WO2022182059A1

WO2022182059A1 - Chimeric antigen receptor specifically binding to hla class ii and use thereof

Info

Publication number: WO2022182059A1
Application number: PCT/KR2022/002352
Authority: WO
Inventors: 진화섭; 전태훈; 양준; 이창희; 이나영; 박인병; 강석진; 박시원; 이현정
Original assignee: 주식회사 이뮤노로지컬디자이닝랩
Priority date: 2021-02-26
Filing date: 2022-02-17
Publication date: 2022-09-01
Also published as: KR20220122831A

Abstract

The present invention relates to: a chimeric antigen receptor specifically binding to HLA class II; a polynucleotide encoding a protein of the chimeric antigen receptor; a vector comprising the polynucleotide; a cell transformed with the vector; and a cell therapy product or a pharmaceutical composition for treating cancer, comprising the cell as an active ingredient. The chimeric antigen receptor according to the present invention comprises extracellular domains (1 to 4) of LAG-3 that specifically binds to HLA class II (HLA-DP, HLA-DQ, HLA-DR). Therefore, in the case of cytotoxic T cells or natural killer cells that are transformed with a vector that can overexpress the chimeric antigen receptor, the cells have cytotoxicity specifically to carcinoma expressing HLA class II (HLA-DP, HLA-DQ, HLA-DR), and thus the cells may be usefully utilized as immune cell therapeutic agents for cancer treatment.

Description

Chimeric antigen receptors specifically binding to HLA CLASS II and uses thereof

The present invention relates to a chimeric antigen receptor (CAR) that specifically binds to HLA class II; a polynucleotide encoding the chimeric antigen receptor protein; a vector comprising the polynucleotide; T cells or natural killer cells transformed with the vector (Natural killer cell, NK cell); It relates to a cell therapeutic agent or a pharmaceutical composition for the treatment of cancer comprising the cell as an active ingredient.

Human major histocompatibility complex (MHC) antigen class II consists of HLA-DP, HLA-DQ, and HLA-DR, and consists of a heterodimer. Integral membrane protein composed of HLA-DPα and HLA-DPβ for HLA-DP, HLA-DQα and HLA-DQβ for HLA-DQ, and HLA-DRα and HLA-DRβ for HLA-DR to be. HLA class II is mainly expressed in professional antigen-presenting cells and plays a role in delivering peptide antigens to CD4 ⁺ T cells (helper T cells). However, HLA class II is expressed abnormally high in various carcinomas when normal cells are tumorigenic. In various carcinomas, HLA class II can block the action of immune checkpoint inhibitors (Johnson et al., 2016; Johnson et al., 2018). That is, HLA class II binds to HLA class II ligand LAG-3 or Fc receptor-like 6 (FCRL6), respectively, to form natural killer cells (NK cells) or cytotoxic T lymphocytes (CTLs). Inhibits the activity of or helper T lymphocytes (Johnson et al., 2016; Johnson et al., 2018).

It has been reported that cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) that directly target cancer cells are important for effective cancer treatment. Until now, studies on anticancer treatment using cytotoxic T cells or natural killer cells have either delivered specific cancer antigens derived from a patient to the patient's cytotoxic T cells to induce activity, or induce antibody-dependent cellular cytotoxicity. (Galluzzi et al., 2018; Lu et al., 2020). However, recently, a new anticancer treatment method using cytotoxic T cells and natural killer cells has been introduced. That is, a single-chain variable fragment (scFv) portion of an antigen-recognizing antibody is grafted onto the cytoplasmic signaling domain of CD3ε or other protein, chimeric antigen receptor, CAR) was tried as a method of delivering. When the chimeric antigen receptor is grafted onto T cells or natural killer cells, the anticancer action of T cells or natural killer cells is enhanced by the specific antigen recognition of single-chain Fv fragments regardless of signal transduction by antigen presenting cells (APCs). It can be activated, and it is not limited to the HLA type, so it can be used as a more efficient treatment method that can be used universally by many people. Indeed, these chimeric antigen receptor-expressing T cells or natural killer cells have shown efficacy in several cancers (Holzinger et al., 2016; Pettitt et al., 2018; Wang et al., 2020).

On the other hand, lymphocyte activation gene-3 (LAG-3; CD223) is an integral membrane protein belonging to the immunoglobulin (Ig) superfamily that is expressed on the cell surface when T cells are activated. LAG-3 expressed in activated T cells is known to inhibit the activity of T cells. LAG-3 is structurally similar to CD4 and binds to MHC class II, but has a much higher affinity for MHC class II than CD4. In addition, according to recent research results, human LAG-3 shows very high affinity to all of human MHC class II, HLA-DP, HLA-DQ, and HLA-DR (Niehrs et al., 2019).

Currently, a chimeric antigen receptor for cancer immunotherapy (Korean Patent Application Laid-Open No. 10-2017-0062446) or a chimeric antigen receptor including a transmembrane region of LAG-3 has been disclosed (US Patent Publication) No. 2017-0275362), the invention of using the LAG-3 extracellular domain having binding specificity for HLA class II-expressing cancer cells as an antigen-binding domain as an immunocancer therapeutic agent has not been described.

Under this background, the present inventors used the fact that HLA class II expression in various cancer cells is confirmed or induced, and that the HLA class II and LAG-3 can bind to the extracellular domain of LAG-3 (No. 1 to 4) were used to prepare cancer-specific chimeric antigen receptor-expressing T cells and natural killer cells expressing HLA class II, and the T cells or natural killer cells specifically target cells expressing HLA class II. It was confirmed through an experiment that it has a toxic ability. At this time, the signal transduction strength was amplified by making a fusion protein linking each of the extracellular domains (No. 1 to No. 4) of LAG-3 with the human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region). That is, the existing chimeric antigen receptor has one site for recognizing an antigen by a single chain Fv fragment, whereas the chimeric antigen receptor designed by the present inventors has the extracellular domain of LAG-3 (No. 1 to No. 4). By linking each with human human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region), two extracellular domains of LAG-3 (No. 1 to No. 4) per chimeric antigen receptor recognize antigen Therefore, it was attempted to amplify the signal transduction strength.

Accordingly, it is an object of the present invention to provide a chimeric antigen receptor that specifically binds to HLA class II.

Another object of the present invention is to provide a polynucleotide encoding the chimeric antigen receptor protein, a vector containing the polynucleotide, and T cells or natural killer cells transformed with the vector.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of cancer that kills cancer cells expressing cell therapeutics or HLA class II, including the transformed T cells or natural killer cells.

In order to achieve the object of the present invention as described above, the present invention provides an antigen-binding domain; transmembrane domain; and an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain (Nos. 1 to 4) of LAG-3 that specifically binds to HLA class II. A phosphorus, chimeric antigen receptor is provided.

In addition, the present invention provides a polynucleotide encoding the chimeric antigen receptor protein, a vector containing the polynucleotide, and T cells or natural killer cells transformed with the vector.

In addition, the present invention provides a pharmaceutical composition for the treatment of cancer comprising the transformed T cells or natural killer cells, a cell therapeutic agent or HLA class II expressing cancer cells.

The chimeric antigen receptor according to the present invention includes an extracellular domain (Nos. 1 to 4) of LAG-3 that specifically binds to HLA class II (HLA-DP, HLA-DQ, HLA-DR). Therefore, in the case of cytotoxic T cells or natural killer cells transformed with a vector capable of overexpressing the chimeric antigen receptor, it is specific for HLA class II (HLA-DP, HLA-DQ, HLA-DR) expressing carcinoma. Because it has cytotoxicity, it can be usefully used as an immune cell therapeutic for cancer treatment.

1 is a schematic diagram showing the cDNA region of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.

2A is a schematic diagram of a lentiviral vector according to an embodiment of the present invention.

2B is a schematic diagram of a retroviral vector according to an embodiment of the present invention.

3 is a schematic diagram of a chimeric antigen receptor expressed on the surface of cytotoxic T cells or natural killer cells according to an embodiment of the present invention.

4 is a view of HLA class II in cancer cells expressing HLA class II (HLA class II positive cells) and cancer cells not expressing HLA class II (HLA class II negative cells), which are targets of the chimeric antigen receptor provided in the present invention. It is a diagram showing the result of measuring the expression level using flow cytometry.

5a is a diagram showing the results of purification by affinity chromatography using a protein A column after making the chimeric antigen receptor provided in the present invention water-soluble and expressing it in Chinese hamster ovary (CHO) cells.

Figure 5b is a water-soluble chimeric antigen receptor according to an embodiment of the present invention using an antibody recognizing the constant region of a human IgG heavy chain (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody) Western blotting It is a diagram showing the results confirmed by .

FIG. 5c is a diagram showing whether the water-soluble chimeric antigen receptor according to an embodiment of the present invention recognizes cancer cells (HLA class II positive cells) expressing the extracellular domain target, HLA class II, using flow cytometry.

Figure 6a is a diagram showing the result of comparing the expression rate of GFP in cytotoxic T cells (LAG-3 CAR-T cell) transduced with the expression vector according to an embodiment of the present invention using a lentiviral system. .

Figure 6b is a diagram showing the result of comparing the expression ratio of LAG-3 in natural killer cells (LAG-3 CAR-NK cells) transduced with the expression vector according to an embodiment of the present invention using a retroviral system to be.

Figure 7a shows HLA using cytotoxic T cells (LAG-3 CAR-T cells) or empty vector-transduced cytotoxic T cells (Mock T cells) as effector cells according to an embodiment of the present invention; It is a diagram showing the results of measuring the cytotoxicity to cancer cells expressing class II (HLA class II positive cells).

7b is HLA class II using natural killer cells (LAG-3 CAR-NK cells) or mock NK cells transduced with an empty vector as effector cells according to an embodiment of the present invention; It is a diagram showing the result of measuring the cytotoxicity to cancer cells (HLA class II positive cells) expressing.

The present invention provides an antigen-binding domain; transmembrane domain; and an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain (Nos. 1 to 4) of LAG-3 that specifically binds to HLA class II. A chimeric antigen receptor is provided.

In the present invention, "chimeric antigen receptor (CAR)" binds to a desired antigen without the mediation of antigen presenting cells (APCs) or antibodies necessary for naturally activating T cells or natural killer cells, thereby producing an antigen-antibody reaction. It refers to a fusion protein for expression in T cells or natural killer cells in order to induce activation of T cells and attack cells expressing the corresponding antigen. That is, when expressed in T cells or natural killer cells, it can be considered as a protein that binds to an antigen and induces activation of these cells. Through this, it may be a protein recognizing an antigen specific to a cell to cause an immune response, and the cell to cause an immune response may refer to a cell existing in a specific tissue or constituting a tissue causing a lesion.

The chimeric antigen receptor protein according to an embodiment of the present invention may include a functional equivalent. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably the amino acid sequence of the chimeric antigen receptor protein as a result of the addition, substitution, or deletion of amino acids. refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity. “Substantially homogenous physiological activity” means that it has an activity capable of specifically binding to HLA class II (HLA-DP, HLA-DQ, HLA-DR).

The present invention also includes fragments, derivatives and analogues of chimeric antigen receptors. As used herein, the terms 'fragment', 'derivative' and 'analog' refer to a polypeptide that retains substantially the same biological function or activity as the chimeric antigen receptor protein of the present invention. Fragments, derivatives and analogs of the present invention include 1) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, wherein the substituted amino acid residues are encoded by the genetic code. may or may not) or 2) a polypeptide having substituent(s) at one or more amino acid residues, or 3) maturation associated with another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol) a polypeptide derived from a polypeptide, or 4) the polypeptide associated with an additional amino acid sequence (eg, a leader sequence, a secretory sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) It may be a polypeptide derived from The fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

Lymphocyte-activation gene 3 (LAG-3) protein, called lymphocyte activation gene-3, belongs to the immunoglobulin (Ig) superfamily, and has an extracellular domain consisting of four domains designated D1 to D4. It is an integral membrane protein.

The antigen _- binding domain is _a dimer ( dimer), but is not limited thereto.

The extracellular domain (No. 1 to No. 4) of the LAG-3 may consist of SEQ ID NO: 1 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

The human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) may consist of SEQ ID NO: 2 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

In the present invention, "HLA class II" is an intrinsic membrane protein that is mainly expressed in professional antigen presenting cells and delivers peptide antigens to CD4+ T cells (helper T cells), but HLA class II is When it becomes oncogenic, it has abnormally high expression in various carcinomas. To date, carcinomas expressing HLA class II have been classified as head and neck squamous cell carcinoma (Meissner et al., 2008), thymoma (Strobel et al., 2008) and esophageal squamous cell carcinoma ( Esophageal squamous cell carcinoma) (Hu et al., 2014), melanoma (Johnson et al., 2016; Rodig et al., 2018; Johnson et al., 2018), breast cancer (Park et al.) al., 2017; Loi et al., 2016; Bartek et al., 1987), colorectal cancer (Michel et al., 2010; Bustin et al., 2001), ovarian cancer (callahan et al.) al., 2008, Turner et al., 2017), prostate cancer (Younger et al., 2008), neuroblstoma (Yazawa et al., 2002), glioma (Soos et al.) al., 2001), non-small cell lung cancer (Yazawa et al., 1999), classic Hodgkin lymphoma (Roemer et al., 2018), T cell leukemia/lymphoma (T-cell leukemia/lymphoma) (Takeuchi et al., 2019), B cell lymphoma (Steidl et al., 2011), chronic myeloid leukemia (Petersdorf et al., 2001) , chronic lymphocytic leukemia (Hojjat-Farsangi et al., 2008), acute bone acute myeloid leukemia (van den Ancker et al., 2014), acute lymphoid leukemia (Hurwitz et al., 1988), and the like. In addition, all HLA class II (HLA-DP, HLA-DQ, HLA-DR) has the characteristic of strongly binding to the extracellular domain of LAG-3 (domains 1 to 4).

In one embodiment, 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. It may include one or more, preferably, a transmembrane domain of CD8, which may consist of SEQ ID NO: 3 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

In the present invention, "intracellular signaling domain" refers to a site that activates an immune response of an immune cell by binding to an antigen-binding domain. In one embodiment, the intracellular domain may be CD28, 4-1BB, CD3 zeta, or a combination thereof, but is not limited thereto.

The chimeric antigen receptor according to the present invention uses CD28, 4-1BB, and CD3 zeta as intracellular signaling domains, thereby exhibiting high activity in cancer cells, particularly HLA class II ((HLA-DP, HLA-DQ, HLA- DR) expressing cancer cells.

The CD28 is SEQ ID NO: 4 or 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid sequence represented by SEQ ID NO: 4 and may consist of an amino acid sequence exhibiting a substantially equivalent function, and 4-1BB (CD137) is SEQ ID NO: 5 or 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% thereof Having the above sequence homology, it may consist of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence shown in SEQ ID NO: 5, CD3 zeta functions as an NK cell activation domain, and SEQ ID NO: 6 or 70 thereof % or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and an amino acid sequence that exhibits a substantially equivalent function to the amino acid sequence represented by SEQ ID NO: 6. can be done

The antigen-binding domain may include a LAG-3 signal peptide, and the LAG-3 signal peptide may consist of SEQ ID NO: 7 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

In another aspect according to the present invention, there is provided a polynucleotide encoding the chimeric antigen receptor protein.

The polynucleotide encoding the antigen receptor of the present invention changes the amino acid sequence of the antigen receptor expressed from the coding region due to codon degeneracy or in consideration of the codon preferred in the organism to express the antigen receptor. Various modifications may be made to the coding region within the range that does not occur, and various modifications or modifications may be made within the range that does not affect the expression of the gene in parts other than the coding region, and such modified genes are also within the scope of the present invention. It will be well understood by those skilled in the art. That is, as long as the polynucleotide of the present invention encodes a protein having an equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.

In addition, as another aspect according to the present invention, there is provided a vector comprising the polynucleotide, and a cell transformed with the vector.

The vector used in the present invention may use a variety of vectors known in the art, and may include a promoter, a terminator, an enhancer, etc., depending on the type of host cell to produce the antigen receptor. Expression control sequences, sequences 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 a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose.

As a preferred embodiment of the present invention, a vector for a lenti-virus or a vector for a retro-virus can be used. pLNCX2 (retro-virus vector) was used, but is not limited thereto.

In one embodiment, a cell can be transformed by introducing a chimeric antigen receptor that specifically binds to HLA class II into the cell through the vector. The cell may be a T cell, a tumor infiltrating lymphocyte, a B cell, a natural killer cell, or an NKT cell, preferably a cytotoxic T cell or a natural killer cell. The cell may be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells or umbilical cord blood, and the cell may be a human cell, but is not limited thereto.

As described above, the transformed cells into which the chimeric antigen receptor of the present invention is introduced recognizes HLA class II ((HLA-DP, HLA-DQ, HLA-DR) as an antigen and strongly binds thereto.

In the present invention, "chimeric antigen receptor T cells (hereinafter abbreviated as 'CAR-T cells')" or "chimeric antigen receptor NK cells (hereinafter referred to as chimeric antigen receptor NK cells)" 'CAR-NK cells') is a chimeric antigen receptor that specifically responds to cancer cells other than the original T cell receptor or NK cell receptor by transducing normal T cells or natural killer cells, etc. refers to T cells or NK cells expressing T cells or NK cells having this receptor induce apoptosis of target cells, thereby exhibiting cytotoxicity.

In the present invention, in particular, CAR-T cells or CAR-NK cells may be cytotoxic T cells or cells in which the chimeric antigen receptor of the present invention is introduced into NK cells. The cells have the advantage of anticancer-specific targeted therapy, which is the existing advantage of CAR-T therapeutics. In particular, the CAR-T cells or CAR-NK cells of the present invention are HLA class II (HLA-DP, HLA-DQ, HLA- It can recognize and effectively destroy cancer cells expressing DR).

Therefore, as another aspect, the present invention provides a cell therapeutic agent comprising the cell or a pharmaceutical composition for the treatment of cancer comprising the same as an active ingredient.

In the present invention, "cell therapeutic" refers to cells and tissues prepared through isolation, culture, and special manipulation from an individual, and as pharmaceuticals used for therapeutic purposes, living autologous, allogeneic, or xenogeneic cells or tissues are restored to function. It refers to a drug used for therapeutic purposes through a series of actions, such as in vitro proliferation and selection or changing the biological properties of cells in other ways.

As used herein, the term “treatment” refers to any action in which symptoms of cancer are improved or beneficially changed by administration of the composition.

The composition may include a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not inhibit the biological activity and properties of the injected compound without irritating the organism. The type of carrier usable in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or may be used in combination of two or more.

The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sucrose, lactose. , gelatin, etc. may be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used.

The composition may be administered in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, infected virus type, and drug. Activity, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field.

The administration means introducing the composition of the present invention to the patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered intranasally, but is not limited thereto.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into two to three times. When the two active ingredients are single drugs, the number of administrations may be the same or different. In addition, the composition of the present invention can be used alone or in combination with other drug treatments for the treatment of HLA class II-expressing cancer. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

The subject is a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig that has or can develop cancer expressing HLA class II. all animals, including If the disease can be effectively treated by administering the pharmaceutical composition of the present invention to the subject, the type of subject is included without limitation.

In the present invention, the types of cancer to be treated include head and neck squamous cell carcinoma, thymoma, esophageal squamous cell carcinoma, melanoma, Breast cancer, colorectal cancer, ovarian cancer, prostate cancer, neuroblstoma, glioma, non-small cell lung cancer , classic Hodgkin lymphoma, T-cell leukemia/lymphoma, B cell lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia), acute myeloid leukemia, or acute lymphoid leukemia, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

실시예 1. 유전자 합성 방법에 의한 키메릭 항원 수용체Example 1. Chimeric antigen receptor by gene synthesis method 유전자의 클로닝cloning of genes

In order to prepare the chimeric antigen receptor of the present invention, a pair of extracellular domains (No. 1 to No. 4) of LAG-3 are each linked to the human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) for antigen recognition The protein coding sequence of the region and the transmembrane domain of CD8α, the intracellular domain of CD28, 4-1BB, and CD3-zeta, respectively, was identified in the gene database. Specific sequence information of extracellular domains 1 to 4 of the LAG-3 is as follows.

LAG-3 세포외 도메인 1번부터 4번의 서열목록Sequence list of LAG-3 extracellular domains 1 to 4
D1D1	CTC CAG CCA GGG GCT GAG GTC CCG GTG GTG TGG GCC CAG GAG GGG GCT CCT GCC CAG CTC CCC TGC AGC CCC ACA ATC CCC CTC CAG GAT CTC AGC CTT CTG CGA AGA GCA GGG GTC ACT TGG CAG CAT CAG CCA GAC AGT GGC CCG CCC GCT GCC GCC CCC GGC CAT CCC CTG GCC CCC GGC CCT CAC CCG GCG GCG CCC TCC TCC TGG GGG CCC AGG CCC CGC CGC TAC ACG GTG CTG AGC GTG GGT CCC GGA GGC CTG CGC AGC GGG AGG CTG CCC CTG CAG CCC CGC GTC CAG CTG GAT GAG CGC GGC CGG CAG CGC GGG GAC TTC TCG CTA TGG CTG CGC CCA GCC CGG CGC GCG GAC GCC GGC GAG TAC CGC GCC GCG GTG CAC CTC AGG GAC CGC GCC CTC TCC TGC CGC CTC CGT CTG CGC CTG GGCCTC CAG CCA GGG GCT GAG GTC CCG GTG GTG TGG GCC CAG GAG GGG GCT CCT GCC CAG CTC CCC TGC AGC CCC ACA ATC CCC CTC CAG GAT CTC AGC CTT CTG CGA AGA GCA GGG GTC ACT TGG CAG CAT CAG CCA GAC AGT GGC CCG C GCT GCC GCC CCC GGC CAT CCC CTG GCC CCC GGC CCT CAC CCG GCG GCG CCC TCC TCC TGG GGG CCC AGG CCC CGC CGC TAC ACG GTG CTG AGC GTG GGT CCC GGA GGC CTG CGC AGC GGG AGG CTG CCC CTG CAG CTGGC GTC CAG CCC CGC GTC GAT GAG CGC GGC CGG CAG CGC GGG GAC TTC TCG CTA TGG CTG CGC CCA GCC CGG CGC GCG GAC GCC GGC GAG TAC CGC GCC GCG GTG CAC CTC AGG GAC CGC GCC CTC TCC TGC CGC CTC CGT CTG CGC CTG GGC	Ig-like V domain (435bp)Ig-like V domain (435bp)
D2D2	CAG GCC TCG ATG ACT GCC AGC CCC CCA GGA TCT CTC AGA GCC TCC GAC TGG GTC ATT TTG AAC TGC TCC TTC AGC CGC CCT GAC CGC CCA GCC TCT GTG CAT TGG TTC CGG AAC CGG GGC CAG GGC CGA GTC CCT GTC CGG GAG TCC CCC CAT CAC CAC TTA GCG GAA AGC TTC CTC TTC CTG CCC CAA GTC AGC CCC ATG GAC TCT GGG CCC TGG GGC TGC ATC CTC ACC TAC AGA GAT GGC TTC AAC GTC TCC ATC ATG TAT AAC CTC ACT GTT CTG GGT CTG GAG CCCCAG GCC TCG ATG ACT GCC AGC CCC CCA GGA TCT CTC AGA GCC TCC GAC TGG GTC ATT TTG AAC TGC TCC TTC AGC CGC CCT GAC CGC CCA GCC TCT GTG CAT TGG TTC CGG AAC CGG GGC CAG GGC CGA GTC CCC GTC CGG GAG T CAT CAC CAC TTA GCG GAA AGC TTC CTC TTC CTG CCC CAA GTC AGC CCC ATG GAC TCT GGG CCC TGG GGC TGC ATC CTC ACC TAC AGA GAT GGC TTC AAC GTC TCC ATC ATG TAT AAC CTC ACT GTT CTG GGT CTG GAG CCC	Ig-like C2 type-1 domain (291bp)Ig-like C2 type-1 domain (291bp)
D3D3	CCA ACT CCC TTG ACA GTG TAC GCT GGA GCA GGT TCC AGG GTG GGG CTG CCC TGC CGC CTG CCT GCT GGT GTG GGG ACC CGG TCT TTC CTC ACT GCC AAG TGG ACT CCT CCT GGG GGA GGC CCT GAC CTC CTG GTG ACT GGA GAC AAT GGC GAC TTT ACC CTT CGA CTA GAG GAT GTG AGC CAG GCC CAG GCT GGG ACC TAC ACC TGC CAT ATC CAT CTG CAG GAA CAG CAG CTC AAT GCC ACT GTC ACACCA ACT CCC TTG ACA GTG TAC GCT GGA GCA GGT TCC AGG GTG GGG CTG CCC TGC CGC CTG CCT GCT GGT GTG GGG ACC CGG TCT TTC CTC ACT GCC AAG TGG ACT CCT CCT GGG GGA GGC CCT GAC CTC CTG GTG ACT GGA GAC AAT GGC GGA GAC GAC TTT ACC CTT CGA CTA GAG GAT GTG AGC CAG GCC CAG GCT GGG ACC TAC ACC TGC CAT ATC CAT CTG CAG GAA CAG CAG CTC AAT GCC ACT GTC ACA	Ig-like C2 type-2 domain (249bp)Ig-like C2 type-2 domain (249bp)
D4D4	TTG GCA ATC ATC ACA GTG ACT CCC AAA TCC TTT GGG TCA CCT GGA TCC CTG GGG AAG CTG CTT TGT GAG GTG ACT CCA GTA TCT GGA CAA GAA CGC TTT GTG TGG AGC TCT CTG GAC ACC CCA TCC CAG AGG AGT TTC TCA GGA CCT TGG CTG GAG GCA CAG GAG GCC CAG CTC CTT TCC CAG CCT TGG CAA TGC CAG CTG TAC CAG GGG GAG AGG CTT CTT GGA GCA GCA GTG TAC TTC ACA GAG CTG TCT AGC CCA GGT GCC CAA CGC TCT GGG AGA GCC CCA GGT GCC CTC CCA GCA GGC CAC CTCTTG GCA ATC ATC ACA GTG ACT CCC AAA TCC TTT GGG TCA CCT GGA TCC CTG GGG AAG CTG CTT TGT GAG GTG ACT CCA GTA TCT GGA CAA GAA CGC TTT GTG TGG AGC TCT CTG GAC ACC CCA TCC CAG AGG AGT TTC TCA GGA CGG CTG GAG GCA CAG GAG GCC CAG CTC CTT TCC CAG CCT TGG CAA TGC CAG CTG TAC CAG GGG GAG AGG CTT CTT GGA GCA GCA GTG TAC TTC ACA GAG CTG TCT AGC CCA GGT GCC CAA CGC TCT GGG AGA GCC CCA GGT GCC CTC CTC GGC CAC CTC	Ig-like C2 type-3 domain (309bp)Ig-like C2 type-3 domain (309bp)

Thereafter, the remaining sequences except for the stop codon portion of the nucleotide sequence constituting the domains were joined together to perform gene synthesis. The accuracy of gene synthesis was confirmed through sequencing after a protein expression plasmid was prepared. A schematic diagram showing the cDNA region of each domain expressing the chimeric antigen receptor is shown in FIG. 1 .

실시예 2. 키메릭 항원 수용체Example 2. Chimeric Antigen Receptor 단백질 발현 플라스미드 제조Protein expression plasmid preparation

Recognizing HLA class II (HLA-DP, HLA-DQ, HLA-DR) and expressing a recombinant protein in which the human immunoglobulin G ₁ heavy chain constant region and the signaling protein domain are fused to cytotoxic T cells and natural killer cells To do this, the gene was cloned into pCDH-CMV-MCS-EF1-copGFP (System Biosciences), a lentivirus-derived expression vector, or pLNCX2 (Addgene), a retrovirus-derived expression vector.

First, for cloning into the pCDH-CMV-MCS-EF1-copGFP vector, a primer that creates a cleavage site sequence of restriction enzyme XbaI at the 5' end was synthesized, and a restriction enzyme cleavage site was created by polymerase chain reaction, and also restricted at the 3' end. Restriction enzyme cleavage site was created by polymerase chain reaction by synthesizing a primer making the cleavage site sequence of the enzyme NotI. After that, the 5' end of the gene having a restriction enzyme cleavage site was treated with XbaI, and the 3' end was treated with NotI due to the polymerase chain reaction. Then, the multicloning site of the expression vector was treated with XbaI and NotI to allow the gene to be inserted. After mixing the restriction enzyme-treated gene with the expression vector, it was ligated by treatment with a ligase.

Next, for cloning into the pLNCX2 vector, a primer that creates a cleavage site sequence of restriction enzyme Bgl II at the 5' end was synthesized to create a restriction enzyme cleavage site by polymerase chain reaction, and the cleavage site sequence of restriction enzyme NotI was also synthesized at the 3' end. A restriction enzyme cleavage site was created by synthesizing the primers to be made and by polymerase chain reaction. After that, the 5' end of the gene having a restriction enzyme cleavage site was treated with BglII, and the 3' end was treated with NotI due to the polymerase chain reaction. And the multicloning site of the expression vector was treated with BglII and NotI to allow the gene to be inserted. After mixing the restriction enzyme-treated gene with the expression vector, it was ligated by treatment with a ligase.

As a result, a recombinant vector LAG-3 that recognizes HLA class II (HLA-DP, HLA-DQ, HLA-DR) and expresses a protein obtained by fusion of a human immunoglobulin G ₁ heavy chain constant region and a signaling protein domain. CAR-T.pCDH-CMV-MCS-EF1-copGFP and LAG-3.CAR-NK.pLNCX2 were completed (see FIGS. 2A and 2B ).

실시예 3. HLA class II를 발현하는 사람 세포주 screeningExample 3. Screening of human cell lines expressing HLA class II

In order to screen human cell lines expressing HLA class II on the cell surface, acute promyelocytic leukemia-derived cell line HL-60 (ATCC) and T cell lymphoma-derived cell line Jurkat (ATCC) Expression of HLA class II was confirmed in the cell line. To confirm expression, the cell lines were treated with an antibody (biolegend) capable of binding to HLA class II to which a fluorescent protein was attached, and then flow cytometry (fluorescence-activated cell sorting) was used. As a result of the analysis, it was confirmed that HLA class II was expressed in the HL-60 cell line, and it was confirmed that the Jurkat cell line did not express HLA class II (see FIG. 4 ).

Based on the above results, the HL-60 cell line expressing HLA class II among the two cells was used as a target cell, and Jurkat cells not expressing HLA class II were used as a negative control.

실시예Example 4. 4. 키메릭chimeric 항원 수용체와 antigen receptors and HLAHLA class II를 발현하는 사람 세포주의 친화도 측정 Affinity measurement of human cell lines expressing class II

In order to confirm whether the constructed chimeric antigen receptor has affinity with _a human cell line expressing HLA class II, each of the extracellular domains (No. IgG ₁ heavy chain constant region), two extracellular domains of LAG-3 (No. 1 to No. 4) per one chimeric antigen receptor made the chimeric antigen receptor water soluble to recognize the antigen, Chinese hamster ovary After expression in (CHO) cells, the protein was purified by affinity chromatography (GE healthcare) using a protein A column (see FIG. 5a ).

In addition, the purified water-soluble chimeric antigen receptor was confirmed by Western blotting using an antibody recognizing the constant region of human IgG heavy chain (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody, abcam) (Fig. 5b).

Thereafter, the purified water-soluble chimeric antigen receptor was treated with a cell line expressing HLA class II (HL-60) and a cell line not expressing HLA class II (Jurkat) to prevent the water-soluble chimeric antigen receptor from binding to the cell. The results confirmed through flow cytometry analysis are shown in FIG. 5c.

As shown in FIG. 5c , it was confirmed that HL-60 cells expressing MHC class II bound to the water-soluble chimeric antigen receptor, but Jurkat cells not expressing MHC class II did not bind to the water-soluble chimeric antigen receptor.

As confirmed from the above results, it was confirmed that the prepared chimeric antigen receptor had a high affinity with the human cell line expressing HLA class II, which was the MHC class II of the immune cell therapy prepared with HL-60 cells and Jurkat cells. This means that it can be used for specific cytotoxicity verification of proteins.

실시예 5. 세포독성 T 세포 분리 및 활성화Example 5. Cytotoxic T cell isolation and activation

To prepare T cells expressing a chimeric antigen receptor according to an embodiment of the present invention, cytotoxic T cells were first isolated from human peripheral blood mononuclear cells. After purchasing human peripheral blood mononuclear cells (Medilab Korea), magnetic-activated cell sorting was used to isolate cytotoxic T cells. Peripheral blood mononuclear cells were combined with an antibody (CD8 ⁺ T cell biotin-conjugated antibody cocktail, Miltenyi Biotec) capable of binding to other immune cells except for cytotoxic T cells, and then the antibodies were re-conjugated with magnetic microbeads (anti- biotin microbead) (Miltenyi Biotec). The microbeads, antibodies and cells attached thereto were passed through a magnetic seperation column (Miltenyi Biotec) to obtain cytotoxic T cells not labeled with the antibody. In order to confirm the purity of the isolated cytotoxic T cells, flow cytometry using CD8 and CD3ε, which are cell surface factors of cytotoxic T cells, was performed, and as a result, the purity was greater than 95%.

For activation of isolated cytotoxic T cells, 1Х10 ⁶ pieces/ml of magnetic beads coated with human CD3 and CD28 antibodies (Thermo Fisher Scientific), and 10% calf serum supplemented with 100 U/μl recombinant human IL-2 Cells were reconstituted in RPMI (Welgene) at a density of 1.5Х10 ⁶ pieces/ml and cultured for 24 hours after inoculation in a 24-well cell culture dish.

실시예 6. 키메릭 항원 수용체 발현 세포독성 T 세포 제작Example 6. Construction of chimeric antigen receptor-expressing cytotoxic T cells

In order to introduce the recombinant vector prepared in Example 2 into cytotoxic T cells, a lentiviral system using 293FT cells (Thermo Fisher Scientific) was used.

First, 293FT cells were inoculated to become 2.5Х10 ⁶ cells in a 100π cell culture dish, and then cultured in DMEM medium containing 10% calf serum. After 24 hours of incubation, when the cells have grown to cover 60-70% of the dish, 20 µg of LAG-3.CAR-T.pCDH-CMV-MCS-EF1-copGFP vector DNA is mixed with 10 µg of psPAX2 (Addgene) and 3 µg pMD2.G (Addgene) vector was crystallized using calcium phosphate and Hepes-buffered solution, and then introduced into 293FT cells. Thereafter, the culture supernatant containing the lentivirus was collected at intervals of 24 hours after 48 hours of the 293FT cells introduced with the expression vector. The collected supernatant was centrifuged at 21,000 rpm in an ultra-high speed centrifuge for 2 hours to concentrate the virus. After the concentrated virus was mixed with 4 μg/ml of polybrene and added to the culture of activated cytotoxic T cells, the transduction was performed by centrifugation at 1,800 g for 75 minutes using a centrifuge. After centrifugation, the cytotoxic T cells were further cultured for 4 hours and then replaced with an RPMI culture medium containing 10% calf serum. After 48 hours, some of the transduced cytotoxic T cells were used to measure the transduction efficiency. Transduction efficiency was measured by using flow cytometry to measure the expression level of GFP inside the cells, and cytotoxic T cells (Mock) transduced with a virus prepared by using the transduced cytotoxic T cells (LAG-3 CAR-T cells) as an empty vector. /T cells) and the results compared with the transduction ratio is shown in Figure 6a.

실시예 7. 키메릭 항원 수용체 발현 자연살해세포 제작Example 7. Preparation of chimeric antigen receptor-expressing natural killer cells

In order to introduce the recombinant vector prepared in Example 2 into NK cells, a retroviral system using 293GPG cells was used.

First, 3Х10 ⁶ 293GPG cells were dissolved in 10 ml of 10 ml of 10% calf serum-containing DMEM culture medium and inoculated in a 100π cell culture dish and then cultured for 24 hours. 20 μg of the previously prepared recombinant vector (LAG-3.CAR-NK.pLNCX2 vector) was crystallized using calcium phosphate and Hepes-buffered solution, and then added to the culture medium of the previously cultured 293GPG cells. Thereafter, the culture medium was changed over 72 hours at 24 hour intervals, and the culture supernatant of 293GPG cells containing retrovirus was collected and stored. The collected retrovirus-containing supernatant was centrifuged at 21,000 rpm for 2 hours using an ultra-high speed centrifuge, and then reconstituted in Myelocult H5100 culture medium (STEMCELL) containing 10% calf serum to be concentrated 100 times compared to before concentration. To transduce the concentrated retrovirus into NK92MI cells (American type culture collection, ATCC), NK92MI cells were inoculated into a 24 well cell culture dish at a density of 5Х10 ⁵ /ml. Then, a mixed solution of 8 ug/ml of polybrene added to the concentrated retrovirus was added to the culture medium of NK92MI cells in culture and cultured for 24 hours. After culturing, the culture medium was replaced with a new culture medium. For the selection of the cells into which the gene was introduced, from 24 hours after the culture medium replacement, the Myelocult H5100 culture medium supplemented with neomycin antibiotic (600 μg/ml) was used to culture the retrovirus-treated NK92MI cells. After 14 days of neomycin selection, NK92MI cells were transferred to fresh culture medium and proliferated for 1 week.

After proliferation, the LAG-3 expression level of NK92MI cells was measured using flow cytometry. NK92MI cells introduced with LAG-3.CAR-NK.pLNCX2 (LAG-3 CAR-NKcell) and NK92MI cells introduced with the empty vector pLNCX2 ( The results of comparing the LAG-3 expression level of Mock CAR-NK cells using a flow cytometer using an anti-human LAG-3 antibody (Novus biologicals) are shown in FIG. 6b .

실시예 8. CAR-T 세포의 MHC class II+ cell 특이적 세포독성 검증Example 8. MHC class II+ cell-specific cytotoxicity verification of CAR-T cells

In order to confirm whether the prepared chimeric antigen receptor-expressing cytotoxic T cells specifically recognize MHC class II cells and show toxicity, the target cells selected in Example 3, HL-60 cells (HLA class II positive cells) and Jurkat cells (HLA class II negative cells) were co-cultured with chimeric antigen receptor-expressing cytotoxic T cells (LAG-3 CAR-T cells) or cytotoxic T cells (Mock T cells) introduced with an empty vector for 6 hours. . A non-radioactive cytotoxicity assay was used to measure the degree of cytotoxicity of cytotoxic T cells to target cells by the amount of lactate dehydrogenase present in the supernatant after co-culture.

First, put cytotoxic T cells (1x10 ⁵ cells) and target cells (1x10 ⁴ cells) into the wells of a 96-well cell culture dish, set the effector: target ratio to 10:1, and inoculate so that the volume per well becomes 100μl Centrifuge for 4 minutes at 250 g using a centrifuge to close the intercellular space. After culturing for 6 hours, 50 μl of the supernatant from each well is removed, transferred to a transparent 96-well dish for absorbance measurement, and treated with an analysis solution and 1M hydrochloric acid solution to proceed and stop the enzyme reaction. After stopping the enzymatic reaction, the absorbance in the 490 nm wavelength band was measured and numerically converted using a fluorescence/luminescence/absorption meter (multi-detection plate reader) to quantify the degree of cytotoxicity of cytotoxic T cells in each well.

As a result, as shown in Figure 7a, the prepared chimeric antigen receptor-expressing cytotoxic T cells (LAG-3 CAR-T cells) showed about 0.5% cytotoxicity to Jurkat cells (HLA class II negative cells), whereas , It was confirmed that HL-60 cells (HLA class II positive cell) showed high cytotoxicity of 22% or more (p<0.01). On the other hand, in the case of cytotoxic T cells introduced with the empty vector as a control, it was confirmed not to have cytotoxicity against Jurkat cells (HLA class II negative cells) or HL-60 cells (HLA class II positive cells).

Through the above results, the chimeric antigen recognition receptor-expressing cytotoxic T cells containing the LAG-3 extracellular domain showed cytotoxicity specific to the MHC class II protein, and thus the related cancer cells using the CAR-T T cells. It was confirmed that treatment was possible.

실시예 9. CAR-NK 세포의 MHC class II+ cell 특이적 세포독성 검증Example 9. MHC class II+ cell-specific cytotoxicity verification of CAR-NK cells

In order to confirm whether the prepared chimeric antigen receptor-expressing NK cells specifically recognize MHC class II cells and show toxicity, HL-60 cells (HLA class II positive cells) and Jurkat cells selected in Example 3 (HLA class II negative cells) were co-cultured with chimeric antigen receptor-expressing NK cells (LAG-3 CAR-NK cells) or NK cells introduced with an empty vector (Mock NK cells).

A non-radioactive cytotoxicity assay was used to measure the degree of cytotoxicity of NK cells to target cells by the amount of lactate dehydrogenase present in the supernatant after co-culture.

First, put natural killer cells (1x10 ⁵ cells) and target cells (1x10 ⁴ cells) into the wells of a 96-well cell culture dish, set the effector: target ratio to 10:1, and inoculate it so that the volume per well becomes 100μl and centrifuge Centrifuge for 4 minutes at 250 g using a separator to close the intercellular space. After culturing for 6 hours, 50 μl of the supernatant from each well is removed and transferred to a transparent 96-well dish for absorbance measurement. After stopping the enzymatic reaction, the absorbance of the 490 nm wavelength band was measured and numerically converted using a fluorescence/luminescence/absorption meter (multi-detection plate reader) to quantify the degree of cytotoxicity of NK cells in each well.

As a result, as shown in FIG. 7b, the prepared chimeric antigen receptor-expressing NK cells (LAG-3 CAR-NK cells) showed almost no cytotoxicity to Jurkat cells (HLA class II negative cells), whereas HL-60 It was confirmed that the cells (HLA class II positive cell) showed a very high cytotoxicity of more than 47% (p<0.01). On the other hand, in the case of cytotoxic T cells introduced with the empty vector as a control, it was confirmed not to have cytotoxicity against Jurkat cells (HLA class II negative cells) or HL-60 cells (HLA class II positive cells).

Through the above results, the chimeric antigen-recognizing receptor-expressing NK cells containing the LAG-3 extracellular domain showed cytotoxicity specific to the MHC class II protein, so that it is possible to treat the related cancer cells using the CAR-NK cells. was able to confirm

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims

antigen binding domain;

transmembrane domain; and

A chimeric antigen receptor (CAR) comprising an intracellular signaling domain comprising:

The antigen-binding domain is a chimeric antigen receptor comprising an extracellular domain (No. 1 to No. 4) of LAG-3 that specifically binds to HLA class II.
According to claim 1,

The antigen binding domain is characterized in that a pair of extracellular domains (No. 1 to No. 4) of LAG-3 are connected to each human immunoglobulin G 1 heavy chain constant region in the form of a dimer (IgG 1 heavy chain constant region) A chimeric antigen receptor.
According to claim 1,

The extracellular domain of the LAG-3 (No. 1 to No. 4) will include the amino acid sequence shown in SEQ ID NO: 1, a chimeric antigen receptor.
3. The method of claim 2,

Human immunoglobulin G 1 heavy chain constant region (IgG 1 heavy chain constant region) is a chimeric antigen receptor comprising the amino acid sequence represented by SEQ ID NO: 2.
According to claim 1,

The transmembrane domain is CD8, the chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO: 3.
The method of claim 1,

The intracellular signaling domain is a chimeric antigen receptor comprising one consisting of CD28, 4-1BB and CD3-zeta or a combination thereof.
7. The method of claim 6,

The CD28 is SEQ ID NO: 4; 4-1BB is SEQ ID NO: 5; And CD3-zeta is a chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO: 6.
According to claim 1,

The antigen binding domain comprises a LAG-3 signal peptide, and the amino acid sequence shown in SEQ ID NO: 7, the chimeric antigen receptor.
A polynucleotide encoding the chimeric antigen receptor protein of any one of claims 1 to 8.
A vector comprising the polynucleotide of claim 9 .
A cell transformed with the vector of claim 10 .
12. The method of claim 11,

The cell, characterized in that the cell is a cytotoxic T cell or NK cell.
A pharmaceutical composition for the treatment of cancer expressing HLA class II comprising the cell of claim 12 as an active ingredient.
14. The method of claim 13,

The cancer expressing the HLA class II is head and neck squamous cell carcinoma, thymoma, esophageal squamous cell carcinoma, melanoma, breast cancer ), colorectal cancer, ovarian cancer, prostate cancer, neuroblstoma, glioma, non-small cell lung cancer, classic Hodgkin Lymphoma (classic Hodgkin lymphoma), T-cell leukemia/lymphoma, B-cell lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, acute A composition selected from the group consisting of acute myeloid leukemia and acute lymphoid leukemia.
A cell therapeutic agent comprising the cell of claim 11 .