KR20190104920A - Natural killer cell expressing anti-cotinine chimeric antigen receptor specifically binding to cotinine - Google Patents

Abstract

The present invention relates to a natural killer cell expressing an anti-cotinine chimeric antigen receptor (CAR) specifically binding to cotinine, and a cell therapeutic agent containing the same. According to the present invention, the natural killer cell expressing the CAR specifically binding to cotinine can effectively migrate to tumor tissues, regardless of types of cancer, depending on a conjugate bound to cotinine. Therefore, the natural killer cell, according to the present invention, can be effectively used as a gene therapy exhibiting a highly efficient anticancer effect.

Description

NATURAL KILLER CELL EXPRESSING ANTI-COTININE CHIMERIC ANTIGEN RECEPTOR SPECIFICALLY BINDING TO COTININE}

The present invention relates to a natural killer cell expressing an anti-cotinine chimeric antigen receptor (CAR) that specifically binds to cotinine, and a cell therapeutic agent comprising the same.

For decades, methods of treating cancer have steadily changed and evolved. From the 1800s to the 1900s, methods such as surgical, chemotherapy, and radiation therapy were mainly performed, but their limitations began to emerge. Most representatively, the existing treatment methods are only effective in the early cases where the cancer does not metastasize, and if metastasis has already progressed, the possibility of recurrence after surgical operation is high. In addition, it has been reported that chemotherapy has a low therapeutic effect in solid tumors and causes a side effect of inhibiting the growth of normal cells other than cancer cells. In order to solve such problems, research on anticancer immunotherapy is being actively conducted in recent years, which means increasing the immune response so that patients themselves can fight cancer cells.

In recent years, there has been increasing interest in cell therapy methods in which immune cells in the body are taken out, strengthened, or genetically engineered to be re-inserted through immune cell therapy. Representative examples thereof include Tumor Infiltrating Lymphocytes (TIL), Chimeric Antigen Receptor (CAR), and T-Cell Receptor (TCR) technology. Research using the receptor CAR is being actively conducted.

The chimeric antigen receptor (CAR) is an artificial receptor designed to transmit antigen specificity to T cells. These include antigen specific components, transmembrane components, and intracellular components selected to activate T cells and provide specific immunity. Chimeric antigen receptor expressing T cells can be used in a variety of therapies, including cancer therapy.

However, while treatments such as CAR-T are effective against tumors, in some cases these treatments have caused side effects due to partially non-specific attacks on healthy tissues. In order to overcome this, research on the 3rd generation CAR-T is currently underway, which has two signal domains that serve as auxiliary stimulation signals and an additional engineered receptor, which increases the'cancer cell antigen recognition ability', thereby preventing normal cells. It is characterized by minimizing the side effects of attacking. Nevertheless, the current CAR-T technology is manufactured to recognize only one protein expressed in cancer cells, so it is too expensive to develop individual therapeutic agents, and once CAR-T is injected, toxic T cells are produced. Even after cancer cells are removed, their function continues to cause toxicity, and if there are normal cells representing the target protein, they also cause non-specific attacks and cause fatal side effects, but the problem that it cannot be reversed is the problem of CAR-T. It is hindering the development of cell therapy products.

Therefore, there is an urgent need for research on a new improved cell therapy that can solve the above problems.

As a result of research to solve the problems of conventional therapeutic agents, the present inventors produced natural killer cells expressing chimeric antigen receptors using an antigen-binding domain that specifically binds to cotinine. It was confirmed that it was possible to easily develop a general-purpose therapeutic agent while solving the problem, and the present invention was completed.

Accordingly, an object of the present invention is to provide a cell therapy using natural killer cells expressing an antigen-binding domain that specifically binds to cotinine, and a cancer treatment method using the same.

In order to solve the above problems, the present invention is a natural killer cell expressing a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is 1) an antigen binding domain, 2) a transmembrane domain, and 3 ) It includes an intracellular signal transduction domain, and the antigen-binding domain is a domain that specifically binds to cotinine, providing a natural killer cell.

In addition, the present invention provides a cell therapy product comprising the natural killer cells.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the natural killer cells as an active ingredient.

In addition, the present invention provides a method for preventing or treating cancer comprising administering the natural killer cells to an individual.

In addition, the present invention provides a pharmaceutical composition for diagnosis of cancer comprising the natural killer cells as an active ingredient.

In addition, the present invention provides a kit for diagnosis of cancer comprising the natural killer cells.

Natural killer cells expressing a chimeric antigen receptor that specifically binds to cotinine according to the present invention can effectively move to tumor tissue regardless of the type of cancer, depending on the conjugated material conjugated to cotinine. Killer cells can be usefully used as gene therapy that exhibits high-efficiency anti-cancer effects.

1 is a diagram showing the pLVX-AcGFP-C1 vector used in the present invention.
2 is a diagram showing a schematic diagram of a cotinine-specific CAR-NK cell and an example of a cotinine conjugate.
3 is a diagram showing the action of Cot-CAR-NK cells, which are cotinin-specific NK cells.
4 is a diagram showing specific related sequences used in the present invention.
5 is a diagram showing an exemplary schematic diagram of an anti-cortinine chimeric antigen receptor and an expression system thereof.
6 is a diagram showing the result of measuring the expression of CAR in NK92 cells by the flow cytometry method.
7 is a diagram confirming the apoptosis effect of cotinine-CAR NK92 of the present invention on AU565 cells.
8 is a diagram showing the results of confirming the expression level of Her2 for various cancer cells through flow cytometry.
9 is a diagram showing the results of confirming the apoptosis effect of cotinine-CAR NK92 of the present invention according to the presence or absence of Her2-cotinine conjugate through the Calcein-AM method.
10 is a diagram showing the result of confirming the degree of cytokine secretion in cotinine-CAR NK92 cells through ELISA.
11 is a diagram showing the results of confirming the expression level of CD107a in cells by flow cytometry.
12 is a diagram showing the result of confirming the phosphorylation of Erk through the Endodomain of Cotinine-CAR NK92 through flow cytometry.
13 is a diagram showing the results of confirming the apoptosis effect of cotinine-CAR NK92 cells through the Calcein-AM method.
14 is a diagram showing the results of confirming the expression levels of Her2 and EGFR in AU565, SK-OV-3, A431 and A459 cells through flow cytometry.
15 is a diagram showing the results of confirming the apoptosis effect of cotinine-CAR NK92 according to the presence or absence of a conjugate through the Calcein-AM method.
16 is a diagram showing the result of confirming the change in cytokine secretion of cancer cells by treatment with cotinine-CAR NK92 and the conjugate through ELISA.
17 is a diagram showing the results of confirming the expression level of CD107a in AU565 cells by flow cytometry.
18 is a diagram showing the results of confirming the expression level of CD107a in A431 cells by flow cytometry.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention is a natural killer cell expressing a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is 1) an antigen binding domain, 2) a transmembrane domain, and 3) a cell It includes an intra-signal transduction domain, and the antigen-binding domain is a domain that specifically binds to cotinine.

Natural killer cells expressing a chimeric antigen receptor that specifically binds to cotinine according to the present invention can effectively move to tumor tissue regardless of the type of cancer, depending on the conjugated material conjugated to cotinine. Killer cells can be usefully used as gene therapy that exhibits high-efficiency anti-cancer effects.

Since the natural killer cells expressing the chimeric antigen receptor according to the present invention are characterized by specifically binding to cotinine, they include an antigen-binding domain that specifically binds to cotinine.

The antigen binding domain may be an antibody or antibody fragment that specifically binds to cotinine. Cancer can be treated by administering NK cells expressing the chimeric antigen receptor together with a cotinine-fused conjugate to a patient.

In particular, the natural killer cells (CAR-NK cells) expressing the chimeric antigen receptor of the present invention are problematic due to the persistent toxicity of cancer immunotherapy using conventional CAR-T therapeutic agents, the risk of autoimmune diseases, and xenogeneic cells. General-purpose therapeutic agent not only can solve the problem of graft-versus-host disease (GVHD) and non-target toxicity problem for transplantation through the switch of reaction start (on) / stop (off), but also target various cancer cells. There is an advantage that can be used as. In addition, since the CAR-NK cells of the present invention can be allografted, high-efficiency cells can be premade compared to CAR-T, which uses the patient's own immune cells, thus shortening the time of administration of the therapeutic agent to increase therapeutic efficacy. According to the development and reduction of treatment cost, it can be usefully used in the development of therapeutic agents for various diseases.

In the present invention, "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and its kind is not particularly limited. In addition, in the present specification, the antibody includes, but is not limited to, fragments of an antibody having antigen-binding ability, such as Fab, Fab', F(ab')2, Fv, and the like.

In the present invention, a "chimeric antibody" refers to an antibody derived from an animal whose variable region or complementarity determining region (CDR) thereof is different from the rest of the antibody. Such an antibody, for example, the antibody variable region may be derived from an animal other than human (eg, mouse, rabbit, poultry, etc.), and the antibody constant region (constant region) may be an antibody derived from human. Such chimeric antibodies can be prepared by methods such as gene recombination known in the art.

As used herein, "heavy chain" refers to a full-length heavy chain and fragments thereof, including variable region domain VH and three constant region domains, CH1, CH2 and CH3, comprising an amino acid sequence of a variable region sufficient to confer antigen specificity. It refers to all.

As used herein, "light chain" refers to both a full-length light chain including a variable region domain VL and a constant region domain CL including an amino acid sequence of a variable region sufficient to confer antigen specificity and a fragment thereof.

The antigen-binding domain constituting the chimeric antigen receptor of the present invention is a site to which the main signal is transmitted, and is located outside the cell membrane and refers to a site that recognizes a cell membrane ligand (a substance that binds to and activates a receptor) of a target cell with a specific antigen.

The transmembrane domain of the present invention is a site that connects the antigen-binding domain, co-stimulatory, and essential signaling domains between cell membranes, and the intracellular signal transduction domain activates the immune response of NK cells by binding of the antigen-binding domain. It means the part to be made.

The chimeric antigen receptor of the present invention is characterized in that the antigen-binding domain specifically binds to cotinine, and the cotinine is a major product of nicotine metabolism, and refers to a substance having the structure of the following formula (1).

[Formula 1]

The cotinine is preferably a conjugate conjugated with a conjugation material, and the conjugation material may be selected from the group consisting of peptides, aptamers, hormones, proteins, and chemicals, and more preferably, the conjugation material is apta. It may be, but is not limited thereto.

In the present invention, the conjugate to which cotinine is conjugated is a dimorphic molecule, and at least one non-polypeptide moiety such as a polymer molecule, a lipophilic compound, a carbohydrate moiety or an organic derivatizing agent, in particular, one or more polypeptides in the polymer moiety, is typical. Can be made by covalently attaching one polypeptide. Additionally, the conjugate may be attached to one or more carbohydrate moieties, in particular using N- or O- glycosylation. Covalent attachment means that the polypeptide and the non-polypeptide moiety are directly covalently linked to each other or indirectly covalently linked to each other through an intermediary moiety or moiety such as a linking bridge, space, linking moiety, or moiety. it means. For example, conjugates in which the conjugation material disclosed herein is conjugated with cotinine are included in this definition.

The conjugate of the conjugation material according to the present invention and the conjugate of cotinine with the anti-cotinine antibody bound to the complex may retain both the conjugated material and the unique properties of the antibody by using cotinine as a hapten. Specifically, the complex may possess the specific reactivity and function of the molecule, complement-mediated cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and a long in vivo half-life, which are characteristics of the antibody.

The conjugation material may be selected from the group consisting of, for example, peptides, aptamers, hormones, proteins, and chemicals, for example, WKYMVm-NH2 peptide (WKYMVm-NH2), wkymvm-NH2 peptide (wkymvm-NH2 ), AS1411 aptamer, pegaptanib, absiksimab and insulin. In some embodiments of the present invention, the protein may be an antibody, specifically an anti-Her2 antibody or an anti-EGFR antibody.

Accordingly, the present invention provides a method of increasing the in vivo half-life of the conjugated substance by binding an anti-cotinine antibody to a conjugate obtained by conjugating a conjugated substance with cotinine.

In addition, the present invention provides a method for causing complement-dependent toxicity to cells to which the conjugated substance binds by binding an anti-cotinine antibody to a conjugate obtained by conjugating a conjugated substance with cotinine.

The antigen-binding domain of the present invention is a substance that specifically binds to cotinine, and may be an antibody or an antibody fragment, and the antibody fragment may be an scFv. Of the antibody or antibody fragment sequence that binds to cotinine, cotinin-scFv may consist of, for example, an amino acid sequence represented by SEQ ID NO: 11, and as a result of modification or substitution thereof, preferably 80% or more of the amino acid sequence, More preferably, it has 90% or more, even more preferably 95% or more sequence homology, and may include, without limitation, a sequence showing substantially the same physiological activity as the amino acid sequence represented by SEQ ID NO: 11.

In addition, cotinine-scFV may be encoded by a nucleotide sequence represented by SEQ ID NO: 17 and a nucleotide sequence having at least preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence homology thereto. have.

In the anti-cotinine chimeric antigen receptor capable of specifically binding to cotinine, the antigen-binding domain may consist of a heavy chain variable region, a linker sequence, and a light chain variable region. The heavy chain variable region includes an amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 1 capable of encoding a functional equivalent to an amino acid expressed by the nucleotide sequence of SEQ ID NO: 1 It may consist of an amino acid sequence encoded by a nucleotide sequence having at least 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more.

The light chain variable region includes an amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence represented by SEQ ID NO: 2 capable of encoding a functional equivalent to an amino acid expressed by the nucleotide sequence of SEQ ID NO: 2 It may consist of an amino acid sequence encoded by a nucleotide sequence having at least 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more.

The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2 and each having 95% or more sequence homology is substantially identical to the amino acid sequence encoded by the nucleotide sequences of SEQ ID NO: 1 and 2 It can show the physiological activity of

The antibody or antibody fragment of the present invention may further include a linker, and the heavy chain variable region and the light chain variable region may be interconnected through the linker. The linker may be used without limitation as long as it is a component capable of forming a VH-linker-VL domain by connecting the heavy chain variable region and the light chain variable region, and is preferably encoded by SEQ ID NO: 3 or a nucleotide sequence showing 95% or more homology thereto. It may consist of an amino acid sequence.

The antigen binding domain of the anti-cotinine chimeric antigen receptor of the present invention may be linked to the transmembrane domain by a hinge region, a spacer region, or a combination thereof. The hinge region or spacer region of the present invention may be one or more selected from Myc epitope, CD8 hinge region, and Fc, and may preferably include Myc epitope and CD8 hinge region. The Myc epitope and CD8 hinge region of the present invention function as a linking domain (spacer).

The CD8 hinge region of the present invention has sequence homology with SEQ ID NO: 12 or 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more with SEQ ID NO: 12, represented by SEQ ID NO: 12. It may consist of an amino acid sequence exhibiting a function substantially equivalent to that of the amino acid sequence being used; Myc epitope is SEQ ID NO: 13 or 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology with SEQ ID NO: 13, and the amino acid sequence represented by SEQ ID NO: 13 It may consist of an amino acid sequence exhibiting substantially equivalent functions. Accordingly, the hinge region or the spacer region of the present invention may include SEQ ID NO: 12 or an amino acid sequence exhibiting 95% or more homology thereto; Or it is preferably composed of an amino acid sequence that shows 95% or more homology to SEQ ID NO: 13 or.

In the present invention, the nucleotide sequence encoding the Myc epitope may include all nucleotide sequences capable of encoding the amino acid sequence of SEQ ID NO: 13, but preferably may be the nucleotide sequence of SEQ ID NO: 7, and human CD8 hinge region The nucleotide sequence encoding is may include all nucleotide sequences capable of encoding the amino acid sequence of SEQ ID NO: 12, but preferably may be the nucleotide sequence of SEQ ID NO: 4.

The transmembrane domain, which is a component of the chimeric antigen receptor of the present invention, is CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. It may include a transmembrane domain of a protein selected from the group consisting of. For example, the transmembrane domain may be a transmembrane domain of CD28, which may be composed of SEQ ID NO: 16 or an amino acid sequence that exhibits 95% or more homology thereto, but is not limited thereto.

In addition, the intracellular signal transduction domain, which is a component of the chimeric antigen receptor of the present invention, may use an intracellular signal transduction domain known in the art without limitation. As an embodiment of the present invention, the intracellular signal transduction domain may be DAP10, CD3 zeta, or a combination thereof, but is not limited thereto.

The chimeric antigen receptor of the present invention can exhibit a killing effect on cancer cells with high activity of NK cells by using DAP10 and CD3 zeta as intracellular signaling domains. In this case, DAP10 functions as a Co-stimulatory domain, and has sequence homology of SEQ ID NO: 14 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more. , It may consist of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 14; CD3 zeta functions as an NK cell activation domain, and has sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% with SEQ ID NO: 15 or As such, it may be composed of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 15. Accordingly, the intracellular signaling domain of the present invention comprises SEQ ID NO: 14 or an amino acid sequence that exhibits 95% or more homology thereto; Alternatively, it may be composed of an amino acid sequence exhibiting 95% or more homology with SEQ ID NO: 15.

In addition, the antigen-binding domain of the present invention may include a signal peptide for domain exposure. The signal peptide may be a CD8 alpha or a mouse light kappa signal peptid, and in the case of a CD8 alpha, the signal peptide of the present invention consists of SEQ ID NO: 10 or an amino acid sequence that exhibits 95% or more homology thereto. I can. In addition, the nucleotide sequence encoding the CD8 alpha region may include all nucleotide sequences capable of encoding the amino acid sequence of SEQ ID NO: 10, but preferably may be the nucleotide sequence of SEQ ID NO: 6.

In addition, the present invention can transform the chimeric antigen receptor into NK cells using a vector containing a polynucleotide capable of encoding (encoding) the anti-cotinine chimeric antigen receptor described above.

Vectors used in the present invention can be used in a variety of vectors known in the art, such as a promoter (promoter), terminator (terminator), enhancer (enhancer) according to the type of host cell to produce the antigen receptor. An expression control sequence, a sequence for membrane targeting or secretion, etc. can be appropriately selected and variously combined according to the purpose. The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable vectors include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be variously prepared according to the purpose.

In the present invention, as a preferred example, a lenti-virus vector (Clontech, 632155) may be used, and specifically, pLVX-AcGFP-C1, which is a vector used in an embodiment of the present invention, is shown in FIG. 1.

In some embodiments, the natural killer cells are obtained or produced from bone marrow, peripheral blood, peripheral blood mononuclear cells, or umbilical cord blood. In some embodiments, the cell is a human cell.

The schematic diagram of NK cells expressing the anti-cotinine chimeric antigen receptor according to the present invention and the state of the cotinine conjugate are shown in FIG. 2.

The NK cells transformed by introducing an antigen receptor as described above can recognize cotinine as an antigen and bind specifically to cotinin, and can express a chimeric antigen receptor specific to cotinine on the cell surface. Specifically, it is possible to induce activation of NK cells and induce tumor-specific death through an intracellular signal transduction domain upon contact and ligation with a tumor antigen such as CAR-NK cell-like activity.

Fig. 3 schematically shows the action of Cot-CAR-NK cells, which are cotinine-specific NK cells according to the present invention.

In the present invention, in particular, CAR-NK cells refer to cells into which a chimeric antigen receptor has been introduced into NK (Natural killer) cells. The cells have the advantage of anticancer-specific targeted treatment, which is the existing advantage of CAR-T therapeutics, and include the chimeric antigen receptor according to the present invention, through a switch function that can turn on/off the therapeutic response, which is an existing problem. In addition to solving the toxicity problem, it can also have the advantage that it can be used as a general-purpose therapeutic agent through the terminal modification of the conjugate fused with cotinine that can bind to the chimeric antigen receptor. That is, NK cells expressing a chimeric antigen receptor that specifically binds to cotinine according to the present invention can effectively move to tumor tissue regardless of the type of cancer, depending on the conjugated material conjugated to cotinine, according to the present invention. Cells can be usefully used as gene therapy that exhibits excellent anticancer effects with high specificity.

Accordingly, another aspect of the present invention is a cell therapy product comprising the natural killer cells, a pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient, or a cancer prevention comprising the step of administering the cells to an individual. Or how to treat it.

In the present invention, the cells can be used for cell therapy such as anticancer therapy. The cells can come from a donor or can be cells obtained from a patient. Such cells can be used, for example, for regeneration to replace the function of diseased cells. The cells can also be modified to express heterologous genes so that biological agents can be delivered to certain microenvironments, such as, for example, diseased bone marrow or metastatic deposits.

In addition, the pharmaceutical composition for preventing or treating cancer of the present invention may additionally include a conjugate in which a conjugate material is fused to cotinine, and the method for preventing or treating cancer is administering a conjugate in which a conjugate material is fused to cotinine. It may further include the step of. Depending on the conjugated material conjugated to cotinine, it may specifically bind to a target cell, thereby exhibiting an excellent anticancer effect.

Specifically, the cells provided in the present invention are natural killer cells expressing a chimeric antigen receptor having an antigen-binding domain capable of specifically binding to cotinine, and the chimeric antigen receptor is, for example, a target cell of a conventional CAR therapeutic agent. The onset (on)/stop (off) response to cotinine can be modulated with conjugates or intermediate mediators fused with cotinine, which can be very beneficial in subsequent cell therapy, situations where the activity of the treated cells needs to be increased or decreased. Includes safety switch. For example, if NK cells expressing a chimeric antigen receptor are provided to a patient, in some circumstances there may be side effects, such as off-target toxicity. Or, for example, the treatment cells may serve to reduce tumor cells, or tumor size, and may no longer be needed. In this situation, cotinine can be regulated so that the treatment cells are no longer activated.

In the present invention, cancer may include all carcinomas known in the art without limitation.

In the context of the present invention, the term "unit dose" refers to physically discrete units suitable as a single dose for a mammal, and includes a predetermined amount of the pharmaceutical composition calculated to obtain the desired immunogen stimulating effect together with the desired diluent. The specific content of the unit dose of the inoculum is influenced by the intrinsic properties of the pharmaceutical composition and the specific immunological effect to be achieved, and is determined accordingly.

An effective amount for a specific application may vary depending on factors such as the disease or condition to be treated, the specific composition to be administered, the size of the subject, and/or the severity of the disease or condition. Without undue experimentation, the effective amount of a particular composition presented herein can be determined empirically.

In addition, another aspect of the present invention is a composition for diagnosis of cancer and a kit for diagnosis of cancer comprising natural killer cells expressing a chimeric antigen receptor (CAR) according to the present invention. Another aspect of the present invention provides information for diagnosis of cancer, comprising the step of contacting a composition comprising a natural killer cell expressing a chimeric antigen receptor (CAR) of the present invention with a sample isolated from an individual. That's how to do it. The composition or kit may additionally include a conjugate in which a conjugate material is fused to cotinine, but is not limited thereto.

In the present invention, the term "diagnosis" refers to determining the susceptibility of an object to a specific disease or disease, determining whether an object currently has a specific disease or disease, as long as it has a specific disease or disease. Determining the prognosis of the subject, or therametrics (eg, monitoring the condition of the subject to provide information on treatment efficacy).

Another aspect of the present invention is for use in the prevention or treatment of cancer of natural killer cells expressing the chimeric antigen receptor (CAR).

The chimeric antigen receptor, natural killer cells expressing it, and its use for preventing or treating cancer are as described above.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Vector backbone

As a vector used in the present invention, a lenti-virus vector (Clontech, 632155) was used, specifically pLVX-AcGFP-C1 shown in FIG. 1 was used. After deleting the Kozak sequence (CTCGAG; n.t. 2801-2806) and AcGFP1 (Aequorea coerulescens green fluorescent protein; n.t. 2807-3604) to be used in the experiment, XhoI was used as a restriction enzyme. Specific related sequences are shown in FIG. 4.

Example 2. Preparation of target antigen and cotinine conjugate

The chemical structure of cotinine (trans4-cotininecarboxylic acid), which is a small molecule substance to be used as a target antigen, is as shown in the following Formula 1, and cotinine was purchased and used from Sigma-Aldrich.

[Formula 1]

In addition, a conjugate in which the cotinine and the conjugation material were fused was prepared by the following method.

Specifically, a conjugate in which cotinine and an anti-HER2 antibody were conjugated was prepared using trastzumab (Genentech, USA), an anti-HER2 antibody, as the HER2-cotinine conjugate. At this time, the conjugate was conjugated using an EDC (1-ethyl-3-[3 dimethylaminopropyl]carbodiimide) connection method. First, the anti-HER2 antibody was prepared by dissolving it in PBS to a concentration of 25 μM. Meanwhile, in 1 ml of MES buffer [0.1 M of MES (2-[morpholino]ethaneusulfonic acid) and 0.5 M of sodium chloride, pH 6.0], trans-4-cotininecarboxylic acid (Sigma-Aldrich) was It was prepared by dissolving to a concentration of 5 mM. 50 mM EDC and 125 mM N-hydroxysulfosuccinimide (Sulfo-NHS, Thermo Scientific, USA) were added thereto, followed by dissolving it with stirring for 15 minutes at room temperature. -An active solution in which the NHS ester was produced was prepared. In order to induce a reaction between the cotinine-NHS ester and the amine group of the protein, sodium hydroxide solution was added, the pH of the active solution was adjusted to 7 or higher, and 1 ml was taken, and an anti-HER2 antibody to be conjugated with cotinine was added thereto. It was added in the same amount as the active solution at a concentration of 25 μM. The mixture was reacted with stirring at room temperature for 3 hours to obtain a cotinine-HER2 antibody conjugate produced through an EDC ligation reaction. The obtained cotinine-HER2 antibody conjugate was dialyzed against PBS using a Slide-A-lyzer™ dialysis cassette (Thermo Fisher Scientific, USA), or the buffer was exchanged with PBS using an Amicon　 Ultra Centrifugal Filter (EMD Millipore, USA). Was used.

In addition, the EGFR-Cotinine conjugate was purchased from Anije Co., Ltd. (Cotinine-zEGFR:95) as a conjugate in which an anti-EGFR affibody was fused with cotinine, and the specific sequence of the used anti-EGFR affibody is as follows.

trsn-4-cotininecarboxylic acid-VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 30)

Example 3. Anti-Cotinine Chimeric Antigen Receptor Derivation

A plasmid containing a nucleic acid encoding each domain of a chimeric antigen receptor that specifically binds to cotinine of the present invention was prepared by the following method.

(1) signal peptide

Based on the Human T-cell surface glycoprotein CD8 alpha chain (GenBank: AK300089.1), using two types of primers (forward primer: SEQ ID NO: 18, reverse primer: SEQ ID NO: 19) each containing the restriction sites of XhoI and XbaI. It was amplified by polymerase chain reaction and then cloned.

(2) target specific recognition domain-scFv

As an antigen-binding domain capable of specifically binding to cotinine, an anti-cotinine chimeric antibody or antibody fragment thereof was to be derived, and ScFv-related information in Korean Patent No. 10-1648960 was referenced for the sequence derivation of ScFv. Specifically, the antigen-binding domain includes the nucleotide sequence represented by SEQ ID NO: 17, and is composed of VH-linker-VL.

(3) Connection domain (spacer)

(A) Myc epitope

In the plasmid (Anti-cotinine 28Z-1 CAR ORF, cot28z-1) possessed by the present inventors, two kinds of primers each containing restriction sites of sfiI and HindIII (forward primer: SEQ ID NO: 20, reverse primer: SEQ ID NO: 21) was amplified by polymerase chain reaction and then cloned.

(B) Human CD8 hinge area

Two kinds of primers each containing a HindIII single restriction enzyme site in the plasmid (Anti-cotinine 28Z-1 CAR ORF, cot28z-1) possessed by the present inventor (forward primer: SEQ ID NO: 22, reverse primer: SEQ ID NO: 23) It was amplified by polymerase chain reaction using and then cloned.

(4) transmembrane area

The cytoplasmic region was used as the membrane permeable region at the hinge of the human CD28 gene. It was prepared by adding the sequence of restriction enzyme BamH1 to the forward primer (SEQ ID NO: 24), and adding the restriction enzyme EcoRI sequence to the reverse primer (SEQ ID NO: 25).

The DNA of the transmembrane region was obtained by performing PCR on the cDNA of Jurkat cells using the above primers.

(5) one or more intracellular signal transduction domains

(A) Co-stimulatory domain

DAP10 was used as a co-stimulatory domain, and the sequence of the restriction enzyme EcoRI was added to the forward primer (SEQ ID NO: 26), and the restriction enzyme NotI sequence was added to the reverse primer (SEQ ID NO: 27).

The cDNA of primary mature NK cells was subjected to PCR using the above primers to create a co-stimulatory domain.

(B) NK cell activation domain

CD3 zeta was used as the NK cell activation domain. Specifically, PCR was performed using two kinds of primers (forward primer: SEQ ID NO: 28, reverse primer: SEQ ID NO: 29) on the cDNA of Jurkat cells to prepare an activation domain.

Each of the domains were sequentially linked using each restriction enzyme, and specific sequence information corresponding to each domain is shown in Table 1 below.

[Table 1]

When the sequence of Table 1 is matched to the structure described above, the signal peptide is represented by SEQ ID NO: 6 corresponding to the human CD8 alpha region, the heavy chain variable region of the target-specific recognition domain is SEQ ID NO: 1, and the linker is SEQ ID NO: 3, the light chain variable region is represented by SEQ ID NO: 2. The linking domain (spacer) was represented by the Myc epitope of SEQ ID NO: 7 or the human CD8 hinge region of SEQ ID NO: 4, and CD28 as the transmembrane region was represented by SEQ ID NO: 5. At least one intracellular signal domain is shown in SEQ ID NO: 8 for DAP-10 as the co-stimulatory domain and CD3 zeta as the NK cell activation domain in SEQ ID NO: 9.

Example 4. Preparation of NK Cells Introduced with Anti-Cotinine Chimeric Antigen Receptor

The polynucleotide encoding the anti-cotinine chimeric antigen receptor shown in Example 3 was introduced into a vector, and a transformed natural killer cell was prepared using this, and a chimeric antigen receptor containing a cotinine-specific antigen-binding domain A schematic diagram of and a schematic diagram of the expression system thereof are shown in FIG. 5.

First, using the vector from which AcGFP was removed from pLVX-AcGFP-C1 of Example 1 as a base vector, using the restriction enzymes of XhoI and XbaI of the MCS in the vector, the anti-Cotinine chimeric antigen receptor of Example 3 (Cotinine -CAR) was inserted into the vector.

Then, a vector containing cotinine-CAR was transformed into HEK293T cells with a viral packaging vector (PMDLg/RRE, RSV/REV, VSVG), and Lentivirus expressing Cotinine-CAR was obtained therefrom. The Lentivirus was concentrated using an ultra-high-speed centrifuge, and the Lentivirus expressing the concentrated Cotinine-CAR was infected with HEK293T or Hela cells, and the amount of myc epitope of cotinine-CAR was confirmed by flow cytometry, and the Infection Unit was calculated. . The number of NK cells and the amount of lentivirus were calculated so that the multiplicity of infection (MOI) was 10, and lentivirus expressing cotinine-CAR was infected with NK cells by spinoculation (360g, 90min, RT). The infected NK cells were cultured for 5 hours at 37° C. and 5% CO ₂ conditions, and then replaced with fresh culture medium. After 3 days, in order to select infected NK cells, puromycin at a concentration of 3 ug/ml was treated to continue cultivation.

As a control, uninfected NK cells were also treated with puromycin, and culture was performed using a culture medium treated with puromycin until the control cells were annihilated by puromycin. At the point when the control cells were annihilated, the infected NK cells were proliferated or expanded by replacing them with a medium without Puromycin. For proliferation or expansion of the selected cells, 12.5% fetal bovine serum containing Alpha-MEM, 12.5% horse serum, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid and 200 U/ml recombinant IL-2 Experiments were carried out using a medium using.

Example 5. Confirmation of expression of CAR in NK cells introduced with anti-cotinine chimeric antigen receptor

Expression of CAR in the NK cells of Example 4 into which the anti-cortinine chimeric antigen receptor of the present invention was introduced was confirmed by flow cytometry.

A Myc antibody (CTS; 9B11) corresponding to the Myc epitope of Cotinine-CAR was reacted to NK cells expressing cotinine-CAR (4°C, 30 minutes in the dark), and the expression of Myc was confirmed through flow cytometry, or Her2 After reacting the cotinine conjugate to which the antibody is fused to NK cells expressing cotinine-CAR for 30 minutes at 4° C., an Fc (eBioscience; 12-4988-82) antibody corresponding to the Fc region of Her2 antibody was secondarily reacted. After that, it was confirmed by flow cytometry. Original NK92 cells that did not express CAR were used as a control, and the results are shown in FIG. 6.

As shown in Fig. 6, it was confirmed that the NK cells of the present invention into which the anti-cotinine chimeric antigen receptor was introduced express the anti-cotinine antibody fragment.

Example 6. Confirmation of the binding specificity of cotinine-CAR NK cells to cotinine

Cotinine polymer cot-SWNT and HER2-tumor cells were used to determine whether or not cotinine ScFv of Cotinine-CAR NK cells specifically binds to cotinie.

Specifically, after staining AU565 (human breast carcinoma) with Calcein-AM (life technologies; C1430), NK cells expressing cotinine-CAR were mixed in 200 ul of RPMI1640 (10% FBS) at a ratio of 1:1, The cotinine conjugate and cot-SWNT in which the Her2 antibody was fused at a concentration of 1 ug/ml were simultaneously treated at different concentrations (0, 0.005, 0.05, 0.5 and 5 mg/ml) to allow competition with the Her2-cotinine conjugate. In addition, in order to confirm the effect of Cot-SWNT alone, the condition of Cot-SWNT (5 mg/ml) alone was added to proceed. After reacting at 37°C and 5% CO ₂ for 4 hours, 100 ul of the supernatant was taken and the amount of calcein present in the supernatant was checked to confirm the killing effect (cell death effect) according to each condition. As a control group, AU565 stained with Calcein was treated with RPMI1640 (10% FBS) only (spontaneous value) and 2% triton X-100 (maximum value), and the killing effect was calculated by the following method. I did.

killing effect (%) = (calcein release value-spontaneous value according to conditions) / (maximum value-spontaneous value) x 100

The results for the resulting killing effect are shown in FIG. 7.

As shown in Figure 7, it was confirmed that the killing effect of cotinine-CAR NK cells against her2-expressing cancer cells decreased as the concentration of cot-SWNT increased, from which the cotinine-CAR NK cells of the present invention It was confirmed that it acts specifically on the target cells depending on the conjugate material of the conjugate.

Example 7. Confirmation of apoptosis effect of cotinine-CAR NK cells by Herceptin (her2)-cotinine conjugate

Using the anti-Her2-cotinine conjugate prepared in Example 3 and the cotinine-CAR NK92 cells of Example 4, the conjugate recognized Her2 on the surface of cancer cells, and thus the anti-cotinine chimeric antigen receptor of the present invention was introduced into NK cells. The apoptosis effect was confirmed.

First, AU565 (human breast carcinoma; RPMI1640 (10% FBS; 200nm HEPEs)), SK-OV-3 (human ovarian carcinoma; RPMI1640 (10% FBS)), SK-BR-3 (human breast carcinoma; DMEM (10 % FBS)) and K562 (chronic myelogenous leukemia; RPMI1640 (10% FBS)) were treated with an anti-Her2 antibody (Invtrogen; BMS120FI) in an amount of 1 ug/100 ul, and at 4° C., 30 in the dark. It was allowed to react for a minute. After the reaction was over, each cell was checked for the degree of Her2 expression in each cell using flow cytometry (BD; FacsCantoII), and the results are shown in FIG. 8.

As shown in Fig. 8, it was confirmed that Her2 was expressed in AU565, SK-OV-3 and SK-BR-3 cells, but Her2 was not expressed in K562 cells.

Then, the apoptosis effect of cotinine-CAR NK cells according to the presence or absence of the Her2-cotinine conjugate was confirmed by the Calcein-AM method. Specifically, the four cell lines of AU565, SK-OV-3, SK-BR-3, and K562 were treated with Calcein-AM at a concentration of 5 ug/ml, and then 37°C, 5% CO ₂ , in the dark conditions. It was reacted for 1 hour. After the reaction was over, the original NK92, NK92 (Puro-92), and cotinine-CAR NK92 (cot-10z-92), each of which a control vector expressing the pLVX empty vector from which AcGFP was deleted, was inserted, and 5: 1, 1:1, 0.5:1 (Effector cell; NK92, Puro-92, Cot-10z-92): Target cell; AU565, SK-OV-3, SK-BR-3, K562) mixed with 200 ul RPMI (10% FBS) and _{reacted for 4 hours at 37°C and 5% CO 2} conditions, and then 100 ul of the supernatant was added. By taking and checking the amount of calcein present in the supernatant, the killing effect according to each condition was confirmed, and the results are shown in FIG. 9.

As shown in Figure 9, cotinine-CAR NK92 cells exhibited apoptotic activity in AU565, SK-OV-3 and SK-BR-3 cells expressing Her2, but not K562 cells not expressing Her2. It was confirmed that it was not. In addition, the Her2-cotinine conjugate did not affect the apoptosis effect of NK92 and Puro-92, but it was confirmed that it had an effect on cotinine-CAR NK92. From these results, it was confirmed that the cotinine-CAR NK cells of the present invention specifically induce apoptosis by the cotinine-conjugate.

Example 8. Confirmation of cell activity of cotinine-CAR NK cells

By confirming the secretion of cytokine and granule of NK cells, the activity of NK cells was confirmed. Specifically, for the secretion of cytokine, the original NK92, PURO-92, and cot-10z-92 were mixed with AU565 and 1:1 in RPMI1640 (10% FBS), respectively, and a cotinine conjugate was added thereto at 37°C and 5% CO ₂ . After reacting for 6 hours, the supernatant was collected and the cytokine IFN-r and TNF-a present in the supernatant were confirmed by ELISA, and the original NK92, PURO-NK92, and cot-10z-92 alone cytokine secretion levels were used as a control. And the results are shown in FIG. 10.

In addition, the expression of CD107a was mixed with the original NK92, PURO-92, and co-10z-92 in RPMI1640 (10% FBS) in a ratio of 1:1 with AU565, respectively, and the cotinine conjugate and the CA107a antibody (BD; 555801) were mixed. After treatment together, it was reacted for 4 hours at 37° C. and 5% CO _2. After staining using CD56 antibody to select NK92 cells from the cells after the reaction, the levels of CD107a expression of original NK92, PURO-NK92, and cot-10z-92 were compared using flow cytometry. The basic levels of CD107a expression of the original NK92, PURO-92, and Cot-10z-92, and the level of expression of CD107a in the absence of a cotinine conjugate mixed with AU565 were used as controls, and the results are shown in FIG. 11.

As shown in FIGS. 10 and 11, it was confirmed that the secretion of cytokines and granules of NK cells responded to cancer cells only in the presence of cotinine-CAR NK cells and her2-cotinine conjugates.

Example 9. Signal change of cotinine-CAR NK cells by Her2-cotinine conjugate

When Cotinine-CAR NK cells exhibited activity, a signal that changes when the NK cells exhibited activity was measured to confirm the activity of NK cells. Specifically, phosphorylation of Erk as a signal through the Endodomain of Cotinine-CAR NK92 was confirmed through flow cytometry, and the results are shown in FIG. 12.

As shown in Fig. 12, phosphorylation of intracellular Erk does not increase when cotinine-CAR NK92 cells are alone (with out; control), but in the presence of cancer cells, by her2-cotinine conjugate (with her2-cot; her2-cot treatment group) cotinine-CAR NK92 cells were found to increase Erk phosphorylation.

Example 10. Confirmation of the specificity of Herceptin (her2)-cotinine conjugate for cotinine-CAR NK cells

In order to confirm the specificity of the Her2-cotinine conjugate of Cotininie-CAR NK cells, the apoptosis effect of the conjugate was confirmed.

First, a respiratory syncytial virus (RSV)-cotinine conjugate was used as a control for the Her2-cotinine conjugate. The RSV-cotinine conjugate was the same method as Example 2 except that an anti-RSV antibody (Palivizumab, Synagisㄾ, AstraZeneca, UK) was used as an antibody to be fused with cotinine in the preparation method of the cotinine conjugate of Example 2. Was prepared.

Then, cotinine-CAR-NK92 cells and calcein-stained AU565 were mixed in RPMI1640 (10% FBS) in a ratio of 5:1, 1:1 and 0.5:1, respectively, and against Her2, an antigen expressed in AU565 cells. Her2-cotinine conjugate and unexpressed respiratory syncytial virus antibody (RSV; Palivizumab; Synagisㄾ, AstraZeneca, UK)-cotinine conjugate were treated at a concentration of 1 ug/ml, respectively, at 37°C and 5% CO ₂ under conditions of 4 After reacting for a period of time, the apoptosis effect of cotinine-CAR NK92 cells was confirmed through the Calcein-AM method, and the results are shown in FIG. 13.

As shown in FIG. 13, it was confirmed that Cotininie-CAR NK cells specifically exhibited activity on the Her2-cotinine conjugate.

Example 11.Confirmation of apoptosis effect of cotinine-CAR NK cells by Herceptin (her2)-cotinine conjugate and EGFR (affibody)-cotinine conjugate

The cotinine conjugate of the present invention by recognizing Her2 or EGFR on the surface of cancer cells using the cotinine-CAR NK92 cells of Example 4 together with the anti-Her2-cotinin conjugate and the anti-EGFR-cotinine conjugate prepared in Example 3 The effect of apoptosis by NK cells into which the chimeric antigen receptor was introduced was confirmed.

First, four of AU565 (human breast carcinoma), SK-OV-3 (human ovarian carcinoma), A431 (human skin carcinoma; DMEM (10% FBS)) and A549 (human lung carcinoma; RPMI1640 (10% FBS)). The cell line was treated with an anti-Her2 antibody and an anti-EGFR antibody (BD;563577) in an amount of 1 ul/100 ul, and after reacting for 30 minutes in the dark at 4°C, the expression level of her2 and EGFR of each cell was measured by flow cytometry. It was confirmed through, and the results are shown in FIG. 14.

As shown in Fig. 14, it was confirmed that the expression levels of Her2 and EGFR of the four cells were different for each cell.

Then, the apoptosis effect of cotinine-CAR NK92 according to the presence or absence of the her2-cotinine conjugate or the EGFR-cotinine conjugate for each of the four cancer cells was confirmed through the Calcein-AM method. Specifically, AU565, SK-OV-3, A431 and A549 cells were stained with calcein, respectively, and then Cot-10z-92 cells and 5:1, 1:1 and 0.5:1 (cot-10z-92: cancer cells) Mix in 200 ul of RPMI1640 (10% FBS) at the rate of, and react only with cot-10z-92 and cancer cell species according to each condition, or her2-cotinine conjugate (1 ug/ml) or EGFR-cotinine conjugate (100 ng/ ml) were treated together and reacted for 4 hours at 37°C and 5% CO _2. The apoptosis effect was confirmed in each condition, and the results are shown in FIG. 15.

As shown in Figure 15, cotinine-CAR NK92 cells exhibit apoptosis effect depending on the expression level of Her2 or EGFR, which is a target antigen, which is the apoptosis effect of cotinine-CAR NK cells of the present invention is dependent on the type of cotinine conjugate. Was confirmed.

Example 12. Confirmation of cell activity of cotinine-CAR NK cells by EGFR-cotinine conjugate

Changes in activity by her2-cotinine conjugate or EGFR-cotinine conjugate against Her2 or EGFR-expressing cancer cells were confirmed through cytokine and granule secretion.

Specifically, AU565 or A431 cells and cot-10z-92 were mixed in RPMI1640 (10% FBS) in a ratio of 1:1, and treated with her2-cotinine conjugate or EGFR-cotinine conjugate, and then 37°C, 5% CO ₂ After reacting for 6 hours under conditions, changes in the secretion of cytokine (IFN-r and TNF-a) in the supernatant were confirmed using ELISA, and the results are shown in FIG. 16.

In addition, the secretion of granule was confirmed by the expression level of CD107a. Specifically, original NK92, PURO-92, and Cot-10z-92 were mixed with AU565 or A431 cells in a ratio of 1:1, respectively, and her2-cotinine conjugate or EGFR- The cotinine conjugate was treated together, and the CD107a antibody was treated together _{and reacted for 4 hours at 37°C and 5% CO 2} conditions. After the reaction, staining with CD56 antibody was performed to select NK92 cells, and then through Flow Cytometry. Confirmed. The results for AU565 and A431 cells are shown in FIGS. 17 and 18, respectively.

16 to 18, it was confirmed that the activity of Cotinine-CAR NK cells was increased by the antibody-cotinine conjugate to the antigen.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Korea Research Institute of Bioscience and Biotechnology SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION <120> NATURAL KILLER CELL EXPRESSING ANTI-COTININE CHIMERIC ANTIGEN RECEPTOR SPECIFICALLY BINDING TO COTININE <130> FPD/201907-0101/C <150> KR 10-2017-0001976 <151> 2017-01-05 <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> heavy variant chain <400> 1 gagctcgatc tgacccagac tccagcctcc gtgtctgcag ctgtgggagg cacagtcacc 60 atcaattgcc agtccagtca gagtccttat agtaacgagt ggttatcctg gtatcagcag 120 aaaccagggc aggctcccaa agtcctaatt tctaggatat ccactctggc atctggggtc 180 tcatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat aagcgacctg 240 gagtgtggcg acgctgccac ttatttctgt gcaggcggtt ataattttgg tttgtttcct 300 ttcggcggag ggaccgagct ggagatccta 330 <210> 2 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> light variant chain <400> 2 agatcttccc agtcggtgaa ggagtccgag ggtcgcctgg tcacgcctgg aggatccctg 60 acactcacct gcacagtctc tggaatcgac ctcagtaggg actggatgaa ctgggtccgc 120 caggctccag gggaggggct ggaatggatc ggagccattg gtagaagtgg agacacatac 180 tacgcgacct gggcgaaagg ccgattcacc atctccaaaa cctcgtcgag gacggtgact 240 ctaacagtca ccgatctgca gcgctcagac acggccacct atttctgtgc cagaattcct 300 tattttggtt ggaataatgg tgacatctgg ggcccaggca ccctggtcac catctcttca 360 360 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> linker <400> 3 tcctctggtg gcggtggctc gggcggtggt gggggtggtt cctct 45 <210> 4 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> Human CD8 hinge region <400> 4 ggggtcaccg tctcttcagc gctgagcaac tccatcatgt acttcagcca cttcgtgccg 60 gtcttcctgc cagcgaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 120 accatcgcgt cgcagcccct gtccctgcgc ccagaggcat gccggccagc ggcggggggc 180 gcagtgcaca cgagggggct gga 203 <210> 5 <211> 252 <212> DNA <213> Artificial Sequence <220> <223> CD28 <400> 5 gtgaaaggga aacacctttg tccaagtccc ctatttcccg gaccttctaa gcccttttgg 60 gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac agtggccttt 120 attattttct gggtgaggag taagaggagc aggctcctgc acagtgacta catgaacatg 180 actccccgcc gccccgggcc cacccgcaag cattaccagc cctatgcccc accacgcgac 240 ttcgcagcct at 252 <210> 6 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Human CD8 alpha region <400> 6 atggccttac cagtgaccgc cttgctcctg ccgctggcct tactgctcca cgccgccagg 60 ccggc 65 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Myc epitope <400> 7 gaacaaaaac tcatctcaga agaggatctg 30 <210> 8 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> DAP10 <400> 8 ctgtgcgcac gcccacgccg cagccccgcc caagaagatg gcaaagtcta catcaacatg 60 ccaggcaggg gc 72 <210> 9 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> CD3 zeta <400> 9 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <210> 10 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Human CD8 alpha region <400> 10 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 11 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Cotinine-ScFv <400> 11 Glu Leu Asp Leu Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Pro Tyr Ser Asn 20 25 30 Glu Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Val 35 40 45 Leu Ile Ser Arg Ile Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu 65 70 75 80 Glu Cys Gly Asp Ala Ala Thr Tyr Phe Cys Ala Gly Gly Tyr Asn Phe 85 90 95 Gly Leu Phe Pro Phe Gly Gly Gly Thr Glu Leu Glu Ile Leu Ser Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg Ser Ser 115 120 125 Gln Ser Val Lys Glu Ser Glu Gly Arg Leu Val Thr Pro Gly Gly Ser 130 135 140 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asp Trp 145 150 155 160 Met Asn Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Ile Gly 165 170 175 Ala Ile Gly Arg Ser Gly Asp Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 180 185 190 Arg Phe Thr Ile Ser Lys Thr Ser Ser Arg Thr Val Thr Leu Thr Val 195 200 205 Thr Asp Leu Gln Arg Ser Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ile 210 215 220 Pro Tyr Phe Gly Trp Asn Asn Gly Asp Ile Trp Gly Pro Gly Thr Leu 225 230 235 240 Val Thr Ile Ser Ser 245 <210> 12 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> Human CD8 hinge region <400> 12 Gly Val Thr Val Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser 1 5 10 15 His Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala 20 25 30 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 35 40 45 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 50 55 60 Arg Gly Leu 65 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Myc epitope <400> 13 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 14 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Dap10 <400> 14 Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val 1 5 10 15 Tyr Ile Asn Met Pro Gly Arg Gly 20 <210> 15 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3 zeta <400> 15 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 16 <211> 84 <212> PRT <213> Artificial Sequence <220> <223> CD28 <400> 16 Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser 1 5 10 15 Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 20 25 30 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 35 40 45 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 50 55 60 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 65 70 75 80 Phe Ala Ala Tyr <210> 17 <211> 735 <212> DNA <213> Artificial Sequence <220> <223> Cotinine-scFv <400> 17 gagctcgatc tgacccagac tccagcctcc gtgtctgcag ctgtgggagg cacagtcacc 60 atcaattgcc agtccagtca gagtccttat agtaacgagt ggttatcctg gtatcagcag 120 aaaccagggc aggctcccaa agtcctaatt tctaggatat ccactctggc atctggggtc 180 tcatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat aagcgacctg 240 gagtgtggcg acgctgccac ttatttctgt gcaggcggtt ataattttgg tttgtttcct 300 ttcggcggag ggaccgagct ggagatccta tcctctggtg gcggtggctc gggcggtggt 360 gggggtggtt cctctagatc ttcccagtcg gtgaaggagt ccgagggtcg cctggtcacg 420 cctggaggat ccctgacact cacctgcaca gtctctggaa tcgacctcag tagggactgg 480 atgaactggg tccgccaggc tccaggggag gggctggaat ggatcggagc cattggtaga 540 agtggagaca catactacgc gacctgggcg aaaggccgat tcaccatctc caaaacctcg 600 tcgaggacgg tgactctaac agtcaccgat ctgcagcgct cagacacggc cacctatttc 660 tgtgccagaa ttccttattt tggttggaat aatggtgaca tctggggccc aggcaccctg 720 gtcaccatct cttca 735 <210> 18 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> signal peptide forward primer <400> 18 ctcgaggcca ggatggcctt accagtgacc gccttgctcc tgccgctggc cttgctgctc 60 cacgccgcca ggccg 75 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> signal peptide reverse primer <400> 19 agatctttag cgagggggca gggcctcccc ctcgtgtgc 39 <210> 20 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Myc epitope forward primer <400> 20 ggcccgggag gccgcgaaca aaaactcatc tcag 34 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Myc epitope reverse primer <400> 21 aagcttcaga tcctcttc 18 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Human CD8 hinge region forward primer <400> 22 aagcttgggg tcaccgtctc ttcagc 26 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human CD8 hinge region reverse primer <400> 23 aagcttatcc agccccctcg tgtgc 25 <210> 24 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CD28 forward primer <400> 24 cgcggatccg tgaaagggaa acacctttgt c 31 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CD28 reverse primer <400> 25 ccggaattca taggctgcga agtcgcg 27 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> DAP10 forward primer <400> 26 ccggaattcc tgtgcgcacg cccacgc 27 <210> 27 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> DAP10 reverse primer <400> 27 ataagaatgc ggccgcgccc ctgcctggca tgttgat 37 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> CD3 zeta forward primer <400> 28 ataagaatgc ggccgctaga gtgaagttca gcaggagcg 39 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD3 zeta reverse primer <400> 29 tgctctagag cattagcgag ggggcagggc 30 <210> 30 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Anti-EGFR affibody <400> 30 Val Asp Asn Lys Phe Asn Lys Glu Met Trp Ala Ala Trp Glu Glu Ile 1 5 10 15 Arg Asn Leu Pro Asn Leu Asn Gly Trp Gln Met Thr Ala Phe Ile Ala 20 25 30 Ser Leu Val Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55

Claims (19)

Natural killer cell expressing chimeric antigen receptor (CAR),
The chimeric antigen receptor comprises 1) an antigen binding domain, 2) a transmembrane domain, and 3) an intracellular signal transduction domain,
The antigen binding domain is a domain that specifically binds to cotinine, natural killer cells.
The natural killer cell of claim 1, wherein the antigen binding domain is an antibody or antibody fragment that specifically binds to cotinine.
The natural killer cell of claim 2, wherein the fragment of the antibody is an scFv.
The natural killer cell of claim 2, wherein the antibody or fragment of the antibody comprises a heavy chain variable region consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1.
The natural killer cell of claim 2, wherein the antibody or fragment of the antibody comprises a light chain variable region consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 2. 4.
The natural killer cell of claim 2, wherein the antibody or fragment of antibody further comprises a linker.
According to claim 6, The linker is a natural killer cell consisting of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3.
The natural killer cell of claim 1, wherein the antigen binding domain is linked to the transmembrane domain by a hinge region, a spacer region, or a combination thereof.
The natural killer cell of claim 8, wherein the hinge region, spacer region or combination thereof is selected from one or more of Myc epitopes, CD8 hinge regions and Fc.
The transmembrane domain of claim 1, wherein the transmembrane domain is selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. A natural killer cell comprising the transmembrane domain of at least one protein.
The cell of claim 10, wherein the transmembrane domain comprises a transmembrane domain of CD28.
The natural killer cell of claim 1, wherein the intracellular signaling domain comprises DAP10, CD3 zeta, or a combination thereof.
The natural killer cell of claim 1, wherein the antigen binding domain comprises a signal peptide.
The natural killer cell of claim 13, wherein the signal peptide is CD8α or mouse light chain kappa signal peptid.
The natural killer cell of claim 1, wherein the cotinine is in the form of a complex conjugated with a conjugate.
The natural killer cell of claim 15, wherein the conjugate is selected from the group consisting of peptides, aptamers, hormones, proteins and chemicals.
Cell therapy comprising the natural killer cells of claim 1.
A pharmaceutical composition for preventing or treating cancer, comprising the natural killer cells of claim 1 as an active ingredient.
A method of providing information for diagnosing cancer, comprising contacting the natural killer cells of claim 1 with a sample isolated from the subject.

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