C. After washing the cells twice with DPBS (+1% FBS), CD107a expression on the surface of NK92 cells was confirmed by flow cytometry to evaluate cytotoxic degranulation.

As a result, as shown in FIG. 6, when NK cells were co-cultured with target cells expressing CD19, the ratio of CD107a-positive cells was increased in NK92 cells into which CAR was introduced compared to blank space (EV) cells. Confirmed. In addition, as a result of comparing NK-CAR-introduced NK92 cells and 2 ^nd CAR-introduced NK92 cells, the level of degranulation of NK92 cells introduced with the NK-CAR of the present invention was higher than that of 2 ^nd CAR-introduced cells, and in particular, NK-activating receptor NK-CAR3-introduced NK92 cells based on the combination (NKG2D+2B4) showed the highest percentage of CD107a-positive cells in the REH-stimulated group and the Ramos-stimulated group at 21.6% and 44.7%, respectively.

The above results show that the NK-CAR of the present invention can induce degranulation of NK cells more effectively than conventional CARs, and thus promote better apoptosis activity.

Example 7. Confirmation of the degree of cytokine production of NK-CAR expressing NK cells

In this Example, it was further verified whether the NK-CAR of the present invention promotes cytokine production by activating NK cells.

Specifically, to evaluate the cytokine production of NK-CAR-introduced NK92 cells, CD19-positive cell lines REH and Ramos cells on day 2 of culture were used as target cells, NK-CAR-introduced NK92 on day 2 of culture, and 2 ^nd CAR transduced NK92 cells were used. In addition, pMXs-IRES-GFP transduced NK92 cells were used as effector cells.

Target cells were suspended in IMDM (+10% FBS, 1% pen/strep) to 1.0×10 ⁵ cells/100 μl/sample and effector cells were 1.0×10 ⁵ cells/100 μl/sample, and then plated in a 96 well plate. Each was aliquoted and co-cultured for 1 _hour in a CO 2 incubator at 37° C. equilibrated with 5.0% CO ₂ gas. Thereafter, brefeldin A (GolgiPlug; BD Biosciences, #51-2301KZ) and a protein transport inhibitor containing monensin (GolgiStop; BD Bioscience, #51-2092KZ) were added, followed by further incubation for 5 hours. After a total of 6 hours, the cell pellet was suspended in DPBS (+1% FBS) by centrifugation and anti-CD3-PerCP (clone SK7, BD Bioscience, #347344), anti-CD56-APC (clone NCAM16.2, BD Bioscience) , #341025) for 35 minutes in the dark at 4°C. After washing the cells twice with DPBS (+1% FBS), the cells were fixed/permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences, #51-2090KZ) for 20 minutes in the dark at 4°C. Then, the cells were washed twice with 1X BD Perm/Wash buffer (BD Biosciences, #51-2091KZ), and incubated with anti-Interferon-γ-PE (clone 25723.11, BD Bioscience, #340452) for 15 hours at 4°C in the dark. dyed. After staining, cells were washed twice with 1X BD Perm/Wash buffer, and cytokine production was evaluated by checking Interferon-γ expression in NK92 cells through flow cytometry.

The results are shown in FIG. 7 . As a result of confirming the production of Interferon-γ in each NK92 cell, when co-cultured with target cells expressing CD19, the ratio of Interferon-γ-positive cells was increased in NK92 cells introduced with CAR compared to cells introduced with blank space (EV). Confirmed. In addition, as a result of comparing the NK-CAR-introduced cells and the ^2nd CAR-introduced cells, the ratio of Interferon-γ-positive cells was higher in the NK92 cells introduced with the NK-CAR of the present invention compared to the ^2nd CAR-introduced NK92 cells. The ratio of Interferon-γ-positive cells in NK-CAR3-introduced NK92 cells based on the NK-activated receptor combination (NKG2D+2B4) was 19.3% and 58.6%, respectively, in the REH-stimulated group and the Ramos-stimulated group, respectively.

The above results show that the NK-CAR of the present invention can promote cytokine production of NK cells more effectively than conventional CARs, and thus induce more excellent apoptosis activity.

As described above, the present inventors derived a natural killer cell-specific chimeric antigen receptor capable of exerting a synergistic effect of activating natural killer cells through a combination of natural killer cell-specific activating receptors. In experiments using various natural killer cells, natural killer cells expressing the chimeric antigen receptor effectively recognized target cells and activated AKT and ERK signals, thereby inducing excellent antitumor activity. In addition, it has been shown that the chimeric antigen receptor activates natural killer cells to promote the production and secretion of cytotoxic substances such as cytokines, and induces degranulation to enhance apoptotic activity. In particular, it was confirmed that the natural killer cell-specific chimeric antigen receptor of the present invention has a higher effect of activating natural killer cells and inducing apoptosis function than T cell-based CAR. Therefore, the natural killer cell-specific chimeric antigen receptor according to the present invention is a novel anti-cancer immunotherapy means, and is expected to exhibit more excellent anti-cancer effects while reducing the risk of side effects of existing anti-cancer therapies.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Specific sequence information related to the present invention is shown in Table 1 below.

division order sequence number Anti-CD19 scFv DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQ VFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS One CD8 hinge domain TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 2 CD28 hinge domain IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP 3 CD28 TM domain FWVLVVVGGVLACYSLLVTVAFIIFWV 4 DAP10 EC domain QTTPGERSSLPAFYPGTSGSCSGCGSLSLP 5 DAP10 TM domain LLAGLVAADAVASLLIVGAVF 6 DAP10 CYP domain LCARPRRSPAQEDGKVYINMPGRG 7 2B4 CYP domain WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS 8 CD3z CYP domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 9 CD8α signal peptide MALPVTALLLPLALLLHAARP 10 Anti-CD19 scFv GATATACAGATGACGCAGAACGTCAAGTCTTTCCGCCAGCTTGGGAGACCGAGTGACTATATCTTGTAGAGCAAGCCAGGATATTTCTAAGTATCTTAACTGGTACCAACAAAAGCCCGATGGAACGGTTAAGCTGCTTATATACCATACCAGTAGACTCCACTCCGGCGTACCATCACGGTTTTCTGGCAGTGGCTCCGGGACCGACTATTCTTTGACGATCTCTAATCTCGAACAAGA GGATATTGCAACATACTTTTGTCAGCAAGGCAATACCTTGCCATATACGTTTGGGGGCGGGACAAAACTTGAGATAACCGGCGGCGGTGGTTCAGGCGGTGGCGGTTCCGGTGGTGGGGGATCAGAGGTTAAGCTTCAGGAATCCGGACCAGGTTTGGTTGCCCCCAGCCAATCTCTCAGCGTTACATGCACGGTTTCAGGCGTCAGTCTCCCCGATTACGGTGTAAGTTGGATTCGGCAACCTCCGCGA AAGGGTCTGGAATGGCTGGGGGTTATTTGGGGGAGTGAGACAACTTATTACAACTCTGCACTTAAGAGTCGGCTTACCATCATCAAGGATAATTCAAAATCACAAGTATTCCTGAAGATGAACTCATTGCAAACAGATGATACAGCTATATACTATTGTGCCAAGCATTACTATTATGGTGGTTCTTATGCAATGGATTACTGGGGGCAAGGCACGTCAGTGACAGTGAGTTCA 11 CD8 hinge domain_NK-CAR1 ACCACAACCCCCAGCACCCCGGCCCCCTACACCTGCACCAACCATCGCCAGCCAGCCTCTGTCCCTGAGGCCAGAGGCCTGCCGCCCCGCCGCCGGCGGAGCAGTGCACAAGGGGCCTGGACTTCGCCTGTGAT 12 CD8 hinge domain_NK-CAR3 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT 13 CD28 hinge domain ATTGAAGTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTTCCCGGACCTTCTAAGCCC 14 CD28 TM domain TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG 15 DAP10 EC domain CAGACAACCCCCGGCGAGCGCAGCTCCCCTGCCAGCCTTTTACCCTGGCACCTCCGGCTCTTGCAGCGGATGTGGCTCCCTGTCTCTGCCT 16 DAP10 TM domain CTGCTGGCCGGCCTGGTGGCCGCCGACGCAGTGGCCTCTCTGCTGATCGTGGGAGCCGTGTTC 17 DAP10 CYP domain CTGTGCGCCAGGCCACGGAGATCCCCAGCACAGGAGGATGGCAAGGTGTATATCAACATGCCTGGCAGGGGA 18 2B4 CYP domain TGGCGGAGAAAGCGGAAGGAGAAGCAGAGCGAGACATCCCCTAAGGAGTTTCTGACCATCTACGAGGACGTGAAGGATCTGAAGACAAGGCGCAATCACGAGCAGGAGCAGACATTCCCCGGCGGAGGCTCCACCATCTATTCTATGATCCAGAGCCAGAGCTCCGCCCCCACATCCCAGGAGCCTGCCTACACCCTGTATTCCCTGATCCAGCCCAGCCGGAAGTCTGGCAGCCGG AAGAGAAACCACTCCCCATCTTTTAATAGCACCATCTACGAAGTGATCGGCAAGTCCCAGCCAAAGGCACAGAACCCAGCAAGGCTGTCTCGCAAGGAGCTGGAGAATTTCGACGTGTATAGC 19 CD3z CYP domain AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 20 CD8α signal peptide ATGGCGCTCCCTGTCACCGCACTGCTTCTTCCGCTGGCACTGCTGCTGCACGCTGCACGGCCT 21 myc-tag GAGCAAAACTTATCTCTGAAGAGGACCTC 22 NK-CAR1_full insert MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGWRRKRKEKQSETSPKEFLTI YEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR 23 NK-CAR2_full insert MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGWRRKRKEKQSETSPKEFLTIY EDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR 24 NK-CAR3_full insert MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVLCARPRRSPAQEDGKVYINMPGRGWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 25 Anti-CD19 scFv H chain_CDR1 GVSLPDYGVS 26 Anti-CD19 scFv H chain_CDR2 VIWGSETTYYNSALKSR 27 Anti-CD19 scFv H chain_CDR3 HYYYGGSYAMDY 28 Anti-CD19 scFv L chain_CDR1 RASQDISKYLN 29 Anti-CD19 scFv L chain_CDR2 SRLHSGV 30 Anti-CD19 scFv L chain_CDR3 QQGNTLPYT 31

Claims (22)

As a natural killer cell-specific chimeric antigen receptor,
(i) an antigen binding domain;
(ii) a CD8 or CD28 hinge domain;
(iii) DAP10 intracellular signaling domain;
(iv) a 2B4 intracellular signaling domain; and
(v) A natural killer cell-specific chimeric antigen receptor comprising a CD3z intracellular signaling domain, wherein the chimeric antigen receptor is expressed in natural killer cells.
According to claim 1,
Characterized in that the antigen-binding domain is a tumor antigen-specific antibody or an antigen-binding fragment thereof, natural killer cell-specific chimeric antigen receptor.
According to claim 1,
The chimeric antigen receptor is a natural killer cell-specific chimeric antigen receptor, characterized in that it satisfies one or more of the following characteristics:
(a) the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 2;
(b) the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 3;
(c) the DAP10 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7;
(d) the 2B4 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 8; and
(e) the CD3z intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 9.
According to claim 1,
The chimeric antigen receptor is a natural killer cell-specific chimeric antigen receptor, characterized in that it further comprises a CD28 transmembrane domain.
According to claim 4,
The CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4, natural killer cell-specific chimeric antigen receptor.
According to claim 1,
The chimeric antigen receptor further comprises at least one selected from the group consisting of a DAP10 extracellular domain and a DAP10 transmembrane domain.
According to claim 6,
The chimeric antigen receptor is a natural killer cell-specific chimeric antigen receptor, characterized in that it satisfies one or more of the following characteristics:
(f) the DAP10 extracellular domain comprises the amino acid sequence of SEQ ID NO: 5; and
(g) the DAP10 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6.
According to claim 1,
The chimeric antigen receptor further comprises a signal peptide, natural killer cell-specific chimeric antigen receptor.
According to claim 8,
Characterized in that the signal peptide is a CD8 signal peptide, natural killer cell-specific chimeric antigen receptor.
According to claim 2,
The tumor antigens include CD19, TAG72, IL13Rα2 (Interleukin 13 receptor alpha-2 subunit), CD52, CD33, CD20, TSLPR, CD22, CD30, GD3, CD171, NCAM (Neural cell adhesion molecule), FBP (Folate binding), Le(Y) (Lewis-Y antigen), PSCA (Prostate stem cell antigen), PSMA (Prostate-specific membrane antigen), CEA (Carcinoembryonic antigen), HER2 (Human epidermal growth factor receptor 2), Mesothelin, CD44v6 (Hyaluronate receptor variant 6), B7-H3, Glypican-3, ROR1 (receptor tyrosine kinase like orphan receptor 1), Survivin, FOLR1 (folate receptor 1), WT1 (Wilm's tumor 1), VEGFR2 (Vascular endothelial growth factor 2), EGFR, And a natural killer cell-specific chimeric antigen receptor, characterized in that at least one selected from the group consisting of KRAS.
According to claim 2,
The antigen-binding fragment is a natural killer cell-specific chimeric antigen receptor, characterized in that selected from the group consisting of scFv, (scFv) ₂ , Fab, Fab' and F (ab') ₂ .
According to claim 2,
The tumor antigen-specific antibody or antigen-binding fragment thereof is a natural killer cell-specific chimeric antigen receptor, characterized in that it comprises the following heavy chain variable region and light chain variable region:
a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28; and
A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 31.
Natural killer cells expressing the natural killer cell-specific chimeric antigen receptor of any one of claims 1 to 12.
According to claim 13,
The natural killer cells are characterized by satisfying one or more characteristics selected from the group consisting of:
(a) increased secretion of one or more selected from the group consisting of MIP-1α, granzyme B, and INF-γ upon antigen recognition;
(b) an increase in one or more activities selected from the group consisting of AKT signaling and ERK signaling upon antigen recognition; and
(c) Increased level of degranulation upon antigen recognition.
A nucleic acid molecule encoding the natural killer cell-specific chimeric antigen receptor of any one of claims 1 to 12.
An expression vector comprising the nucleic acid molecule of claim 15 .
An isolated cell into which the expression vector of claim 16 has been introduced.
A pharmaceutical composition for preventing or treating cancer, comprising natural killer cells expressing the natural killer cell-specific chimeric antigen receptor of any one of claims 2 to 12 as an active ingredient.
According to claim 18,
The cancers include colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharynx cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, and stomach cancer. , bone cancer, lymphoma, hematological cancer, squamous cell carcinoma, adenocarcinoma of the lung, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, A pharmaceutical composition, characterized in that at least one selected from the group consisting of soft tissue sarcoma, urethral cancer, gastrointestinal cancer, glioblastoma, ovarian cancer, endometrial cancer, salivary gland cancer, vulvar cancer, and head and neck cancer.
A cell therapy agent for preventing or treating cancer, comprising natural killer cells expressing the natural killer cell-specific chimeric antigen receptor of any one of claims 2 to 12 as an active ingredient.
(S1) introducing the expression vector of claim 16 into the isolated cells; and
(S2) A method for producing a natural killer cell-specific chimeric antigen receptor comprising culturing cells into which the expression vector has been introduced.
A method for producing natural killer cells expressing a natural killer cell-specific chimeric antigen receptor, comprising introducing the expression vector of claim 16 into isolated natural killer cells.

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