RU2832189C2 - Antigen receptor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to an antigen receptor containing a GITRL domain on the C-terminal side through a self-splitting peptide domain, as well as to a polynucleotide coding said antigen receptor. Also disclosed is a cell expressing said antigen receptor, wherein the cell is a T-cell with cytotoxic activity or NK-cell, and a pharmaceutical composition.

EFFECT: invention provides creation of antigen receptor with high therapeutic or preventive effect on cancer.

11 cl, 23 dwg, 12 ex

Description

Field of technology

[0001] The present invention relates to an antigen receptor and the like.

State of the art

[0002] In addition to surgery, radiotherapy, and chemotherapy, immunotherapy has attracted attention as a fourth method for treating cancer. Examples of immunotherapy currently available or to be approved in the future for use in Japan include immune checkpoint inhibitors and chimeric antigen receptor (CAR) gene-modified T-cell therapy. CAR T-cell therapy targeting CD19, which is expected to be effective against intractable B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL), has been approved in the United States, Europe, and other countries since 2017, and was approved in Japan in March 2019. Although this therapy has a remarkable effect on intractable cases resistant to conventional treatment including hematopoietic stem cell transplantation, there are many relapsed and refractory cases; Thus, its further improvement is desirable. It is expected that CAR-T cell therapy for solid tumors will be developed in the future.

[0003] In order to enhance the effect of CAR-T cell therapy, studies are being conducted on, for example, combined use with immune checkpoint inhibitors and improving metabolism in the tumor microenvironment. It is noteworthy that CD19 CAR-T cells with the ligand of the costimulatory molecule 4-1BB bound to CD28-CD3ξ as an intracellular domain have high resistance and stronger antitumor effects (Non-patent Document 1).

List of references cited

Non-patent document

[0004] NPL 1: Zhao Z. et al. Cancer Cell, 28(4): 415-428, 2015.

The essence of the invention

Technical task

[0005] The aim of the present invention is to create a new antigen receptor. Preferably, the aim of the present invention is to create an antigen receptor with a high therapeutic or preventive effect on cancer.

Solving a technical problem

[0006] The present inventors have conducted extensive studies in connection with the above-mentioned problem and found that the problem can be solved by using an antigen receptor that contains a GITRL domain at a position closer to the C-terminus through a self-cleaving peptide domain. The present inventors have conducted further studies based on this discovery and completed the present invention. In particular, the present invention includes the following subject matter.

[0007] Item 1. An antigen receptor comprising a GITRL domain at a position closer to the C-terminus via a self-cleaving peptide domain.

[0008] Item 2. The antigen receptor of item 1, comprising a core domain comprising a variable region of an antibody or T-cell receptor, a transmembrane domain and an intracellular domain of the TCR.

[0009] Item 3. The antigen receptor of item 2, comprising a GITRL domain at a position closer to the C-terminus of the core domain via a self-cleaving peptide domain.

[0010] Item 4. The antigen receptor according to item 2 or 3, wherein the core domain further comprises an intracellular costimulator domain.

[0011] Item 5. The antigen receptor of item 4, wherein the costimulator is CD28.

[0012] Item 6. The antigen receptor according to any one of items 1-5, which is a chimeric antigen receptor or a T-cell receptor.

[0013] Item 7. The antigen receptor of any one of items 2-6, wherein the variable region is capable of binding to CD19.

[0014] Item 8. A polynucleotide encoding an antigen receptor according to any one of items 1-7.

[0015] Item 9. A cell containing a polynucleotide according to item 9.

[0016] Item 10. The cell according to item 9, which is an αβ T cell, a γδ T cell, or a NK cell.

[0017] Item 11. A pharmaceutical composition comprising a cell according to item 10.

[0018] Item 12. A pharmaceutical composition according to item 11, intended for use in the treatment or prevention of cancer.

Advantageous effects of the invention

[0019] The present invention relates to a new antigen receptor. Using the antigen receptor, cancer can be effectively treated or prevented.

Brief description of the figures

[0020]

In fig. Figure 1 schematically shows the structure of the 28z CAR.

Fig. 2 shows the nucleotide sequence of CD19 28z CAR.

Fig. 3 shows the nucleotide sequence and amino acid sequence of CD19 28z GITRL CAR.

Fig. 4 shows the results of Experimental Example 3 (CAR expression in αβ cells when the intracellular domain is CD28). The relative number of cells expressing CAR is shown as a percentage. NGMC indicates cells with the unmodified gene (the same applies to other figures).

Fig. 5 shows the results of Experimental Example 3 (CAR expression in γδ cells when the intracellular domain is CD28). The relative number of cells expressing CAR is shown as a percentage.

Fig. 6 shows the results of Experimental Example 3 (CAR expression in αβ cells when the intracellular domain is GITR). The relative number of cells expressing CAR is shown as a percentage.

Fig. 7 shows the results of Experimental Example 3 (CAR expression in γδ cells when the intracellular domain is GITR). The relative number of cells expressing CAR is shown as a percentage.

Fig. 8 shows the results of Experimental Example 4 (IFN-γ expression in αβ cells when the intracellular domain is CD28). The relative number of CAR-expressing cells and the relative number of IFN-γ-producing cells in CAR-expressing cells are shown as percentages.

Fig. 9 shows the results of Experimental Example 4 (IFN-γ expression in γδ cells when the intracellular domain is CD28). The relative number of CAR-expressing cells and the relative number of IFN-γ-producing cells in CAR-expressing cells are shown as percentages.

Fig. 10 shows the results of Experimental Example 4 (IFN-γ expression in αβ cells when the intracellular domain is GITR). The relative number of CAR-expressing cells and the relative number of IFN-γ-producing cells in CAR-expressing cells are shown as percentages.

Fig. 11 shows the results of Experimental Example 4 (IFN-γ expression in γδ cells when the intracellular domain is GITR). The relative number of CAR-expressing cells and the relative number of IFN-γ-producing cells in CAR-expressing cells are shown as percentages.

Fig. 12 shows a summary of the results of Experimental Examples 3 and 4 for αβ cells (CAR expression and IFNγ production).

Fig. 13 shows a summary of the results of Experimental Examples 3 and 4 for γδ cells (CAR expression and IFNγ production).

Figure 14 shows the average ratios of GITRL-bound to GITRL-unbound (GITRL-bound/GITRL-unbound) in the results in the bottom row of each of Figures 12 and 13.

Fig. 15 shows the results of Experimental Example 5 (results for αβ cells). The top row shows the cytotoxicity results analyzed over time using each type of effector cell. The middle row shows the cytotoxicity results after 5, 20, 40, 60, 80, 100, and 120 h. The bottom row shows the time required to damage half of the target cells. In the top and middle rows, the vertical axis represents the cytotoxic activity (%) measured using the xCELLigence assay, and the horizontal axis represents the time since the addition of the effector cells. The bars on the left of each histogram show a treatment in which effector cells were not allowed to function, a treatment in which effector cells injected with CD19 28z were allowed to function, and a treatment in which effector cells injected with CD19 28z-GITRL were allowed to function.

Fig. 16 shows the results of Experimental Example 5 (γδ cell results). The top row shows the cytotoxicity results analyzed over time using each type of effector cell. The middle row shows the cytotoxicity results after 5, 20, 40, 60, 80, 100, and 120 h. The bottom row shows the time required to damage half of the target cells. In the top and middle rows, the vertical axis represents the cytotoxic activity (%) measured using the xCELLigence assay, and the horizontal axis represents the time since the addition of the effector cells. The bars on the left of each histogram show a treatment in which effector cells were not allowed to function, a treatment in which effector cells injected with CD19 28z were allowed to function, and a treatment in which effector cells injected with CD19 28z-GITRL were allowed to function.

Fig. 17 shows the results of Experimental Example 6. PBS indicates the case where PBS was administered without administering CAR T cells, NGM-αβT indicates the case where NGM (non-genome-modified) T cells were administered, CD19 28z-αβT indicates the case where T cells administered with CD19 28z were administered, CD19 28z-GITRL-αβT indicates the case where T cells administered with CD19 28z-GITRL were administered.

Fig. 18 shows the results of Experimental Example 7. The relative number of IFN-γ-positive cells in CD19CAR-positive cells is shown in each of the CD8 and CD4 fractions (yellow, red letters in the box).

Fig. 19 shows the results of Experimental Example 8. The ratios of effector memory T cells to central memory T cells are shown in each of the TMRM low and TMRM high fractions.

Fig. 20 shows the results of Experimental Example 9. The vertical axis represents the cytotoxic activity (%) measured using the xCELLigence assay, and the horizontal axis represents the time elapsed since the addition of effector cells.

Fig. 21 shows the results of Experimental Example 10. The vertical axis shows the ratio of the abundance of infused cells in the peripheral blood.

Fig. 22 shows the results of Experimental Example 11. The results are the results of the analysis of the peripheral blood of the mouse 38 days after the cell therapy, i.e., 10 days after the second inoculation of NALM6 cells (NALM6/luc 5×10 ⁶ cells i.v./mouse, effector:target ratio=1:1).

Fig. 23 shows the results of Experimental Example 12. They represent the results of the analysis of the peripheral blood of mouse #2, which received 28z-GITRL CAR-T cells, 55 days after the cell therapy, i.e., 27 days after the second inoculation of NALM6 leukemia cells to induce tumors in the mouse.

Detailed description of embodiments of the invention

[0021] As used herein, the terms "comprising," "consisting," and "including" include the concepts of "comprising," "consisting," "consisting essentially of," and "consisting of."

[0022] 1. Definitions

For the purposes of the present invention, the terms "comprising," "consisting," and "including" include the concepts of "comprising," "consisting," "consisting essentially of," and "consisting of."

[0023] In the context of the present invention, the term "identity" of amino acid sequences refers to the degree to which two or more different amino acid sequences correspond to each other. Thus, the higher the degree of coincidence between two amino acid sequences, the higher the identity or similarity of these sequences. The level of identity of an amino acid sequence is determined, for example, using FASTA (a sequence analysis tool) with default parameters. Alternatively, the level of amino acid sequence identity can be determined using the BLAST algorithm of Karlin and Altschul (Karlin S., Altschul S.F. Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes, Proc. Natl. Acad. Sci. USA, 87: 2264-2268(1990), Karlin S., Altschul S.F. Applications and statistics for multiple high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. USA, 90: 5873-7(1993)). Based on this BLAST algorithm, a program called "BLASTX" has been developed. The specific methods for these analysis methods are known and can be found on the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/). The "identity" of nucleotide sequences is also determined in a similar manner as described above.

[0024] As used herein, the term "conservative substitution" means a substitution of an amino acid residue with another amino acid residue having a similar side chain. For example, a substitution between amino acid residues having a basic side chain, such as lysine, arginine, or histidine, is considered a conservative substitution. The following substitutions between other amino acid residues are also considered conservative substitutions: a substitution between amino acid residues having an acidic side chain, such as aspartic acid or glutamic acid; a substitution between amino acid residues having an uncharged polar side chain, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine; a substitution between amino acid residues having a non-polar side chain, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; a substitution between amino acid residues having a beta-branched side chain, such as threonine, valine, or isoleucine; and a substitution between amino acid residues having an aromatic side chain, such as tyrosine, phenylalanine, tryptophan, or histidine.

[0025] In the context of the present invention, the term "CDR" is an abbreviation for complementarity determining region. CDR is a region in the variable regions of immunoglobulins or T-cell receptors, and is actively involved in the specific binding of an antibody or T-cell receptor to its antigen. The expression "light chain CDR" refers to a CDR present in the variable regions of the light chain of immunoglobulins, and the expression "heavy chain CDR" refers to a CDR present in the variable regions of the heavy chain of immunoglobulins. Also in T-cell receptors there is an α-chain, β-chain, γ-chain, δ-chain and the like chains, and also refers to the CDR of these chains.

[0026] In the context of the present invention, the term "variable region" refers to a region comprising CDR1-CDR3 (hereinafter simply "CDR 1-3"). The order in which these CDR 1-3 are arranged is not limited; however, the variable region preferably refers to a region in which CDR1, CDR2 and CDR3 are arranged in the specified order from the N-terminus to the C-terminus or in the reverse order, either sequentially or via other specified amino acid sequences as "framework regions" (FR), which will be described below. The term "heavy chain variable region" refers to a region in which CDR 1-3 of the heavy chain are located, and the term "light chain variable region" refers to a region in which CDR 1-3 of the light chain are located. Also in T-cell receptors there is an α-chain, β-chain, γ-chain, δ-chain and similar chains, and also refers to the CDR of these chains.

[0027] Regions other than CDRs 1-3 in each variable region are referred to as "framework regions" (FRs), as defined above. Specifically, the region between the N-terminus and CDR1 of the variable region is defined as FR1, the region between CDR1 and CDR2 as FR2, the region between CDR2 and CDR3 as FR3, and the region between CDR3 and the C-terminus of the variable region is defined as FR2 as FR4.

[0028] 2. Antigen receptor

In one embodiment, the present invention relates to an antigen receptor comprising a GITRL domain at a position closer to the C-terminus via a self-cleaving peptide domain (which may be referred to as the "antigen receptor of the present invention" in the present specification). The antigen receptor of the present invention is described below.

[0029] The antigen receptor may be any antigen receptor and is, for example, a chimeric antigen receptor (CAR). A chimeric antigen receptor (CAR) is typically a chimeric protein, a single-chain antibody (scFv), which consists of a light chain (VL) linked in tandem with a heavy chain (VH) of a variable region of a monoclonal antibody located closer to the N-terminus as a domain responsible for its ability to bind to an antigen, and its T cell receptor (TCR) ξ-chain located in a position closer to the C-terminus. T cells expressing CAR are called "CAR-T cells".

[0030] The antigen receptor is not limited to a chimeric antigen receptor and may be, for example, a T-cell receptor.

[0031] The antigen receptor of the present invention may be any antigen receptor that contains a GITRL domain at a position closer to the C-terminus via a self-cleaving peptide domain. This can increase the efficiency of expression of the antigen receptor or the cytotoxic activity of T cells containing the antigen receptor. Expression of the antigen receptor of the present invention results in cleavage of the self-cleaving peptide domain, which allows for intracellular expression of GITRL in its free form.

[0032] As used herein, the term "self-cleaving peptide" refers to a peptide sequence with cleaving activity located between two amino acid residues in the peptide sequence. Examples of self-cleaving peptides include 2A peptides and 2A-like peptides. For example, in 2A peptides or 2A-like peptides, cleavage occurs between a glycine residue and a proline residue of the peptides. This occurs through a "ribosome skipping mechanism" in which the normal peptide bond between the glycine residue and the proline residue is not formed during translation, and this does not affect downstream translation. The ribosome skipping mechanism is known in the art and is used to express multiple proteins encoded by a single messenger RNA (mRNA) molecule. The self-cleaving peptide for use in the present invention may be derived from 2A peptides of viruses or 2A-like peptides having equivalent functionality. For example, the self-cleaving peptide may be selected from the group consisting of 2A peptides derived from foot-and-mouth disease virus (FMDV) (F2A), 2A peptides derived from equine rhinitis virus A (ERAV) (E2A), peptides derived from porcine teschovirus (PTV-1) (P2A), and 2A peptides derived from Thosea asigna virus (TaV) (T2A). The domain of the self-cleaving peptide may be mutated, provided that the activity of the self-cleaving peptide domain is not significantly impaired.

[0033] The GITRL domain may be any domain that contains the amino acid sequence GITRL.

[0034] The GITRL for use may be various types of GITRL obtained from mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits. Of these, a GITRL obtained from a target organism (e.g., a human) is preferred.

[0035] Amino acid sequences of GITRL obtained from various organisms are known. In particular, examples include proteins having the amino acid sequence shown in SEQ ID NO: 4. GITRL can also be a protein with a deleted N-terminal signal peptide.

[0036] GITRL may be mutated, such as by substitution, deletion, addition or insertion of amino acids, so long as GITRL retains the ability to bind to its receptor, GITR. The mutation is preferably a substitution and more preferably a conservative substitution in view of the lower likelihood that GITRL will lose its ability to bind to GITR.

[0037] A preferred specific example of an amino acid sequence of GITRL is at least one member selected from the group consisting of the amino acid sequence described in the following paragraph (a) and the amino acid sequence described in the following paragraph (b):

(a) the amino acid sequence shown in any part of SEQ ID NO: 4, and

(b) an amino acid sequence that has at least 85% identity to the amino acid sequence shown in any part of SEQ ID NO: 4 and that is a protein capable of binding to GITR.

[0038] In (b) above, the identity is preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and particularly preferably at least 99%.

[0039] The inducibility of GITRL to bind to GITR can be determined according to a known method or a similar method. The expression "capable of binding to GITR" means that GITRL is capable of binding to GITR, for example, to a degree of at least 50%, at least 70%, at least 80%, at least 90%, or at least 95% of the binding ability of GITRL before mutation.

[0040] An example of an amino acid sequence described in (b) above is (b') an amino acid sequence that has a substitution, deletion, addition or insertion of one or more amino acids in a region of the amino acid sequence shown in any part of SEQ ID NO: 4, which is a protein capable of binding to GITR.

[0041] In item (b') above, “several amino acids” means, for example, 2 to 15 amino acids, preferably 2 to 10 amino acids, more preferably 2 to 5 amino acids, and more preferably 2 to 3 amino acids.

[0042] The antigen receptor of the present invention usually comprises a domain responsible for the ability to bind to its antigen (antigen-binding domain). The antigen is not particularly limited, and examples thereof include various proteins or complexes that are expressed on the surface of tumor cells or T cells. Specific examples of antigens include CD19, GD2, GD3, CD20, CEA, HER2, EGFR, mutant EGFR type III, CD38, BCMA, MUC-1, PSMA, WT1, testicular cancer antigens (e.g., NY-ESO-1 and MAGE-A4), mutant peptides (e.g., k-ras, h-ras and p53), hTERT, PRAM, TYRP1, mesothelin, PMEL, mucin and complexes of fragments of these antigens with MHC (pMHC). Of these, the antigen is preferably CD19 or GD2, and more preferably CD19. The antigen-binding domain typically comprises one or more, preferably two, three, four or five or more CDRs, or more preferably all six CDRs of an antibody or T cell receptor for an antigen (e.g., in the case of antibody CDRs, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3). The antigen-binding domain more preferably comprises a variable region of an antibody or T cell receptor for an antigen (e.g., in the case of antibodies, heavy chain variable regions and/or light chain variable regions).

[0043] The antigen binding domain preferably has an scFv structure. The linker that links the variable region of the heavy chain to the variable region of the light chain may be any linker that maintains the functionality of the antigen receptor. The linker is preferably a GS linker (typically a linker having a repeating sequence containing GGGGS (SEQ ID NO: 5) as a structural unit). The number of amino acid residues of the linker is, for example, 5 to 30, preferably 10 to 20, and more preferably 15. The location of the variable region of the heavy chain and the variable region of the light chain is not limited. The variable region of the light chain may be at a position closer to the N-terminus, or the variable region of the heavy chain may be at a position closer to the N-terminus. The first location is preferred.

[0044] The antigen receptor of the present invention typically comprises a core domain comprising a variable region of an antibody or a T-cell receptor (e.g., an scFv domain comprising a heavy chain variable region and a light chain variable region), a transmembrane domain, and a TCR intracellular domain. In the core domain, the variable region, transmembrane domain, and TCR intracellular domain are arranged in this order from the N-terminus directly or through other domains.

[0045] The antigen receptor of the present invention comprising a core domain preferably comprises a GITRL domain at a position closer to the C-terminus of the core domain via a self-cleaving peptide domain.

[0046] The transmembrane domain may be of any type that does not adversely affect the functionality of the antigen receptor. For example, CD28, CD3ξ, CD4, or CD8α, which are expressed in cells such as T cells, may be used. These transmembrane domains may be mutated, provided that the functionality of the chimeric antigen receptor is not impaired.

[0047] The intracellular domain of the TCR may be, for example, an intracellular domain derived from CD3, which is also called the "TCR ξ chain". CD3 may be mutated, provided that the functionality of the chimeric antigen receptor is not impaired. The mutation of CD3 is preferably carried out in such a way that CD3 contains an ITAM (immunoreceptor tyrosine-based activation motif).

[0048] The antigen receptor of the present invention preferably has a spacer sequence between the variable region and the transmembrane domain. The spacer sequence may be of any length and may be formed from any amino acid residues, provided that the functionality of the antigen receptor is not impaired. For example, the spacer sequence may be designed to contain from about 10 to about 200 amino acid residues. The spacer sequence used is preferably a light chain constant region sequence.

[0049] The core domain in the antigen receptor of the present invention preferably further comprises an intracellular domain of a costimulator. The intracellular domain of the costimulator may be any intracellular domain derived from a costimulator of cells such as T cells. For example, at least one member selected from the group consisting of OX40, 4-1BB, GITR, CD27, CD278, CD28 and the like can be suitably selected and used. Of these, CD28 is preferable in view of its substantially high efficiency due to its combination with the GITRL domain. The intracellular domain of these costimulators may be mutated as long as the functionality of the antigen receptor is not impaired. The position of the intracellular domain of the costimulator is not particularly limited as long as the intracellular domain is at a position closer to the C-terminus of the transmembrane domain; the intracellular domain may be closer to the N-terminus or the C-terminus of the intracellular domain of the TCR.

[0050] In a particularly preferred embodiment of the present invention, the variable region, spacer sequence, transmembrane domain, intracellular costimulator domain, intracellular TCR domain, self-cleaving peptide domain and GITRL domain are arranged in this order from the N-terminus directly or via other domains.

[0051] The antigen receptor of the present invention may comprise a ligand domain, such as a GITRL domain, a 4-1BBL domain, or an ICOSL domain, at a position closer to the C-terminus of the core domain via a self-cleaving peptide domain.

[0052] The antigen receptor of the present invention may be a molecule formed by one type of polypeptide, or a molecule formed by a complex of two or more types of polypeptides. The antigen receptor of the present invention may also be a molecule formed by a polypeptide or a complex of polypeptides, or a molecule formed by a polypeptide or a complex of polypeptides to which another compound (e.g., a fluorescent compound, a radioactive compound, or an inorganic particle) is attached.

[0053] The antigen receptor of the present invention may be chemically modified. The polypeptide that constitutes the antigen receptor of the present invention may have a carboxyl group (-COOH), a carboxylate group (-COO-), an amide group (-CONH ₂ ) or an ester group (-COOR) at the C-terminus. "R" in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl; a C _3-8 cycloalkyl group such as cyclopentyl or cyclohexyl; a C _6-12 aryl group such as phenyl or α-naphthyl; a phenyl-C _1-2 alkyl group such as benzyl or phenethyl; C _7-14 aralkyl group such as α-naphthyl-C _1-2 alkyl group such as α-naphthylmethyl; or pivaloyloxymethyl group. The polypeptide that constitutes the antigen receptor of the present invention may have an amidated or esterified carboxyl group (or carboxylate) that is not a carboxyl group at the C-terminus. The ester in this case may be, for example, the ester at the C-terminus described above. The polypeptide that constitutes the antigen receptor of the present invention further includes polypeptides having an amino group of an N-terminal amino acid residue protected by a protecting group (e.g., a C _1-6 acyl group including a C _1-6 alkanoyl such as a formyl group and an acetyl group), polypeptides having a pyroglutaminated N-terminal glutamine residue that can be formed as a result of cleavage in vivo; and polypeptides having a substituent (e.g., -OH, -SH, amino group, imidazole group, indole group and guanidino group) on the substitution side of an amino acid in a molecule protected by an appropriate protecting group (e.g., C _1-6 acyl group, including C _1-6 alkanoyl group such as formyl group and acetyl group).

[0054] The antigen receptor of the present invention may have an added protein or peptide (e.g., a known protein tag or signal sequence). Examples of protein tags include biotin, a His tag, a FLAG tag, a Halo tag, an MBP tag, an HA tag, a Myc tag, a V5 tag, a PA tag, and a fluorescent protein tag.

[0055] The antigen receptor of the present invention may be a pharmaceutically acceptable salt formed with an acid or a base. The salt may be any pharmaceutically acceptable salt and may be either an acidic or a basic salt. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate and phosphate; organic acid salts such as acetate, propionate, tartarate, fumarate, maleate, malate, citrate, methanesulfonate and p-toluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; and alkaline earth metal salts such as calcium salts and magnesium salts.

[0056] The antigen receptor of the present invention may be in the form of a solvate. The solvent may be any pharmaceutically acceptable solvent, such as water, ethanol, glycerol, or acetic acid.

[0057] Methods for obtaining an antigen receptor and a CAR-T cell that expresses the antigen receptor are known. Antigen receptors and CAR-T cells can be obtained according to a known method or a similar method.

[0058] 3. Polynucleotide

In one embodiment, the present invention relates to a polynucleotide encoding an antigen receptor of the present invention (which may be referred to as a "polynucleotide of the present invention" in this specification). The polynucleotide of the present invention is described below.

[0059] The polynucleotide of the present invention may comprise other sequences in addition to the sequence encoding the antigen receptor of the present invention. Preferably, the polynucleotide of the present invention precisely comprises the sequence of the antigen receptor of the present invention. Other sequences include promoter sequences, enhancer sequences, repressor sequences, insulator sequences, origins of replication, sequences encoding a reporter protein (e.g., fluorescent proteins), and sequences encoding a drug resistance gene. The polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide (e.g., a vector). The vector may be a plasmid vector or a viral vector (e.g., adenovirus or retrovirus). The vector may also be, for example, a cloning vector or an expression vector. An expression vector includes vectors for prokaryotic cells such as Escherichia coli or actinomycetes, and vectors for eukaryotic cells such as yeast cells, insect cells, or mammalian cells.

[0060] The polynucleotide of the present invention includes not only DNA and RNA but also known chemically modified DNA or RNA described below. In order to prevent degradation by hydrolases such as nucleases, a phosphate residue (phosphate) of each nucleotide can be replaced with, for example, a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate or phosphorodithionate. The hydroxyl group at position 2 of ribose of each ribonucleotide can also be replaced with -OR (R is, for example, CH ₃ (2'-O-Me), CH ₂ CH ₂ OCH ₃ (2'-O-MOE), CH ₂ CH ₂ NHC(NH)NH ₂ , CH ₂ CONHCH ₃ or CH ₂ CH ₂ CN). In addition, the base group (pyrimidine, purine) can be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base or by replacing the carbonyl group at the 2-position with thiocarbonyl. In addition, the polynucleotides of the present invention also include, but are not limited to, polynucleotides formed by modifying a phosphate group or a hydroxyl group, for example, with biotin, an amino group, a lower alkylamino group, or an acetyl group. The term "polynucleotide" includes not only natural nucleic acids, but also BNA (bridged nucleic acids), LNA (closed nucleic acids), and PNA (peptide nucleic acids).

[0061] 4. Cell

In one embodiment, the present invention relates to a cell comprising a polynucleotide of the present invention (which may be referred to as a "cell of the present invention" herein). The cell of the present invention is described below.

[0062] The cells from which the cells of the present invention are derived are not particularly limited. For the purpose of using the cell of the present invention to produce the antigen receptor of the present invention, cells that can be used for protein expression (e.g., insect cells, eukaryotic cells, mammalian cells) can be used as source cells, for example.

[0063] The cell of the present invention is preferably a T cell (e.g., an αβ T cell or a γδ T cell) or an NK cell. These cells are preferably cells expressing the antigen receptor of the present invention. In a more specific embodiment of the cells of the present invention, the antigen receptor of the present invention is expressed on the cell membrane, and is preferably expressed in a state such that the antigen-binding domain is exposed outside the cell membrane.

[0064] A T cell or the like expressing an antigen receptor recognizes an antigen in the antigen-binding domain, and then intracellularly transfers a recognition signal to activate a signal that induces cytotoxic activity. In this regard, the cell "attacks" other cells or tissues expressing the antigen, or exhibits cytotoxic activity.

[0065] When the cell exhibiting such function is a CTL, such a cell is called a "chimeric antigen receptor T cell" ("CAR-T cell"). Cells that can potentially exhibit cytotoxic activity, such as NK cells, can also exhibit cytotoxic activity when the antigen-binding domain binds to an antigen, such as a T cell chimeric antigen receptor. Thus, a host cell containing a polynucleotide encoding an antigen receptor (in particular, a host cell having cytotoxic activity) is useful as an active ingredient in pharmaceutical compositions.

[0066] Such T cells and the like are useful for the treatment or prevention of cancer and the like because they specifically recognize cancer tissue (tumor tissue). The type of cancer is not particularly limited and includes a solid tumor and blood cancer. Examples of solid cancer include lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma and the like.

[0067] The cell of the present invention can be obtained by introducing the polynucleotide of the present invention into the cells. If necessary, the cells containing the polynucleotide of the present invention can be concentrated or can be concentrated using a specific marker (a CD antigen such as CD8) as an indicator.

[0068] 5. Pharmaceutical composition

In one embodiment, the present invention relates to a pharmaceutical composition comprising a T cell or NK cell of the present invention (which may be referred to as the “pharmaceutical composition of the present invention” in this specification). The pharmaceutical composition of the present invention is described below.

[0069] The cell content of the pharmaceutical composition can be appropriately set in consideration of the type of the target disease (e.g., solid cancer), the desired therapeutic effects, the administration route, the treatment period, the age of the patient, the body weight of the patient, etc. The cell content of the pharmaceutical composition can be, for example, about 1 cell/mL to 10 ⁴ cells/mL.

[0070] The administration form of the pharmaceutical composition is not particularly limited as long as the desired effects are achieved. The pharmaceutical composition can be administered to mammals including humans by any of the following administration routes: oral administration and parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, cutaneous administration, and local administration). Since the active ingredient is a cell, the administration form is preferably parenteral administration, and more preferably intravenous injection. Dosage forms for oral administration and parenteral administration and methods for producing them are well known to those skilled in the art. The pharmaceutical composition can be produced by a conventional method, for example, by mixing the antibody or cell of the present invention with a pharmaceutically acceptable carrier, etc.

[0071] Examples of dosage forms for parenteral administration include injections (e.g., intravenous drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, and endodermal injections), external preparations (e.g., ointments, cataplasms, and lotions), suppositories, inhalants, eye drops, eye ointments, nasal drops, ear drops, liposomal agents, and the like. For example, an injection preparation can be prepared by dissolving or suspending an antibody or cells in distilled water for injection and optionally adding a solubilizer, a buffer, a pH adjuster, an isotonic agent, a soothing agent, a preservative, a stabilizer, etc. The pharmaceutical composition can also be used as a lyophilized preparation prepared before use.

[0072] The pharmaceutical composition may additionally contain other drugs effective for the treatment or prevention of diseases. The pharmaceutical composition may also contain components such as sterilizers, anti-inflammatory agents, cell activators, vitamins and amino acids, if necessary.

[0073] As a carrier used for formulating a pharmaceutical composition, excipients, binders, disintegrants, lubricants, colorants and flavorings which are commonly used in this field of technology can be used; and stabilizers, emulsifiers, absorption enhancers, surfactants, pH regulators, antiseptics, antioxidants, fillers, humectants, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents and the like can also be optionally used.

[0074] The type of disease treated or prevented using the pharmaceutical composition is not particularly limited as long as the treatment or prevention can be achieved. Examples of specific target diseases include tumors. The type of tumor is not particularly limited and includes solid cancer and blood cancer. Examples of solid cancer include lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, etc.

[0075] The target subject for administration (test subject) of the pharmaceutical composition is, for example, an animal suffering from the disease described above or an animal with a potential risk of developing such a disease. "Potential risk of developing such a disease" can be determined using a known diagnostic method. The animal is, for example, a mammal and preferably a human.

[0076] The dose of the pharmaceutical composition may be determined by the attending physician taking into account various factors such as the route of administration, the type of disease, the severity of symptoms, the age, sex and body weight of the patient, the severity of the disease, pharmacological data such as pharmacokinetics and toxicological characteristics, the use or non-use of a drug delivery system and whether the composition is administered as part of a combination drug with other drugs. For example, if the active ingredient is a cell, the dose may be from about 10 ⁴ cells/kg (body weight) to 10 ⁹ cells/kg (body weight). The administration schedule of the pharmaceutical composition may also be determined taking into account the same factors as in determining the dose. For example, the composition may be administered from once a day to once a month at the daily dose given above.

Examples

[0077] The present invention is described in detail below with reference to examples. However, the present invention is not limited to the examples given.

[0078] Experimental Example 1: Construction of CAR structure

The anti-CD19 (FMC-63)-28z CAR or anti-CD19 (FMC-63)-28z-GITRL CAR construct was recombined with pMS3 to obtain a plasmid vector, and the plasmid vector was introduced into Plat-A cells (Cell Biolabs) with FuGENE (Promega) to produce retroviruses. Fig. 1 shows the structures of CD19 28z CAR and CD19 28z GITRL CAR. Fig. 2 shows the nucleotide sequence (SEQ ID NO: 1) of CD19 28z CAR, and Fig. 3 shows the nucleotide sequence (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 3) of CD19 28z GITRL CAR.

[0079] Retroviruses were also generated using the anti-CD19(FMC-63)-zG or anti-CD19(FMC-63)-zG-GITRL CAR construct. Specifically, the retroviruses were generated in a similar manner except that the N-terminal side-CD28 intracellular domain-CD3ξ domain region in the CD19 28z CAR and CD19 28z GITRL CAR was replaced with the N-terminal side-CD3ξ domain-GITR domain region.

[0080] Experimental Example 2: Obtaining an Effector Cell

αβ cells (obtained by culturing peripheral blood mononuclear cells isolated using Ficoll-Paque in GT-T551 medium supplemented with 0.6% inactivated human plasma and IL-2 at a final concentration of 600 IU/ml in a 12-well plate containing 2 μg OKT3 and 10 μg retronectin immobilized thereon, and harvesting the cells on day 4) and γδ cells (obtained by culturing peripheral blood mononuclear cells in YM-AB medium containing a new bisphosphonate drug (PTA), supplemented with 25 ng/ml IL-7 and 25 ng/ml IL-15, and harvesting the cells on day 4) were infected with the above immobilized retroviruses.

[0081] Experimental Example 3: Confirmation of CAR Expression

CAR expression was determined using a flow cytometer. After interaction with human κ antibody (rabbit IgG), the cells were reacted with Alexa 488-labeled anti-rabbit IgG (goat polyclonal antibody) (Invitrogen). In addition, staining with APC/Cy7-labeled anti-human CD8 antibody (BD) and APC-labeled anti-human CD4 antibody (BioLegend) was added for αβ cells and staining with PE-labeled anti-human Vδ2 antibody was added for γδ cells, followed by measurement using FACSCanto analysis. The expression level of CAR molecules (recognized by anti-human κ antibody) in the CD4 and CD8 fractions of αβ cells and in the Vδ2-positive fraction of γδ cells was calculated.

[0082] For CD19 28z and CD19 28z-GITRL used for gene transfer, CD19 CAR expression was higher in cells injected with CD19 28z-GITRL in both αβ cells (five gene transfer experiments) and γδ cells (four gene transfer experiments) (Figs. 4 and 5 and top row of each of Figs. 12 and 13).

[0083] For each of CD19 zG and CD19 zG-GITRL, whose intracellular domain is zG, one gene transfer experiment was performed using αβ cells, and one gene transfer experiment was performed using γδ cells. As with 28z, CD19 CAR expression was higher using the GITRL-linked form (Figs. 6 and 7, and the far right column in the top row of each of Figs. 12 and 13).

[0084] Experimental Example 4: Recognition of a Target Cell by an Effector Cell

After addition of APC-labeled anti-human CD107a antibody (BD), co-culture with CD19-positive B-type acute lymphocytic leukemia cell line NALM6 (CD19-negative line K562 was used as a control) was performed for 4 h. Thereafter, CAR-T cell surface antigen was stained similarly as described above, and cytokines in the cells after Cytofix/Cytoperm treatment (BD) were stained with V450-labeled anti-human IFN-γ antibody and PE/Cy7-labeled anti-human TNF-α antibody, followed by analysis by FACSCanto II. The levels of CD107a, IFN-γ, and TNF-α-positive CAR-positive cells in the CD4 and CD8 αβ cell fractions and in the Vδ2-positive γδ cell fraction were determined.

[0085] The relative number of IFN-γ-producing cells in CD19CAR-positive cells after 4 h of co-culture with the target cell line (NALM6) tended to be higher in both αβ cells (28z: Fig. 8, zG: Fig. 10) and γδ cells (28z: Fig. 9 and zG: Fig. 11) when using the GITRL-linked form, but there was no statistically significant difference (middle row in each of Figs. 12 and 13). However, the number of IFN-γ-producing CD19CAR-positive cells was higher in cells in the GITRL-linked transduced form (both αβ cells and γδ cells) (bottom row in each of Figs. 12 and 13). On average, the value in cells in the GITRL-bound transduced form was approximately twofold higher (Fig. 14).

[0086] Experimental Example 5: Confirmation of the Cytotoxic Action of the Effector Cell

The cytotoxic activity over time was measured by real-time CTL assay (xCELLigence) through co-culture with NALM6. Specifically, anti-CD9 antibody (ACEA Bioscience kit, catalog no.: 8710247) adjusted to a final concentration of 4 μg/mL using binding buffer (catalog no.: 8718617) was immobilized in each well of E-plate 16. Three hours later, washing with PBS was performed, and NALM6 target cells (6.0× ¹⁰⁴ ) suspended in 100 μL of RPMI 1640 medium supplemented with 10% FCS were added and left at room temperature in a clean bench for 0.5 h, followed by culture in an incubator under conditions of 0.5% _CO2 and 37°C for 24 h or more. Each effector cell type (6×10 ⁴ ) was added and left at room temperature on a clean bench for 0.5 h, followed by additional culturing in an incubator under CO ₂ and 37°C for 5-7 days. Changes in electrical resistance over time were analyzed for cytotoxicity to NALM6 cells (xCELLigence assay, ACEA Bioscience). CD19 28zCAR-transduced αβ T cells or γδ T cells, or GITRL-coexpressing CD19 28zCAR-transduced αβ T cells or γδ T cells were used as effector.

[0087] In a 120 h real-time CTL assay, both CD19 28z-GITRL-transduced αβ cells and CD19 28z-GITRL-transduced γδ cells showed higher cytotoxic activity than CD19 28z-transduced cells. Specifically, CD19 28z-GITRL was superior in the level of cytotoxic activity, duration/sustainability of maximal cytotoxic activity, and rapidity, as indicated by the time required to reach half of maximal cytotoxic activity (Figures 15 and 16).

[0088] Experimental Example 6: Study of Antitumor Effect

The antitumor effect of NALM6 cells transplanted into human immunodeficient (NOG) mice was investigated. After irradiation with 2 Gy, NOG mice were transplanted and inoculated with NALM6 (1×10 ⁶ ) into which the luciferase gene was introduced, then each kind of CD19 CAR-T cells (5×10 ⁶ ) were injected. Images were obtained using IVIS time-lapse imaging to observe the kinetics of NALM6 cells. Specifically, the experiment was carried out as follows.

[0089] Immediately after 6-week-old immunodeficient female mice (NOD/Shi-scid/IL-2Rgammanull) were exposed to total body irradiation (TBI) at a dose of 2 Gy, each mouse was injected via the tail vein with luciferase gene-engineered CD19-positive leukemia cells (NALM6/Luc) at a concentration of 1×10 ⁶ cells/mouse (on day -7). One week later (day 0), mice were injected with 200 μl/mouse PBS via the tail vein (untreated group; two mice), and mice were injected with T cells from the same donor without CD19 CAR gene transfer via the tail vein (NGM-αβ T cells-treated group; 5× ¹⁰⁶ cells/mouse; two mice) (both of these groups serve as negative controls); and the control group (three mice) was intravenously injected with conventional CD19-28z CAR-transduced αβ T cells, and the experimental group (three mice) was intravenously injected with desired CD19 28z-GITRL CAR-transduced γδT cells. The number of remaining leukemic cells luminescent upon luciferin administration was analyzed over time using the PerkinElmer in vivo imaging system (IVIS).

[0090] As shown in Fig. 17 , the animals in the untreated group (PBS-treated group) lost their tumors within 3 weeks, and the animals in the group receiving unmodified T cells (NGM-T-treated group) also lost their tumors within 4 weeks. In contrast, in both the 28z CAR-T-treated group and the 28z-GITRL CAR-T-treated group, leukemia cells were eliminated within 1 week after administration, and leukemia relapse was not observed until 7 weeks after administration. However, the survival rate in the 28z-CAR-T-treated group was lower than that in the 28z-GITRL CAR-T-treated group.

[0091] Experimental Example 7: Assay for IFN-γ Production

After co-culture of CD19-positive leukemia cell line NALM6 (2×10 ⁵ ) with CD19 28z CAR-T cells or GITRL-coexpressing CD19 28z CAR-T cells (2×10 ⁵ ) for 4 h, NALM6-reactive IFN-γ production of each effector cell type was analyzed using a flow cytometer (BD FACSCanto II).

[0092] Figure 18 shows the results. CD19 CAR-T (αβ) cells co-expressing GITRL showed increased IFN-γ production after co-culture with target leukemia cells (NALM6).

[0093] Experimental Example 8: Analysis of Central Memory Cells

Staining of CD8-positive αβ T cells injected with CD19 28z CAR or GITRL-coexpressing CD19 28z CAR was performed using tetramethylrhodamine methyl ester (TMRM), which is an indicator of mitochondrial membrane potential, in addition to antibody against CD45RA and CD62L, both of which are indicators of cell differentiation stage.

[0094] Fig. 19 shows the results. Compared with 28z CAR-transduced CD8-positive T cells, an increase in the immunological memory cell group (CD62L-CD45RA-, effector memory T cells (T _EM ) and CD62L+CD45RA-, central memory T cells (T _CM )) was observed in the 28z-GITRL CAR-transduced CD8-positive T cell group. This trend was more pronounced in T _CM . In addition, these immunological memory cells were confirmed to be immunological memory cells by their metabolic characteristics, which were accompanied by an increase in mitochondrial membrane potential and a dependence on oxidative phosphorylation for their energy. These properties are thought to promote long-term engraftment of CAR-T cells injected into the patient, indicating the superiority of the GITRL gene-coexpressing CAR.

[0095] Experimental Example 9: Analysis of Long-Term Cytotoxic Activity

CEA-positive BxPC-3 cells (7000 cells) and CEA-negative MIA Paca-2 cells (7000 cells) were cultured in an E-plate and γδ cell-derived effector cells (21000 cells) were added. Co-culture was performed and cytotoxicity was analyzed over 24 h over time. After completion of co-culture, effector cells were collected and co-cultured with new target cells for 24 h, after which changes over time were analyzed. A third co-culture was performed similarly and changes over time were analyzed.

[0096] Fig. 20 shows the results. The cytotoxic activity was higher when using GITRL co-expressing CEA 28z CAR-T cells than when using CEA 28z CAR-T cells alone. It was noted that the difference in cytotoxic activity tended to increase as the number of cultures performed increased.

[0097] Experimental Example 10: Analysis of 1 Remaining Cells

CEA-positive BxPC-3 cells (5×10 ⁶ ) were subcutaneously transplanted into NOG mice to induce tumor development in mice. Subsequently, GITRL co-expressing CEA 28z CAR-T cells, γδ-derived CEA 28z CAR-T cells, and blank control cells (each at 1×10 ⁷ ) were injected into animals individually intravenously. The abundance ratio of infused cells in peripheral blood at 2 weeks and 3 weeks was examined using anti-Vd2 antibody staining and biotinylated CEA staining. The numbers of mice used in the experiment were 2, 2, and 3.

[0098] Figure 21 shows the results. In NOG mice, 3 weeks after effector cell infusion, the number of cell survivors was higher when GITRL-coexpressing γδ CEA 28z CAR-T cells were infused than when γδ CEA 28z CAR-T cells were infused.

[0099] Experimental Example 11: Analysis of the 2 Remaining Cells

On day 7 after intravenous inoculation of luciferase-labeled NALM6 leukemia cells into NOG mice, CD19 28 CAR-T cells (αβ) or CD19 28z-GITRL CAR-T cells (αβ) were injected to induce tumor development in the mice. Tumor cell clearance was confirmed 14 days after infusion, and the same number of NALM6 cells were inoculated to induce tumor development in the mice on day 28. On day 38, venous blood from the mice was analyzed. In both CD19 28 CAR-T cell-treated mice and CD19 28z-GITRL CAR-T cell-treated mice, NALM6 was again rejected, indicating that the antileukemic effect was maintained. Regarding the remaining infused CAR-T cells stained with anti-kappa antibody, the number of remaining CD8+CAR+ cells was 0.002% and the number of remaining CD4+CAR+ cells was 0.017% in the peripheral blood of 28z CAR-T cell-infused mice, whereas in the two mice injected with 28z-GITRL CAR-T cells, the number of remaining CD8+CAR+ cells was 0.01% and 0.024% and the number of remaining CD4+CAR+ cells was 0.033% and 0.032% in the peripheral blood. Both CD8+CAR+ cells and CD4+CAR+ cells remained more numerous in the mice injected with 28z-GITRL CAR-T cells (Fig. 22).

[0100] Experimental Example 12: Analysis of Grafted Cells

Peripheral blood was analyzed 55 days after cell therapy in mouse #2, which received 28z-GITLR CAR-T cells (αβ) in the NALM6 cell-bearing NOG mouse model (described above). Human lymphocytes were detected in the mouse peripheral blood lymphocytes (1 in Fig. 23). Human CD3-positive T cells were detected in 20.3% of the lymphocytes, and CD19-positive NALM6 human leukemia cells were not detected (2 in Fig. 23). CAR-T cells were detected in 3.4% of the human T cells (3 in Fig. 23). When peripheral blood from this mouse was again cocultured with NALM6 cells in vitro for 18 h, all CAR-positive human lymphocytes retained their NALM6-reactive IFN-γ production capacity (reactivity to leukemia cells) (4 in Fig. 23).

[0101] Conclusion

Compared with CD19 28z, CD19 28z-GITRL provides efficient expression of CAR molecule in both αβ and γδ T cells. Even when the intracellular domain is zG instead of 28z, the GITRL-bound form provides high efficiency of CAR molecule expression. Moreover, although no advantage of GITRL-bound form was found in cytokine production when co-cultured with target cells for a short period of time (4 h), the GITRL-bound form showed potent, rapid and long-lasting target-specific cytotoxic activity in both αβ and γδ T cells in a real-time CTL assay. Based on these results, it is suggested that GITRL-linked CAR-T cells induce enhanced antitumor activity largely through enhanced activation and prolongation of T cell activation process after antigen recognition rather than through antigen recognition process. Thus, GITRL-linked CAR-T cells are expected to have clinical impact in refractory and relapsed cases, which are challenges for current CD19 CAR-T (28z) cell therapy.

[0102] Moreover, a similar trend as described above was observed when targeting GD2.

--->

List of sequences

<110> MIE UNIVERSITY

<120> ANTIGENE RECEPTOR

<130> P20-149WO

<150> JP 2019-142357

<151> 2019-08-01

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 1693

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of CD19 28z CAR

<400> 1

gcggccgccc agatccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct 60

gctccacgcc gccaggccgg acatccagat gacacagact acatcctccc tgtctgcctc 120

tctggggagac agagtcacca tcagttgcag ggcaagtcag gacattagta aatatttaaa 180

ttggtatcag cagaaaccag atggaactgt taaactcctg atctaccata catcaagatt 240

acactcagga gtcccatcaa ggttcagtgg cagtgggtct ggaacagatt attctctcac 300

cattagcaac ctggagcaag aagatattgc cacttacttt tgccaacagg gtaatacgct 360

tccgtacacg ttcggagggg ggaccaagct ggagatcaca ggaggtggag gttctggtgg 420

aggaggttca ggtggaggtg gatcagaggt gaaactgcag gagtcaggac ctggcctggt 480

ggcgccctca cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta 540

tggtgtaagc tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg 600

gggtagtgaa accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga 660

caactccaag agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat 720

ttactactgt gccaaacatt attactacgg tggtagctat gctatggact actggggcca 780

aggaacctca gtcaccgtct cctcacgaac tgtggctgca ccatctgtct tcatcttccc 840

gccatctgat gagcagttga aatctggaac tgcctctgtt gtgtgcctgc tgaataactt 900

ctatcccaga gaggccaaag tacagtggaa ggtggataac gccctccaat cgggtaactc 960

ccaggagagt gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct 1020

gacgctgagc aaagcagact acgagaaaca caaagtctac gcctgcgaag tcacccatca 1080

gggcctgagc tcgcccgtca caaagagctt caacagggga gagtgtacta gattttgggt 1140

gctggtggtg gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat 1200

tattttctgg gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac 1260

tccccgccgc cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt 1320

cgcagcctat cgctccctga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca 1380

gcagggccag aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt 1440

tttggacaag agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa 1500

ccctcaggaa ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga 1560

gattgggatg aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct 1620

cagtacagcc accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta 1680

atcgattctc gag 1693

<210> 2

<211> 2356

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence CD19 28z GITRL CAR

<400> 2

gcggccgccc agatccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct 60

gctccacgcc gccaggccgg acatccagat gacacagact acatcctccc tgtctgcctc 120

tctggggagac agagtcacca tcagttgcag ggcaagtcag gacattagta aatatttaaa 180

ttggtatcag cagaaaccag atggaactgt taaactcctg atctaccata catcaagatt 240

acactcagga gtcccatcaa ggttcagtgg cagtgggtct ggaacagatt attctctcac 300

cattagcaac ctggagcaag aagatattgc cacttacttt tgccaacagg gtaatacgct 360

tccgtacacg ttcggagggg ggaccaagct ggagatcaca ggaggtggag gttctggtgg 420

aggaggttca ggtggaggtg gatcagaggt gaaactgcag gagtcaggac ctggcctggt 480

ggcgccctca cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta 540

tggtgtaagc tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg 600

gggtagtgaa accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga 660

caactccaag agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat 720

ttactactgt gccaaacatt attactacgg tggtagctat gctatggact actggggcca 780

aggaacctca gtcaccgtct cctcacgaac tgtggctgca ccatctgtct tcatcttccc 840

gccatctgat gagcagttga aatctggaac tgcctctgtt gtgtgcctgc tgaataactt 900

ctatcccaga gaggccaaag tacagtggaa ggtggataac gccctccaat cgggtaactc 960

ccaggagagt gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct 1020

gacgctgagc aaagcagact acgagaaaca caaagtctac gcctgcgaag tcacccatca 1080

gggcctgagc tcgcccgtca caaagagctt caacagggga gagtgtacta gattttgggt 1140

gctggtggtg gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat 1200

tattttctgg gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac 1260

tccccgccgc cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt 1320

cgcagcctat cgctccctga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca 1380

gcagggccag aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt 1440

tttggacaag agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa 1500

ccctcaggaa ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga 1560

gattgggatg aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct 1620

cagtacagcc accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcgg 1680

atcccagtgc accaattacg ctcttctgaa gctggccggc gacgtggaga gcaaccccgg 1740

ccccatgacc ctgcacccca gccccatcac ctgcgagttc ctgttcagca ccgccctgat 1800

cagccccaag atgtgcctga gccacctgga gaacatgccc ctgagccaca gcagaaccca 1860

gggcgcccag agaagcagct ggaagctgtg gctgttctgc agcatcgtga tgctgctgtt 1920

cctgtgcagc ttcagctggc tgatcttcat cttcctgcag ctggagaccg ccaaggagcc 1980

ctgcatggcc aagttcggcc ccctgcccag caagtggcag atggccagca gcgagccccc 2040

ctgcgtgaac aaggtgagcg actggaagct ggagatcctg cagaacggcc tgtacctgat 2100

ctacggccag gtggccccca acgccaacta caacgacgtg gcccccttcg aggtgagact 2160

gtacaagaac aaggacatga tccagaccct gaccaacaag agcaagatcc agaacgtggg 2220

cggcacctac gagctgcacg tgggcgacac catcgacctg atcttcaaca gcgagcacca 2280

ggtgctgaag aacaacacct actggggcat catcctgctg gccaaccccc agttcatcag 2340

ctaatcgatt ctcgag 2356

<210> 3

<211> 775

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence of CD19 28z GITRL CAR

<400> 3

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe

260 265 270

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

275 280 285

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

290 295 300

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

305 310 315 320

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

325 330 335

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

340 345 350

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

355 360 365

Glu Cys Thr Arg Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala

370 375 380

Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg

385 390 395 400

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

405 410 415

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

420 425 430

Arg Asp Phe Ala Ala Tyr Arg Ser Leu Arg Val Lys Phe Ser Arg Ser

435 440 445

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

450 455 460

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

465 470 475 480

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro

485 490 495

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

500 505 510

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

515 520 525

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

530 535 540

Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gln Cys Thr Asn

545 550 555 560

Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro

565 570 575

Met Thr Leu His Pro Ser Pro Ile Thr Cys Glu Phe Leu Phe Ser Thr

580 585 590

Ala Leu Ile Ser Pro Lys Met Cys Leu Ser His Leu Glu Asn Met Pro

595 600 605

Leu Ser His Ser Arg Thr Gln Gly Ala Gln Arg Ser Ser Trp Lys Leu

610 615 620

Trp Leu Phe Cys Ser Ile Val Met Leu Leu Phe Leu Cys Ser Phe Ser

625 630 635 640

Trp Leu Ile Phe Ile Phe Leu Gln Leu Glu Thr Ala Lys Glu Pro Cys

645 650 655

Met Ala Lys Phe Gly Pro Leu Pro Ser Lys Trp Gln Met Ala Ser Ser

660 665 670

Glu Pro Pro Cys Val Asn Lys Val Ser Asp Trp Lys Leu Glu Ile Leu

675 680 685

Gln Asn Gly Leu Tyr Leu Ile Tyr Gly Gln Val Ala Pro Asn Ala Asn

690 695 700

Tyr Asn Asp Val Ala Pro Phe Glu Val Arg Leu Tyr Lys Asn Lys Asp

705 710 715 720

Met Ile Gln Thr Leu Thr Asn Lys Ser Lys Ile Gln Asn Val Gly Gly

725 730 735

Thr Tyr Glu Leu His Val Gly Asp Thr Ile Asp Leu Ile Phe Asn Ser

740 745 750

Glu His Gln Val Leu Lys Asn Asn Thr Tyr Trp Gly Ile Ile Leu Leu

755 760 765

Ala Asn Pro Gln Phe Ile Ser

770 775

<210> 4

<211> 199

<212> Protein

<213> Homo sapiens

<400> 4

Met Thr Leu His Pro Ser Pro Ile Thr Cys Glu Phe Leu Phe Ser Thr

1 5 10 15

Ala Leu Ile Ser Pro Lys Met Cys Leu Ser His Leu Glu Asn Met Pro

20 25 30

Leu Ser His Ser Arg Thr Gln Gly Ala Gln Arg Ser Ser Trp Lys Leu

35 40 45

Trp Leu Phe Cys Ser Ile Val Met Leu Leu Phe Leu Cys Ser Phe Ser

50 55 60

Trp Leu Ile Phe Ile Phe Leu Gln Leu Glu Thr Ala Lys Glu Pro Cys

65 70 75 80

Met Ala Lys Phe Gly Pro Leu Pro Ser Lys Trp Gln Met Ala Ser Ser

85 90 95

Glu Pro Pro Cys Val Asn Lys Val Ser Asp Trp Lys Leu Glu Ile Leu

100 105 110

Gln Asn Gly Leu Tyr Leu Ile Tyr Gly Gln Val Ala Pro Asn Ala Asn

115 120 125

Tyr Asn Asp Val Ala Pro Phe Glu Val Arg Leu Tyr Lys Asn Lys Asp

130 135 140

Met Ile Gln Thr Leu Thr Asn Lys Ser Lys Ile Gln Asn Val Gly Gly

145 150 155 160

Thr Tyr Glu Leu His Val Gly Asp Thr Ile Asp Leu Ile Phe Asn Ser

165 170 175

Glu His Gln Val Leu Lys Asn Asn Thr Tyr Trp Gly Ile Ile Leu Leu

180 185 190

Ala Asn Pro Gln Phe Ile Ser

195

<210> 5

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> GS Linker

<400> 5

Gly Gly Gly Gly Ser

1 5

<---

Claims (13)

1. An antigen receptor containing a GITRL domain at the C-terminal end via a self-cleaving peptide domain and a tumor antigen-binding domain, wherein the antigen receptor has the ability to bind to its antigen. 2. The antigen receptor according to claim 1, comprising a core domain comprising a variable region of an antibody or T-cell receptor, a transmembrane domain and an intracellular domain of the TCR. 3. The antigen receptor according to claim 2, comprising a GITRL domain at the C-terminal side of the core domain via a self-cleaving peptide domain. 4. The antigen receptor according to claim 2 or 3, wherein the core domain comprises an intracellular costimulator domain. 5. The antigen receptor of claim 4, wherein the costimulator is CD28. 6. An antigen receptor according to any one of claims 1 to 5, which is a chimeric antigen receptor or a T-cell receptor. 7. The antigen receptor according to any one of claims 2 to 6, wherein the variable region is capable of binding to CD19. 8. A polynucleotide encoding an antigen receptor according to any one of claims 1 to 7, wherein the amino acid sequence of the GITRL domain is at least one member selected from the group consisting of the amino acid sequence described below (a) and the amino acid sequence described below (b): (a) the amino acid sequence shown in any part of SEQ ID NO: 4, and (b) an amino acid sequence that has at least 85% identity to the amino acid sequence shown in any part of SEQ ID NO: 4 and that is a protein capable of binding to GITR. 9. A cell comprising a polynucleotide according to claim 8, wherein the cell expresses an antigen receptor according to any one of claims 1-7, and wherein the cell is an αβ T cell with cytotoxic activity, a γδ T cell with cytotoxic activity, or a NK cell. 10. A pharmaceutical composition consisting of a cell according to claim 9 and a pharmaceutically acceptable carrier, which is intended for use in the treatment or prevention of cancer. 11. The pharmaceutical composition according to claim 10, wherein the cancer is blood cancer or pancreatic cancer.

Images