Polynucleotides

Abstract

Provided is a technique for improving the proliferative capacity of CAR-expressing cells. The invention relates to reduction of the expression of the CUL5 gene.

Description

Technical Field

[0001] The present invention relates to polynucleotides, etc., that can be used to produce cells expressing chimeric antigen receptors. Background Art

[0002] Chimeric Antigen Receptor (hereinafter also referred to as "CAR") binds to the antigen according to the specificity of the antibody, thereby causing damage to the target cells, cytokine release, and T cell division after stimulation. Through gene introduction, T cells can be given specificity, and the preparation of cells is generally easier than the case of culturing and amplifying endogenous tumor-specific T cell receptor (TCR) positive T cells. In addition, the treatment method using CAR (CAR-T therapy) is superior to antibody therapy in that it shows high cytotoxic activity and can be autonomously amplified by antigen stimulation with a single infusion, so multiple treatments are not required. In TCR gene introduction, since the complex of HLA and the peptide presented thereon is the target, treatment can only be performed if the patient has a specific HLA (HLA restriction), and CAR is more advantageous in that there is no HLA restriction. That is, as long as the target antigen is positive on tumor cells, all patients who are positive for the target antigen can be treated.

[0003] Chimeric antigen receptor gene-transferred T cell (CAR-T cell) therapy has been approved as a CD19CAR-T drug in various countries around the world. It has high clinical efficacy and is expected to be used clinically for various malignant tumors (e.g., Patent Document 1).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent No. 5312721 Summary of the invention

[0007] Technical problem to be solved by the invention

[0008] It is believed that the problem of recurrence and treatment resistance after CAR-T therapy is that CAR-T cells survive and function in the patient's body for a short time. In order to eliminate this problem, it is hoped to improve the proliferation ability of CAR-T cells (especially the proliferation ability after contact with the target antigen). In addition, it is also important to obtain CAR-T cells effectively.

[0009] Therefore, the problem of the present invention is to provide a technology for improving the proliferation ability of CAR-expressing cells. In addition, the problem of the present invention is to provide a technology for efficiently obtaining CAR-expressing cells.

[0010] Technical solutions for solving technical problems

[0011] The inventors of the present invention have conducted in-depth research in view of the above-mentioned problems, and found that by reducing the expression of the CUL5 gene, the proliferation ability of CAR-expressing cells is improved. Based on this finding, the inventors of the present invention have further studied and completed the present invention. That is, the present invention includes the following schemes.

[0012] Item 1. A polynucleotide comprising an expression cassette for a polynucleotide that inhibits CUL5 gene expression and an expression cassette for a chimeric antigen receptor.

[0013] Item 2. The polynucleotide according to Item 1, wherein the CUL5 gene expression inhibitory polynucleotide is at least one selected from the group consisting of a CUL5-specific siRNA, a CUL5-specific miRNA, and a CUL5-specific antisense polynucleotide.

[0014] Item 3. The polynucleotide according to Item 1, wherein the polynucleotide for inhibiting CUL5 gene expression is a CUL5-specific siRNA.

[0015] Item 4. The polynucleotide according to Item 1, which is a vector.

[0016] Item 5. The polynucleotide according to Item 1, which is a viral vector or a viral genome.

[0017] Item 6. The polynucleotide according to Item 1, wherein the target antigen of the chimeric antigen receptor is a tumor antigen.

[0018] Item 7. A cell comprising the polynucleotide according to any one of Items 1 to 6.

[0019] Item 8. The cell according to Item 7, wherein the expression of the CUL5 gene is reduced by the CUL5 gene expression inhibitory polynucleotide, and the cell expresses a chimeric antigen receptor.

[0020] Item 9. The cell according to Item 8, which is a lymphocyte.

[0021] Item 10. A pharmaceutical composition comprising the cell according to Item 7.

[0022] Item 11. The pharmaceutical composition according to Item 10, which is used for treating, preventing or ameliorating cancer.

[0023] Item 12. A cell altered so that expression of the CUL5 gene is reduced and expresses a chimeric antigen receptor.

[0024] Item 13. The cell according to Item 12, which is a lymphocyte.

[0025] Item 14. A pharmaceutical composition comprising the cell according to Item 12 or 13.

[0026] Item 15. The pharmaceutical composition according to Item 14, which is used for treating, preventing or ameliorating cancer.

[0027] Effects of the Invention

[0028] According to the present invention, a technology for improving the proliferation ability of CAR-expressing cells can be provided. In addition, according to a preferred embodiment of the present invention, a technology for effectively obtaining CAR-expressing cells can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram showing the CAR construct obtained in Experimental Example 1.

[0030] Figure 2 An outline of the GW-CRISPR screening (primary screening) of Experimental Example 2 is shown.

[0031] Figure 3 The results of the secondary screening of Experiment 2 are shown. day represents the number of days that have passed since the initial stimulation. The vertical axis represents the value (%) obtained by dividing the GFP-positive cell ratio (%) of day 30 by the GFP-positive cell ratio (%) of day 0. The horizontal axis represents the gRNA expressed by the cells.

[0032] Figure 4 The results of the secondary screening of Test Example 2 are shown. The vertical axis represents the ratio of GFP-positive cells (%). On the horizontal axis, D represents the number of days elapsed from the initial stimulation. *** represents P<0.001 between groups.

[0033] Figure 5 The results of Western blotting in the secondary screening of Experiment 2 show the expression level of CUL5 when gRNA targeting CUL5 is expressed. The vertical axis shows the ratio of the expression level of CUL5 to the expression level of β-actin. The horizontal axis shows the gRNA expressed. * indicates P < 0.05 between groups, and ** indicates P < 0.01 between groups.

[0034] Figure 6 The degree of cell division measured in Test Example 3 is shown. The vertical axis represents the fluorescence intensity. The lower the fluorescence intensity, the more enhanced the division. In the horizontal axis, Ctrl represents the case where the control gRNA is expressed, and CUL5 KO represents the case where the CUL5 gRNA is expressed. * indicates that P < 0.05 between groups.

[0035] Figure 7 The ratio of intracellular cytokine-positive cells measured in Test Example 3 (vertical axis) is shown. The horizontal axis represents stimulated cells. In the two columns of each stimulated cell, the left side represents the case where the control gRNA is expressed, and the right side represents the case where the CUL5 gRNA is expressed. * indicates P<0.05 between groups.

[0036] Figure 8 The left figure shows the effector memory T cells (T EM : CD45RA(-)CCR7(-)) and right panel: central memory T cells (T CM : The ratio of CD45RA(-)CCR7(+) (vertical axis). On the horizontal axis, Ctrl indicates the case where the control gRNA was expressed, and CUL5 KO indicates the case where the CUL5 gRNA was expressed. ** indicates P<0.01 between groups.

[0037] Fig. 9 The luminescence (cold light) emitted by tumor cells measured in Test Example 5 (vertical axis) is shown. The horizontal axis represents the number of days since tumor cell inoculation. Ctrl CAR represents the case where CAR-T cells expressing control gRNA are administered, CUL5KO CAR represents the case where CAR-T cells expressing CUL5 gRNA are administered, and tEGFR-T represents the case where T cells expressing tEGFR are administered.

[0038] Fig.10 It represents the survival rate (vertical axis) measured in Test Example 5. The horizontal axis represents the number of days since the inoculation of tumor cells. Ctrl CAR represents the situation where CAR-T cells expressing control gRNA were administered, CUL5KO CAR represents the situation where CAR-T cells expressing CUL5 gRNA were administered, and tEGFR-T represents the situation where T cells expressing tEGFR were administered. * indicates P < 0.05 between groups, *** indicates P < 0.001 between groups, and **** indicates P < 0.0001 between groups.

[0039] Fig.11 A schematic diagram showing the Two-in-one vector obtained in Experimental Example 6.

[0040] Fig.12 The diagram shows the scheme for preparing CAR-T cells using a two-in-one vector (before stimulation begins).

[0041] Fig.13 The number of CAR-T cells measured in Experimental Example 7 (vertical axis) is shown. The left column shows the case of method 1, and the right column shows the case of method 2 (the case of using two-in-one vector). *** indicates P<0.001 between groups.

[0042] Fig.14The results of Western blotting for measuring the expression level of CUL5 in Test Example 7 are shown. Ctrl indicates the case where shRNA against GFP was expressed, and CUL5 KD indicates the case where shRNA against CUL5 was expressed.

[0043] Fig.15 The figure shows the proliferation of CAR-T cells measured in Test Example 7. The vertical axis shows the number of CAR-T cells. The horizontal axis shows the number of days from the initial stimulation. ** indicates P<0.01 between groups.

[0044] Fig.16 The luminescence (vertical axis) emitted by tumor cells measured in Experimental Example 8 is shown. The horizontal axis indicates the number of days since tumor cell inoculation. Ctrl indicates the case where CAR-T cells expressing shRNA against GFP were administered, and CUL5KD indicates the case where CAR-T cells expressing shRNA against CUL5 were administered. Mock indicates the case where CAR-T cells were not administered.

[0045] Fig.17 The tumor volume measured in Test Example 9 (vertical axis) is shown. The horizontal axis indicates the number of days from the inoculation of tumor cells. shGFPCD19CAR-T indicates the expression of shRNA for GFP, shCUL5CD19CAR-T indicates the expression of shRNA for CUL5, and tEGFR-T indicates the administration of T cells expressing tEGFR. * indicates P < 0.05 between groups, and ** indicates P < 0.01 between groups.

[0046] Fig.18 The vertical axis represents the survival rate measured in Test Example 9. The horizontal axis represents the number of days from the inoculation of tumor cells. shGFPCD19CAR-T represents the case where shRNA targeting GFP is expressed, shCUL5CD19CAR-T represents the case where shRNA targeting CUL5 is expressed, and tEGFR-T represents the case where T cells expressing tEGFR are administered. * indicates that P < 0.05 between groups.

[0047] Fig.19 The luminescence images of Experimental Example 11 are shown. The left side of the photo shows the number of days that have passed since tumor cell inoculation. The top of the photo shows the intracellular domain and shRNA used.

[0048] Fig. 20 The vertical axis represents the tumor volume measured in Test Example 11. The horizontal axis represents the number of weeks from tumor cell inoculation. The legend represents the intracellular domain and shRNA used. DETAILED DESCRIPTION

[0049] In the present specification, the expressions about “containing” and “comprising” include the concepts of “containing”, “including”, “consisting essentially of” and “consisting only of”.

[0050] 1. Definition

[0051] In the present specification, the expressions about “containing” and “comprising” include the concepts of “containing”, “including”, “consisting essentially of” and “consisting only of”.

[0052] The "identity" of an amino acid sequence refers to the degree of consistency of two or more amino acid sequences that can be compared, relative to each other. Therefore, the higher the consistency of two amino acid sequences, the higher the identity or similarity of these sequences. The level of identity of an amino acid sequence can be determined, for example, using the sequence analysis tool FASTA, using default parameters. Alternatively, the algorithm BLAST of Karlin and Altschul (KarlinS, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87: 2264-2268 (1990), KarlinS, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90: 5873-7 (1993)) can be used to determine. A program called BLASTX based on such an algorithm of BLAST has been developed. The specific methods of these analysis methods are well known, and can be found on the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ). In addition, the "identity" of the base sequence is also defined as above.

[0053] In this specification, "conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine are conservative substitutions. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also conservative substitutions.

[0054] In this specification, "nucleic acid" and "polynucleotide" are not particularly limited, including any natural or artificial ones. Specifically, in addition to DNA, RNA, etc., "nucleic acid" and "polynucleotide" that have been subjected to known chemical modifications as exemplified below can also be used. In order to prevent decomposition caused by hydrolases such as nucleases, the phosphate residues (phosphates) of each nucleotide can be replaced by chemically modified phosphate residues such as thiophosphates (PS), methylphosphonates, dithiophosphates, etc. In addition, the hydroxyl group at the 2nd position of the sugar (ribose) of each ribonucleotide can also be replaced by -OR (R, for example, represents CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, CH2CONHCH3, CH2CH2CN, etc.). In addition, chemical modifications can also be applied to the base part (pyrimidine, purine), for example, the introduction of a methyl or cationic functional group to the 5th position of the pyrimidine base, or the substitution of the carbonyl group at the 2nd position by a thiocarbonyl group, etc. can be cited. In addition, examples include, but are not limited to, products in which the phosphate part or the hydroxyl part is modified by, for example, biotin, amino group, lower alkylamino group, acetyl group, etc. In addition, BNA (LNA) etc. can be used in which the conformation of the sugar part is fixed to the N type by cross-linking the 2' oxygen and 4' carbon of the sugar part of the nucleotide.

[0055] In this specification, "CDR" means C omplementarity D eTermining RCDR is the abbreviation of complementarity determining region, also known as complementarity determining region. CDR refers to the region present in the variable region of immunoglobulin or T cell receptor, and is a region closely related to the specific binding of antibodies or T cell receptors to antigens. In addition, "light chain CDR" refers to the CDR present in the light chain variable region of immunoglobulin, and "heavy chain CDR" refers to the CDR present in the heavy chain variable region of immunoglobulin. In T cell receptors, there are also α chain, β chain, γ chain, δ chain, etc., and the same is true for these CDRs.

[0056] In this specification, "variable region" refers to a region containing CDR1 to CDR3 (hereinafter referred to as "CDRs1-3"). The order of configuration of these CDRs1-3 is not particularly limited, and preferably refers to a region configured in the direction from the N-terminal side to the C-terminal side, in the order of CDR1, CDR2 and CDR3, or in the opposite order, continuously or via other amino acid sequences referred to as framework regions (FR) described later. In addition, a "heavy chain variable region" refers to a region in which the above-mentioned heavy chain CDRs1-3 are configured in an immunoglobulin, and a "light chain variable region" refers to a region in which the above-mentioned light chain CDRs1-3 are configured in an immunoglobulin. In T cell receptors, there are also α chains, β chains, γ chains, δ chains, etc., and the same is true for these CDRs.

[0057] The regions other than the above CDR1-3 of each variable region are referred to as framework regions (FR) as described above. In particular, the region between the N-terminus of the variable region and the above CDR1 is defined as FR1, the region between CDR1 and CDR2 is defined as FR2, the region between CDR2 and CDR3 is defined as FR3, and the region between CDR3 and the C-terminus of the variable region is defined as FR4.

[0058] 2. Polynucleotides

[0059] In one embodiment, the present invention relates to a polynucleotide (also referred to as "polynucleotide of the present invention" in this specification) comprising an expression cassette for a polynucleotide for inhibiting CUL5 gene expression and an expression cassette for a chimeric antigen receptor. This will be described below.

[0060] The polynucleotide of the present invention comprises an expression cassette of a polynucleotide that inhibits the expression of the CUL5 gene. By using the polynucleotide of the present invention comprising an expression cassette of a polynucleotide that inhibits the expression of the CUL5 gene to prepare CAR-expressing cells, the proliferation ability of CAR-expressing cells can be improved. The CUL5 gene is a gene that expresses Cullin-5 mRNA / protein. Cullin-5 protein is a core component of multiple SCF-like ECS (Elongin-Cullin 2 / 5-SOCS-boxprotein) E3 ubiquitin-protein ligase complexes, mediating the ubiquitination of target proteins and subsequent proteasomal degradation. CUL5 gene expression inhibition is to inhibit the expression of Cullin-5 mRNA / protein. There is no particular limitation on the source biological species of the CUL5 gene, and various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer can be cited. The human CUL5 gene is a gene with NCBI gene ID: 8065.

[0061] The amino acid sequences of Cullin-5 proteins and the base sequences of Cullin-5 mRNA from various biological species are well known. Specifically, for example, as human Cullin-5 protein, a protein consisting of the amino acid sequence shown in sequence number 30 (NCBI Reference Sequence: NP_003469.2) can be listed, and as human Cullin-5 mRNA, an mRNA consisting of the base sequence shown in sequence number 31 can be listed (NCBI Reference Sequence: NM_003478.6). In addition, as Cullin-5 protein and Cullin-5 mRNA, the above-mentioned splicing variants can also be included.

[0062] The Cullin-5 protein expressed by the CUL5 gene to be inhibited may form an E3 ubiquitin-protein ligase complex having its original activity, i.e., ubiquitin E3 ligase activity, and may have amino acid mutations such as substitution, deletion, addition, and insertion. As the mutation, from the viewpoint that the activity is less likely to be impaired, substitution is preferred, and conservative substitution is more preferred.

[0063] Cullin-5 mRNA expressed from the CUL5 gene to be inhibited may have base mutations such as substitution, deletion, addition, and insertion, as long as the protein translated from the mRNA forms an E3 ubiquitin-protein ligase complex having its original activity, i.e., ubiquitin E3 ligase activity. As mutations, mutations that do not cause amino acid substitutions or mutations that cause conservative amino acid substitutions in the protein translated from the mRNA are preferred.

[0064] Preferred specific examples of the Cullin-5 protein expressed by the CUL5 gene to be inhibited include at least one selected from the group consisting of the proteins described in (A) below and the proteins described in (B) below:

[0065] (A) a protein consisting of the amino acid sequence represented by any one of SEQ ID NO: 30, and

[0066] (B) A protein consisting of an amino acid sequence having 85% or more identity with the amino acid sequence represented by any one of SEQ ID NO: 30, and forming an E3 ubiquitin-protein ligase complex having ubiquitin E3 ligase activity.

[0067] In the above (B), the identity is more preferably 90% or more, further preferably 95% or more, and even more preferably 98% or more.

[0068] Examples of the protein described in (B) above include:

[0069] (B') A protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted into the amino acid sequence represented by any one of SEQ ID NO: 30, and which forms an E3 ubiquitin-protein ligase complex having ubiquitin E3 ligase activity.

[0070] In the above (B'), a plurality of means, for example, 2 to 50, preferably 2 to 20, more preferably 2 to 10, and further preferably 2 or 3.

[0071] Preferred specific examples of Cullin-5 mRNA expressed from the CUL5 gene to be inhibited include at least one selected from the group consisting of mRNA described in (C) below and mRNA described in (D) below:

[0072] (C) an mRNA consisting of a base sequence represented by any one of SEQ ID NO: 31, and

[0073] (D) An mRNA consisting of a base sequence having 85% or more identity with the base sequence represented by any one of SEQ ID NO: 31 and encoding a protein that forms an E3 ubiquitin-protein ligase complex having ubiquitin E3 ligase activity.

[0074] In the above (D), the identity is more preferably 90% or more, further preferably 95% or more, and even more preferably 98% or more.

[0075] Examples of the mRNA described in (D) above include:

[0076] (D') mRNA consisting of a base sequence in which one or more bases are substituted, deleted, added or inserted into the base sequence represented by any one of SEQ ID NO: 31, and encoding a protein that forms an E3 ubiquitin-protein ligase complex having ubiquitin E3 ligase activity.

[0077] In the above (D′), a plurality of means, for example, 2 to 200, preferably 2 to 100, more preferably 2 to 50, and further preferably 2 to 10.

[0078] The CUL5 gene expression inhibiting polynucleotide is not particularly limited as long as it can inhibit the expression level of CUL5 protein, CUL5 mRNA, etc., and examples thereof include CUL5-specific small interfering RNA (siRNA), CUL5-specific microRNA (miRNA), and CUL5-specific antisense polynucleotides.

[0079] Here, expression inhibition refers to suppressing the expression level of Cullin-5 protein to, for example, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, and also includes reducing the expression level to zero.

[0080] As long as the CUL5-specific siRNA is a double-stranded RNA molecule that specifically inhibits the expression of the CUL5 gene, there is no particular limitation. In one embodiment, the siRNA is preferably, for example, more than 18 bases, more than 19 bases, more than 20 bases, or more than 21 bases in length. In addition, the siRNA is preferably, for example, less than 25 bases, less than 24 bases, less than 23 bases, or less than 22 bases in length. The upper and lower limits of the length of the siRNA described herein can be conceived of any combination. For example, one can imagine a length with a lower limit of 18 bases and an upper limit of 25 bases, 24 bases, 23 bases or 22 bases; a length with a lower limit of 19 bases and an upper limit of 25 bases, 24 bases, 23 bases or 22 bases; a length with a lower limit of 20 bases and an upper limit of 25 bases, 24 bases, 23 bases or 22 bases; a combination of lengths with a lower limit of 21 bases and an upper limit of 25 bases, 24 bases, 23 bases or 22 bases.

[0081] siRNA can be shRNA (small hairpin RNA). shRNA can be designed to form a stem-loop structure in part. For example, if the sequence of a certain region of shRNA is used as sequence a and the complementary strand to sequence a is used as sequence b, it can be designed in the order of sequence a, spacer, and sequence b, and these sequences exist on one RNA strand so that the whole becomes 45 to 60 bases in length. Sequence a is a sequence of a part of the base sequence of CUL5 encoding the target, and the target region is not particularly limited, and any region can be used as a candidate. And the length of sequence a is 19 to 25 bases, preferably 19 to 21 bases.

[0082] CUL5-specific siRNA may have an added base at the 5' or 3' end. The length of the added base is usually about 2 to 4 bases. The added base may be DNA or RNA, but when DNA is used, the stability of the nucleic acid can sometimes be improved. As the sequence of such added bases, for example, sequences such as ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', uuuuu-3' can be cited, but are not limited to these.

[0083] siRNA can have an overhang sequence (overhang) at the 3' end, and specifically, siRNA with added dTdT (dT represents deoxythymidine) can be listed. In addition, it can also be a smooth end (blunt end) without added ends. In siRNA, the sense strand and the antisense strand can have different numbers of bases, and for example, "asymmetrical interfering RNA, asymmetric interfering RNA (aiRNA)" can be listed in which the antisense strand has an overhang sequence (overhang) at the 3' end and the 5' end. In a typical aiRNA, the antisense strand is composed of 21 bases, the sense strand is composed of 15 bases, and there are overhang structures of 3 bases at both ends of the antisense strand.

[0084] The position of the target sequence of CUL5-specific siRNA is not particularly limited, but in one embodiment, the target sequence is preferably selected from the region of about 50 bases from the 5'-UTR and the start codon, and the region outside the 3'-UTR. For the candidate group of the selected target sequence, it is preferred to use homology search software such as BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) to investigate whether the continuous sequence of 16-17 bases in the mRNA other than the target has homology, and confirm the specificity of the selected target sequence. For the target sequence confirmed to be specific, a double-stranded RNA consisting of a sense strand with a 3' end overhang of TT or UU in the 19-21 bases after AA (or NA), and an antisense strand with a sequence complementary to the 19-21 bases and a 3' end overhang of TT or UU can be designed as siRNA. In addition, shRNA, which is a precursor of siRNA, can be designed by appropriately selecting an arbitrary linker sequence (eg, about 5 to 25 bases) that can form a loop structure and linking the sense strand and antisense strand via the linker sequence.

[0085] The sequences of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such websites include the following.

[0086] siRNA Target Finder provided by Ambion (http: / / www.ambion.com / jp / techlib / misc / siRNA_finder.html) pSilencer (registered trademark) Expression Vector Insert Design Tool (http: / / www.ambion.com / jp / techlib / misc / psilencer_converter.html) GeneSeer provided by RNAi Codex (http: / / codex.cshl.edu / scripts / newsearchhairpin.cgi).

[0087] siRNA can be prepared by synthesizing the sense strand and antisense strand of the target sequence on mRNA using a DNA / RNA automatic synthesizer, denaturing at about 90°C to about 95°C for about 1 minute in an appropriate annealing buffer, and then annealing at about 30°C to about 70°C for about 1 to about 8 hours. Alternatively, shRNA, which is a precursor of siRNA, can be synthesized and cleaved with an RNA-cleaving protein endoribonuclease (dicer).

[0088] CUL5-specific miRNA can be any as long as it inhibits the translation of the CUL5 gene. For example, miRNA does not cut the target mRNA like siRNA, but can pair with the 3' untranslated region (UTR) of the target to inhibit its translation. The miRNA can be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA) and mature miRNA. The length of miRNA is not particularly limited. The length of pri-miRNA is usually hundreds to thousands of bases, the length of pre-miRNA is usually 50 to 80 bases, and the length of mature miRNA is usually 18 to 30 bases. In one embodiment, CUL5-specific miRNA is preferably pre-miRNA or mature miRNA, more preferably mature miRNA. Such CUL5-specific miRNA can be synthesized by a known method or purchased from a company that provides synthetic RNA.

[0089] CUL5 specific antisense polynucleotide refers to a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the mRNA of the CUL5 gene or a portion thereof, and having the function of inhibiting the synthesis of the CUL5 protein by forming a specific and stable double-stranded chain and binding with the mRNA. The antisense polynucleotide can be, for example, DNA, RNA or a DNA / RNA chimera. In the case where the antisense polynucleotide is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) and causes the selective decomposition of the target RNA. Therefore, in the case of antisense DNA directed to decompose by RNase H, the target sequence can be not only a sequence in the mRNA, but also a sequence in the intron region of the primary translation product of the CUL5 gene. The intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the CUL5 gene using homology detection programs such as BLAST and FASTA.

[0090] The target region of the CUL5-specific antisense polynucleotide is a region that inhibits translation to the CUL5 protein as a result of hybridization by the antisense polynucleotide, and its length is not limited. The CUL5-specific antisense polynucleotide can be the entire sequence of the mRNA encoding CUL5 or a partial sequence. If the ease of synthesis and antigenicity, intracellular transferability, etc. are considered, an oligonucleotide consisting of about 10 to about 40 bases, especially about 15 to about 30 bases, is preferably used, but is not limited to these. More specifically, the 5' hairpin loop, 5' untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3' untranslated region, 3' palindromic region or 3' hairpin loop of the CUL5 gene can be selected as the preferred target region of the antisense polynucleotide, but is not limited to them.

[0091] The CUL5-specific antisense polynucleotide may be a polynucleotide (antigene) that not only hybridizes with the mRNA or primary transcript of the CUL5 gene to inhibit translation into protein but also binds to the gene as a double-stranded DNA to form a triplex and is capable of inhibiting transcription into RNA.

[0092] CUL5-specific siRNA, CUL5-specific miRNA, and CUL5-specific antisense polynucleotides can be prepared by determining the target sequence of mRNA or primary transcription product based on the cDNA sequence or genomic DNA sequence of the CUL5 gene, and synthesizing a complementary sequence thereto using a commercially available DNA / RNA automatic synthesizer. In addition, various modified antisense polynucleotides can also be chemically synthesized by known methods.

[0093] The expression cassette (expression cassette 1) of the CUL5 gene expression inhibiting polynucleotide comprises a promoter and a CUL5 gene expression inhibiting polynucleotide coding sequence under the control of the promoter. In order to be under the control of the promoter, the coding sequence is usually configured downstream of the promoter. As promoters that can be used, for example, RNA polymerase II (poII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, etc.; RNA polymerase III (polIII) promoters such as mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, etc., among which, from the viewpoint of being able to correctly transcribe short RNA, polIII promoters are preferred. The promoter is operably connected to the coding sequence. Among them, "the promoter is operably connected to the coding sequence" has the same meaning as "the coding sequence is configured under the control of the promoter", and usually, the coding sequence is connected directly or via other sequences on the 3' end side of the promoter. A poly A addition signal sequence is placed downstream of the coding sequence. Transcription is terminated by using the poly A addition signal sequence. As the poly A addition signal sequence, the poly A addition sequence of SV40, the poly A addition sequence from the bovine growth hormone gene, etc. can be used.

[0094] The polynucleotide of the present invention comprises an expression cassette of a chimeric antigen receptor. By preparing CAR-expressing cells using the polynucleotide of the present invention comprising an expression cassette of a chimeric antigen receptor, CAR-expressing cells can be efficiently obtained.

[0095] There are no particular limitations on chimeric antigen receptors as long as they can bind to a target antigen and transmit signals required for activation of immune cells into cells through binding to the target antigen. Chimeric antigen receptors typically include an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0096] As long as the antigen-binding domain is configured outside the cell when the chimeric antigen receptor is configured on the cell membrane, and recognizes the antigen, the domain that can bind to the antigen can be used without particular limitation. As antigens, specifically, for example, CD19, GD2, GD3, CD20, CD37, CEA, HER2, EGFR, type III mutant EGFR, CD38, BCMA, MUC-1, PSMA, WT1, cancer testis antigen (cancer testis antigen) (such as NY-ESO-1, MAGE-A4, etc.), mutant peptides (mutation peptide) (such as k-ras, h-ras, p53, etc.), hTERT, PRAM, TYRP1, mesothelin, PMEL, mucin, etc., and complexes of these fragments and MHC (pMHC), etc., can be cited. Among these, in one embodiment of the present invention, CD19 is preferably cited. The antigen-binding domain generally has one or more CDRs of an antibody or T cell receptor for the antigen (for example, if it is an antibody CDR, it is heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3), preferably 2, 3, 4, 5 or more, and more preferably all 6. The antigen-binding region more preferably comprises a variable region of an antibody or T cell receptor for the antigen (for example, a heavy chain variable region and / or a light chain variable region in the case of an antibody).

[0097] The antigen-binding domain preferably adopts a single-chain antibody structure, and more preferably adopts an scFv structure. In the case where the scFv structure includes a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are usually connected via a linker. The linker is not particularly limited as long as it does not significantly impair the antigen binding, and is arbitrary. As a linker, a linker composed of glycine or glycine and serine (GGS linker, GS linker, GGG linker, etc.) is preferably used. The length of the linker is not particularly limited. The number of amino acid residues in the linker is, for example, 5 to 30, preferably 10 to 25. There is no particular limitation on the configuration relationship between the heavy chain variable region and the light chain variable region, and both the mode in which the N-terminal side is the light chain variable region and the case in which the N-terminal side is the heavy chain variable region can be adopted. The configuration relationship is preferably the former.

[0098] As long as the transmembrane domain is a domain configured in the cell membrane when the chimeric antigen receptor of the present invention is configured on the cell membrane, it can constitute a chimeric antigen receptor, without particular limitation. As a transmembrane domain, for example, transmembrane regions such as CD28, CD3ε, CD8α, CD3, CD4, 4-1BB can be used. The transmembrane region of each factor is known, or can be easily determined by known sequence information (such as using a prediction program for a transmembrane region, etc.). In addition, an artificially constructed transmembrane domain composed of a polypeptide can also be used. These transmembrane domains can also be appropriately introduced into mutations as long as they do not significantly damage the function of the chimeric antigen receptor.

[0099] As the transmembrane domain, the transmembrane region of CD28 can be preferably used. As a specific example of the transmembrane region of CD28, the amino acid sequence described in (a) below or the amino acid sequence described in (b) below can be cited:

[0100] (a) the amino acid sequence shown in SEQ ID NO: 3, or

[0101] (b) An amino acid sequence that has 85% or more identity with the amino acid sequence of SEQ ID NO: 3 and that can be located in the cell membrane when the chimeric antigen receptor of the present invention is located on the cell membrane.

[0102] In the above (b), the identity is preferably 90% or more, more preferably 95% or more, further preferably 98% or more, and particularly preferably 99% or more.

[0103] An example of the amino acid sequence described in (b) above includes:

[0104] (b') An amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted into the amino acid sequence of SEQ ID NO: 3 and which can be located in the cell membrane when the chimeric antigen receptor of the present invention is located on the cell membrane.

[0105] In the above (b'), a plurality of means, for example, 2 to 5, preferably 2 to 3, and more preferably 2.

[0106] In one embodiment of the present invention, the antigen-binding domain and the transmembrane domain are preferably connected directly or via a relatively short spacer. In the chimeric antigen receptor of the present invention, the number of constituent amino acid residues between the antigen-binding domain and the transmembrane domain can be, for example, 400 or less, 300 or less, or 250 or less, and particularly preferably 100 or less. The number of constituent amino acid residues is preferably 0 to 60, more preferably 0 to 40, and further preferably 0 to 30. In a particularly preferred embodiment of the present invention, the number of amino acid residues is 1 or more, 4 or more, or 8 or more, and further 25 or less, 20 or less, or 15 or less, and further 1 to 25, 4 to 20, or 8 to 15. The sequence of the spacer is not particularly limited, and sequences such as a hinge portion or a portion thereof of IgG, preferably human IgG (e.g., subtype IgG1 or IgG4), a hinge portion and a portion of CH2, or a portion of a factor (e.g., CD28) used in a transmembrane domain can be used in the spacer. In addition, by utilizing a hinge portion or a portion thereof, it is possible to expect a flexible spacer.

[0107] The intracellular signaling domain is a domain that is configured in the cell when the chimeric antigen receptor of the present invention is configured on the cell membrane, and is a domain that can transmit the signal necessary for the effector function of the immune cell, that is, when the antigen binding domain binds to the antigen, the signal required for the activation of the immune cell can be transmitted. The intracellular signaling domain preferably includes an intracellular activation domain. As an intracellular activation domain, for example, in addition to CD3ζ, intracellular domains such as FcεRIγ can also be used. CD3ζ is preferably used. CD3 can be appropriately introduced into mutations as long as it does not hinder the function of the antigen receptor. In the case of introducing mutations in CD3, it is preferably carried out in a manner comprising ITAM (immunoreceptortyrosine-basedactivation motif, immunoreceptor tyrosine activation motif).

[0108] Specific examples of the intracellular activation domain include the amino acid sequence described in (c) below or the amino acid sequence described in (d) below:

[0109] (c) the amino acid sequence shown in SEQ ID NO: 5, or

[0110] (d) An amino acid sequence that has 85% or more identity with the amino acid sequence shown in SEQ ID NO: 5 and has an immune cell activating effect.

[0111] In the above (d), the identity is preferably 90% or more, more preferably 95% or more, further preferably 98% or more, and particularly preferably 99% or more.

[0112] An example of the amino acid sequence described in (d) above includes:

[0113] (d') An amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted into the amino acid sequence of SEQ ID NO: 5 and which has an immune cell activating effect.

[0114] In the above (d'), the plurality of elements is, for example, 2 to 15, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 to 3.

[0115] The intracellular signaling domain preferably includes the intracellular domain of a costimulatory factor. The intracellular domain of a costimulatory factor is not particularly limited as long as it is the intracellular domain of a costimulatory factor from T cells, etc. For example, one or more intracellular domains selected from OX40, 4-1BB, GITR, CD79a, CD40, CD27, CD278, and CD28, etc., can be appropriately selected for use. In one embodiment of the present invention, a domain formed by connecting the intracellular domain of CD79a to the intracellular domain of CD40 in series can be used.

[0116] Specific examples of the intracellular domain include the amino acid sequence described in (e) below or the amino acid sequence described in (f) below:

[0117] (e) the amino acid sequence represented by any one of SEQ ID NOs: 4 and 30 to 32, or

[0118] (f) An amino acid sequence that has 85% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 4 and 30 to 32 and has an immune cell activating effect.

[0119] In the above (f), the identity is preferably 90% or more, more preferably 95% or more, further preferably 98% or more, and particularly preferably 99% or more.

[0120] An example of the amino acid sequence described in (f) above includes:

[0121] (f') An amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted into the amino acid sequence of SEQ ID NO: 4 and which has an immune cell activating effect.

[0122] In the above (f'), the plurality of elements is, for example, 2 to 15, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 to 3.

[0123] The chimeric antigen receptor of the present invention may include other regions other than those described above. As other regions, for example, a leader sequence (signal peptide) (e.g., a leader sequence of a GM-CSF receptor) for promoting the transport of CAR to the cell membrane, a spacer / linker (e.g., between a transmembrane region and an intracellular signaling domain, between domains within an intracellular signaling domain), etc. can be cited.

[0124] In addition, there are several reports on experiments and clinical studies using CAR (e.g., Rossig C, et al. Mol Ther 10:5-18, 2004; Dotti G, et al. Hum Gene Ther 20:1229-1239, 2009; Ngo MC, et al. Hum Mol Genet 20(R1):R93-99, 2011; Ahmed N, et al. Mol Ther 17:1779-1787, 2009; Pule MA, et al. Nat Med 14:1264-1270, 2008; Louis CU, et al. Blood 118:6050-6056, 2011; Kochenderfer JN, et al. Blood 116:4099-4102, 2010; Kochenderfer JN, et al. Blood 119:2709-2720,2012; Porter DL,et al.N Engl J Med 365:725-733,2011; Kalos M,et al.Sci Transl Med 3:95ra73,2011; Brentjens RJ,et al.Blood 118:4817-4828,2011; Brentjens RJ,et al.Sci Transl Med 5:177ra38,2013), the chimeric antigen receptor of the present invention can be constructed with reference to these reports.

[0125] A particularly preferred embodiment of the chimeric antigen receptor of the present invention is a chimeric antigen receptor comprising a core region in which an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain are sequentially arranged, wherein the domains are connected directly or via a spacer / linker.

[0126] The chimeric antigen receptor of the present invention may be a receptor of a protein or peptide to which a known protein tag, signal sequence, etc. is added. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, fluorescent protein tag, etc.

[0127] The expression cassette (expression cassette 2) of the chimeric antigen receptor comprises a promoter and a chimeric antigen receptor coding sequence under the control of the promoter. In order to be under the control of the promoter, the coding sequence is usually configured downstream of the promoter. As a promoter that can be used, for example, RNA polymerase II (poII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, etc. can be cited. The promoter is operably connected to the coding sequence. Here, "the promoter is operably connected to the coding sequence" has the same meaning as "the coding sequence is configured under the control of the promoter". Usually, the coding sequence is connected directly or via other sequences on the 3' end side of the promoter. Poly A is configured downstream of the coding sequence. By using the Poly A addition signal sequence, transcription is terminated. As the Poly A addition signal sequence, the Poly A addition sequence of SV40, the Poly A addition sequence from the growth hormone gene of cattle, etc. can be used.

[0128] The polynucleotide of the present invention may contain other sequences in addition to expression cassette 1 and expression cassette 2. Examples of other sequences include enhancer sequences, repressor sequences, insulator sequences, replication origins, reporter protein (e.g., fluorescent protein, etc.) coding sequences, and drug resistance gene coding sequences.

[0129] The expression cassette may also contain detection genes (reporter genes, cell or tissue specific genes, selection marker genes, etc.), enhancer sequences, WRPE sequences, etc. The detection gene can be used in the introduction of the expression cassette or the efficiency determination, the expression detection of the CAR gene or the determination of the expression efficiency, the selection and separation preparation of cells expressing the CAR gene, etc. On the other hand, the expression efficiency can be improved by using the enhancer sequence. As detection genes, the neo gene that confers resistance to neomycin, the npt gene (Herrera Estrella, EMBO J. 2 (1983), 987-995) or nptII gene (Messing & Vierra. Gene 1 9:259-268 (1982)) that confers resistance to kanamycin and the like, the hph gene (Blochinger & Digglmann, Mol Cell Bio 4:2929-2931) that confers resistance to hygromycin, the dhfr gene (Bourouis et al., EMBO J. 2 (7)) that confers resistance to methotrexate, etc. (these are marker genes), the luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), the β-glucuronidase (GUS) gene, GFP (Gerdes, FEBS Lett.389 (1996), 44-47) or its variants (EGFP or d2EGFP, etc.) and other fluorescent protein genes (the above are reporter genes), genes lacking the intracellular domain of epithelial growth factor receptor (EGFR) gene, etc. The detection gene is connected to the CAR gene, for example, via a bicistronic control sequence (such as an internal ribosomal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is a 2A peptide (T2A) from Thosea asignavirus (bright vein flat moth virus), but is not limited to this. As self-cleaving peptides, 2A peptides (F2A) from foot disease virus (FMDV), 2A peptides (E2A) from horse armor rhinitis virus (ERAV), and 2A peptides (P2A) from porcine teschovirus (PTV-1) are known.

[0130] The polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide (vector, etc.). The vector may be a plasmid vector or a viral vector. In addition, the vector may be, for example, a cloning vector or an expression vector. As expression vectors, vectors for prokaryotic cells such as Escherichia coli or actinomycetes, or vectors for eukaryotic cells such as yeast cells, insect cells or mammalian cells may be cited. More specifically, as viral vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, Sendai virus vectors, etc. may be cited. As examples of non-viral vectors, various plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417, 1987), YAC vectors, BAC vectors may be cited.

[0131] From the viewpoint of being able to reduce the damage caused to the cells when the cells of the present invention are obtained as described later, the polynucleotide of the present invention is preferably a viral vector or a viral genome. The polynucleotide of the present invention is more preferably a retroviral vector or a retroviral genome, or a lentiviral vector or a lentiviral genome. The polynucleotide of the present invention is particularly preferably a lentiviral vector or a lentiviral genome.

[0132] 3. Cells

[0133] The present invention, in one embodiment, relates to a cell comprising a polynucleotide of the present invention (sometimes referred to as "cell 1 of the present invention" in this specification). In addition, the present invention, in one embodiment, relates to a cell that has been altered so that the expression of the CUL5 gene is reduced and that expresses a chimeric antigen receptor (sometimes referred to as "cell 2 of the present invention" in this specification). In this specification, cell 1 of the present invention and cell 2 of the present invention are sometimes collectively referred to as "cells of the present invention". These are described below.

[0134] 3-1. Cell 1 of the present invention

[0135] The cell 1 of the present invention can be obtained by a method including the step of introducing the polynucleotide of the present invention into a cell. In one embodiment of the cell, the expression of the CUL5 gene is reduced by the CUL5 gene expression-inhibiting polynucleotide, and the chimeric antigen receptor is expressed. The expression level of the Cullin-5 protein in the cell 1 of the present invention can be, for example, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less relative to the expression level of the Cullin-5 protein in a control cell (a cell treated under the same conditions except that the polynucleotide of the present invention is not introduced).

[0136] The method for introducing the polynucleotide of the present invention into cells is not particularly limited, and from the perspective of reducing damage to cells and further improving the production efficiency and proliferation ability of CAR-expressing cells, it is preferably a method of introducing a virus containing the polynucleotide of the present invention. Thus, it is possible to introduce without using methods such as electroporation or lipofection that may damage cells.

[0137] As cells (CAR expression cells) for introducing the polynucleotides of the present invention, it is possible to list: CD4 positive CD8 negative T cells, CD4 negative CD8 positive T cells, T cells prepared from iPS cells, αβ-T cells, γδ-T cells, NK cells, NKT cells, etc. As long as lymphocytes or progenitor cells as described above are included, various cell populations can be used. PBMC (peripheral blood mononuclear cells) collected from peripheral blood are one of the preferred target cells. That is, in a preferred embodiment, PBMC is subjected to a gene introduction operation. PBMC can be prepared according to or based on conventional methods.

[0138] It is preferred that the CAR expression cells are activated in advance before the polynucleotide of the present invention is introduced. For example, anti-CD3 antibodies and anti-CD28 antibodies can be used to stimulate the CAR expression cells. For example, it is possible to culture the culture container (e.g., a culture dish) coated with anti-CD3 antibodies and anti-CD28 antibodies on the culture surface, and apply the stimulation brought by anti-CD3 antibodies and anti-CD28 antibodies. The stimulation can also be performed using magnetic beads coated with anti-CD3 antibodies and anti-CD28 antibodies (e.g., Dynabeads T-Activator CD3 / CD28 provided by VERITAS).

[0139] In order to improve the survival rate / proliferation rate of cells, it is preferred to use a culture solution to which a T cell proliferation factor has been added during the activation treatment. As T cell proliferation factors, IL-2, IL-15, IL-7, etc. can be used. T cell proliferation factors such as IL-2, IL-15, IL-7 can be prepared according to a conventional method. In addition, commercially available products can also be used. Although the use of T cell proliferation factors of animal species other than humans is not excluded, T cell proliferation factors generally use factors from humans (which may be recombinants).

[0140] Typically, for its application (administration to a patient), the cells after the polynucleotide of the present invention is introduced (the polynucleotide of the present invention is introduced into lymphocytes) are proliferated. For example, the polynucleotide of the present invention is cultured into lymphocytes (subcultured as needed) using a culture fluid to which a T cell proliferation factor is added. It is also possible to perform the same treatment (reactivation) as in the case of activation of CAR expression cells on the basis of this culture.

[0141] In addition, the culture of CAR expression cells and polynucleotides of the present invention into lymphocytes can also use a medium supplemented with serum (human serum, fetal bovine serum, etc.), but by using a serum-free medium, it is possible to prepare cells with high safety during clinical application and less prone to differences in culture efficiency due to differences in serum batches. In the case of using serum, it is preferred to use autologous serum, i.e., serum collected from patients who have received CAR gene introduction into lymphocytes.

[0142] 3-2. Cell 2 of the present invention

[0143] By changing the cell in a manner that reduces the expression of the CUL5 gene and then making it express a chimeric antigen receptor, the cell 2 of the present invention can be obtained. The expression amount of the Cullin-5 protein in the cell 2 of the present invention relative to the expression amount of the Cullin-5 protein in the control cell (cells obtained by treatment under the same conditions except that the expression of the CUL5 gene is not changed in a manner that reduces the expression of the CUL5 gene) can be, for example, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10%. By reducing the expression of the CUL5 gene, the proliferation ability of CAR-expressing cells can be improved. As changes that reduce the expression of the CUL5 gene, for example, gene defects (gene disruption), mutations in protein coding regions, mutations in splicing regulatory regions, mutations in expression control regions (such as promoters, activators, enhancers, etc.) can be listed. This change can be carried out, for example, using a gene editing system such as the CRSPR / Cas system.

[0144] The method for expressing the chimeric antigen receptor is not particularly limited. For example, the chimeric antigen receptor can be expressed by introducing a polynucleotide containing an expression cassette for the chimeric antigen receptor into cells.

[0145] For matters not described above, the description of "3-1. Cell 1 of the present invention" is cited.

[0146] 3-3. Cells of the present invention

[0147] The source cells of the cells of the present invention are not particularly limited. As long as the cells of the present invention are used for purification of the chimeric antigen receptor of the present invention, the source cells include cells that can be used for protein expression (e.g., insect cells, eukaryotic cells, mammalian cells, etc.).

[0148] The cells of the present invention are preferably lymphocytes (e.g., T cells (e.g., CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, αβ-T cells, γδ-T cells, etc.), NK cells, NKT cells, etc.). These cells are preferably cells expressing the chimeric antigen receptor of the present invention. In a more specific embodiment, in these cells, the chimeric antigen receptor of the present invention is expressed on the cell membrane, and preferably the chimeric antigen receptor of the present invention is expressed in a state where the antigen-binding domain is exposed outside the cell membrane.

[0149] After T cells expressing chimeric antigen receptors recognize antigens in the antigen-binding region, the recognition signal is transmitted to the interior of the T cells, activating the signal that induces cytotoxic activity. In conjunction with this, the cells can attack other cells or tissues expressing the antigen or exert cytotoxic activity.

[0150] In the case where the cell that performs such a function is CTL, it is referred to as a chimeric antigen receptor T cell (CAR-T cell). NK cells and other cells that have the possibility of performing cytotoxic activity are also like chimeric antigen receptor T cells, and can perform cytotoxic activity by binding to antigens through antigen binding regions. Therefore, a host cell (particularly a host cell with cytotoxic activity) comprising a polynucleotide encoding a chimeric antigen receptor of the present invention is useful as an active ingredient of a pharmaceutical composition.

[0151] 4. Pharmaceutical compositions

[0152] The present invention, in one embodiment, relates to a pharmaceutical composition containing the cells of the present invention (sometimes referred to as "the pharmaceutical composition of the present invention" in this specification). This is described below.

[0153] The pharmaceutical composition of the present invention can be widely used as a cell preparation for the treatment, prevention or improvement of tumors / cancers (target diseases) expressing target antigens of antigen-binding domains. Target diseases include solid cancers and blood cancers. As target diseases. For example, various B-cell malignant lymphomas (B-cell acute lymphoblastic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.), myeloproliferative diseases, myelodysplastic / myeloproliferative tumors (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndrome, acute myeloid leukemia, multiple myeloma, lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumors, gastric cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, etc. can be cited. "Treatment" includes alleviation of the characteristic symptoms or accompanying symptoms of the target disease (mildening), prevention or delay of symptom deterioration, etc. "Prevention" refers to preventing or delaying the onset / manifestation of a disease (disorder) or its symptoms, or reducing the risk of onset / manifestation. On the other hand, "improvement" refers to alleviation (mildening), improvement, remission or cure (including partial cure) of a disease (disorder) or its symptoms.

[0154] The pharmaceutical composition of the present invention contains a therapeutically effective amount of the cells of the present invention. For example, for a single administration, 1×10 4 ~1×10 10 Cells. For the purpose of protecting cells, dimethyl sulfoxide (DMSO) or serum albumin may be included in the cell preparation. For the purpose of preventing bacterial contamination, antibiotics may be included in the cell preparation. For the purpose of activating, proliferating or inducing differentiation of cells, various ingredients (vitamins, cytokines, growth factors, steroids, etc.) may be included in the cell preparation.

[0155] The administration route of the CAR gene introduced into lymphocytes or cell preparations of the present invention is not particularly limited. For example, administration is performed by intravenous injection, intra-arterial injection, intra-portal vein injection, intradermal injection, subcutaneous injection, intramuscular injection or intraperitoneal injection. Not only systemic administration but also local administration is possible. As a local administration, direct injection into target tissues, organs, and organs can be exemplified. The administration schedule can be prepared considering the gender, age, weight, condition, etc. of the subject (patient). In addition to single administration, continuous or regular multiple administrations can also be performed.

[0156] Example

[0157] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[0158] Experimental Example 1. Design and preparation of CAR constructs

[0159] Design of CAR constructs ( Figure 1 , amino acid sequence: sequence number 8, base sequence: sequence number 16), the CAR construct is configured in order from the N-terminal side:

[0160] Anti-CD19 scFv domain (amino acid sequence: SEQ ID NO: 1, base sequence: SEQ ID NO: 9),

[0161] IgG4 hinge (amino acid sequence: SEQ ID NO: 2, base sequence: SEQ ID NO: 10), CD28 transmembrane domain (amino acid sequence: SEQ ID NO: 3, base sequence: SEQ ID NO: 11),

[0162] CD28 intracellular domain (amino acid sequence: SEQ ID NO: 4, base sequence: SEQ ID NO: 12),

[0163] CD3ζ intracellular domain (amino acid sequence: SEQ ID NO: 5, base sequence: SEQ ID NO: 13),

[0164] 5 amino acid spacer,

[0165] The CAR coding sequence (base sequence: sequence number 16) is composed of a T2A sequence (amino acid sequence: sequence number 6, base sequence: sequence number 14) and a Truncated EGFR domain (amino acid sequence: sequence number 7, base sequence: sequence number 15). The restriction endonuclease recognition sequences added to both ends of the CAR are integrated into a retroviral vector (vector name: pLZRS-BMN-Z).

[0166] In addition, the sequences of the anti-CD19 scFv domain and the Truncated EGFR domain contain a GM-CSF receptor leader sequence added to the N-terminus.

[0167] Experimental Example 2. Screening of CAR-T cell proliferation factors

[0168] Screening of CAR-T cell proliferation factors was performed through GW (Genome Wide)-CRISPR screening. Figure 2 The specific steps are as follows.

[0169] Blood was collected from three healthy donors, and CD8-positive lymphocytes were separated and prepared using immunomagnetic beads (Myltenii). The CD8-positive cells were stimulated with CD3 / CD28 beads to induce proliferation (Day0). On Day1 (Day 1), the lentivirus expressing GW-gRNA was introduced, and the Cas9 protein was introduced by electroporation on Day2 (Day 2). On Day3 (Day 3), a retrovirus containing a CAR construct (Test Example 1) consisting of CD19CAR and EGFR (truncated EGFR, tEGFR) lacking the intracellular domain as a gene introduction marker was introduced. On Day7 (Day 7), tEGFR was used as a gene introduction marker, and tEGFR-positive cells (CD19CAR-positive cells) were purified using biotin (biotin) EGFR antibodies and anti-biotin microbeads (anti-biotin microbeads) for positive cells. Continue to culture until Day 10 (Day 10), then repeat the stimulation three times every 10 days with the Raji cell line (CD19+, a cell line from Burkitt's lymphoma) treated with 100Gy radiation, and collect the remaining cells on Day 40 (Day 40). Using DNA collected from Day 10 (Day 10) CAR-T cells and Day 40 (Day 40) CAR-T cells, the gRNA sequence was determined and quantified. The knockout genes were ranked for each donor using GFOLD analysis. Based on the comprehensive GFOLD analysis, 6 genes were selected as the top 10 genes reported to be expressed in T cells.

[0170] Next, secondary screening was performed. Instead of the GW-gRNA Library, a lentiviral vector (vector name: pLV-EGFP-U6) containing an expression cassette of any one of the two gRNAs targeting each of the six genes selected above was used. Figure 2 CAR-T cells were prepared according to the protocol. The target sequence of gRNA is shown below.

[0171] SMCO2 gene (NCBI gene ID: 341346)

[0172] gRNA1: SEQ ID NO: 17

[0173] gRNA2: SEQ ID NO: 18

[0174] TSR2 gene (NCBI gene ID: 90121)

[0175] gRNA1: SEQ ID NO: 19

[0176] gRNA2: SEQ ID NO: 20

[0177] PKM gene (NCBI gene ID: 5315)

[0178] gRNA1: SEQ ID NO: 21

[0179] gRNA2: Sequence number 22

[0180] CUL5 gene (NCBI gene ID: 8065)

[0181] gRNA1 (#4690): SEQ ID NO: 23

[0182] gRNA2 (#8377): SEQ ID NO: 24

[0183] KLRC1 gene (NCBI gene ID: 3821)

[0184] gRNA1: SEQ ID NO: 25

[0185] gRNA2: SEQ ID NO: 26

[0186] ING3 gene (NCBI gene ID: 54556)

[0187] gRNA1: SEQ ID NO: 27

[0188] gRNA2: SEQ ID NO: 28

[0189] The results are expressed in Figure 3 and Figure 4 Only when CUL5 was inhibited, the GFP positive ratio increased. In addition, the results of measuring the CUL5 expression level on Day 7 (Day 7) by Western blotting are shown in Figure 5 .

[0190] Test Example 3. Analysis of the Effect of CUL5 Knockout 1

[0191] Cells from five donors were used, and a lentiviral vector containing an expression cassette of any of two gRNAs targeting CUL5 (Test Example 2) was used instead of the GW-gRNA Library. Figure 2 The CAR-T cells were prepared according to the protocol.

[0192] CAR-T cells stimulated with irradiated Raji cells on Day 10 (Day 10) on Day 4 (Day 14 in total) were stained with CellTrace Violet, and the extent of cell division after 72 hours (Day 17 in total) was measured by diluting CellTrace Violet using a flow cytometer. The results are shown in Figure 6It was found that cell division was enhanced by knocking out CUL5.

[0193] The intracellular cytokine production after stimulation was measured by flow cytometry using intracellular cytokine staining. Control sgRNA was introduced into CAR-T and CUL5KO CAR-T, and stimulated with K562 (CD19 negative) and Raji (CD19 positive), respectively, and intracellular cytokines (IFN-γ) were stained 4 hours later. The results are shown in Figure 7 It was found that cytokine production was enhanced by knocking out CUL5.

[0194] Test Example 4. Analysis 2 of the effect of CUL5 knockout

[0195] Cells from four donors were used, and a lentiviral vector containing an expression cassette of any of two gRNAs targeting CUL5 (Test Example 2) was used instead of the GW-gRNA Library. Figure 2 The CAR-T cells were prepared according to the protocol.

[0196] After the first stimulation of CAR-T cells on Day 10 (Day 10) with CD3 / 28 beads and Raji cells, the surface characteristics on Day 4 were measured by flow cytometry to count the effector memory T cells (T EM :CD45RA(-)CCR7(-)) and central memory T cells (T CM : The ratio of CD45RA(-)CCR7(+). The results are shown in Figure 8 It was found that knockout of CUL5 increased effector memory T cells.

[0197] Experimental Example 5. Analysis of the Effect of CUL5 Knockout 3

[0198] Instead of GW-gRNA Library, a lentiviral vector containing an expression cassette of any of the two gRNAs targeting CUL5 (Test Example 2) was used. Figure 2 The CAR-T cells were prepared according to the protocol.

[0199] For NOD-Scid shi common gamma chain knockout mouse (NOD-Scid shi common gamma chain gene knockout mouse, NOG mouse), 5.0x10e5 Raji cells (Raji / Luc cells) stably expressing firefly luciferase and GFP were intravenously injected on day 0 (Day 0), and 1.0x10e6 various T cells (CAR-T cells expressing control gRNA, CAR-T cells expressing CUL5 gRNA, T cells expressing tEGFR) were intravenously administered on day 7 (Day 7). Then, luciferin was administered every week, and the luminescence (light) emitted by the tumor cells was photographed.

[0200] The results are expressed in Fig. 9 and Fig.10 . By knocking out CUL5, the bioluminescence in CAR-T cells showed low values ​​over time, and the survival was also significantly improved. It should be noted that tEGFR-T, n = 6; control CAR-T and CUL5KO CAR-T, n = 10, the experiment used T cells prepared from 3 different donors.

[0201] Experimental Example 6. Design and Production of Two-in-one Vector 1

[0202] The following will be configured from the N-terminal side:

[0203] Anti-CD19 scFv domain (amino acid sequence: SEQ ID NO: 1, base sequence: SEQ ID NO: 9),

[0204] IgG4 hinge (amino acid sequence: SEQ ID NO: 2, base sequence: SEQ ID NO: 10), CD28 transmembrane domain (amino acid sequence: SEQ ID NO: 3, base sequence: SEQ ID NO: 11),

[0205] CD28 intracellular domain (amino acid sequence: SEQ ID NO: 4, base sequence: SEQ ID NO: 12),

[0206] CD3ζ intracellular domain (amino acid sequence: SEQ ID NO: 5, base sequence: SEQ ID NO: 13),

[0207] 5 amino acid spacer,

[0208] A CAR expression cassette composed of a T2A sequence (amino acid sequence: SEQ ID NO: 6, base sequence: SEQ ID NO: 14), a Truncated EGFR domain (amino acid sequence: SEQ ID NO: 7, base sequence: SEQ ID NO: 15), and

[0209] The expression cassette of shRNA targeting CUL5 (base sequence: sequence number 29) was integrated into a lentiviral vector (vector name: pGreenPuro_shRNA(EF1)) using restriction endonuclease recognition sequences added to both ends. The promoter of the CAR expression cassette is the EF-1α promoter, and the promoter of the shRNA expression cassette is the H1 promoter. The schematic diagram of the resulting CAR construct is shown in Fig.11 In addition, the sequences of the anti-CD19 scFv domain and the Truncated EGFR domain contained a GM-CSF receptor leader sequence added at the N-terminus.

[0210] Experimental Example 7. Analysis of the Effect of Two-in-one Vector

[0211] Method 1

[0212] Cells from three donors were used, and a lentiviral vector containing an expression cassette of any of two gRNAs targeting CUL5 (Test Example 2) was used instead of the GW-gRNA Library. Figure 2 The CAR-T cells were prepared according to the protocol.

[0213] Method 2

[0214] On the other hand, Figure 2 Part of the plan (the plan before the stimulus started) was changed to Fig.12 Scheme to prepare CAR-T cells. Specifically, blood was collected from three healthy donors, and CD8-positive lymphocytes were separated and prepared using immunomagnetic beads (Myltenii), and the CD8-positive cells were stimulated with CD3 / CD28 beads to induce proliferation (Day0). On the first day (Day1), the lentivirus obtained from the Two-in-one vector (Test Example 6) was introduced into the gene. On the fifth day (Day5), tEGFR was used as a gene introduction marker, and tEGFR-positive cells (CD19CAR-positive cells) were purified using biotinylated EGFR antibodies and anti-biotin microbeads for positive cells. Continue to culture until the 10th day (Day10), then, every 10 days, the Raji cell line (CD19+, a cell line from Burkitt's lymphoma) treated with 100Gy radiation was repeatedly stimulated 3 times, and the remaining cells were collected on the 40th day (Day40).

[0215] Method 3

[0216] In addition, as a control of Method 2, CAR-T cells were prepared in the same manner as Method 2, except that a vector containing an expression cassette for shRNA for GFP was used instead of the expression cassette for shRNA for CUL5.

[0217] In any of the above methods, CAR-T cells are prepared starting from 1x10e6 CD8 positive cells.

[0218] The number of CAR-T cells was determined by trypan blue staining. The number of CAR-T cells on day 7 (Day 7) is shown in Fig.13 In addition, the results of Western blotting for the expression of CUL5 on Day 10 are shown in Fig.14 In addition, the proliferation of CAR-T cells is expressed in Fig.15 It was found that by producing CUL5 knockdown CAR-T cells using a polynucleotide comprising an expression cassette for a CUL5 gene expression inhibition polynucleotide and an expression cassette for a chimeric antigen receptor as in method 2, the production efficiency and proliferation of CAR-T cells can be greatly improved.

[0219] Experimental Example 8. Analysis of the Effect of Two-in-one Vector 2

[0220] CAR-T cells were prepared according to Method 2 and Method 3 of Experimental Example 7.

[0221] For NOD-Scid shi common gamma chain knockout mouse (NOG mouse, n=8-10), 2.0x10e6 Raji cells (Raji / Luc cells) stably expressing firefly luciferase and GFP were intravenously injected on day 0 (Day 0), and 1.0x10e6 various T cells were intravenously administered on day 7 (Day 7). Then, luciferin was administered every week, and the luminescence emitted by the tumor cells was photographed.

[0222] The results are expressed in Fig.16 In CAR-T cells in which CUL5 was knocked down by two-in-one vector, bioluminescence showed low values ​​over time.

[0223] Experimental Example 9. Analysis of the Effect of Two-in-one Vector 3

[0224] CAR-T cells were prepared according to Method 2 and Method 3 of Experimental Example 7.

[0225] In NOD-Scid shi common gamma chain knockout mouse (NOG mouse), 2.0x10e6 Raji cells (Raji / Luc cells) stably expressing firefly luciferase and GFP were intravenously injected on day 0 (Day 0), and 1.0x10e6 of various T cells were intravenously administered on day 10 (Day 10). Then, luciferin was administered every week, and the luminescence emitted by tumor cells was photographed to measure the tumor volume. In addition, when the tumor volume exceeded 1500mm 3 In the event of euthanasia,

[0226] The tumor volume measurements were expressed as Fig.17 , the survival rate is expressed as Fig.18 In CAR-T cells in which CUL5 was knocked down by Two-in-one vector, tumor growth was significantly inhibited, and the survival rate was greatly improved.

[0227] Experimental Example 10. Design and Production of Two-in-one Vector 2

[0228] A lentiviral vector was obtained in the same manner as in Experimental Example 6, except that the anti-Eva1 scFv domain was used instead of the anti-CD19 scFv domain, and the tandemly connected domain of the 4-1BB intracellular domain (amino acid sequence: sequence number 30, base sequence: sequence number 33) or the CD79a intracellular domain (amino acid sequence: sequence number 31, base sequence: sequence number 34)-CD40 intracellular domain (amino acid sequence: sequence number 32, base sequence: sequence number 35) was used instead of the CD28 intracellular domain as the intracellular domain.

[0229] Test Example 11. Analysis of the Effect of Two-in-one Vector 4

[0230] CAR-T cells were prepared in the same manner as Method 2 and Method 3 of Experimental Example 7, except that the Two-in-one vector of Experimental Example 10 was used.

[0231] 1x10e6 NCI-H1975 (NCI-H1975-firefly luciferase-GFP, cells stably expressing firefly luciferase and GFP) was injected subcutaneously into mice. 14 days later, 0.5x10e5 / mouse of various T cells (shCUL5-4-1BB-Eva1CAR-T, shGFP-4-1BB-Eva1CAR-T, shCUL5-CD79A / 40-Eva1CAR-T, shGFP-CD79A / 40-Eva1CAR-T) were intravenously administered. Then, luciferin was administered at regular intervals, and the luminescence emitted by tumor cells was photographed to measure the tumor volume. In addition, when the tumor volume exceeded 2000mm 3 euthanasia in case of

[0232] Luminescence images are displayed on Fig.19 The tumor volume measurement results are shown in Fig. 20 .

Claims

1. A polynucleotide, characterized in that: An expression cassette comprising a polynucleotide for inhibiting CUL5 gene expression and an expression cassette for a chimeric antigen receptor.

2. The polynucleotide according to claim 1, characterized in that: The CUL5 gene expression inhibition polynucleotide is at least one selected from the group consisting of CUL5-specific siRNA, CUL5-specific miRNA, and CUL5-specific antisense polynucleotide.

3. The polynucleotide according to claim 1, characterized in that: The CUL5 gene expression inhibition polynucleotide is a CUL5-specific siRNA.

4. The polynucleotide according to claim 1, characterized in that: It is a carrier.

5. The polynucleotide according to claim 1, wherein: It is a viral vector or a viral genome.

6. The polynucleotide according to claim 1, wherein: The target antigen of the chimeric antigen receptor is a tumor antigen.

7. A cell, characterized in that: A polynucleotide comprising the polynucleotide according to any one of claims 1 to 6.

8. The cell according to claim 7, characterized in that: The expression of the CUL5 gene is decreased by the CUL5 gene expression inhibitory polynucleotide, and the chimeric antigen receptor is expressed.

9. The cell according to claim 8, characterized in that: They are lymphocytes.

10. A pharmaceutical composition, characterized in that: Containing the cell according to claim 7.

11. The pharmaceutical composition according to claim 10, characterized in that: It is used for the treatment, prevention or improvement of cancer.

12. A cell altered so that expression of the CUL5 gene is reduced and expresses a chimeric antigen receptor.

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