Chimeric antigen receptor, polynucleotide, vector, cell, method for producing cell, and pharmaceutical composition

Abstract

Provided are a chimeric antigen receptor, a polynucleotide, a vector, a cell, a method for producing a cell, and a pharmaceutical composition that recognize a wide range of cancer cells, can be used for treatment, have a high function, and are effective for treatment of many kinds of intractable cancers. The present invention pertains to a chimeric antigen receptor which includes an immunoglobulin domain of NKp44, an extracellular hinge domain of NKp44, a transmembrane domain, a costimulatory signal transduction domain, and an activation signal transduction domain, and in which the costimulatory signal transduction domain includes CD79a and CD40 domains. The present invention also pertains to a polynucleotide, a vector, a cell, a method for producing a cell, and a pharmaceutical composition.

Description

Chimeric antigen receptor, polynucleotide, vector, cell, method for producing cell, and pharmaceutical composition

[0001] The present invention relates to a chimeric antigen receptor, a polynucleotide encoding the chimeric antigen receptor, a vector using the same, a cell expressing the chimeric antigen receptor, a method for producing the cell, and a pharmaceutical composition containing the cell. This application claims priority based on Japanese Patent Application No. 2023-172267, filed October 3, 2023, the contents of which are incorporated herein by reference.

[0002] In recent years, adoptive immunotherapy using T cells (CAR-T) transfected with a chimeric antigen receptor (CAR) gene has attracted attention as a cancer treatment method. CAR is an artificial receptor that uses a single-chain antibody or the like as an antigen-binding site, and the concept of CAR was first established by Eshhar et al. in Israel in 1993 (Non-Patent Document 1). CAR has a ligand-binding domain, a transmembrane domain, and an activation signaling domain, and an activation signal is transmitted into the T cell when a ligand binds to the ligand-binding domain.

[0003] As for the clinical efficacy of CAR, it has been reported that T cells transfected with a CAR using a single chain variable fragment (scFv) that binds to CD19 in the ligand-binding domain (Patent Document 1, Non-Patent Document 2) showed dramatic efficacy against refractory / relapsed B-cell leukemia (Non-Patent Document 3). However, CD19 is an antigen expressed only on B cells and cannot be applied to solid tumors, etc. Furthermore, research into CARs targeting other tumor antigens is also underway, but sufficient clinical efficacy has not yet been achieved.

[0004] In order to develop a CAR that demonstrates clinical efficacy against malignant tumors, including solid tumors, antigen selection is important. The antigen targeted by the CAR must be an antigen present on the surface of tumor cells, and must be an antigen that is not expressed or expressed at extremely low levels on normal cells, or whose expression is restricted to cells whose loss does not affect the survival of the organism. CAR-T exhibits extremely high cytotoxic activity against cells that have the target antigen on their cell surface. Therefore, even low-level expression on normal cells, which is not a problem with antibody drugs, may cause serious side effects in CAR-T therapy. Therefore, even antigens that have already been put into practical use as targets for antibody drugs are limited in number and can be used as target antigens in CAR-T therapy. This lack of target antigens poses a major obstacle to the clinical application of CAR-T therapy.

[0005] Furthermore, scFv that binds to a target antigen has conventionally been used as the ligand-binding domain of a CAR, and therefore, when attempting to create a CAR that targets a new antigen, it has been necessary to first obtain a monoclonal antibody that specifically binds to the antigen, and then create an scFv from the variable region of the monoclonal antibody.

[0006] Meanwhile, a method has been reported in which, instead of using scFv as the ligand-binding domain, the ligand-binding domain of NKp30, a type of natural cytotoxicity receptor (NCR) of natural killer (NK) cells, is used as the ligand-binding domain of a CAR (Patent Document 2). In addition to NKp30, NKp44 and NKp46 are known NCRs, which play important roles in the cytotoxicity of NK cells against tumor cells. When a ligand binds to these NCRs, an activation signal is transmitted into the NK cell, activating the NK cell and causing the release of cytotoxic granules and the production of cytokines. Using the ligand-binding domain of NKp30 has the advantage that it is not necessary to obtain scFv when creating a CAR.

[0007] The present inventors have reported that they have created CARs based on the NKp44 molecule as CAR-T cells or CAR-NK cells that can recognize and attack various types of cancer cells (Non-Patent Document 4). Furthermore, Julamanee et al. have reported the usefulness of CD79a / CD40 as the intracellular domain of CAR (Non-Patent Document 5).

[0008] The present inventors have also created and reported a novel CAR gene that targets the NKp44 ligand. First, they developed and reported a chimeric antigen receptor comprising the immunoglobulin domain of NKp44, the extracellular hinge domain of NKp44, the transmembrane domain of CD28, and the activation signaling domain of CD3ζ (Patent Document 3). They also developed and reported a chimeric antigen receptor comprising the immunoglobulin domain, transmembrane domain, and activation signaling domain of NKp44 (Patent Document 4). These technologies can contribute to the realization of CAR-T therapy or CAR gene-transduced NK cell therapy (CAR-NK therapy), which can recognize and attack various types of cancer cells. These technologies avoid the use of mouse-derived single-chain antibodies by using the immunoglobulin-like domain of the NKp44 receptor as the ligand binding site.

[0009] U.S. Patent No. 8,399,645 International Publication No. 2013 / 033626 Japanese Patent No. 6,842,688 Japanese Patent No. 7,054,181

[0010] Eshhar Z, et al. Proc Natl Acad Sci USA. 1993, 90(2): 720-4.Imai C, et al. Leukemia 2004, 18(4): 676-84.Maude SL, et al. N Engl J Med. 2014, 371(16): 1507-17.Kasahara Y, et al. Clin Transl Immunol 2020; 9(7): e1147. Julamanee J, et al. Mol Ther. 2021; 29(9): 2677-2690.

[0011] As described above, CARs targeting CD19 have been shown to be highly effective in treating malignant tumors derived from B lymphocytes. However, their application to other malignant tumors has unfortunately not been sufficiently effective. One of the barriers to their application to other malignant tumors, including solid tumors, is the limited number of targetable antigens. Furthermore, monoclonal antibody genes have traditionally been used as the antigen recognition site for CARs. Monoclonal antibodies have been established for antigens present on the surface of solid tumors, and hybridomas producing these monoclonal antibodies have been required. Furthermore, the efficacy of CAR-T cells is directly linked to their amplification by tens of thousands of times in the patient's body after administration. However, the optimal CAR receptor structure and signal domains for achieving this and for CAR-T cells to remain in the body for a long period of time have not yet been established.

[0012] The present inventors discovered the 4-1BB type CAR as a suitable example of a second-generation CAR among the conventional NKp44 CARs described in Patent Documents 3 and 4. However, subsequent studies led them to believe that a specific structure combining functional domains of several other immune molecules might further improve functionality, and they therefore continued to study new structures.

[0013] The present invention was made in consideration of the above-mentioned circumstances, and its object is to provide chimeric antigen receptors, polynucleotides, vectors, cells, methods for producing cells, and pharmaceutical compositions that can recognize and be used in treatment of a wide range of cancer cells, have high functionality, and are effective in treating many types of intractable cancers.

[0014] In order to achieve the above object, the present invention has the following aspects. (1) A chimeric antigen receptor comprising an immunoglobulin domain of NKp44, an extracellular hinge domain of NKp44, a transmembrane domain, a costimulatory signaling domain, and an activation signaling domain, wherein the costimulatory signaling domain comprises the costimulatory signaling domains of CD79a and CD40. (2) The chimeric antigen receptor according to (1), wherein the activation signaling domain comprises the activation signal domain of CD3ζ. (3) The chimeric antigen receptor according to (1) or (2), wherein the transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of NKp44, CD8α, and CD28. (4) A polynucleotide encoding the chimeric antigen receptor according to any one of (1) to (3). (5) A vector comprising the polynucleotide according to (4). (6) A cell expressing the chimeric antigen receptor according to (1) or (2). (7) The cell according to (6), which is a T cell or a natural killer cell. (8) A method for producing cells expressing a chimeric antigen receptor, comprising the step of introducing the polynucleotide according to (5) into cells. (9) The method for production according to (8), wherein the cells are T cells or natural killer cells. (10) A pharmaceutical composition comprising the cells according to (6) or (7). (11) A pharmaceutical composition for treating or preventing a tumor, comprising the cells according to (6) or (7).

[0015] According to the present invention, there are provided chimeric antigen receptors, polynucleotides, vectors, cells, methods for producing cells, and pharmaceutical compositions that can recognize a wide range of cancer cells and be used in therapy, have high functionality, and are effective in treating many types of intractable cancers.

[0016] FIG. 1 is a schematic diagram of a CAR construct created in this example. FIG. 2 is a graph showing the results of flow cytometry analysis of T cells in this example. FIG. 3 is a graph showing the results of flow cytometry analysis of human NK cells in this example. FIG. 4 is a graph showing the results of a cytotoxicity evaluation test in this example. FIG. 5 is a graph showing the results of the antitumor effect at 72 hours in this example. FIG. 6 is a graph showing the results of the antitumor effect at 1 week in this example. FIG. 7 is a graph showing another result of the antitumor effect at 1 week in this example. FIG. 8 is a schematic diagram showing the structure of a comparative example in this example. FIG. 9 is a diagram showing the results of cell surface expression in the examples and comparative examples in this example. FIG. 10 is a graph showing the influence of different structures on the cytocidal effect of CARs in this example. FIG. 11 is a graph showing the relationship between tumor volume and days after administration in CAR-T-administered mice. FIG. 12 is a graph showing the survival rate in CAR-T-administered mice.

[0017] The chimeric antigen receptor, polynucleotide, vector, cell, method for producing a cell, and pharmaceutical composition according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0018] [Chimeric Antigen Receptor] The chimeric antigen receptor of this embodiment comprises an immunoglobulin domain of NKp44, an extracellular hinge domain of NKp44, a transmembrane domain, a costimulatory signaling domain, and an activation signaling domain, and the costimulatory signaling domain comprises CD79a and CD40 domains.

[0019] As used herein, the term "chimeric antigen receptor (CAR)" refers to a chimeric protein comprising at least one ligand-binding domain, at least one transmembrane domain, and at least one activation signal transduction domain. A chimeric protein refers to a protein comprising regions derived from at least two or more heterologous proteins. As used herein, the term "CAR" is not limited to CARs comprising only the above three domains, but also encompasses CARs comprising other domains. Generally, CARs having only the activation signal transduction domain of the T cell receptor (TCR)-CD3 complex as an intracellular domain are referred to as first-generation CARs, CARs having a costimulatory signal transduction domain derived from a costimulatory molecule in addition to the activation signal transduction domain are referred to as second-generation CARs, and CARs having multiple costimulatory signal transduction domains in addition to the activation signal transduction domain are referred to as third-generation CARs. The term "CAR" as used herein encompasses all of these.

[0020] <Immunoglobulin domain of NKp44> The CAR of this embodiment comprises an immunoglobulin domain of NKp44 as a ligand-binding domain.

[0021] NKp44 is a type of NCR on NK cells and plays an important role in the cytotoxic activity of NK cells against tumor cells. When a ligand binds to NKp44, an activation signal is transmitted into the NK cell, activating the NK cell and resulting in the release of cytotoxic granules and cytokine production. NKp44 ligands have been reported to be expressed in a wide range of tumor cells, including hematopoietic tumors, and NK cell cytotoxic activity against these NKp44 ligand-expressing tumor cells is expressed through the binding of the ligand to NKp44. To date, MLL5 (Baycheiler F, et al. Blood 2013, 122(17): 2935-42) and PCNA (Rosental B, et al. J Immunol 2011, 187: 5693-5702) have been reported as NKp44 ligands. Examples of the amino acid sequence of human MLL5 include GenBank IDs NP_061152 (nucleotide sequence: NM_018682.3) and NP_891847.1 (nucleotide sequence: NM_182931.2), and examples of the amino acid sequence of human PCNA include, but are not limited to, GenBank IDs NP_002583.1 (nucleotide sequence: NM_002592.2) and NP_872590.1 (nucleotide sequence: NM_182649.1). NKp44 is not expressed in non-activated NK cells, but is expressed in activated NK cells. As used herein, the term "NKp44" encompasses all proteins having the above-described properties.

[0022] In the CAR of this embodiment, the organism from which NKp44 is derived is not particularly limited, and may be NKp44 derived from humans or NKp44 from non-human mammals such as mice, rats, cows, sheep, horses, dogs, pigs, or monkeys, although human NKp44 is preferred. Examples of the amino acid sequence of human NKp44 include, but are not limited to, GenBank IDs NP_004819.2 (isoform 1), NP_001186438.1 (isoform 2), and NP_001186439.1 (isoform 3). Human NKp44s consisting of these amino acid sequences are variants of human NKp44, and all of them have the characteristics of NKp44 described above. However, NKp44 is not limited to those having these amino acid sequences and also includes mutants and interspecies homologues thereof. In addition to the above isoforms 1 to 3, the protein may have other amino acid sequences registered in sequence databases such as GenBank and DDBJ, or may be a mutant thereof.

[0023] Polynucleotide sequences encoding the above amino acid sequences have been registered in GenBank as NM_004828.3 (isoform 1), NM_001199509.1 (isoform 2), and NM_001199510.1 (isoform 3). As typical examples of the amino acid sequence of human NKp44, the nucleotide sequence and amino acid sequence of isoform 1 (NM_004828.3, NP_004819.2) are shown in SEQ ID NOs: 3 and 4, respectively.

[0024] Examples of the human NKp44 mutant include proteins that have approximately 1 to 50, 1 to 30, 1 to 20, or 1 to 10 mutations compared to the amino acid sequence set forth in SEQ ID NO: 4, and that retain the above-mentioned NKp44 function. The mutations may be deletions, substitutions, insertions, or additions of amino acid residues, or a combination thereof. Other examples of the human NKp44 mutant include proteins that have an amino acid sequence that has 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity (homology) with the amino acid sequence set forth in SEQ ID NO: 4, and that retain the above-mentioned NKp44 function. The sequence identity between amino acid sequences is determined by aligning two amino acid sequences, with gaps inserted at the positions corresponding to insertions and deletions, so that the number of corresponding amino acids is greatest, and then calculating the percentage of identical amino acids in the resulting alignment relative to the total amino acid sequence excluding gaps. The sequence identity between amino acid sequences can be determined using various homology search software known in the art.

[0025] The immunoglobulin domain of NKp44 functions as a ligand-binding domain of NKp44 and has the ability to bind to NKp44 ligands. The immunoglobulin domain of NKp44 is located in the extracellular region of NKp44 and has an immunoglobulin-like structure. An example of the amino acid sequence of the immunoglobulin domain of human NKp44 is one having the amino acid sequence from 22 to 130 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 4. However, the immunoglobulin domain of NKp44 is not limited to those containing the above amino acid sequence, and includes all polypeptides that have the ability to bind to NKp44 ligands.

[0026] Examples of the immunoglobulin domain of NKp44 include, but are not limited to, polypeptides described in any of the following (i) to (iv): (i) a polypeptide comprising the amino acid sequence of the 22nd to 130th amino acids from the N-terminus of the amino acid sequence of isoform 1 (SEQ ID NO: 4); (ii) a polypeptide having about 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 mutations compared to the amino acid sequence of (i) and having binding ability to an NKp44 ligand (the mutations may be deletions, substitutions, insertions, or additions, or a combination thereof); (iii) a polypeptide having an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity (homology) with the amino acid sequence of (i) and having binding ability to an NKp44 ligand; and (iv) A polypeptide consisting of the amino acid sequence of (i) in which one or more amino acids are deleted, substituted or added, and having the ability to bind to an NKp44 ligand (the "multiple" means 2 to 30, preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5).

[0027] <Extracellular hinge domain> The CAR of this embodiment comprises the extracellular hinge domain of NKp44. As used herein, the term "extracellular hinge domain" refers to a domain that links the extracellular ligand-binding domain and the transmembrane domain.

[0028] The extracellular hinge domain of NKp44 can be selected from the various NKp44 sequences described above. For example, 50 to 70 amino acids near the N-terminus, preferably 60 amino acids from the N-terminus, can be selected from the various NKp44 sequences described above. Specific examples include, but are not limited to, the amino acid sequence from positions 131 to 190 from the N-terminus of the amino acid sequence of isoform 1 (SEQ ID NO: 4) described above.

[0029] Furthermore, the extracellular hinge domain may be a mutant of the extracellular hinge domain of NKp44 as described above. Examples of mutants of the extracellular hinge domain of NKp44 include, for example, polypeptides described in any of the following (i) to (iii): (i) a polypeptide having about 1 to 20, 1 to 10, 1 to 5, or 1 to 3 mutations compared to the amino acid sequence of the extracellular hinge domain of NKp44, and capable of linking an NKp44 immunoglobulin domain and a transmembrane domain (the mutations may be deletions, substitutions, insertions, or additions, or a combination thereof); (ii) a polypeptide comprising an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity (homology) with the amino acid sequence of the extracellular hinge domain of NKp44, and capable of linking an NKp44 immunoglobulin domain and a transmembrane domain; and (iii) A polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the extracellular hinge domain of NKp44, and which is capable of linking the NKp44 immunoglobulin domain and the transmembrane domain (the "multiple" means 2 to 30, preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5).

[0030] <Transmembrane domain> The CAR of this embodiment has at least one transmembrane domain. The transmembrane domain that can be used for the CAR of this embodiment is not particularly limited as long as it is a polypeptide that has the function of penetrating a cell membrane. The transmembrane domain may be derived from a natural protein or may be artificially designed. A transmembrane domain derived from a natural protein can be obtained from any membrane-binding protein or transmembrane protein. In the CAR of this embodiment, the transmembrane domain can transmit an activation signal to the activation signaling domain in response to binding of a ligand to the NKp44 immunoglobulin domain.

[0031] Examples of proteins from which transmembrane domains usable in the CAR of this embodiment are derived include the α chain and β chain of T cell receptor, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, NKp30, NKp44, and NKp46. Preferred examples include CD28, CD8α, and NKp44, and more preferred examples include CD28 and CD8α. The transmembrane domain comprised by the CAR of this embodiment may include the transmembrane domain of these proteins, or may consist of the transmembrane domain of these proteins.

[0032] Examples of the amino acid sequences of the transmembrane domains of CD28, CD8α, and NKp44 include, but are not limited to, the amino acid sequence of positions 153 to 179 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence of positions 182 to 206 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 6, and the amino acid sequence of positions 191 to 213 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 4.

[0033] The transmembrane domain may also be a mutant of a transmembrane domain derived from a naturally occurring protein as described above. Examples of mutant transmembrane domains derived from naturally occurring proteins include the polypeptides described in any of (i) to (iii) below. (i) a polypeptide having approximately 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 mutations compared to the amino acid sequence of a transmembrane domain derived from a naturally occurring protein, and having membrane-penetrating ability (the mutations may be deletions, substitutions, insertions, or additions, or a combination thereof); (ii) a polypeptide having membrane-penetrating ability, comprising an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity (homology) with the amino acid sequence of a transmembrane domain derived from a naturally occurring protein; and (iii) a polypeptide having membrane-penetrating ability, consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of a transmembrane domain derived from a naturally occurring protein (the "multiple" refers to 2 to 30, preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5).

[0034] <Costimulatory signaling domain> In one aspect, the CAR of the present embodiment may comprise a costimulatory signaling domain. It is known that costimulatory molecules expressed on T cells transmit costimulatory signals intracellularly upon binding to ligands specific to the respective costimulatory molecules expressed on antigen-presenting cells, thereby assisting T cell activation (secondary signaling). As used herein, the term "costimulatory signaling domain" (costimulatory signaling domain) refers to an intracellular domain involved in the costimulatory signal transmission of the above-mentioned costimulatory molecules.

[0035] (Costimulatory signaling domain of CD79a and CD40) The costimulatory signaling domain of the CAR of this embodiment comprises the costimulatory signaling domain of CD79a and CD40. The costimulatory signaling domain of CD79a and CD40 of this embodiment can be selected from the intracellular domain of CD79a (nucleotide sequence Genebank: NM_001783: SEQ ID NO: 27; amino acid sequence NCBI Seq: NP_017774.1: SEQ ID NO: 28) or CD40 (nucleotide sequence Genbank: NM_001250: SEQ ID NO: 29; amino acid sequence NCBI Seq: NP_001241.1: SEQ ID NO: 30). As the intracellular domain of CD79a, for example, amino acid numbers 166 to 226 of the full-length amino acid sequence of CD79a can be used. As the intracellular domain of CD40, for example, amino acid numbers 216 to 277 of the full-length amino acid sequence of CD40 can be used. The costimulatory signaling domain of the CAR of this embodiment may contain these sequences or may have around several amino acid residues therein, and may also have homology to these sequences in the range of 70% or more but less than 100%.

[0036] (Other Costimulatory Signaling Domains) The costimulatory signaling domain may comprise other sequences and is not particularly limited as long as it is an intracellular domain involved in costimulatory signaling of a costimulatory molecule. Examples of costimulatory molecules include CD2, CD4, CD5, CD8α, CD8β, CD28, OXO40 (CD134), 4-1BB (CD137), ICOS, CD154, and the like. The CAR of this embodiment may comprise the costimulatory signaling domain of these costimulatory molecules, or may consist of the costimulatory signaling domain of these costimulatory molecules. Preferred examples of the costimulatory signaling domain comprised in the CAR of this embodiment include the costimulatory signaling domains of 4-1BB, CD28, and OXO40, and a more preferred example is the costimulatory signaling domain of 4-1BB. An example of the amino acid sequence of the 4-1BB costimulatory signaling domain includes, but is not limited to, the amino acid sequence from positions 214 to 255 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 10.

[0037] The costimulatory signaling domain may also be a mutant of a costimulatory signaling domain derived from a naturally occurring protein as described above. Examples of mutant costimulatory signaling domains derived from naturally occurring proteins include the polypeptides described in any of (i) to (iii) below. (i) a polypeptide having about 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 mutations compared to the amino acid sequence of a costimulatory signaling domain derived from a naturally occurring protein, and having costimulatory signaling ability (the mutations may be deletions, substitutions, insertions, or additions, or a combination thereof); (ii) a polypeptide having costimulatory signaling ability, comprising an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity (homology) with the amino acid sequence of a costimulatory signaling domain derived from a naturally occurring protein; and (iii) a polypeptide having costimulatory signaling ability, consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of a costimulatory signaling domain derived from a naturally occurring protein (the "multiple" refers to 2 to 30, preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5).

[0038] The number of costimulatory signaling domains that the CAR of this embodiment can comprise is not limited to the two, CD79a and CD40, and the CAR may comprise additional costimulatory signaling domains. The CAR of this embodiment may further comprise, for example, 1 to 5, 1 to 3, or 1 or 2 costimulatory signaling domains. When the CAR of this embodiment comprises multiple costimulatory signaling domains, the multiple costimulatory signaling domains may be the same or different. For example, the CAR may comprise a costimulatory signaling domain of another costimulatory molecule in addition to the costimulatory signaling domain of 4-1BB.

[0039] <Activation Signaling Domain> The CAR of this embodiment comprises at least one activation signaling domain. When an MHC-peptide complex binds to the TCR of a T cell, a signal is transmitted intracellularly via the TCR-CD3 complex, inducing various phosphorylation signals (primary signaling). As used herein, the term "activation signaling domain" refers to a domain of a protein involved in the primary signaling induced by the binding of an antigen peptide to the TCR, as described above, that is involved in the signaling.

[0040] The activation signaling domain that can be used in the CAR of this embodiment is not particularly limited, as long as it is an activation signaling domain of a protein involved in primary signaling. It is known that primary signaling is involved in immunoreceptor tyrosine-based activation motif (ITAM). Therefore, proteins having an ITAM are suitable examples of proteins from which activation signaling domains are derived. Examples of proteins having an ITAM include CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Activation signaling domains containing ITAM of these proteins are suitable examples of activation signaling domains used in the CAR of this embodiment. The activation signaling domain contained in the CAR of this embodiment may include or consist of the activation signaling domain of these proteins.

[0041] Among the above, a more preferred example is an activation signaling domain containing ITAM of CD3ζ. An example of the amino acid sequence of the activation signaling domain of CD3ζ includes, but is not limited to, the amino acid sequence of 52 to 163 amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 12.

[0042] The activation signaling domain may also be a mutant of the activation signaling domain derived from the above-mentioned naturally occurring protein. Examples of mutant activation signaling domains derived from naturally occurring proteins include the polypeptides described in any of (i) to (iii) below. (i) a polypeptide having approximately 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 mutations compared to the amino acid sequence of an activation signaling domain derived from a naturally occurring protein, and having primary signaling ability (the mutations may be deletions, substitutions, insertions, or additions, or a combination thereof); (ii) a polypeptide having primary signaling ability, comprising an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity (homology) with the amino acid sequence of an activation signaling domain derived from a naturally occurring protein; and (iii) a polypeptide having primary signaling ability, consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of an activation signaling domain derived from a naturally occurring protein (the "multiple" refers to 2 to 30, preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5).

[0043] The activation signaling domain may also be artificially designed to contain one or more naturally occurring ITAMs or modified ITAMs derived from naturally occurring ITAMs. The number of ITAMs is not particularly limited as long as the activation signaling domain has primary signaling ability, but may be, for example, about 1 to 5 or about 1 to 3.

[0044] Furthermore, the number of activation signaling domains contained in the CAR of this embodiment is not limited to one, and the CAR may contain multiple activation signaling domains. In this case, the multiple activation signaling domains contained in the CAR may be the same or different. For example, the CAR may contain an activation signaling domain derived from another protein in addition to the activation signaling domain of CD3ζ. The CAR of this embodiment may contain, for example, 1 to 5, 1 to 3, or 1 or 2 activation signaling domains.

[0045] <Other Domains> The CAR of this embodiment may comprise other domains in addition to the immunoglobulin domain of NKp44, the extracellular hinge domain of NKp44, the transmembrane domain, the costimulatory signaling domain, and the activation signaling domain. Examples of other domains include, but are not limited to, a signal peptide.

[0046] (Signal Peptide) In one aspect, the CAR of this embodiment may comprise a signal peptide. A signal peptide is a peptide that directs the localization of a membrane protein or a secretory protein. A signal peptide is generally a peptide consisting of about 5 to 60 amino acids present at the N-terminus of a membrane protein, and is removed in a mature protein that has completed localization.

[0047] The signal peptide usable in the CAR of this embodiment is preferably a signal peptide that directs localization to the cell membrane, and is preferably a signal peptide of a membrane protein. Examples of signal peptides include signal peptides of the α chain and β chain of the T cell receptor, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, NKp30, NKp44, and NKp46. A preferred example is the signal peptide of NKp44. An example of the amino acid sequence of the NKp44 signal peptide is the amino acid sequence from the 1st to 21st amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 4, but is not limited to this.

[0048] <Overall structure of CAR> In the CAR of this embodiment, the NKp44 immunoglobulin domain, the NKp44 extracellular hinge domain, the transmembrane domain, and the activation signaling domain are arranged in the above order from the N-terminus. These domains may be linked to each other directly or via other domains, spacer sequences, etc.

[0049] The costimulatory signaling domain is positioned between the transmembrane domain and the activation signaling domain or on the C-terminal side of the activation signaling domain. When the CAR of this embodiment comprises a plurality of either or both of the activation signaling domain and the costimulatory signaling domain, these domains can be positioned in any order on the C-terminal side of the transmembrane domain. Preferably, the costimulatory signaling domain is positioned between the transmembrane domain and the activation signaling domain.

[0050] When the CAR of this embodiment contains a signal peptide, the signal peptide is located at the N-terminus of the CAR.

[0051] In a preferred aspect, the CAR of this embodiment has a signal peptide, an NKp44 immunoglobulin domain, an extracellular hinge domain, a transmembrane domain, a costimulatory signaling domain, and an activation signaling domain, arranged in the above order from the N-terminus. When the CAR matures and translocates to the cell membrane, the signal peptide is cleaved. Therefore, in another preferred aspect, the CAR of this embodiment has an NKp44 immunoglobulin domain, an extracellular hinge domain, a transmembrane domain, a costimulatory signaling domain, and an activation signaling domain, arranged in the above order from the N-terminus.

[0052] Preferred examples of the CAR of this embodiment include, but are not limited to, structures shown in either (a) or (b) below. The order of the domains in (a) and (b) corresponds to the order of arrangement of the domains from the N-terminus. (a) A CAR comprising an immunoglobulin domain of NKp44, an extracellular hinge domain of NKp44, a transmembrane domain of CD8α, costimulatory signaling domains of CD79a and CD40, and an activating signaling domain of CD3ζ. (b) A CAR comprising an immunoglobulin domain of NKp44, an extracellular hinge domain of NKp44, a transmembrane domain of CD28, costimulatory signaling domains of CD79a and CD40, and an activating signaling domain of CD3ζ.

[0053] An example of the amino acid sequence of the CAR (a) is the amino acid sequence of SEQ ID NO: 1. A CAR comprising the amino acid sequence of SEQ ID NO: 1 is a suitable example of the CAR of this embodiment. Furthermore, a CAR consisting of the amino acid sequence of SEQ ID NO: 1 is a more suitable example of the CAR of this embodiment. In the amino acid sequence of SEQ ID NO: 1, the sequence of amino acids 1 to 21 from the N-terminus is a signal peptide, which is removed during localization to the cell membrane. Therefore, the CAR of this embodiment may be a polypeptide consisting of or containing the amino acid sequence of amino acids 22 to 130 from the N-terminus of the amino acid sequence of SEQ ID NO: 1.

[0054] An example of the amino acid sequence of the CAR of (b) is the amino acid sequence of SEQ ID NO: 2. A CAR comprising the amino acid sequence of SEQ ID NO: 2 is a suitable example of the CAR of this embodiment. Furthermore, a CAR consisting of the amino acid sequence of SEQ ID NO: 2 is a more suitable example of the CAR of this embodiment. In the amino acid sequence of SEQ ID NO: 2, the sequence of amino acids 1 to 21 from the N-terminus is a signal peptide, which is removed during localization to the cell membrane. Therefore, the CAR of this embodiment may be a polypeptide consisting of or including the amino acid sequence of amino acids 22 to 130 from the N-terminus of the amino acid sequence of SEQ ID NO: 2.

[0055] [Polynucleotide Encoding Chimeric Antigen Receptor] This embodiment provides a polynucleotide encoding the CAR of the above embodiment.

[0056] The polynucleotide of this embodiment comprises a nucleotide sequence encoding an NKp44 immunoglobulin domain, a nucleotide sequence encoding a transmembrane domain, and a nucleotide sequence encoding an activation signaling domain. In addition to these nucleotide sequences, it may optionally comprise a nucleotide sequence encoding an extracellular hinge domain, a nucleotide sequence encoding a costimulatory signaling domain, a nucleotide sequence encoding a signal peptide, etc. The polynucleotide of this embodiment preferably comprises a nucleotide sequence encoding an NKp44 immunoglobulin domain, a nucleotide sequence encoding an extracellular hinge domain, a nucleotide sequence encoding a transmembrane domain, a nucleotide sequence encoding a costimulatory signaling domain, and a nucleotide sequence encoding an activation signaling domain.

[0057] The proteins from which each of the above domains is derived are as described above in the section [Chimeric Antigen Receptor], and known amino acid sequences of these domains can be used without particular limitation. Furthermore, the amino acid sequences of each of the above domains can also be obtained from sequence databases such as GenBank and DDBJ. Furthermore, known nucleotide sequences encoding each of the above domains can also be used, and those registered in sequence databases such as GenBank and DDBJ can also be used.

[0058] Furthermore, the base sequence encoding each of the above domains is not limited to known ones, and any base sequence encoding each of the above domains can be used. Due to the degeneracy of the genetic code, there are multiple codons corresponding to one amino acid. Therefore, there are many base sequences encoding the same amino acid sequence. The base sequence of the polynucleotide of this embodiment may be any of multiple base sequences resulting from the degeneracy of the genetic code, as long as it encodes the CAR of the above embodiment.

[0059] In the polynucleotide of this embodiment, an example of a base sequence encoding the immunoglobulin domain of NKp44 is a base sequence encoding the amino acid sequence from positions 22 to 130 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 4. Furthermore, when the transmembrane domain of any of NKp44, CD8α, and CD28 is used as the transmembrane domain, examples of base sequences encoding these domains include a base sequence encoding the amino acid sequence from positions 191 to 213 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 4, a base sequence encoding the amino acid sequence from positions 181 to 206 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 6, and a base sequence encoding the amino acid sequence from positions 153 to 179 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 8. Furthermore, when the activation signaling domain of CD3ζ is used as the activation signaling domain, an example of a base sequence encoding this domain is a base sequence encoding the amino acid sequence from positions 52 to 163 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 12.

[0060] Furthermore, when the polynucleotide of this embodiment comprises a nucleotide sequence encoding an extracellular hinge domain and the extracellular hinge domain of any one of NKp44, CD8α, and CD28 is used as the domain, examples of the nucleotide sequences encoding this domain include a nucleotide sequence encoding the amino acid sequence from positions 131 to 190 of the amino acid sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding the amino acid sequence from positions 128 to 182 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 6, and a nucleotide sequence encoding the amino acid sequence from positions 113 to 152 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 8. Furthermore, when the polynucleotide of this embodiment comprises a costimulatory signaling domain and the costimulatory signaling domains of CD79a and CD40 are used as the domain, examples of the nucleotide sequences encoding the domain include a nucleotide sequence encoding the amino acid sequence from positions 22 to 130 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 1 and a nucleotide sequence encoding the amino acid sequence from positions 22 to 130 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 2.

[0061] Furthermore, when the polynucleotide of this embodiment contains a signal peptide and the signal peptide is that of NKp44, an example of a base sequence encoding the signal peptide is a base sequence encoding the 1st to 21st amino acids from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 4.

[0062] Among the polynucleotides encoding each of the above domains, the polynucleotide encoding the immunoglobulin domain of NKp44 can be obtained by amplifying DNA extracted from NK cells as a template using PCR or the like. Alternatively, it can be obtained by synthesizing cDNA by reverse transcription using RNA extracted from NK cells as a template, and then amplifying the synthesized cDNA as a template using PCR or the like.

[0063] Furthermore, a polynucleotide encoding a transmembrane domain or a polynucleotide encoding an activation signal transduction domain can be obtained by amplifying DNA extracted from T cells as a template using PCR or the like. Alternatively, the polynucleotide can be obtained by synthesizing cDNA by reverse transcription using RNA extracted from T cells as a template, and then amplifying the synthesized cDNA as a template using PCR or the like.

[0064] A polynucleotide encoding an extracellular hinge domain or a polynucleotide encoding a costimulatory signaling domain can also be obtained by amplifying DNA extracted from T cells as a template using PCR or the like. Alternatively, the polynucleotide can be obtained by synthesizing cDNA by reverse transcription using RNA extracted from T cells as a template, and then amplifying the synthesized cDNA as a template using PCR or the like.

[0065] A polynucleotide encoding a signal peptide can also be obtained from DNA or RNA extracted from NK cells, T cells, etc. When a short signal peptide is used, it may be synthesized by a known nucleic acid synthesis method such as the phosphoramidite method.

[0066] Primers for amplifying polynucleotides encoding each of the above domains can be designed from the known gene sequences of each domain. Examples of primers include, but are not limited to, those described in the Examples section of this specification.

[0067] When the polynucleotide of this embodiment contains a base sequence encoding a domain other than those described above, the polynucleotide encoding the domain of interest can be obtained by extracting DNA or RNA from cells expressing the domain of interest, designing appropriate primers, and amplifying the DNA or RNA using PCR or the like. The polynucleotide encoding each domain obtained as described above may be modified by substitution, deletion, addition, or the like, as long as the function of each domain after translation is not lost.

[0068] The polynucleotide of this embodiment can be obtained by linking the polynucleotides encoding each domain obtained as described above, directly or via a spacer. Preferably, the polynucleotides encoding each domain are linked directly without an intervening sequence such as a spacer. Linking of the polynucleotides encoding each domain can be performed using a known method such as overlap extension PCR.

[0069] A suitable example of the polynucleotide of this embodiment is a polynucleotide comprising a base sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and more preferably a polynucleotide consisting of a base sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0070] The polynucleotide of this embodiment may include, in addition to the base sequence encoding the CAR of the above embodiment, a regulatory sequence such as a promoter, an enhancer, or a poly A addition signal, a base sequence encoding another protein, and the like.

[0071] [Vector Comprising a Polynucleotide Encoding a Chimeric Antigen Receptor] This embodiment provides a vector comprising the polynucleotide of the above embodiment.

[0072] In this embodiment, the type of vector is not particularly limited, and commonly used expression vectors, etc. Examples of vectors include viral vectors, plasmid vectors, episomal vectors, and artificial chromosome vectors.

[0073] Examples of viral vectors include Sendai virus vectors, retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors.

[0074] Examples of the plasmid vector include plasmid vectors for expression in animal cells, such as pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0075] An episomal vector is a vector capable of autonomous replication outside of a chromosome. Specific means for using an episomal vector are known (Yu et al., Science, 324, 797-801 (2009)). Examples of episomal vectors include vectors that contain sequences necessary for autonomous replication derived from EBV, SV40, etc. as vector elements. Specific examples of vector elements necessary for autonomous replication include a replication origin and a gene encoding a protein that binds to the replication origin and controls replication. For example, in the case of EBV, the replication origin oriP and the EBNA-1 gene, and in the case of SV40, the replication origin ori and the SV40LT gene are mentioned.

[0076] Examples of artificial chromosome vectors include YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, and PAC (P1-derived artificial chromosome) vectors.

[0077] The vector of this embodiment may include, in addition to the base sequence encoding the CAR of the above embodiment, a control sequence such as a promoter, an enhancer, or a poly A addition signal, a replication origin, a base sequence encoding a protein that binds to the replication origin and controls replication, a base sequence encoding another protein, etc.

[0078] The base sequence encoding the CAR of the above embodiment is preferably placed in a vector so that it is expressed under the control of an appropriate promoter. Examples of promoters include the SRα promoter, SV40 early promoter, retroviral LTR, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF1α promoter, metallothionein promoter, and heat shock promoter. An enhancer of the IE gene of human CMV may also be used together with the promoter. An example is the CAG promoter (containing a cytomegalovirus enhancer, chicken β-actin promoter, and a poly(A) signal site of the β-globin gene).

[0079] Furthermore, examples of the base sequence encoding the other protein include marker genes such as a fluorescent protein gene, a reporter gene, and a drug resistance gene, and genes encoding molecules involved in T cell activation such as cytokines. Furthermore, the base sequence encoding the other protein may be a base sequence encoding a suicide gene such as thymidine kinase, in order to appropriately induce apoptosis of the administered CAR-expressing cells in the body of a cancer patient or the like to which the CAR-expressing cells have been administered.

[0080] The vector of this embodiment can be obtained by inserting a polynucleotide encoding the CAR of the above embodiment into a vector having an appropriate base sequence such as a promoter. The polynucleotide encoding the CAR is preferably inserted at the 3' end of the promoter sequence. A commercially available expression vector or the like may be used to create the vector of this embodiment.

[0081] [Cells expressing chimeric antigen receptors] This embodiment provides cells expressing the CAR of the above embodiment.

[0082] The cell of this embodiment can be obtained by introducing the polynucleotide or vector of the above embodiment into a cell. Accordingly, in one aspect, the present invention provides a method for producing a cell that expresses a CAR, the method comprising the step of introducing the polynucleotide or vector of the above embodiment into a cell.

[0083] In this embodiment, the cells are preferably mammalian-derived cells. For example, human-derived cells or cells derived from non-human mammals such as mice, rats, cattle, sheep, horses, dogs, pigs, and monkeys can be used. The type of cells is not particularly limited, and cells collected from body fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, tumors, cancerous ascites, etc. can be used. Preferred examples include immune cells, such as peripheral blood mononuclear cells isolated from peripheral blood. Particularly preferred cells among peripheral blood mononuclear cells include T cells, NK cells, and their precursor cells. The type of T cells is not particularly limited, and may be any T cell, such as αβ T cells, γδ T cells, CD8-positive T cells, CD4-positive T cells, tumor-infiltrating T cells, or natural killer cells (e.g., natural killer T cells).

[0084] Methods for introducing the polynucleotide or vector of the above embodiment into cells include lipofection, microinjection, calcium phosphate method, DEAE-dextran method, electroporation, particle gun method, and the like.

[0085] Furthermore, for example, when the vector of the above embodiment is a retroviral vector, appropriate packaging cells may be selected based on the LTR sequence and packaging signal sequence possessed by the vector, and used to prepare retroviral particles. Examples of packaging cells include PG13, PA317, GP+E-86, GP+envAm-12, and Psi-Crip. Also, 293 cells and 293T cells, which have high transfection efficiency, can be used as packaging cells. Various retroviral vectors and packaging cells that can be used to package these vectors are widely available commercially, and these commercially available products may be used.

[0086] When introducing the polynucleotide or vector of the above embodiment into cells, a functional substance that improves the introduction efficiency can also be used. Examples of such functional substances include substances that have the activity of binding to viral vectors, such as fibronectin and fibronectin fragments. For example, when introducing a retroviral vector, RetroNectin (registered trademark), a fibronectin fragment with a heparin-binding site, can be preferably used. These functional substances can be used in a state immobilized on an appropriate solid phase (e.g., a plate, a Petri dish, a conical tube, a microtube, etc.) or a carrier (e.g., microbeads, etc.).

[0087] After the polynucleotide or vector of the above embodiment is introduced into a cell, the expression of CAR in the cell can be confirmed by a known method such as flow cytometry, RT-PCR, Northern blotting, Western blotting, ELISA, or fluorescent immunostaining.

[0088] The cell of this embodiment expresses the CAR of the above embodiment on its cell membrane. When an NKp44 ligand binds to the NKp44 immunoglobulin domain of the CAR, the cell of this embodiment is activated, resulting in the release of cytotoxic granules and the production of cytokines. These cytotoxic granules and cytokines destroy the NKp44 ligand-expressing cells.

[0089] [Pharmaceutical composition comprising chimeric antigen receptor-expressing cells] This embodiment provides a pharmaceutical composition comprising the cells of the above embodiment.

[0090] The cells of the above embodiments exhibit specific cytotoxicity against NKp44 ligand-expressing cells. Therefore, the cells of the above embodiments can be used to treat or prevent diseases involving NKp44 ligand-expressing cells. Because NKp44 ligands are expressed in a wide range of tumor cells, including hematopoietic tumors, pharmaceutical compositions containing the cells of the above embodiments can be provided as pharmaceutical compositions for treating or preventing tumors. More specifically, the pharmaceutical composition of the present embodiment is suitable as a pharmaceutical composition for treating or preventing tumors that express an NKp44 ligand. For example, lung cancer, malignant melanoma, cervical cancer, colon cancer, B-cell lymphoma, T-cell leukemia, pancreatic cancer, breast cancer, neuroblastoma, liver cancer including hepatocellular carcinoma, chronic myeloid leukemia, acute myeloid leukemia, and the like have been reported to express NKp44 ligands (Byrd A, et al. Plos One 2007, 2(12): e1339; Baychelier F, et al. Blood 2013, 122(17): 2935-2942). Therefore, the pharmaceutical composition of this embodiment can be suitably used for treating or preventing these tumors. Note that the pharmaceutical composition of this embodiment is not limited to the tumors described above, and can be applied to any tumor that expresses an NKp44 ligand. Whether or not a tumor expresses an NKp44 ligand can be confirmed by performing known methods such as flow cytometry, Western blotting, ELISA, and fluorescent immunostaining using an anti-NKp44 ligand antibody, a fusion protein of the NKp44 ligand binding domain and an immunoglobulin constant domain, or the like.

[0091] The pharmaceutical composition of this embodiment may contain, in addition to the cells of the above embodiment, other components such as a pharmaceutically acceptable carrier. Examples of other components include, but are not limited to, a pharmaceutically acceptable carrier, a T cell activating factor such as a cytokine, an immunostimulant, an immune checkpoint inhibitor, cells expressing other CARs, and an anti-inflammatory agent. Examples of pharmaceutically acceptable carriers include cell culture media, physiological saline, phosphate buffer, and citrate buffer.

[0092] The pharmaceutical composition of this embodiment can be administered to a patient preferably by injection or infusion. The administration route is preferably intravenous administration, but is not limited thereto, and administration may also be by intratumoral injection, etc.

[0093] The pharmaceutical composition of this embodiment may contain a therapeutically effective amount of the cells of the above embodiment. The dosage and administration interval may be appropriately selected depending on the age, sex, weight, etc. of the subject, the type, progression, symptoms, etc. of the disease, and the administration method, etc. The dosage may be a therapeutically effective amount, and for example, 1 x 10 cells may be administered in one administration. ３ ~1x10 １０ pieces / kg (body weight), preferably 1 x 10 ４ ~1x10 ９ pieces / kg (body weight), more preferably 1 x 10 ５ ~1x10 ８ It can be expressed as pieces / kg (body weight).

[0094] The administration interval of the pharmaceutical composition of this embodiment can be, for example, once a week, every 10 to 30 days, once a month, every 3 to 6 months, or once a year. Furthermore, since the cells of the above embodiment can grow autonomously in the body of the administration subject, they can be administered only once. Furthermore, the number of CAR-expressing cells in the body may be monitored after administration, and the administration timing may be determined based on the results.

[0095] In another aspect, the present invention provides use of the cells of the above embodiments in the manufacture of a pharmaceutical composition for treating or preventing tumors. In another aspect, the present invention provides a method for treating or preventing tumors, comprising administering the cells of the above embodiments to a subject. In another aspect, the present invention provides the cells of the above embodiments for treating or preventing tumors.

[0096] <Effects of this embodiment> According to this embodiment, it is possible to provide a chimeric antigen receptor, a polynucleotide, a vector, a cell, a method for producing a cell, and a pharmaceutical composition that can recognize a wide range of cancer cells and be used for treatment, have high functionality, and are effective in treating many types of intractable cancers.

[0097] The present inventors have found that when the NKp44 CAR gene is introduced into human lymphocytes (T cells or NK cells) using a method such as a retroviral vector, the corresponding artificial receptor (NKp44 CAR) is expressed on the lymphocyte surface. These lymphocytes (T cells or NK cells) can antigen-specifically recognize cancer cells bearing an NKp44 ligand, activate the cells, and kill the cancer cells. NKp44 ligands have been reported to be expressed in a wide range of cancer cells, including hematopoietic tumors, and therefore have a wide range of applications. The novel CAR gene discovered by the present inventors that targets the NKp44 ligand can contribute to the realization of CAR-T therapy, which can recognize and attack a wide variety of cancer cells, or CAR gene-introduced NK cell therapy (CAR-NK therapy).

[0098] The inventors have now investigated the structure and effects of CAR genes and found that a CAR having an immunoglobulin domain and an extracellular hinge domain of NKp44, and a costimulatory signaling domain region of CD79a and CD40 as a costimulatory signaling domain, exhibits high activity. In conventional techniques, effective sequences have been found by using sequences other than NKp44, such as CD8α, as the extracellular hinge domain. However, the inventors have now found that when a costimulatory signaling domain region of CD79a and CD40 is present, a wild-type NKp44 or a structure having a certain length or longer is effective. The ability to use a wild-type, or so-called natural form, of NKp44 is also thought to affect the range of applicability of CARs.

[0099] The newly constructed CAR gene with a novel structure further improved CAR-T cell function, suggesting its potential for use in treating various types of intractable human cancers.

[0100] <Other Embodiments> The chimeric antigen receptor, polynucleotide, vector, cell, method for producing a cell, and pharmaceutical composition of this embodiment can be used in methods for treating cancer-related diseases. The chimeric antigen receptor, polynucleotide, vector, cell, method for producing a cell, and pharmaceutical composition of this embodiment can be used for producing a pharmaceutical composition for treating cancer-related diseases.

[0101] Examples are shown below, but the present invention is not limited to these examples.

[0102] [Experimental Example 1] Preparation of NKp44-based CAR gene The gene sequences in the Examples and Comparative Examples were prepared by cloning as described below or by direct synthesis.

[0103] (Direct Synthesis Method) Gene constructs were created by direct synthesis (chemical synthesis and PCR) using gene sequences encoding the amino acid sequences of Example 1 (SEQ ID NO: 1) and Example 2 (SEQ ID NO: 2) outsourced to a nucleotide synthesis company. In Examples 1 and 2, elements from NKp44 were used for the extracellular hinge domain, elements derived from CD8α and CD28 were used for the transmembrane domain, and elements derived from CD79a and CD40 were used for the costimulatory signaling domain. Elements derived from CD3ζ were used for the activation signaling domain. For Comparative Example 2, the extracellular hinge domain of SEQ ID NO: 1 (amino acid sequence from 131 to 190 from the N-terminus of SEQ ID NO: 1) was replaced with the extracellular hinge domain of CD8α (amino acid sequence from 182 to 206 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 6), and synthesis was similarly performed by direct synthesis. In Comparative Example 3, a structure in which the extracellular hinge domain of SEQ ID NO: 1 was shortened, i.e., the extracellular hinge domain of SEQ ID NO: 1 was changed to a fragment (15 amino acids) of the hinge region of an immunoglobulin heavy chain, and synthesis was similarly performed by the direct synthesis method.

[0104] (Cloning of NKp44 gene) The sequence of Comparative Example 1 (conventional example) was created by cloning. In all CARs created in Comparative Example 1, an immunoglobulin domain, which is the ligand binding site of NKp44, was used as the antigen recognition site. Note that, hereinafter, a CAR using the ligand binding site of NKp44 as the antigen recognition site is referred to as an "NKp44-based CAR." Cloning of the immunoglobulin domain of NKp44 was performed as follows. With informed consent, 10 mL of peripheral blood was collected from a healthy donor, and mononuclear cells were isolated by Ficoll density gradient centrifugation. The isolated mononuclear cells were co-cultured with feeder cells (K562-mb15-41BBL) for one week to amplify natural killer (NK) cells. RNA was extracted from the amplified NK cells using NucleoSpin™ RNA (TaKaRa). Complementary DNA (cDNA) was synthesized from the extracted RNA by reverse transcription using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen). Using this cDNA as a template, wild-type NKp44 gene isoform 1 (NCBI Reference Sequence: protein: NP_004819.2 (SEQ ID NO: 4), mRNA: NM_004828.3 (SEQ ID NO: 3)) was cloned by polymerase chain reaction (PCR) using PrimeSTAR® HS DNA Polymerase (TaKaRa) as the enzyme. Three isoforms of NKp44 are known, and the IDs of the other isoforms are as follows: isoform 2: NP_001186438.1, NM_001199509.1; isoform 3: NP_001186439.1, NM_001199510.1.

[0105] (Cloning of each element sequence of CAR other than antigen recognition site) Elements constituting CAR include an antigen recognition domain (immunoglobulin domain, IG), as well as an extracellular hinge domain (EH), a transmembrane domain (TM), a costimulatory signaling domain (CS), and an activation signaling domain (AS). In a comparative example, an extracellular hinge domain derived from CD8α and a costimulatory signaling domain derived from 4-1BB were also used.

[0106] RNA was extracted from human peripheral blood T cells stimulated with phytohemagglutinin (PHA), and cDNA was obtained by reverse transcription. Using the resulting cDNA as a template, the sequences of the extracellular hinge domain of the wild-type NKp44 gene isoform 1 and the extracellular hinge domain and transmembrane domain of CD8α (NP_001759.3: SEQ ID NO: 6, NM_001768.6: SEQ ID NO: 5) were cloned by PCR. Similarly, the sequences of the costimulatory signaling domain or activation signaling domain of 4-1BB (NP_001552.2: SEQ ID NO: 10, NM_001561.5: SEQ ID NO: 9) and CD3ζ (NP_000725.1: SEQ ID NO: 12, NM_000734.3: SEQ ID NO: 11) were cloned. The primers used for the costimulatory signaling domain (CS) of 4-1BB were SEQ ID NOs: 23 and 24, and the primers used for the activation signaling domain (AS) of CD3ζ were SEQ ID NOs: 25 and 26.

[0107] The sequences of the extracellular hinge domain and transmembrane domain of CD28 (NP_006130.1: SEQ ID NO: 8, NM_006139.3: SEQ ID NO: 7) were cloned by PCR using RNA extracted from the T-cell leukemia cell line Jurkat and cDNA obtained by reverse transcription as a template. RNA extraction, reverse transcription, and PCR were performed in the same manner as described above in "(Cloning of the NKp44 gene)." The sequences of the primers used for cloning each element are shown in Table 1. The position of each cloned element in the full-length amino acid sequence is shown in Table 2.

[0108]

[0109]

[0110] (Construction of CAR gene constructs) Multiple types of NKp44-based CAR genes with different combinations of extracellular hinge domains, transmembrane domains, costimulatory signaling domains, and activation signaling domains were created by splicing together the CAR elements without inserting any extra nucleotides between them using overlap extension-PCR. Because the sequences of the elements do not contain any extra nucleotides between them, the CARs synthesized from the created CAR genes do not contain any extra amino acids between the elements.

[0111] A CAR without a costimulatory signaling domain is called a first-generation CAR, while one with both a costimulatory signaling domain and an activating signaling domain is called a second-generation CAR. A first-generation CAR was first created using the NKp44-based CAR gene, followed by a second-generation CAR. A CAR (truncated CAR) that has the immunoglobulin domain of NKp44 in its antigen-recognition domain but does not have the activating signaling domain was also created as a control.

[0112] A schematic diagram of the prepared CAR construct is shown in Figure 1. The sequences used in each Example and Comparative Example are shown in Table 3, in the order of immunoglobulin domain 1, extracellular hinge domain 2, transmembrane domain 3, costimulatory signaling domain 4, and activation signaling domain 5 in the diagram. Examples 1 and 2 represent the technology developed by the present inventors. Comparative Example 1 shows a CAR based on a conventional technology (Patent Document 2) previously developed by the present inventors. Comparative Example 2 is similar to the present invention, but uses CD8α as the extracellular hinge domain.

[0113]

[0114] (Preparation of vector for CAR gene transformation) All of the prepared NKp44-based CAR DNAs have a restriction enzyme EcoRI recognition site at the 5' end and an XhoI recognition site at the 3' end. After cleaving the NKp44-based CAR DNA with EcoRI and XhoI, ligation was performed with a Murine stem cell virus (MSCV) retroviral vector. Competent cells were transformed using the ligation product. Escherichia coli colonies formed on LB medium were collected, and the sequences were confirmed by sequence analysis. The collected E. coli were then cultured in a large volume of LB liquid medium to obtain a high-concentration NKp44-based CAR plasmid. The resulting CAR plasmid, viral structural protein plasmid (pEQ-PAM3-E), and envelope plasmid (RDF) were transiently infected into 293T cells by lipofection (3-plasmid system). After 2 to 4 days, the supernatant was collected to prepare a retroviral solution of NKp44-based CAR. The titer of the retroviral solution was determined by measuring the infection efficiency in HeLa cells in the presence of polybrene.

[0115] Experimental Example 2: Introduction of CAR gene into human T cells and human primary NK cells (Introduction of CAR gene into human T cells) Mononuclear cells were separated from peripheral blood of a healthy donor by Ficoll density gradient centrifugation. CD3-positive cells were isolated from the separated mononuclear cells using RosetteSep™ human T Cell (STEMCELL technology). T cells were stimulated for 48 to 72 hours using beads for activating and stimulating T cell proliferation (Dynabeads (registered trademark) Human T-Activator CD3 / CD28, Gibco) and rIL-2. On the other hand, the titered NKp44-based CAR retrovirus solution was added to a 15 mL round-bottom tube onto which RetroNectin (registered trademark) (TaKaRa) had been adsorbed, and the tube was centrifuged at 32°C and 1200 to 1300 g for 120 minutes to adsorb the retrovirus onto the conical tube (RetroNectin-bound virus infection method). ５100 T cells were added to 2-3 mL of medium (RPMI1640, Sigma-Aldrich) containing fetal bovine serum (FBS) at a final concentration of 10%, and the cells were cultured in an incubator at 37°C and 5% CO2 to introduce the NKp44-based CAR gene into the T cells. After CAR gene introduction, 200 U / mL rIL-2 was added every 2-3 days.

[0116] (Transduction of CAR gene into human NK cells) Mononuclear cells isolated from peripheral blood of healthy donors were co-cultured with feeder cells (K562-mb15-41BBL) under administration of rIL-2 to expand NK cells. One week later, T cells were removed using EasySep (registered trademark) CD3 positive selection cocktail (STEMCELL technology), and the CAR gene was then introduced into NK cells by the RetroNectin-bound virus infection method. The gene-transduced NK cells were cultured in RPMI 1640 medium supplemented with FBS at a final concentration of 10%, and 100 U / mL rIL-2 was added every 2 to 3 days.

[0117] (Evaluation of Expression of Transduced CAR Gene) The MSCV retroviral vector used in this study contained a green fluorescent protein (GFP) gene integrated downstream of an internal ribosome entry site (IRES) sequence. The efficiency of CAR gene transduction into T cells was determined by measuring the signal intensity at FL1, the detection range for GFP fluorescence wavelengths, using flow cytometry (BD FACSCalibur™, BD Sciences). One week after CAR gene transduction, NKp44 present on the cell surface of T cells or NK cells was stained with a monoclonal antibody (NKp44-PE, BECKMAN COULTER) and measured by flow cytometry to compare the expression levels of NKp44-based CAR.

[0118] Figure 2 is a graph showing the results of flow cytometry analysis of T cells. Mock indicates cells not transfected with a CAR, and each comparative example and example indicates cells transfected with the respective CAR. In Comparative Example 2, in which CD8α was used for the extracellular hinge domain, the results were almost the same as in Comparative Example 1, which is a conventional technology (described in Patent Document 2), but in Examples 1 and 2, the results showed that cell surface expression of NKp44 was enhanced. In human T cells, the gene transfer efficiency calculated based on GFP expression was 88.2% (n = 3, 77.4 to 98.4%). In both T cells and NK cells, the gene construct that showed the best expression of NKp44-based CAR on the cell surface was [EH(p44)-TM(CD8)-CS(CD79a / 40)-AS(CD3ζ)], a construct using NKp44 in the extracellular hinge domain (EH), CD8 in the transmembrane domain (TM), CD79a / 40 in the costimulatory signaling domain (CS), and CD3ζ in the activating signaling domain (AS). Furthermore, [EH(p44)-TM(CD28)-CS(CD79a / CD40)-AS(CD3ζ)] also showed good expression of NKp44-based CAR on the cell surface. Based on these results, human T cells transfected with these constructs were used in the cytotoxicity evaluation test described below.

[0119] Figure 3 is a graph showing the results of flow cytometry analysis of human NK cells. (a) shows the results of flow cytometry, and (b) shows a quantitative comparison of cell surface expression levels. NK cells obtained from three different healthy adult donors were transfected with the CARs of each Example and Comparative Example, and the NKp44 expression levels on the cell surface were compared. Comparative Example 2 was equivalent to Comparative Example 1, which is a conventional technology, but Examples 1 and 2 showed significantly enhanced expression compared to the conventional type. Furthermore, when comparing the Examples, Example 1 tended to have higher expression.

[0120] [Experimental Example 3] Evaluation of cytotoxicity of CAR-introduced T cells In the cytotoxicity evaluation test, myeloid leukemia cell lines (K562, THP-1) and lymphoma cell line (U937), which have been confirmed to express NKp44 ligand on their surfaces, were used as target cells. The cytotoxicity of T cells (NKp44 CAR-T) transfected with the genes of the above-mentioned Examples and Comparative Examples against these target cells was evaluated one week or later after gene transfection. The target cells were stained with CellTrace™ Calcein Red-Orange AM (Thermofisher), and then NKp44 CAR-T and target cells were incubated at 37°C, 5% CO at various effector-target ratios (E:T ratios). ２ The cells were co-cultured for 6 hours in a 96-well U-bottom plate in an incubator under the conditions of [Illegible]. Wells containing only target cells were cultured for the specified time and used as controls. For comparison, T cells transfected with an empty vector (mock) (mock-T) were used. Furthermore, to confirm the possibility of attacking normal cells of both the self and allogeneic subjects, autologous activated T cells (Auto-T cells) cultured, stimulated, and expanded under administration of rIL-2 were used as the control cells for the cytotoxicity test. Similarly, peripheral T cells from a healthy donor other than the T cell donor were isolated and used as the control cells (Allo-T cells).

[0121] Figure 4 is a graph showing the results of the cytotoxicity evaluation test. The Y axis represents the proportion of residual tumor cells. (a) shows the results for K562 cells, and 8b) shows the results for U937 cells. The ratio at the bottom of the graph represents the E:T ratio. For all target cells, the cytocidal effect of CAR-T cells co-cultured for 18 hours was comparable in Comparative Example 2 to that of Comparative Example 1, which is the conventional technology, but was significantly enhanced in Examples 1 and 2, demonstrating a marked increase in cytotoxicity against each cell line. When comparing the Examples, Example 1 had a greater cytocidal effect.

[0122] [Experimental Example 4] Antitumor effect of CAR-introduced T cells over 72 hours Using K562 as target cells, the antitumor effect (effect of suppressing tumor cell proliferation) of CAR-T cells was investigated by co-culture for 72 hours under the same conditions as in Experimental Example 3, using CAR-T cells derived from two different healthy individuals.

[0123] Figure 5 is a graph showing the results of the antitumor effect at 72 hours. Both are for K562 cells, and (a) and (b) show different donors at the time of producing CAR-T cells. The ratio at the bottom of the figure indicates the E:T ratio. For both donors, Comparative Example 2 had the same effect as Comparative Example 1 of the prior art, but Examples 1 and 2 had significantly enhanced effects. When comparing the Examples, Example 1 had a greater antitumor effect.

[0124] [Experimental Example 5] Evaluation of cytotoxicity of CAR-introduced T cells for one week Using K562 as target cells, the antitumor effect (effect of suppressing tumor cell proliferation) of CAR-T cells was investigated by co-culture for one week (7 days) under the same conditions as in Experimental Example 4.

[0125] Figure 6 is a graph showing the results of the antitumor effect after one week. The ratio at the bottom of the graph indicates the E:T ratio. Compared to Comparative Example 1, which is a conventional technology, Comparative Example 2 had a similar effect, but Examples 1 and 2 showed significantly enhanced effects. When comparing the Examples, Example 1 had a stronger antitumor effect. From the results in Figures 4 to 6, Comparative Example 2, Examples 1, and 2, which have the CD79a / CD40 domain, showed enhanced cytotoxicity against the K562 and U937 cell lines compared to Mock. In particular, when compared to the conventional type, Comparative Example 2 showed equivalent results, but Examples 1 and 2 showed a strong enhancement of cytotoxicity at both culture times.

[0126] Experimental Example 6 Antitumor effect of long-term culture on a cervical cancer cell line (HeLa) Using a cervical cancer cell line (HeLa) as target cells, the antitumor effect (effect of suppressing tumor cell proliferation) of CAR-T cells was investigated by co-culture for 1 week (7 days) under the same conditions as in Experimental Example 4.

[0127] Cytotoxicity against a solid cell tumor line (cervical cancer: HeLa) was examined for Comparative Example 2, Example 1, and Example 2. For comparison, T cells (mock-T) transfected with an empty vector (mock) were used. A certain number of solid tumor cell lines (2 x 10 ４100 μL). NKp44 CAR-T and mock-T cells were added to each solid tumor cell line at an effector-target ratio (E:T ratio) of 1:1. Each well contained 200 μL of medium prepared by adding recombinant human IL-2 (final concentration: 10 U / ml) to RPMI 1640 medium containing 10% bovine serum. Additionally, wells containing only 200 μL of the medium were prepared for blank absorbance measurements. Additionally, wells containing only the solid tumor cell line without CAR-T were prepared as controls for the cytotoxicity test. All conditions were tested in triplicate. After one week of co-culture, 20 μL of Cell Counting Kit-8 (Dojindo Laboratories) was added to each well, and the cells were allowed to react in a CO2 incubator. 120 minutes after addition, the absorbance at 450 nm was measured using a microplate reader. The absorbance at 650 nm was measured as a control wavelength, and the target absorbance was calculated by calculating the difference from the absorbance at 450 nm. For each well, the cell viability was calculated using the following formula (1), and the average value for three wells was calculated.

[0128]

[0129] In the above formula (1), (+)T 450 : Absorbance at 450 nm in wells where solid tumor cell lines and CAR-T were co-cultured (+)T 650 : Absorbance at 650 nm in wells where solid tumor cell lines and CAR-T were co-cultured (-)T 450 : Absorbance at 450 nm in wells where only solid tumor cell lines were cultured (-)T 650 B: Absorbance at 650 nm in wells where only solid tumor cell lines were cultured 450 B: Absorbance at 450 nm in blank well 650 : absorbance at 650 nm in blank wells. The survival rate (%) of tumor cells was shown as "residual tumor (%)."

[0130] 7 is a graph showing the results of the antitumor effect after 1 week. Compared to Comparative Example 1 of the prior art, Comparative Example 2 had an equivalent effect, but Examples 1 and 2 had significantly enhanced effects. When comparing the Examples, Example 1 had a slightly higher antitumor effect. In other words, compared to the mock CAR used as a control, NKp44 CAR-T exhibited potent cytotoxic activity.

[0131] [Experimental Example 7] Structure and cell surface expression of NKp44 CAR having a shortened hinge As Comparative Example 3, a CAR having a structure in which the extracellular hinge domain was shortened, i.e., a fragment (15 amino acids) of the hinge region of an immunoglobulin heavy chain in the extracellular hinge domain, was prepared, and its cell surface expression was investigated. The gene of Comparative Example 3 was prepared by the method described above, and was expressed in T cells from a healthy subject.

[0132] FIG. 8 is a schematic diagram showing the structure of Comparative Example 3 in comparison with the above-mentioned Comparative Example 1 and Example 1. FIG. 9 is a diagram showing the results of cell surface expression. The results of flow cytometry for Comparative Example 1, Example 1, and Comparative Example 3 are shown. The results in the upper part (APC) of FIG. 9 show that the cell surface expression of NKp44 CAR was lower in Comparative Example 3 than in Comparative Example 1, which is the conventional technology. The results in the lower part (area of ​​the Count graph) of FIG. 9 also showed that the cell surface expression of NKp44 CAR was lower in Comparative Example 3 than in Comparative Example 1. That is, compared to the previously reported Construct 2G-d (Comparative Example 1, having 4-1BB in the costimulatory domain) and the newly created Construct B (Example 1), the surface expression and cytotoxic activity were significantly lower. Comparative Example 3 has the CD79a and CD40 costimulatory signaling domains found in Examples 1 and 2, but only has a shortened extracellular hinge domain. From the above results, it is believed that in order to exhibit the activity of Examples 1 and 2, in addition to the CD79a and CD40 costimulatory signaling domains, a sufficiently long sequence of the NKp44 extracellular hinge domain (close to the wild type) is required.

[0133] [Experimental Example 8] Effect of different structures on cytocidal effect of CARs Using a synovial sarcoma cell line (STS-163) as target cells, co-culture was performed for 24 hours under the same conditions as in Experimental Example 4, and the residual tumor was measured to examine the cytocidal effect of CAR-T cells.

[0134] 10 is a graph showing the effect of CARs with different structures on the cytocidal effect. The ratio in parentheses in the figure indicates the E:T ratio. Compared with Comparative Example 1, which is a conventional technique, the cytocidal effect was significantly enhanced in Example 1. On the other hand, the cytocidal effect in Comparative Example 3 tended to be lower than that of Comparative Example 1.

[0135] Experimental Example 9: Efficacy evaluation test of NKp44 CAR-T in NCI-H1975-Luc subcutaneously transplanted mice BRJ mice (BALB / c mice lacking both the Rag2 gene and the Jak3 gene), 18 to 19 weeks old, male, were used. BRJ mice are severely immunodeficient mice with a complete deficiency of T cells, B cells, and NK cells, and a reduced number of NKT cells (J Biomed Biotechnol. 2011; 2011: 539748.). The donor for CAR-T was a 58-year-old male, and the source was peripheral blood.

[0136] A retrovirus for expressing CAR was produced and stored in an ultra-low temperature freezer at −150° C. NKp44 CAR-T was produced using the above retrovirus.

[0137] To generate the luciferase-expressing human lung adenocarcinoma cell line NCI-H1975-Luc, NCI-H1975 was purchased from ATCC and genetically modified to express luciferase and GFP. Cells were sorted by FACS based on GFP fluorescence. After expansion, stocks were prepared and stored in an ultra-low temperature freezer at -150°C. Subcultured cells were used prior to testing.

[0138] The reagents used are as follows. The catalog number and manufacturer are shown in parentheses.RPMI-1640 (R8758-500ML, Sigma-Aldrich) FBS:LOT:19L00G (175012, NICHIREI) RPMI-1640 (R5886, Sigma-Aldrich) Human Serum (H4522, Sigma-Aldrich) L-glutamine 200 mM (G7513, Sigma-Aldrich) 2-mercaptoethanol (21418-42, Nacalai Tesque) Penicillin-Streptomycin Sterile Solution (P4333, Sigma-Aldrich) RPMI-1640: 4,500 mg / L glucose with L-glutamine, phenol red, HEPES, and sodium pyruvate (187-02705, Wako); Penicillin-Streptomycin Mixed Solution, Stabilized (09367-34, Nacalai Tesque); D-PBS (-), hereinafter referred to as PBS (14249-95, Nacalai Tesque); 2.5 g / L trypsin / 1 mmol / L EDTA solution (32777-15, Nacalai Tesque); Trypan Blue Solution. 0.4% (15250061, Gibco) RosetteSep Human T Cell Enrichment Cocktail (ST-15061, STEMCELL Technologies) Dynabeads (TM) Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (11131D, Gibco) Dynabeads (TM) Human T-Expander CD3 / CD28 (11141D, Gibco) Ficoll-Paque PLUS (17144002, GLSTJ) 50 IU / μL recombinant interleukin 2, IL-2 (200-02, PeproTech), Retronectin (T100B, Takara Bio), heparin Na, 5000 units / 5mL (-, Mochida Pharmaceutical Co., Ltd.), D-Luciferin K+ salt (LK10000, OZ Biosciences).

[0139] The equipment and instruments used are as follows. The numbers in parentheses indicate the model or catalog number and manufacturer. Direct heat type CO ２Incubator (SCA-165DS, Astec) Bioclean bench (Airstream Pro, ESCO) Inverted culture microscope (ECLIPS Ts2, Nikon) High-speed refrigerated centrifuge (6000, Kubota Shoji) Universal refrigerated centrifuge (CAX-571, TOMY) Ultra-low temperature freezer, -152°C (MDF-1156ATN-PJ, Panasonic) Biomulticooler (KGT-4010 HC, Nippon Freezer) Incubator (SM-05N, TAITEC) Flow cytometer (BD accuri C6 Plus, Becton & Dickinson) 15ml Conical Centrifuge Tube, Bulk, Pack of 50 (339650, Thermo Fisher Scientific) 50ml Conical Centrifuge Tube, Bulk, Pack of 25 (339652, Thermo Fisher Scientific) 1.5mL Hypermicrotube, Round Bottom, Soft Touch (131-7155CS-N, WATSON) Cell Culture Dish (VTC-D100, VIOLAMO) Falcon® Round Tube, 14mL, Polystyrene, Two-Position, with Cap (352057, Corning) 24-well Sterile, Non-Pyrogenic Cell Culture Plate Polystyrene (TR5002, Trueline) Tissue Culture Test Plate, 6-well (92006, TPP) Terumo syringe 50 mL, slip tip, side opening (SS-50ESZ, TERUMO) Terumo syringe 10 mL, slip tip, center opening (SS-10SZ, TERUMO) Vacuum filtration "rapid"-Filtermax, 0.22 μm (99500, TPP) Hawatch Sterilized PES syringe filter, 0.22 μm, 25 mm diameter (SLPES2522S, Hawatch Scientific) EasySep Magnet (ST-18000, Veritas) IVIS Spectrum (-, PerkinElmer) 1.5 mL light-shielding black microtube (131-915BL, WATSON)

[0140] (Preparation of medium) Medium for NKp44 CAR-T was prepared as follows and stored at 4°C after preparation. RPMI-1640 500 mL FBS (Fatal Bovine Serum) 50 mL, final concentration 10%

[0141] NCI-H1975-Luc medium was prepared as follows and stored at 4°C after preparation: RPMI-1640 (4,500 mg / L Glucose) with L-Glutamine, Phenol Red, HEPES, and Sodium Pyruvate, 500 mL; FBS (Fatal Bovine Serum), 55 mL, final concentration 10%; Penicillin-Streptomycin Mixed Solution (Stabilized), 5 mL, final concentration 1%.

[0142] (Cell Culture) Cryopreserved NCI-H1975-Luc was thawed and seeded. ２ Incubator (37°C, 5% CO ２ For subculture, the cells were washed with PBS and then treated with trypsin for 5 minutes to detach the cells.

[0143] (CAR-T Preparation) Details of CAR-T preparation are shown below. <Day 2> Approximately 40 mL of blood was collected from the donor, and 5 mL of the collected whole blood (approximately 40 mL) was placed in a 15 mL conical tube. 250 μL of RosetteSep (for T cells) was added per tube. The tubes were left to stand at room temperature for 20 minutes in the dark. Approximately 4 mL of Ficoll solution was dispensed into 16 15 mL tubes. The blood mixed with 10 mL of RosetteSep was diluted with 10 mL of PBS. 5 mL of blood diluted with PBS was slowly added to the tube containing the Ficoll solution, taking care not to mix, and the tube was centrifuged at 20°C, 1500 rpm, 30 minutes with Decel off. During this time, RetroNectin-coated tubes were prepared.

[0144] After centrifugation, the buffy coat was placed in a new 15 mL tube and suspended in RPMI (without FBS). The cells were centrifuged at 20°C and 1500 rpm for 5 minutes, the supernatant was decanted, and the cells were resuspended in 2 mL of RPMI + 10% FBS. The cells were then counted. Fifty μL of the solution was dispensed into FCM tubes, and the phenotype was confirmed by flow cytometry (FCM).

[0145] 25 μL / 1 x 10 in a sterilized round tube ６ Dynabeads containing T cells were placed in the wells. Approximately 2 mL of PBS was added, the wells were vortexed, and then placed in a magnetic magnet. After 1 minute, the liquid was aspirated, the wells were removed from the magnet, and the same amount of RPMI + 10% FBS was added. The Dynabeads with the replaced solution were added to 2 mL of T cell solution and pipetted. The resulting solution was placed in a 24-well plate at 1000 μL / well (2 wells were used), to which 1000 μL of RPMI + 10% FBS was added to make a total of 2000 μL / well, and 8 μL of IL-2 (50 IU / μL) was added. The wells were incubated at 37°C.

[0146] <Day -1> Retronectin (Takara: T1008) 2.5 mL was dispensed into 500 μL aliquots and stored at -20°C. Retronectin stock solution (1 mg / mL) was diluted 10-fold (=100 μg / mL). 500 μL of Retronectin was added to a 14 mL round tube in a clean bench, and the tube was left to stand overnight in a refrigerator.

[0147] <Day 0> Retronectin was collected, and 500 μL of PBS + 2% BSA (FBS can be used instead) was added. The tube was left to stand at room temperature for 30 minutes for blocking. The PBS + 2% BSA was removed, and the tube was washed once with PBS and sealed with parafilm. The virus solution was added to the Retronectin tube so that the MOI was > 2, and the tube was centrifuged at 32°C, 2000 rpm, for 120 minutes. After centrifugation, the supernatant was discarded, and an appropriate amount of PBS was added and discarded. 2 x 10 ５ T cells were added and mixed with RPMI (+10% FBS) to make a total volume of 1 mL. 4 μL of IL-2 (50 IU / μL) was added and culture was started at 37°C.

[0148] <Day 2 and after> After 48 to 72 hours, the cells were transferred from the round tube to a 24-well plate, and medium was added so that the total volume was approximately 1 to 2 mL. IL-2 (1 μl / mL) was added once every 2 to 3 days while the culture was continued. Around Day 7, the cells were transferred to a 6-well plate. The gene transfer rate was confirmed on Day 7. The phenotype was measured on Day 14.

[0149] (Subcutaneous transplantation of NCI-H1975-Luc) BRJ mice were housed in a cage with a maximum of five mice, and were housed in an environment set up in a breeding room with a 12-hour lighting schedule (7:00-19:00). During the breeding period, the mice were allowed free access to food and sterilized water. Subcutaneous transplantation of NCI-H1975-Luc was performed on day 14 after NKp44 CAR-T production. At this time, the hair on the backs of the BRJ mice was shaved with clippers.

[0150] NCI-H1975-Luc was collected from the culture flask using trypsin and resuspended in RPMI-1640 medium (without additives such as FBS). The GFP-positive rate was confirmed by FCM, the cell number was counted, and the cells were resuspended in RPMI-1640 medium at 1 × 10 ７ The prepared NCI-H1975-Luc was dispensed into 1.5 mL tubes in 110 μL aliquots and placed on ice.

[0151] BRJ mice were anesthetized by inhaling isoflurane. 110 μL of the dispensed NCI-H1975-Luc was subcutaneously implanted into the right side of the mouse, approximately 5 mm from the spine, using a syringe with a tuberculin needle. ６ The volume of the solution was 100 μL / mouse. The needle was slowly withdrawn while pressing the area around the needle. After subcutaneous transplantation, the mice were housed in cages with a maximum of five mice per cage, while monitoring the state of tumor formation. The conditions of food, lighting, and drinking water were maintained unchanged from before transplantation.

[0152] (Administration of CAR-T to mice) CAR-T on day 21 after preparation was administered to mice. The day before CAR-T administration, tumor diameter and bioluminescence intensity (BLI) were measured using an IVIS (in vivo imaging system), and the mice were divided into groups to minimize variability. CAR-T was centrifuged at 1500 rpm at 4°C for 5 minutes, after which the supernatant was discarded and resuspended in D-PBS(-), and the cell number was counted. CAR-positive cells were 9 x 10 ６ The cells were centrifuged at 1500 rpm at 4°C for 5 minutes and resuspended in 110 µL of PBS. CAR-T was administered intraperitoneally using a syringe with an insulin needle.

[0153] The structures of the CARs used were: (CAR-T designation: B (Example 1)) immunoglobulin-like domain: NKp44 extracellular hinge domain: NKp44 transmembrane domain: CD8 costimulatory domain: composite CD79A / CD40 costimulatory endodomain (CAR-T designation: 2G-d (Comparative Example 1)) immunoglobulin-like domain: NKp44 extracellular hinge domain: CD8 transmembrane domain: CD8 costimulatory domain: 4-1BB.

[0154] The administration group composition is as follows: B CAR-T group: number of administered cells 9 x 10 ６ cells / mouse 3 animals 2G-d CAR-T group: number of administered cells 9 x 10 ６ cells / mouse: 3 mice No treatment (control without administration): 3 mice

[0155] (Weight measurement, tumor diameter measurement, euthanasia) Body weight and tumor volume were measured three times a week (Monday, Wednesday, Friday). Tumor volume was measured by measuring the tumor diameter with a caliper and then calculating the "minor diameter". ２ The tumor volume was calculated using the formula: x long diameter x 1 / 2. ３ Mice exceeding this age were euthanized due to ethical considerations.

[0156] 11 is a graph showing the relationship between tumor volume and days after administration in CAR-T-administered mice. As shown in the figure, the increase in tumor volume in the B CAR-T-administered group mice was smaller than that in the untreated mice and the 2G-d CAR-T-administered group, and one out of three mice reached a tumor volume of 1,500 mm, which was the euthanasia criterion, after 28 days. ３ Of the remaining two, one exceeded 400-700 mm between 12 and 49 days. ３ There was no significant increase in tumor volume from the beginning to the end, and one mouse showed a decrease in tumor volume from the 28th. Furthermore, the increase in tumor volume in the mice in the 2G-d CAR-T administration group was less than in the untreated mice, and the two mice that exceeded the euthanasia criteria reached the criteria on the 35th and 42nd days, confirming a significant life-extension effect compared to the untreated group. On the other hand, the tumor volume in the untreated mice increased, and both mice reached a tumor volume of 1500 mm3 early on. ３ exceeded.

[0157] FIG. 12 is a graph showing the survival rate of CAR-T-administered mice. The survival rate of B CAR-T-administered mice decreased in the late 20s (one mouse was euthanized), but continued to decrease until the 35th day, and two mice survived until the 50th day, when the observation period ended. The survival rate of 2G-d CAR-T-administered mice decreased due to euthanasia on the 35th and 42nd days, but one mouse survived until the end of the observation period. On the other hand, the survival rate of untreated mice decreased from the early 20s onwards, and none of them survived until the 33rd day. These results demonstrated that the increase in tumor volume was suppressed and the survival rate was maintained in CAR-T-administered mice compared to untreated mice. In particular, superior effects were confirmed in mice administered with B CAR-T.

[0158] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims.

[0159] According to the present invention, there are provided chimeric antigen receptors, polynucleotides, vectors, cells, methods for producing cells, and pharmaceutical compositions that can recognize a wide range of cancer cells and be used in therapy, have high functionality, and are effective in treating many types of intractable cancers.

[0160] 1. Immunoglobulin domain 2. Extracellular hinge domain 3. Transmembrane domain 4. Costimulatory signaling domain 5. Activation signaling domain

Claims

1. A chimeric antigen receptor comprising an immunoglobulin domain of NKp44, an extracellular hinge domain of NKp44, a transmembrane domain, a costimulatory signaling domain, and an activation signaling domain, wherein the costimulatory signaling domain comprises the costimulatory signaling domains of CD79a and CD40.

2. The chimeric antigen receptor of claim 1, wherein the activation signaling domain comprises the activation signal domain of CD3ζ.

3. The chimeric antigen receptor of claim 1 or 2, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of NKp44, CD8α, and CD28.

4. A polynucleotide encoding the chimeric antigen receptor of claim 1 or 2.

5. A vector comprising the polynucleotide of claim 4.

6. A cell expressing the chimeric antigen receptor of claim 1 or 2.

7. The cell of claim 6, which is a T cell or a natural killer cell.

8. A method for producing a cell expressing a chimeric antigen receptor, comprising the step of introducing the polynucleotide according to claim 4 into a cell.

9. The method of claim 8, wherein the cells are T cells or natural killer cells.

10. A pharmaceutical composition comprising the cells described in claim 6.

11. A pharmaceutical composition for treating or preventing a tumor, comprising the cells according to claim 6.

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