KR102122546B1 - Natural killer cell expressing anti-cotinine chimeric antigen receptor specifically binding to cotinine - Google Patents

Abstract

The present invention relates to natural killer cells expressing an anti-cortinine chimeric antigen receptor (CAR) that specifically binds to cotinine and a cell therapeutic agent comprising the same. Natural killer cells expressing a chimeric antigen receptor that specifically binds to cotinine according to the present invention can effectively migrate to tumor tissue regardless of the type of cancer, depending on the conjugated substance conjugated to cotinine, and thus Killer cells can be usefully used as a gene therapy for high-efficiency anti-cancer effects.

Description

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

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

For decades, the methods of treating cancer have steadily changed and developed. From the 1800s to the 1900s, methods such as surgical, chemotherapy, and radiation therapy were predominant, but limitations on these began to emerge. Most representatively, the existing treatment methods are effective only in the early stages when the cancer has not metastasized, and if the metastasis has already progressed, the likelihood of recurrence after surgery is high. In addition, chemotherapy has been reported to cause a side effect that has a low therapeutic effect in solid tumors and also inhibits the growth of normal cells other than cancer cells. In order to solve this problem, studies on anti-cancer immunotherapy have been actively conducted, which means that the immune response is increased so that patients themselves can fight cancer cells.

In recent years, interest in cell therapy methods that take out immune cells from the body with immune cell therapy, enhance or genetically modify them and put them back is increasing. Typical examples include tumor infiltrating lymphocytes (TIL), chimeric antigen receptor (CAR), and T-cell receptor (TCR) technology. Research using the CAR, a receptor, has been actively conducted.

The chimeric antigen receptor (CAR) is an artificial receptor designed to deliver 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, treatments such as CAR-T are effective against tumors, but in some cases these treatments have caused side effects due to partially non-specific attacks on healthy tissue. In order to overcome this, research is currently underway on the third generation CAR-T, which adds two signal domains that serve as auxiliary stimulus signals and an additional engineered receptor to increase normal'cancer cell antigen recognition ability'. It is characterized by trying to minimize the side effects of attack. Nevertheless, current CAR-T technology is manufactured to recognize only one protein expressed in cancer cells, and with the limitation that it is too expensive to develop an individual therapeutic agent, once CAR-T is injected, toxic T cells CAR-T has a problem in that its function persists even after the cancer cells are removed, causing toxicity, and in the presence of normal cells showing the target protein, it also causes a non-specific attack and causes fatal side effects. It is inhibiting the development of cell therapy products.

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

As a result of research in order to solve the problems of the conventional therapeutic agents, the present inventors produced natural killer cells expressing the chimeric antigen receptor using an antigen binding domain that specifically binds to cotinine, so that the existing CAR-T therapeutic agent had It solved the problem and at the same time, it was confirmed that it was possible to easily develop a general-purpose therapeutic agent, and the present invention was completed.

Accordingly, an object of the present invention is to provide a cell therapeutic agent 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 ) Intracellular signal transduction domain, wherein the antigen-binding domain is a domain that specifically binds to cotinine, providing natural killer cells.

In addition, the present invention provides a cell therapeutic agent 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 the diagnosis of cancer comprising the natural killer cells as an active ingredient.

In addition, the present invention provides a kit for diagnosing 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 migrate to tumor tissue regardless of the type of cancer, depending on the conjugated substance conjugated to cotinine, and thus Killer cells can be usefully used as a gene therapy for high-efficiency anti-cancer effects.

1 is a view showing a pLVX-AcGFP-C1 vector used in the present invention.
FIG. 2 is a schematic diagram of cotinine-specific CAR-NK cells and an example of a cotinine conjugate.
Figure 3 is a diagram showing the working state of Cotine-specific NK cells Cot-CAR-NK cells.
4 is a view showing a specific related sequence used in the present invention.
5 is an exemplary schematic diagram of an anti-cortinine chimeric antigen receptor and its expression system.
6 is a view showing the results of measuring the expression of CAR in a NK92 cell by a 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 Her2 expression level for various cancer cells through flow cytometry.
9 is a diagram showing the results of confirming the cell death effect of the cotinine-CAR NK92 of the present invention with or without the Her2-cotinine conjugate through the Calcein-AM method.
10 is a diagram showing the results of confirming the cytokine secretion level of 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 results of confirming 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 Her2 and EGFR expression levels of AU565, SK-OV-3, A431 and A459 cells through flow cytometry.
15 is a diagram showing the results of confirming the cell death effect of cotinine-CAR NK92 with or without the conjugate through the Calcein-AM method.
16 is a view showing the results of confirming the change in the secretion of Cytokine in cancer cells through ELISA by treating cotinine-CAR NK92 and the conjugate.
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. My signal transduction domain, and the antigen-binding domain is a domain that specifically binds to cotinine, and relates to natural killer cells.

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

Natural killer cells expressing the chimeric antigen receptor according to the present invention are characterized by specifically binding to cotinine, and thus include an antigen binding domain specifically binding 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 conjugate fused with cotinine to a patient.

In particular, natural killer cells (CAR-NK cells) expressing the chimeric antigen receptor of the present invention have problems caused by persistent toxicity of cancer immunotherapy using conventional CAR-T therapeutic agents, risk of autoimmune diseases, and xenogeneic cells The problem of non-target toxicity and graft-versus-host disease (GVHD) for transplantation can be solved through the switch of reaction on (on)/stop (off), as well as targeting various cancer cells. There is an advantage that can be utilized as. In addition, since the CAR-NK cells of the present invention are capable of allograft, it is possible to premade high-efficiency cells compared to CAR-T using the patient's own immune cells, thereby shortening the administration time of the therapeutic agent and increasing the therapeutic efficacy. It can be usefully used to develop therapeutic agents for various diseases according to the reduction of development and treatment costs.

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

In the present invention, "chimeric antibody" refers to an antibody originated in an animal in which the antibody variable region or its complementarity determining region (CDR) is different from the rest of the antibody. Such antibodies may be, for example, antibody variable regions derived from animals other than humans (eg, mice, rabbits, poultry, etc.), and antibody constant regions may be antibodies derived from humans. Such chimeric antibodies may be produced by methods such as gene recombination known in the art.

“Heavy chain” herein refers to a full-length heavy chain comprising the variable region domain VH and the three constant region domains CH1, CH2 and CH3 and fragments thereof, which contain the amino acid sequence of a variable region sufficient to confer specificity to the antigen. Refers to them all.

“Light chain” as used herein refers to both a full-length light chain comprising the variable region domain VL and the constant region domain CL, and fragments thereof, which contain the amino acid sequence of a variable region sufficient to confer specificity to the antigen.

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

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

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 Formula 1 below.

[Formula 1]

Preferably, the cotinine is a conjugate conjugated to a conjugate substance, and the conjugate substance may be selected from the group consisting of peptides, aptamers, hormones, proteins, and chemical substances, and more preferably, the conjugate substance is an apta May be, but is not limited to.

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

The complex in which the anti-cortinine antibody is bound to the conjugate of the conjugate material and cotinine according to the present invention can retain all of the properties of the conjugate material and the antibody by using cotinine as hapten. Specifically, the complex may possess the specific reactivity and function of the molecule, complement-mediated cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and long half-life in vivo that are characteristic of antibodies.

The conjugation material may be selected from the group consisting of peptides, aptamers, hormones, proteins, and chemicals, for example, WKYMVm-NH2 peptide (WKYMVm-NH2), wkymvm-NH2 peptide (wkymvm-NH2) ), AS1411 aptamer, pegaptanib, aptiximab and insulin. In some embodiments of the invention, the protein can 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 a conjugated substance by binding an anti-cotinine antibody to a conjugate conjugated with a conjugated substance.

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

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 fragment of an antibody may be scFv. Of the antibody or antibody fragment binding sequence of the cotinine, cotinine-scFv may be composed of, for example, an amino acid sequence represented by SEQ ID NO: 11, and preferably 80% or more of the amino acid sequence as a result of modification or substitution thereof. More preferably, it has a sequence homology of 90% or more, even more preferably 95% or more, and may include, without limitation, a sequence showing physiological activity substantially identical to the amino acid sequence represented by SEQ ID NO: 11.

In addition, cotinine-scFV can be encoded by a base sequence represented by SEQ ID NO: 17 and a sequence having at least preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. 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 is the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 1 or the amino acid sequence expressed by the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 1 that can encode a functional equivalent It may be composed of an amino acid sequence encoded by a base 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 is 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 the amino acid expressed by the nucleotide sequence of SEQ ID NO: 2 It may be composed of an amino acid sequence encoded by a base 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 with 95% or more sequence homology is substantially the same as the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 and 2 It can indicate the physiological activity of.

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

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

The CD8 hinge region of the present invention has SEQ ID NO: 12 or a sequence homology of 70% or more thereof, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. It may be composed of an amino acid sequence showing a function substantially equivalent to the amino acid sequence; The Myc epitope has SEQ ID NO: 13 or a sequence homology of 70% or more thereof, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and the amino acid sequence represented by SEQ ID NO: 13 It may consist of amino acid sequences that exhibit substantially equivalent functions. Therefore, the hinge region or the spacer region of the present invention comprises SEQ ID NO: 12 or an amino acid sequence showing 95% or more homology to it; Or it is preferably composed of SEQ ID NO: 13 or an amino acid sequence showing 95% or more homology to it.

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

The transmembrane domains that are one component of the chimeric antigen receptors of the invention are CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. It may be to include a transmembrane domain of a protein selected from the group consisting of. As an 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 showing 95% or more homology, 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, can use an intracellular signaling 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 SEQ ID NO: 14 or a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. , May be made of an amino acid sequence showing 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 a sequence homology of SEQ ID NO: 15 or more and 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more. That is, it may be made of an amino acid sequence showing a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 15. Therefore, the intracellular signaling domain of the present invention comprises SEQ ID NO: 14 or an amino acid sequence showing 95% or more homology to it; Or it may be made of an amino acid sequence showing a homology of SEQ ID NO: 15 or more than 95%.

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 chain kappa signal peptid, and in the case of CD8 alpha, the signal peptide of the present invention consists of SEQ ID NO: 10 or an amino acid sequence showing 95% or more homology to the signal peptide Can be. In addition, the nucleotide sequence encoding the CD8 alpha region may include all of the nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 10, but may preferably 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-cortinine chimeric antigen receptor described above.

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

As a preferred example in the present invention, a lenti-virus vector (Clontech, 632155) may be used, and specifically, pLVX-AcGFP-C1, which is a vector used in Examples of the present invention, is illustrated in FIG. 1.

In some embodiments, the natural killer cells are obtained or prepared 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 the NK cell expressing the anti-cotinine chimeric antigen receptor according to the present invention and the state of the cotinine conjugate are shown in FIG. 2.

As described above, NK cells transformed by introducing the antigen receptor can recognize cotinine as an antigen and specifically bind cotinine, and express a chitin antigen receptor specific for 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 activities such as CAR-NK cells, such as tumor antigens.

The working state of the cotine-specific NK cell Cot-CAR-NK cell according to the present invention is shown schematically in FIG. 3.

In the present invention, in particular, CAR-NK cells refer to cells in which chimeric antigen receptors are introduced into NK (Natural killer) cells. The cells have the advantage of anti-cancer specific target treatment, which is the existing advantage of the CAR-T therapeutic, and the existing problem is through the switch function that can turn on/off the therapeutic response, including the chimeric antigen receptor according to the present invention. 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 terminal modification of a conjugate fused with cotinine that can bind to a chimeric antigen receptor. That is, NK cells expressing a chimeric antigen receptor that specifically binds to cotinine according to the present invention can effectively migrate to tumor tissue regardless of the type of cancer according to the conjugated substance conjugated to cotinine, and accordingly, according to the present invention Cells with high specificity can be usefully used as a gene therapy to show excellent anti-cancer effects.

Therefore, another aspect of the present invention is a cell therapeutic agent comprising the natural killer cells, a pharmaceutical composition for the prevention or treatment of cancer comprising the same as an active ingredient, or preventing cancer comprising administering the cells to an individual Or a treatment.

In the present invention, the cells can be used for cell therapy such as anti-cancer treatment. Cells can come from donors or can be cells obtained from a patient. The cells can be used, for example, for regeneration, replacing the function of diseased cells. The cells can also be modified to express heterologous genes so that biological agents can be delivered to a specific microenvironment, such as diseased bone marrow or metastatic deposits.

In addition, the pharmaceutical composition for preventing or treating cancer of the present invention may further include a conjugate in which a conjugate substance is fused to cotinine, and the method for preventing or treating the cancer administers a conjugate in which a conjugate substance is fused to cotinine. It may further include a step. According to the conjugation material conjugated to the cotinine, it can specifically bind to target cells and exhibit excellent anticancer effects.

Specifically, the cell provided in the present invention is a natural killer cell expressing a chimeric antigen receptor having an antigen binding domain capable of specifically binding to cotinine, wherein the chimeric antigen receptor is, for example, a target cell of a conventional CAR therapeutic agent Response on (on)/stop (off) can be regulated with conjugates or intermediates fused with cotinine, which can be very beneficial in situations where subsequent cell therapy, the activity of therapeutic cells needs to be increased or decreased. Safety switch included. For example, if NK cells expressing a chimeric antigen receptor are provided to a patient, in some situations, there may be side effects, such as off-target toxicity. Or, for example, a therapeutic cell may act to reduce tumor cells, or tumor size, and may no longer be needed. In this situation, it is possible to control the therapeutic cells to be no longer activated through the regulation of cotinine.

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

In 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 a pharmaceutical composition calculated to achieve the desired immunogen stimulating effect with a desired diluent. The specific content of the unit dose of the inoculum is influenced by and determined according to the unique properties of the pharmaceutical composition and the specific immunological effect to be achieved.

The 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. The effective amount of a particular composition presented herein can be determined empirically without undue experimentation.

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

In the present invention, the term "diagnosis" is to determine the susceptibility (susceptibility) of an object to a specific disease or condition, to determine whether an object currently has a specific disease or condition, as long as the disease or condition Determining the prognosis of the object, or terrametrics (eg, monitoring the condition of the object to provide information about treatment efficacy).

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

Chimeric antigen receptors, natural killer cells expressing them, and their 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, and 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), a small molecule substance to be used as a target antigen, is as shown in Chemical Formula 1 below, and cotinine was purchased from Sigma-Aldrich.

[Formula 1]

In addition, a conjugate in which the cotinine and the conjugate substance were fused was prepared in the following manner.

Specifically, the HER2-cotinine conjugate was prepared using an anti-HER2 antibody, trastuzumab (Genentech, USA), to prepare a conjugate in which cotinine and the anti-HER2 antibody were conjugated. At this time, the conjugate was conjugated by 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. On the other hand, trans-4-cotininecarboxylic acid (Sigma-Aldrich) was added to 1 ml of MES buffer [0.1 M MES (2-[morpholino]ethaneusulfonic acid) and 0.5 M sodium chloride, pH 6.0]. It was prepared by dissolving to a concentration of 5 mM. After adding EDC at a concentration of 50 mM and N-hydroxysulfosuccinimide at a concentration of 125 mM (N-hydroxysulfosuccinimide, Sulfo-NHS, Thermo Scientific, USA), this was stirred at room temperature for 15 minutes to dissolve and coat cotinine. -An active solution in which NHS ester was produced was prepared. Sodium hydroxide solution was added to induce the reaction between the cotinine-NHS ester and the amine group of the protein, 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 linkage reaction. The obtained cotinine-HER2 antibody conjugate is dialyzed with PBS using Slide-A-lyzer™ dialysis cassette (Thermo Fisher Scientific, USA), or the buffer is exchanged with PBS using Amicon　Ultra Centrifugal Filter (EMD Millipore, USA) Was used.

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

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

Example 3. Derivation of anti-cortinine chimeric antigen receptors

Plasmids containing nucleic acids encoding each domain of the chimeric antigen receptor that specifically bind to cotinine of the present invention were prepared in the following manner.

(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 XhoI and XbaI restriction enzyme sites. Amplified by polymerase chain reaction and then cloned.

(2) target specific recognition domain (target specific recognition domain)-scFv

As an antigen-binding domain capable of specifically binding to cotinine, an attempt was made to derive an anti-cotinine chimeric antibody or an antibody fragment thereof. For the sequence derivation of ScFv, scFv-related information of Korean Patent Registration No. 10-1648960 was referred. Specifically, the antigen-binding domain contains the nucleotide sequence represented by SEQ ID NO: 17, and is composed of VH-linker-VL.

(3) connection domain (spacer)

(A) Myc epitope

Two primers each containing sfiI and HindIII restriction enzyme sites in the plasmid (Anti-cotinine 28Z-1 CAR ORF, cot28z-1) possessed by the present inventors (forward primer: SEQ ID NO: 20, reverse primer: SEQ ID NO: 21) was used to amplify by polymerase chain reaction and then cloned.

(B) Human CD8 hinge region

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

(4) Transmembrane area

The cytoplasmic region was used as a transmembrane region in the hinge of the human CD28 gene. It was prepared by adding the sequence of the 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).

PCR was performed on the cDNA of Jurkat cells using the primers above to obtain DNA of the transmembrane region.

(5) one or more intracellular signaling domains

(A) Co-stimulatory domain

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

PCR was performed on the cDNA of primary mature NK cells using the above primer to construct 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 types of primers (forward primer: SEQ ID NO: 28, reverse primer: SEQ ID NO: 29) to the cDNA of Jurkat cells to prepare an activation domain.

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

[Table 1]

When the sequence of Table 1 corresponds to the structure described above, the signal peptide was 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 shown in 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 the transmembrane region of CD28 by SEQ ID NO: 5. One or more intracellular signaling domains are represented by DAP-10 as a co-stimulatory domain in SEQ ID NO: 8 and CD3 zeta as a NK cell activation domain in SEQ ID NO: 9.

Example 4. Preparation of NK cells introduced with anti-cortinine chimeric antigen receptor

The polynucleotide encoding the anti-cortinine chimeric antigen receptor shown in Example 3 was introduced into a vector, and the transformed spontaneous killer cells were prepared using the same, and a chimeric antigen receptor comprising a cotinine specific antigen binding domain 5 is a schematic diagram of and a schematic diagram of its expression system.

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

Then, a vector containing cotinine-CAR was transformed into HEK293T cells together with viral packaging vector (PMDLg/RRE, RSV/REV, VSVG), from which a Lentivirus expressing Cotinine-CAR was obtained. 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 to calculate the Infection Unit. . The number of NK cells and the amount of lentivirus were calculated so that the multiplicity of infection (MOI) was 10, and the lentivirus expressing cotinine-CAR was infected with NK cells by spinoculation method (360g, 90min, RT). After culturing the infected NK cells at 37°C and 5% CO ₂ for 5 hours, they were replaced with fresh culture medium. After 3 days, the culture was continued by treating puromycin at a concentration of 3 ug/ml for the selection of infected NK cells.

As a control, puromycin was also treated to uninfected NK cells, and culture was performed using a culture medium treated with puromycin until control cells were annihilated by puromycin. At the time when the control cells were wiped out, the infected NK cells were exchanged with Puromycin-free medium to proliferate or expand. Alpha-MEM containing 12.5% fetal calf serum, 12.5% equine serum, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid and 200 U/ml recombinant IL-2 for proliferation or expansion of the selected cells The experiment was performed using a medium using.

Example 5. Confirmation of CAR expression in NK cells introduced with anti-cortinine 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 a flow cytometry method.

The Myc antibody (CTS; 9B11) corresponding to the Myc epitope of Cotinine-CAR was reacted with NK cells expressing cotinine-CAR (4°C, 30 minutes in the cow), and the expression of Myc was confirmed through flow cytometry, or Her2 The antibody-fused cotinine conjugate was reacted to NK cells expressing cotinine-CAR at 4°C for 30 minutes, followed by secondary reaction of the Fc (eBioscience; 12-4988-82) antibody corresponding to the Fc region of Her2 antibody. After that, it was confirmed by flow cytometry. As a control, original NK92 cells that did not express CAR were used, 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 binding specificity of cotinine-CAR NK cells to cotinine

It was confirmed whether cotinine ScFv of Cotinine-CAR NK cells specifically binds to cotinie using cot-SWNT and HER2-tumor cells, which are Cotinine polymers.

Specifically, after AU565 (human breast carcinoma) was stained 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, Cortinine conjugates fused with Her2 antibody at a concentration of 1 ug/ml and cot-SWNT were treated simultaneously at concentrations (0, 0.005, 0.05, 0.5, and 5 mg/ml) to compete with Her2-cotinine conjugates. In addition, in order to confirm the effect of Cot-SWNT alone, treatment was performed by adding Cot-SWNT (5 mg/ml) alone treatment conditions. After reacting at 37°C and 5% CO ₂ for 4 hours, 100 ul of 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, a group treated with RPMI1640 (10% FBS) only (spontaneous value) and a group treated with 2% triton X-100 (maximum value) on AU565 stained with Calcein was used, and the killing effect was calculated by the following method. Did.

killing effect (%) = (conditional calcein release value-spontaneous value) / (maximum value-spontaneous value) x 100

The result of the killing effect is shown in FIG. 7.

As shown in FIG. 7, as the concentration of cot-SWNT increased, it was confirmed that the killing effect of cotinine-CAR NK cells on cancer cells expressing her2 decreased, from which the cotinine-CAR NK cells of the present invention were It was confirmed that it acts specifically on the target cell according to 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-cortinin conjugate prepared in Example 3 and the cotinine-CAR NK92 cells of Example 4, the conjugate recognizes Her2 on the surface of the cancer cell, thereby introducing NK cells into the anti-cortinine chimeric antigen receptor of the present invention. 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 anti-Her2 antibody (Invtrogen; BMS120FI) in an amount of 1 ug/100 ul, 4° C., 30 in cows The reaction was carried out for a minute. After the reaction was completed, each cell was confirmed by using flow cytometry (BD; FacsCantoII) to express Her2 expression level of each cell, and the results are shown in FIG. 8.

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

Then, the apoptosis effect of cotinine-CAR NK cells with or without Her2-cotinine conjugate was confirmed by Calcein-AM method. Specifically, after treatment with Calcein-AM at a concentration of 5 ug/ml on the four cell lines of AU565, SK-OV-3, SK-BR-3, and K562, at 37°C, 5% CO ₂ , and cow conditions The reaction was carried out for 1 hour. After the reaction, NK92 (Puro-92), and cotinine-CAR NK92 (cot-10z-92), respectively, were inserted into the cell lines with a control vector expressing the original NK92, AcGFP-depleted pLVX empty vector, respectively 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 in 200 ul RPMI (10% FBS) and reacted at 37°C and 5% CO ₂ for 4 hours, 100 ul of supernatant It was taken to confirm the amount of calcein present in the supernatant to confirm the Killing effect according to each condition, and the results are shown in FIG. 9.

As shown in Figure 9, cotinine-CAR NK92 cells show apoptosis activity in AU565, SK-OV-3 and SK-BR-3 cells expressing Her2, but do not show activity in K562 cells where Her2 is not expressed. Was confirmed. In addition, Her2-cotinine conjugate did not affect the apoptotic effects of NK92 and Puro-92, but was confirmed to have 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 cellular activity of cotinine-CAR NK cells

The activity of NK cells was confirmed by confirming the secretion of cytokine and granule of NK cells. Specifically, the secretion of cytokine was mixed with original NK92, PURO-92, and cot-10z-92 in RPMI1640 (10% FBS) 1:1 with AU565, respectively, and put the cotinine conjugate at 37°C and 5% CO ₂ conditions. 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 cytokine secretion amount of NK92, PURO-NK92, and cot-10z-92 was used as a control. The results are shown in Figure 10.

In addition, the expression of CD107a was mixed with original NK92, PURO-92, and co-10z-92 in AU565 and RPMI1640 (10% FBS) in a ratio of 1:1, and the cortinine conjugate and CA107a antibody (BD;555801) were mixed. After treating together, the mixture was reacted at 37°C and 5% CO ₂ for 4 hours. After staining with CD56 antibody so that NK92 cells can be selected from the cells after the reaction, the degree of CD107a expression of the original NK92, PURO-NK92, and cot-10z-92 was compared using flow cytometry. The original CDNKa expression level of NK92, PURO-92, and Cot-10z-92 and the expression level of CD107a in the absence of cotinine conjugate mixed with AU565 were used as a control, and the results are shown in FIG. 11.

As shown in FIGS. 10 and 11, it was confirmed that the secretion of Cytokine and granule of NK cells responds to cancer cells only when cotinine-CAR NK cells and her2-cotinine conjugates are present.

Example 9. Her2 - cotinine By conjugate cotinine -CAR NK Cell signal change

When the Cotinine-CAR NK cells showed activity, a signal that was changed was measured to confirm the activity of the 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 when there are cancer cells by her2-cotinine conjugate (with her2-cot; Her2-cot treatment group) It was confirmed that the Erk phosphorylation of cotinine-CAR NK92 cells increased.

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

In order to confirm the specificity of the Her2-cotinine conjugate of Cotininie-CAR NK cells, the apoptosis effect according to 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 as Example 2 except that the anti-RSV antibody (Palivizumab, Synagisㄾ, AstraZeneca, UK) was used as an antibody to be fused with cotinine in the method of preparing the cotinine conjugate of Example 2. It was prepared.

Then, cotinine-CAR-NK92 cells and calcein-stained AU565 were mixed in RPMI1640 (10% FBS) at 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 non-expressing respiratory syncytial 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 at 4 After the reaction for a time, the apoptosis effect of the 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 exhibit activity specifically for 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

Using the cotinine-CAR NK92 cells of Example 4 together with the anti-Her2-cortinine conjugate and anti-EGFR-cotinine conjugate prepared in Example 3, the anti-cortinine of the present invention by recognizing the Her2 or EGFR of the cancer cell surface The apoptosis effect 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)) Anti-Her2 antibody and anti-EGFR antibody (BD;563577) were treated in a cell line in an amount of 1 ul/100 ul, and reacted for 30 minutes at 4° C. in a cow, and then the flow rate of her2 and EGFR expression of each cell was measured. It was confirmed through, the results are shown in Figure 14.

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

Then, the cell death effect of cotinine-CAR NK92 according to the presence or absence of her2-cotinine conjugate or EGFR-cotinine conjugate for each of the four cancer cells was confirmed through the Calcein-AM method. Specifically, after staining AU565, SK-OV-3, A431 and A549 cells with calcein, respectively, 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 a ratio of, and react only with cot-10z-92 and cancer cell species depending on each condition, or her2-cotinine conjugate (1 ug/ml) or EGFR-cotinine conjugate (100 ng/ ml) and reacted for 4 hours at 37°C and 5% CO ₂ conditions. The apoptosis effect was confirmed under each condition, and the results are shown in FIG. 15.

As shown in Fig. 15, cotinine-CAR NK92 cells show apoptosis effect depending on the expression level of Her2 or EGFR, the target antigen, and the apoptosis effect of cotinine-CAR NK cells of the present invention depends on the type of cotinine conjugate. Was confirmed.

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

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

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

In addition, the secretion of granule was confirmed by the expression level of CD107a. Specifically, native NK92, PURO-92, and Cot-10z-92 were mixed with AU565 or A431 cells in a ratio of 1:1, and her2-cotinine conjugate or EGFR- The cotinine conjugate was treated together, and the CD107a antibody was treated together to react at 37°C and 5% CO ₂ for 4 hours. After the reaction, staining with a CD56 antibody was performed to select NK92 cells, followed by 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 is increased by the antibody-cotinine conjugate to the antigen.

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

<110> SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION <120> NATURAL KILLER CELL EXPRESSING ANTI-COTININE CHIMERIC ANTIGEN RECEPTOR SPECIFICALLY BINDING TO COTININE <130> P17-196-REA-KRI <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)

As a natural killer cell expressing a 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, a natural killer cell.
According to claim 1, wherein the antigen-binding domain is an antibody or antibody fragment that specifically binds to cotinine, natural killer cells.
The natural killer cell of claim 2, wherein the fragment of the antibody is scFv.
According to claim 2, The antibody or fragment of the antibody comprises a heavy chain variable region consisting of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, natural killer cells.
According to claim 2, wherein the antibody or fragment of the antibody comprises a light chain variable region consisting of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, natural killer cells.
The natural killer cell of claim 2, wherein the antibody or fragment of the antibody further comprises a linker.
The natural killer cell of claim 6, wherein the linker consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3.
According to claim 1, The antigen-binding domain is a natural killer cell that is connected 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, the spacer region, or a combination thereof is one or more selected from Myc epitope, CD8 hinge region, and Fc.
The transmembrane domain of claim 1 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. Natural killer cell comprising a transmembrane domain of one or more proteins.
The natural killer cell of claim 10, wherein the transmembrane domain comprises a transmembrane domain of CD28.
The 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 cell of claim 13, wherein the signal peptide is CD8α or mouse light kappa signal peptid.
The natural killer cell of claim 1, wherein the cotinine is in the form of a complex conjugated with a conjugate.
16. The cell of claim 15, wherein the conjugation material is selected from the group consisting of peptides, aptamers, hormones, proteins and chemicals.
A cell therapeutic agent comprising the natural killer cells of claim 1.
A pharmaceutical composition for the prevention or treatment of cancer, comprising the natural killer cells of claim 1 as an active ingredient.
A method of providing information for the diagnosis of cancer comprising contacting the natural killer cell of claim 1 with a sample isolated from the individual.

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