JP2023174589A - polynucleotide - Google Patents

Abstract

To provide a technique that enhances the proliferative ability of CAR-expressing cells.SOLUTION: The expression of the CUL5 gene is reduced.SELECTED DRAWING: None

Description

There is an application for exception to loss of novelty.

The present invention relates to polynucleotides and the like useful for producing cells expressing chimeric antigen receptors.

Chimeric Antigen Receptor (hereinafter also referred to as "CAR") binds to antigen according to the specificity of the antibody, and can thereby cause target cell damage, cytokine release, and T cell division after stimulation. . It is possible to impart specificity to T cells by gene transfer, and the preparation of cells is generally easier than when culturing and expanding endogenous tumor-specific T cell receptor (TCR)-positive T cells. In addition, treatment using CAR (CAR-T therapy) shows higher cytotoxic activity than antibody therapy, and requires multiple treatments because it is autonomously amplified by antigen stimulation with a single infusion. It is excellent in that it is not. TCR gene transfer targets the complex of HLA and the peptide presented on it, so it can only be treated if the patient has a specific HLA (HLA-restricted), but CAR has HLA-restricted It is more advantageous in that it has no characteristics. That is, if the tumor cells are positive for the target antigen, it is possible to treat all patients who are positive for the target antigen.

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

Patent No. 5312721

Recurrence and treatment resistance after CAR-T therapy are thought to be due to the short time that CAR-T cells remain viable and active within the patient's body. In order to solve this problem, it is desirable to increase the proliferation ability of CAR-T cells (particularly the proliferation ability after contact with a target antigen). It is also important to efficiently obtain CAR-T cells.

Therefore, an object of the present invention is to provide a technique for increasing the proliferation ability of CAR-expressing cells. Another objective of the present invention is to provide a technique for efficiently obtaining CAR-expressing cells.

In view of the above-mentioned problems, the present inventors conducted intensive research and found that the proliferation ability of CAR-expressing cells was increased by lowering the expression of the CUL5 gene. The present inventor conducted further research based on this knowledge and completed the present invention. That is, the present invention includes the following aspects.

Item 1. A polynucleotide comprising an expression cassette for a CUL5 gene expression suppressing polynucleotide and an expression cassette for a chimeric antigen receptor.

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

Item 3. Item 2. The polynucleotide according to Item 1, wherein the CUL5 gene expression suppressing polynucleotide is a CUL5-specific siRNA.

Item 4. Item 2. The polynucleotide according to item 1, which is a vector.

Item 5. Item 2. The polynucleotide according to item 1, which is a viral vector or a viral genome.

Item 6. Item 2. The polynucleotide according to Item 1, wherein the target antigen of the chimeric antigen receptor is a cancer antigen.

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

Section 8. 8. The cell according to item 7, wherein the CUL5 gene expression is reduced by the CUL5 gene expression suppressing polynucleotide and expresses a chimeric antigen receptor.

Item 9. The cell according to item 8, which is a lymphocyte cell.

Item 10. A pharmaceutical composition containing the cell according to item 7.

Item 11. Item 11. The pharmaceutical composition according to item 10, which is used for treating, preventing, or improving cancer.

Item 12. A cell that has been modified to reduce expression of the CUL5 gene and expresses a chimeric antigen receptor.

Item 13. 13. The cell according to item 12, which is a lymphocyte cell.

Section 14. A pharmaceutical composition containing the cell according to item 12 or 13.

Item 15. Item 15. The pharmaceutical composition according to item 14, which is used for treating, preventing, or improving cancer.

According to the present invention, a technique for increasing the proliferation ability of CAR-expressing cells can be provided. Furthermore, according to a preferred embodiment of the present invention, a technique for efficiently obtaining CAR-expressing cells can be provided.

A schematic diagram of the CAR construct obtained in Test Example 1 is shown. An overview of GW-CRISPR screening (primary screening) of Test Example 2 is shown. The results of the secondary screening of Test Example 2 are shown. Day indicates the number of days that have passed since the first stimulation. The vertical axis is the value (%) obtained by dividing the GFP-positive cell percentage (%) on day 0 from the GFP-positive cell percentage (%) on day 30. The horizontal axis shows gRNA expressed in cells. The results of the secondary screening of Test Example 2 are shown. The vertical axis is the GFP-positive cell percentage (%). On the horizontal axis, D indicates the number of days that have passed since the initial stimulation. *** indicates P<0.001 between groups. The results of measuring the CUL5 expression level by Western blotting when gRNA for CUL5 was expressed in the secondary screening of Test Example 2 are shown. The vertical axis shows the ratio of CUL5 expression level to β-actin expression level. The horizontal axis shows expressed gRNA. * indicates P<0.05 between groups, ** indicates P<0.01 between groups. The degree of cell division measured in Test Example 3 is shown. The vertical axis shows fluorescence intensity. The lower the fluorescence intensity, the more accelerated the division. On the horizontal axis, Ctrl indicates the case where control gRNA was expressed, and CUL5 KO indicates the case where CUL5 gRNA was expressed. * indicates P<0.05 between groups. The percentage of intracellular cytokine-positive cells measured in Test Example 3 (vertical axis) is shown. The horizontal axis shows stimulated cells. Of the two columns for each stimulated cell, the left side shows the case where control gRNA was expressed, and the right side shows the case where CUL5 gRNA was expressed. * indicates P<0.05 between groups. Left figure: Percentage of effector memory T cells (T _EM :CD45RA(-)CCR7(-)) and right figure: Central memory T cells (T _CM :CD45RA(-)CCR7(+)) measured in Test Example 4. (vertical axis). On the horizontal axis, Ctrl indicates the case where control gRNA was expressed, and CUL5 KO indicates the case where CUL5 gRNA was expressed. ** indicates P<0.01 between groups. The luminescence emitted by tumor cells (vertical axis) measured in Test Example 5 is shown. The horizontal axis indicates the number of days elapsed since tumor cell inoculation. Ctrl CAR indicates administration of CAR-T cells expressing control gRNA, CUL5KO CAR indicates administration of CAR-T cells expressing CUL5 gRNA, and tEGFR-T indicates administration of CAR-T cells expressing tEGFR. The case where cells were administered is shown. The survival rate (vertical axis) measured in Test Example 5 is shown. The horizontal axis indicates the number of days elapsed since tumor cell inoculation. Ctrl CAR indicates administration of CAR-T cells expressing control gRNA, CUL5KO CAR indicates administration of CAR-T cells expressing CUL5 gRNA, and tEGFR-T indicates administration of CAR-T cells expressing tEGFR. The case where cells were administered is shown. * indicates P<0.05 between groups, *** indicates P<0.001 between groups, **** indicates P<0.0001 between groups. A schematic diagram of the two-in-one vector obtained in Test Example 6 is shown. A scheme of CAR-T cell adjustment using a two-in-one vector (before stimulation starts) is shown. The number of CAR-T cells (vertical axis) measured in Test Example 7 is shown. The left column shows the case of method 1, and the right column shows the case of method 2 (when using two-in-one vector). *** indicates P<0.001 between groups. The results of measuring CUL5 expression level by Western blotting 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. It represents the proliferation of CAR-T cells measured in Test Example 7. The vertical axis shows the number of CAR-T cells. The horizontal axis indicates the number of days that have passed since the initial stimulation. ** indicates P<0.01 between groups. The luminescence emitted by tumor cells (vertical axis) measured in Test Example 8 is shown. The horizontal axis indicates the number of days elapsed 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. The tumor volume (vertical axis) measured in Test Example 9 is shown. The horizontal axis indicates the number of days elapsed since tumor cell inoculation. shGFP CD19CAR-T shows the case where shRNA against GFP was expressed, shCUL5 CD19CAR-T shows the case where shRNA against CUL5 was expressed, and tEGFR-T shows the case where T cells expressing tEGFR were administered. * indicates P<0.05 between groups, ** indicates P<0.01 between groups. The survival rate (vertical axis) measured in Test Example 9 is shown. The horizontal axis indicates the number of days elapsed since tumor cell inoculation. shGFP CD19CAR-T shows the case where shRNA against GFP was expressed, shCUL5 CD19CAR-T shows the case where shRNA against CUL5 was expressed, and tEGFR-T shows the case where T cells expressing tEGFR were administered. * indicates P<0.05 between groups. A photographed image of luminescence in Test Example 11 is shown. The number of days elapsed since tumor cell inoculation is shown on the left side of the photo. The intracellular domain and shRNA employed are shown above the photograph. The tumor volume (vertical axis) measured in Test Example 11 is shown. The horizontal axis shows the number of weeks that have passed since tumor cell inoculation. The legend indicates the intracellular domain and shRNA used.

In this specification, the expressions "contain" and "including" include the concepts of "containing", "comprising", "consisting essentially" and "consisting only".

1. Definitions In this specification, the expressions "contain" and "including" include the concepts of "containing,""including,""consisting essentially of," and "consisting only of."

"Identity" of amino acid sequences refers to the extent to which two or more comparable amino acid sequences match each other. Therefore, the higher the similarity between two certain amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using the sequence analysis tool FASTA using default parameters. Alternatively, the algorithm BLAST by Karlin and Altschul (Karlin S, 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), Karlin S, Altschul SF. It can be determined using “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on the BLAST algorithm. Specific techniques for these analysis methods are publicly known, and the website of the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) may be referred to. Furthermore, "identity" of base sequences is also defined according to the above.

As used herein, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, conservative substitutions include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine. In addition, amino acid residues with acidic side chains such as aspartic acid and glutamic acid; amino acid residues with uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with nonpolar side chains such as proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains such as threonine, valine, and isoleucine; and aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine. Substitutions between amino acid residues are also considered conservative substitutions.

In this specification, "nucleic acid" and "polynucleotide" are not particularly limited, and include both natural and artificial ones. Specifically, in addition to DNA, RNA, etc., materials that have been subjected to known chemical modifications may be used, as exemplified below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues of each nucleotide are replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. I can do it. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is replaced by -OR (R is, for example, CH3 (2´-O-Me), CH2CH2OCH3 (2´-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.) may be substituted. Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, such as introducing a methyl group or cationic functional group to the 5-position of the pyrimidine base, or replacing the carbonyl group at the 2-position with thiocarbonyl. can be mentioned. Further examples include, but are not limited to, those in which the phosphoric acid moiety or hydroxyl moiety is modified with biotin, an amino group, a lower alkylamine group, an acetyl group, or the like. Furthermore, BNA (LNA), etc., in which the conformation of the sugar part of the nucleotide is fixed to N-type by cross-linking the 2' oxygen and 4' carbon of the sugar part of the nucleotide, can also be used.

As used herein, "CDR" is an abbreviation for complementarity determining region , and is also referred to as a complementarity determining region. CDRs are regions present in the variable regions of immunoglobulins or T cell receptors, and are deeply involved in specific binding of antibodies or T cell receptors to antigens. Note that "light chain CDR" refers to a CDR present in the light chain variable region of an immunoglobulin, and "heavy chain CDR" refers to a CDR present in the heavy chain variable region of an immunoglobulin. T cell receptors also have α chains, β chains, γ chains, δ chains, etc., and the same applies to CDRs of these chains.

As used herein, the term "variable region" refers to a region containing CDR1 to CDR3 (hereinafter simply referred to as "CDRs1-3"). The arrangement order of these CDRs1-3 is not particularly limited, but preferably CDR1, CDR2, and CDR3 are arranged in the order from the N-terminal side to the C-terminal side, or in the reverse order, consecutively or in the frames described below. It refers to a region located through another amino acid sequence called a working region (FR). In addition, "heavy chain variable region" is a region in which the above-mentioned heavy chain CDRs 1-3 are arranged in an immunoglobulin, and "light chain variable region" is a region in which the above-mentioned light chain CDRs 1-3 are arranged in an immunoglobulin. This is the area where . T cell receptors also have α chains, β chains, γ chains, δ chains, etc., and the same applies to CDRs of these chains.

The regions other than CDR1-3 in 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 CDR1 above is FR1, the region between CDR1 and CDR2 is FR2, the region between CDR2 and CDR3 is FR3, and the region between CDR3 and the C-terminus of the variable region is Each is defined as FR4.

2. Polynucleotide In one aspect, the present invention provides polynucleotides (herein referred to as "polynucleotides of the present invention") comprising an expression cassette for a CUL5 gene expression suppressing polynucleotide and an expression cassette for a chimeric antigen receptor. ). This will be explained below.

The polynucleotide of the present invention includes an expression cassette of a polynucleotide that suppresses CUL5 gene expression. By preparing CAR-expressing cells using the polynucleotide of the present invention containing an expression cassette of a polynucleotide that suppresses CUL5 gene expression, the proliferation ability of CAR-expressing cells can be increased. 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-box protein) E3 ubiquitin-protein ligase complexes that mediate the ubiquitination and subsequent proteasomal degradation of target proteins. Suppression of CUL5 gene expression means suppressing the expression level of Cullin-5 mRNA/protein. The species of origin of the CUL5 gene is not particularly limited, and includes various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. The human CUL5 gene is a gene with NCBI gene ID:8065.

The amino acid sequences of Cullin-5 proteins derived from various biological species and the base sequences of Cullin-5 mRNA are known. Specifically, for example, human Cullin-5 protein includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 30 (NCBI Reference Sequence: NP_003469.2), and human Cullin-5 mRNA includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 31. An example of this is mRNA (NCBI Reference Sequence: NM_003478.6), which consists of the base sequence of In addition, the Cullin-5 protein and Cullin-5 mRNA may also include the above-mentioned splicing variants.

The Cullin-5 protein expressed from the CUL5 gene, whose expression is to be suppressed, is susceptible to substitutions, deletions, additions, insertions, etc. as long as it forms an E3 ubiquitin-protein ligase complex that has its original activity, that is, ubiquitin E3 ligase activity. may have an amino acid mutation. Preferably, the mutation is a substitution, and more preferably a conservative substitution, from the viewpoint that the activity is less likely to be impaired.

As long as the Cullin-5 mRNA expressed from the CUL5 gene, which is the target of expression suppression, the protein translated from the mRNA forms an E3 ubiquitin-protein ligase complex that has its original activity, that is, ubiquitin E3 ligase activity. It may have base mutations such as substitutions, deletions, additions, and insertions. Preferable mutations include mutations that do not result in amino acid substitutions or mutations that result in conservative amino acid substitutions in the protein translated from the mRNA.

Preferred specific examples of the Cullin-5 protein expressed from the CUL5 gene whose expression is to be suppressed include the protein described in (A) below and the protein described in (B) below:
(A) A protein consisting of an amino acid sequence shown in any of SEQ ID NO: 30, and (B) A protein consisting of an amino acid sequence having 85% or more identity with any of the amino acid sequences shown in SEQ ID NO: 30, and ubiquitin E3 At least one type selected from the group consisting of proteins that form an E3 ubiquitin-protein ligase complex having ligase activity is included.

In (B) above, the identity is more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more.

An example of the protein described in (B) above is, for example, one or more amino acids have been substituted, deleted, added, or inserted into the amino acid sequence shown in (B') SEQ ID NO: 30. Examples include proteins that form an E3 ubiquitin-protein ligase complex consisting of an amino acid sequence and having ubiquitin E3 ligase activity.

In the above (B'), the plural number is, for example, 2 to 50, preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 or 3.

Preferred specific examples of Cullin-5 mRNA expressed from the CUL5 gene whose expression is to be suppressed include the mRNA described in (C) below and the mRNA described in (D) below:
(C) mRNA consisting of a base sequence shown in any of SEQ ID NO: 31, and (D) mRNA consisting of a base sequence having 85% or more identity with any of SEQ ID NO: 31, and ubiquitin E3 mRNA encoding a protein that forms an E3 ubiquitin-protein ligase complex with ligase activity
At least one selected from the group consisting of:

In the above (D), the identity is more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more.

As an example of the mRNA described in (D) above, for example, one or more bases have been substituted, deleted, added, or inserted to the base sequence shown in (D') SEQ ID NO: 31. Examples include mRNA that encodes a protein that consists of a base sequence and forms an E3 ubiquitin-protein ligase complex that has ubiquitin E3 ligase activity.

In the above (D'), the plural number is, for example, 2 to 200, preferably 2 to 100, more preferably 2 to 50, and even more preferably 2 to 10.

The CUL5 gene expression suppressing polynucleotide is not particularly limited as long as it is a polynucleotide that can suppress the expression level of CUL5 protein, CUL5 mRNA, etc., such as CUL5-specific small interfering RNA (siRNA), CUL5-specific microRNA (miRNA) , CUL5-specific antisense polynucleotides, and the like.

Note that expression suppression 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. It also includes setting the expression level to 0.

CUL5-specific siRNA is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of CUL5 gene. In one embodiment, the siRNA preferably has a length of, for example, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more. Further, the siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. It is envisaged that the upper and lower limits of the length of siRNA described herein may be combined arbitrarily. For example, lengths where the lower limit is 18 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 19 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases. A length; a lower limit of 20 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 21 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; A combination of lengths that are bases is envisaged.

siRNA may be shRNA (small hairpin RNA). shRNA can be designed so that a portion thereof forms a stem-loop structure. For example, in shRNA, if the sequence of a certain region is sequence a, and the complementary strand to sequence a is sequence b, then these sequences exist in one RNA strand in the order of sequence a, spacer, and sequence b. It can be designed to have a total length of 45 to 60 bases. Sequence a is a partial region of a base sequence encoding target CUL5, and the target region is not particularly limited, and any region can be used as a candidate. The length of sequence a is 19 to 25 bases, preferably 19 to 21 bases.

CUL5-specific siRNA may have additional bases at the 5' or 3' end. The length of the additional base is usually about 2 to 4 bases. The additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Sequences of such additional bases include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3. Examples include, but are not limited to, sequences such as ', uuuuu-3'.

The siRNA may have a protrusion sequence (overhang) at the 3' end, and specific examples include those to which dTdT (dT represents deoxythymidine) is added. Alternatively, it may have a blunt end without any terminal addition. The sense strand and the antisense strand of siRNA may have different numbers of bases. For example, siRNA is an asymmetrical interfering RNA (asymmetrical interfering RNA) in which the antisense strand has overhangs at the 3' and 5' ends. aiRNA). A typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and an overhang structure of 3 bases at each end of the antisense strand.

Although the location of the target sequence of CUL5-specific siRNA is not particularly limited, in one embodiment, the target sequence is selected from the 5'-UTR and up to about 50 bases from the start codon, and from a region other than the 3'-UTR. It is desirable to do so. For the selected target sequence candidate group, BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) to confirm the specificity of the selected target sequence. For the target sequence for which specificity has been confirmed, a sense strand with a 3'-terminal overhang of TT or UU at bases 19-21 after AA (or NA), a sequence complementary to the 19-21 bases, and a sequence of TT or A double-stranded RNA consisting of a UU strand and an antisense strand having a 3'-terminal overhang may be designed as siRNA. In addition, for shRNA, which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5 to 25 bases) that can form a loop structure is appropriately selected, and the sense strand and antisense strand are connected via the linker sequence. It can be designed by connecting them.

The sequences of siRNA and/or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include the following:
siRNA Target Finder provided by Ambion (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) Insert Design Tool for pSilencer® Expression Vector (http://www.ambion.com/ jp/techlib/misc/psilencer_converter.html) GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).

siRNA is produced by synthesizing the sense and antisense strands of the target sequence on mRNA using an automatic DNA/RNA synthesizer, denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute, and then It can be prepared by annealing at about 30 to about 70°C for about 1 to about 8 hours. Alternatively, it can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving this using the RNA cutting protein dicer.

CUL5-specific miRNA is optional as long as it inhibits translation of the CUL5 gene. For example, miRNAs may pair with the 3' untranslated region (UTR) of a target and inhibit its translation, rather than cleaving the target mRNA as siRNAs do. miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA. The length of miRNA is not particularly limited; pri-miRNA length is usually several hundred to several thousand bases, pre-miRNA length is usually 50-80 bases, and mature miRNA length is usually 18 ~30 bases. In one embodiment, the CUL5-specific miRNA is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA. Such CUL5-specific miRNA may be synthesized by a known method or purchased from a company that provides synthetic RNA.

A CUL5-specific antisense polynucleotide is a nucleic acid that contains a base sequence that is complementary or substantially complementary to the base sequence of the mRNA of the CUL5 gene, or a part thereof, and that has a specific and stable double base sequence with the mRNA. It is a nucleic acid that has the function of suppressing CUL5 protein synthesis by forming chains and binding together. Antisense polynucleotides can be, for example, DNA, RNA, or DNA/RNA chimeras. When the antisense polynucleotide is DNA, the RNA:DNA hybrid formed by the target RNA and antisense DNA is recognized by endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only a sequence in mRNA but also a sequence in the intron region of the early translation product of the CUL5 gene. The intron sequence can be determined by comparing the genome sequence and the cDNA base sequence of the CUL5 gene using a homology search program such as BLAST or FASTA.

The length of the target region of the CUL5-specific antisense polynucleotide is not limited as long as hybridization of the antisense polynucleotide results in inhibition of translation into CUL5 protein. The CUL5-specific antisense polynucleotide may be the entire sequence or a partial sequence of mRNA encoding CUL5. In consideration of ease of synthesis, antigenicity, intracellular distribution, etc., oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred, but are not limited thereto. More specifically, the 5' end hairpin loop, 5' untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3' untranslated region, 3' palindromic region, or 3' end of the CUL5 gene. ' terminal hairpin loops and the like may be selected as preferred target regions for antisense polynucleotides, but are not limited thereto.

CUL5-specific antisense polynucleotides not only hybridize with CUL5 gene mRNA and early transcripts to inhibit protein translation, but also bind to these genes, which are double-stranded DNA, to form triple-stranded (triplex) genes. It may also be a substance (antigene) that can form a complex (antigene) and inhibit transcription into RNA.

CUL5-specific siRNA, CUL5-specific miRNA, CUL5-specific antisense polynucleotide, etc. are produced by determining the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of the CUL5 gene, and then using commercially available DNA/RNA. It can be prepared by synthesizing a complementary sequence to this using an automatic synthesizer. Furthermore, antisense polynucleotides containing various modifications can also be chemically synthesized by known techniques.

The expression cassette for the CUL5 gene expression suppressing polynucleotide (expression cassette 1) includes a promoter and a CUL5 gene expression suppressing polynucleotide coding sequence under the control of the promoter. The coding sequence is usually placed downstream of the promoter so that it is under the control of the promoter. Examples of promoters that can be used include RNA polymerase II (poII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, and CAG promoter; mouse and human U6-snRNA promoters, and human Examples include RNA polymerase III (polIII) promoters such as H1-RNase P RNA promoter and human valine-tRNA promoter. Among these, polIII promoters are preferred from the viewpoint of being able to accurately transcribe short RNAs. . A promoter is operably linked to a coding sequence. Here, "the promoter is operably linked to the coding sequence" is synonymous with "the coding sequence is placed under the control of the promoter," and is usually directly attached to the 3' end of the promoter. Or the coding sequences will be linked via other sequences. A polyA addition signal sequence is placed downstream of the coding sequence. Transcription is terminated by the use of a polyA addition signal sequence. As the poly A addition signal sequence, the poly A addition sequence of SV40, the poly A addition sequence of the bovine growth hormone gene, etc. can be used.

The polynucleotide of the invention comprises a chimeric antigen receptor expression cassette. CAR-expressing cells can be efficiently obtained by preparing CAR-expressing cells using the polynucleotide of the present invention containing an expression cassette for a chimeric antigen receptor.

The chimeric antigen receptor is not particularly limited as long as it can bind to the target antigen and, upon binding to the target antigen, can transmit signals necessary for activation of immune cells into the cells. Chimeric antigen receptors typically include an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

The antigen-binding domain is a domain that is placed outside the cell when the chimeric antigen receptor is placed on the cell membrane, and there are no particular restrictions as long as it is a domain that can recognize and bind to the antigen. Not done. Specifically, the antigen includes, for example, CD19, GD2, GD3, CD20, CD37, CEA, HER2, EGFR, type III mutant EGFR, CD38, BCMA, MUC-1, PSMA, WT1, cancer testis antigen (for example, NY-ESO -1, MAGE-A4, etc.), mutation peptides (e.g. k-ras, h-ras, p53, etc.), hTERT, PRAM, TYRP1, mesothelin, PMEL, mucin, etc., and complexes of these fragments with MHC ( pMHC), etc. Among these, in one embodiment of the present invention, preferred is CD19. The antigen-binding domain usually includes the CDRs of an antibody or T cell receptor for an antigen (for example, for antibody CDRs, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR). chain CDR3), preferably two, three, four, five or more, more preferably all six. The antigen-binding region more preferably includes the variable region of an antibody or T cell receptor against the antigen (in the case of an antibody, for example, a heavy chain variable region and/or a light chain variable region).

The antigen-binding domain preferably has a single-chain antibody structure, more preferably an scFv structure. When a scFv structure includes a heavy chain variable region and a light chain variable region, the heavy chain variable region and light chain variable region are usually connected via a linker. The linker is not particularly limited and is optional as long as it does not significantly impair antigen binding. The linker is preferably glycine or a linker composed of glycine and serine (GGS linker, GS linker, GGG linker, etc.). 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. The positional relationship between the heavy chain variable region and the light chain variable region is not particularly limited, and either an embodiment where the N-terminal side is the light chain variable region or a case where the N-terminal side is the heavy chain variable region can be adopted. The arrangement relationship is preferably the former.

The transmembrane domain is a domain that is placed within the cell membrane when the chimeric antigen receptor of the present invention is placed on the cell membrane, and is not particularly limited as long as it can constitute the chimeric antigen receptor. As the transmembrane domain, for example, transmembrane regions such as CD28, CD3ε, CD8α, CD3, CD4, and 4-1BB can be used. The transmembrane region of each factor is known or can be easily determined from known sequence information (for example, using a transmembrane region prediction program, etc.). Alternatively, a transmembrane domain consisting of an artificially constructed polypeptide may be used. Suitable mutations may be introduced into these transmembrane domains as long as they do not significantly inhibit the function of the chimeric antigen receptor.

As the transmembrane domain, preferably the transmembrane region of CD28 can be employed. Specific examples of the transmembrane region of CD28 include the amino acid sequence described in (a) below or the amino acid sequence described in (b) below:
(a) the amino acid sequence shown in SEQ ID NO: 3, or (b) has 85% or more identity with the amino acid sequence shown in SEQ ID NO: 3, and the chimeric antigen receptor of the present invention is arranged on the cell membrane. Examples include amino acid sequences that can sometimes be located within cell membranes.

In (b) above, the identity is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, particularly preferably 99% or more.

An example of the amino acid sequence described in (b) above is, for example, (b') an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted with respect to the amino acid sequence shown in SEQ ID NO: 3. and which can be located within the cell membrane when the chimeric antigen receptor of the present invention is located on the cell membrane.

In (b') above, the plural number is, for example, 2 to 5, preferably 2 to 3, and more preferably 2.

In one aspect of the present invention, the antigen-binding domain and the transmembrane domain are preferably linked 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 preferably 100 or less. Particularly preferred. The number of constituent amino acid residues is preferably 0 to 60, more preferably 0 to 40, and still more preferably 0 to 30. In particularly preferred embodiments of the invention, the number of amino acid residues is 1 or more, 4 or more, or 8 or more, and 25 or less, 20 or less, or 15 or less, and 1-25, 4-20, or 8 to 15. The sequence of the spacer is not particularly limited, but includes, for example, the hinge region of IgG, preferably human IgG (e.g., subtypes IgG1 and IgG4) or a part thereof, the hinge region and a part of CH2, or a factor used in the transmembrane domain (e.g. A sequence such as a part of CD28) can be used as a spacer. Note that by utilizing the arrangement of the hinge portion or a portion thereof, it can be expected that a highly flexible spacer will be constructed.

The intracellular signal domain is a domain that is placed inside the cell when the chimeric antigen receptor of the present invention is placed on the cell membrane, and is used to transmit signals necessary for the effector function of immune cells to be exerted. When the antigen-binding domain binds to an antigen, it is a domain that can transmit signals necessary for activation of immune cells. The intracellular signaling domain preferably includes an intracellular activation domain. As the intracellular activation domain, for example, in addition to CD3ζ, intracellular domains such as FcεRIγ can be used. Preferably, CD3ζ is used. CD3 may be appropriately mutated as long as it does not inhibit the function of the antigen receptor. When introducing mutations into CD3, it is preferable to include ITAM (immunoreceptor tyrosine-based activation motif).

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:
(c) the amino acid sequence shown in SEQ ID NO: 5, or (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 activation effect on immune cells.

In (d) above, the identity is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, particularly preferably 99% or more.

An example of the amino acid sequence described in (d) above is, for example, (d') an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted to the amino acid sequence shown in SEQ ID NO: 5. Examples include amino acid sequences that have the effect of activating immune cells.

In the above (d'), the plural number is, for example, 2 to 15, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 to 3.

The intracellular signaling domain preferably comprises an intracellular domain of a co-stimulatory factor. The intracellular domain of the costimulatory factor is not particularly limited as long as it is an intracellular domain derived from a costimulatory factor possessed by T cells and the like. For example, one or more intracellular domains selected from the group consisting of OX40, 4-1BB, GITR, CD79a, CD40, CD27, CD278, CD28, etc. can be appropriately selected and used. In one embodiment of the present invention, a domain in which a CD79a intracellular domain and a CD40 intracellular domain are linked in tandem can be employed.

Specific examples of the intracellular domain include the amino acid sequence described in (e) below or the amino acid sequence described in (f) below:
(e) has an amino acid sequence shown in any of SEQ ID NOs: 4 and 30-32, or (f) has 85% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 4 and 30-32, and Examples include amino acid sequences that have an activation effect on immune cells.

In (f) above, the identity is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, particularly preferably 99% or more.

An example of the amino acid sequence described in (f) above is, for example, (f') an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted to the amino acid sequence shown in SEQ ID NO: 4. Examples include amino acid sequences that have the effect of activating immune cells.

In the above (f'), the plural number is, for example, 2 to 15, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 to 3.

The chimeric antigen receptor of the present invention can contain other regions than those described above. Other regions include leader sequences (signal peptides) (e.g. GM-CSF receptor leader sequences), spacers/linkers (e.g. transmembrane regions and intracellular signal domains) to facilitate transport of CAR onto the cell membrane. (between each domain within the intracellular signal domain), etc.

There have been several reports of experiments and clinical studies using CAR (for example, 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 5:177 ra38, 2013), and the chimeric antigen receptor of the present invention can be constructed with reference to these reports.

A particularly preferred embodiment of the chimeric antigen receptor of the present invention includes a core region in which an antigen-binding domain, a transmembrane domain, and an intracellular signal domain are arranged in this order. Each domain is linked directly or via a spacer/linker.

The chimeric antigen receptor of the present invention may be one to which a protein or peptide such as a known protein tag or signal sequence 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, and the like.

The expression cassette for the chimeric antigen receptor (expression cassette 2) comprises a promoter and the chimeric antigen receptor coding sequence under the control of the promoter. The coding sequence is usually placed downstream of the promoter so that it is under the control of the promoter. Examples of promoters that can be used include RNA polymerase II (poII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, and CAG promoter. A promoter is operably linked to a coding sequence. Here, "the promoter is operably linked to the coding sequence" is synonymous with "the coding sequence is placed under the control of the promoter," and is usually directly attached to the 3' end of the promoter. Or the coding sequences will be linked via other sequences. A polyA addition signal sequence is placed downstream of the coding sequence. Transcription is terminated by the use of a polyA addition signal sequence. As the poly A addition signal sequence, the poly A addition sequence of SV40, the poly A addition sequence of the bovine growth hormone gene, etc. can be used.

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

The expression cassette may include a detection gene (reporter gene, cell- or tissue-specific gene, selection marker gene, etc.), an enhancer sequence, a WRPE sequence, and the like. The detection gene is used for determining the success or failure and efficiency of introduction of the expression cassette, detecting the expression of the CAR gene or determining the expression efficiency, and selecting and sorting cells in which the CAR gene is expressed. On the other hand, expression efficiency can be improved by using an enhancer sequence. Genes for detection include the neo gene that confers resistance to neomycin, the npt gene that confers resistance to kanamycin, etc. (Herrera Estrella, EMBO J. 2 (1983), 987-995), and nptII gene (Messing & Vierra. Gene 1). 9:259-268 (1982)), the hph gene that confers resistance to hygromycin (Blochinger & Diggl mann, Mol Cell Bio 4:2929-2931), and the dhfr gene that confers resistance to metrexate (Bourouis et al. , EMBO J. 2(7)) etc. (marker genes), luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), β - Glucuronidase (GUS) gene, fluorescent protein genes (reporter genes) such as GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) and its variants (EGFP, d2EGFP, etc.), and intracellular domains. A gene lacking such as the epidermal growth factor receptor (EGFR) gene can be used. The detection gene is linked to the CAR gene via, for example, a bicistronic control sequence (eg, internal ribosomal recognition sequence (IRES)) or a sequence encoding a self-cleavable peptide. An example of a self-cleavable peptide is, but is not limited to, the 2A peptide (T2A) from Thosea asigna virus. Known self-cleavable peptides include 2A peptide (F2A) derived from foot disease virus (FMDV), 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), and 2A peptide (P2A) derived from porcine teschovirus (PTV-1). It is being

The polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide (such as a vector). The vector can be a plasmid vector or a viral vector. The vector can also be, for example, a cloning vector or an expression vector. Expression vectors include vectors for prokaryotic cells such as Escherichia coli and actinomycetes, and vectors for eukaryotic cells such as yeast cells, insect cells, and mammalian cells. More specifically, viral vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, Sendai virus vectors, etc., and examples of non-viral vectors include various plasmid vectors, Liposome vectors, positively charged liposome vectors (Felgner, P.L., Gadek, T.R., Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, and BAC vectors. be able to.

The polynucleotide of the present invention is preferably a viral vector or a viral genome from the viewpoint of reducing damage to cells when obtaining the cells of the present invention described below. The polynucleotide of the present invention is more preferably a retroviral vector, a retroviral genome, a lentiviral vector, or a lentiviral genome. The polynucleotide of the invention is particularly preferably a lentiviral vector or a lentiviral genome.

3. Cell In one aspect, the present invention relates to a cell (herein sometimes referred to as "cell 1 of the present invention") containing the polynucleotide of the present invention. In one embodiment, the present invention also provides cells (herein referred to as "cells of the present invention 2") that have been modified to reduce the expression of the CUL5 gene and express a chimeric antigen receptor. ). In this specification, the cell 1 of the present invention and the cell 2 of the present invention may be collectively referred to as "the cell of the present invention." These will be explained below.

3-1. Cells of the invention 1
Cell 1 of the present invention can be obtained by a method that includes introducing the polynucleotide of the present invention into the cell. In one embodiment of the cell, the expression of the CUL5 gene is reduced by the CUL5 gene expression suppressing polynucleotide, and the cell expresses a chimeric antigen receptor. The expression level of Cullin-5 protein in cell 1 of the present invention is, for example, 70% of the expression level of Cullin-5 protein in control cells (cells treated under the same conditions except that the polynucleotide of the present invention was not introduced). It can be less than or equal to 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%.

The method of introducing the polynucleotide of the present invention into cells is not particularly limited, but from the viewpoint of reducing damage to cells and further increasing the production efficiency and proliferation ability of CAR-expressing cells, a virus containing the polynucleotide of the present invention can be introduced into cells. It is preferable to use a method of introduction. This makes it possible to introduce cells without using methods such as electroporation or lipofection that may cause damage to cells.

Cells into which the polynucleotide of the present invention is introduced (CAR-expressing cells) include 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. Various cell populations can be used as long as they contain lymphocytes or progenitor cells as described above. PBMC (peripheral blood mononuclear cells) collected from peripheral blood are one of the preferred target cells. That is, in one preferred embodiment, gene introduction operation is performed on PBMC. PBMC can be prepared according to or analogously to a conventional method.

It is preferable to activate CAR-expressing cells before introducing the polynucleotide of the present invention. For example, CAR-expressing cells can be activated by stimulation with anti-CD3 antibodies and anti-CD28 antibodies. For example, stimulation by anti-CD3 antibodies and anti-CD28 antibodies can be applied by culturing in a culture container (eg, a culture dish) whose culture surface is coated with 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 (for example, Dynabeads T-Activator CD3/CD28 provided by VERITAS).

In order to increase the survival rate/proliferation rate of cells, it is preferable to use a culture medium to which T cell growth factors are added during the activation treatment. IL-2, IL-15, IL-7, etc. can be used as T cell growth factors. T cell growth factors such as IL-2, IL-15, and IL-7 can be prepared according to conventional methods. Moreover, commercially available products can also be used. Although this does not exclude the use of T cell growth factors from animal species other than humans, human-derived T cell growth factors (which may be recombinant) are usually used.

Typically, for its application (administration to a patient), cells (lymphocytes introduced with the polynucleotide of the invention) after introduction of the polynucleotide of the invention are expanded. For example, the polynucleotide-introduced lymphocytes of the present invention are cultured using a culture medium supplemented with T cell growth factors (subcultured if necessary). In addition to this culture, the same treatment (reactivation) as in the case of activation of CAR-expressing cells may be performed.

Note that a medium supplemented with serum (human serum, fetal bovine serum, etc.) may be used to culture the CAR-expressing cells and the polynucleotide-introduced lymphocytes of the present invention, but by employing a serum-free medium, It becomes possible to prepare cells that are highly safe for clinical application and have the advantage that differences in culture efficiency due to differences between serum lots are less likely to occur. When using serum, it is preferable to use autologous serum, ie, serum collected from a patient receiving CAR gene-transduced lymphocytes.

3-2. Cells of the invention 2
Cell 2 of the present invention can be obtained by modifying the cell so that expression of the CUL5 gene is reduced and further expressing a chimeric antigen receptor. The expression level of Cullin-5 protein in Cell 2 of the present invention is compared to the expression level of Cullin-5 protein in control cells (cells treated under the same conditions except that the expression of CUL5 gene is not modified to decrease). For example, it can be 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. By decreasing the expression of the CUL5 gene, the proliferation ability of CAR-expressing cells can be increased. Examples of modifications that reduce the expression of the CUL5 gene include gene deletion (gene disruption), mutations in the protein coding region, mutations in the splicing regulatory region, and mutations in the expression regulatory region (e.g., promoter, activator, enhancer, etc.). It will be done. The modification can be performed using a gene editing system such as the CRSPR/Cas system.

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

Regarding matters not explained above, the explanation in "3-1. Cells of the present invention 1" is cited.

3-3. Cells of the Invention The cells from which the cells of the invention are derived are not particularly limited. If the purpose of using the cells of the present invention is to purify the chimeric antigen receptor of the present invention, the derived cells may include cells that can be used for protein expression (e.g., insect cells, eukaryotic cells, mammalian cells, etc.). Can be mentioned.

The cells of the present invention are preferably lymphoid cells (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, and in a more specific embodiment, these cells have the chimeric antigen receptor of the present invention expressed on the cell membrane, Preferably, the chimeric antigen receptor of the present invention is expressed with the antigen-binding domain exposed outside the cell membrane.

After T cells expressing chimeric antigen receptors recognize antigens with their antigen-binding regions, they transmit the recognition signal to the inside of the T cells, activate signals that induce cytotoxic activity, and act in conjunction with this. The cells can then exert attacking or cytotoxic activity against other cells or tissues expressing the antigen.

When cells that exert such functions are CTLs, they are called chimeric antigen receptor T cells (CAR-T cells). Cells that have the potential to exhibit cytotoxic activity, such as NK cells, can also exhibit cytotoxic activity when their antigen-binding regions bind to antigens, similar to chimeric antigen receptor T cells. Therefore, a host cell (particularly a host cell having cytotoxic activity) containing a polynucleotide encoding the chimeric antigen receptor of the present invention is useful as an active ingredient of a pharmaceutical composition.

Four. Pharmaceutical Composition In one aspect, the present invention relates to a pharmaceutical composition (sometimes referred to herein as "the pharmaceutical composition of the present invention") containing the cells of the present invention. This will be explained below.

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) that express the target antigen of the antigen-binding domain. Target diseases include solid cancers and blood cancers. Target diseases include 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, myelodysplasia/myeloproliferative neoplasms (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, multiple myeloma, lung cancer, colorectal cancer, ovarian cancer Examples include breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma. "Treatment" includes alleviating symptoms characteristic of the target disease or accompanying symptoms (alleviation), preventing or delaying the worsening of symptoms, and the like. "Prevention" refers to preventing or delaying the onset/expression of a disease (disorder) or its symptoms, or reducing the risk of onset/expression. On the other hand, "improvement" refers to alleviation (alleviation), improvement, remission, or cure (including partial cure) of a disease (disorder) or its symptoms.

The pharmaceutical composition of the present invention contains a therapeutically effective amount of the cells of the present invention. For example, a single administration can contain 1×10 ⁴ to 1×10 ¹⁰ cells. Dimethyl sulfoxide (DMSO) and serum albumin are used to protect cells; antibiotics are used to prevent bacterial contamination; various ingredients (vitamins, , cytokines, growth factors, steroids, etc.) can be included in the cell preparation.

The route of administration of the CAR gene-transduced lymphocytes or cell preparations of the present invention is not particularly limited. For example, it is administered by intravenous, intraarterial, intraportal, intradermal, subcutaneous, intramuscular, or intraperitoneal injection. Instead of systemic administration, local administration may also be used. An example of local administration is direct injection into the target tissue, organ, or organ. The administration schedule may be created taking into consideration the gender, age, weight, pathological condition, etc. of the subject (patient). In addition to single administration, it may be administered continuously or periodically multiple times.

The present invention will be described in detail below based on Examples, but the present invention is not limited to these Examples.

Test example 1. Design and production of CAR construct
From the N-terminal side,
Anti-CD19 scFv domain (amino acid sequence: SEQ ID NO: 1, nucleotide sequence: SEQ ID NO: 9), IgG4 hinge region (amino acid sequence: SEQ ID NO: 2, nucleotide sequence: SEQ ID NO: 10),
CD28 transmembrane domain (amino acid sequence: SEQ ID NO: 3, nucleotide sequence: SEQ ID NO: 11),
CD28 intracellular domain (amino acid sequence: SEQ ID NO: 4, nucleotide sequence: SEQ ID NO: 12),
CD3ζ intracellular domain (amino acid sequence: SEQ ID NO: 5, nucleotide sequence: SEQ ID NO: 13),
5 amino acid spacer,
A CAR construct (Figure 1, amino acid sequence) consisting of a T2A sequence (amino acid sequence: SEQ ID NO: 6, nucleotide sequence: SEQ ID NO: 14) and a Truncated EGFR domain (amino acid sequence: SEQ ID NO: 7, nucleotide sequence: SEQ ID NO: 15) : SEQ ID NO: 8, nucleotide sequence: SEQ ID NO: 16), and the coding sequence of the CAR (nucleotide sequence: SEQ ID NO: 16) was added to both ends of the restriction enzyme recognition sequence to create a retrovirus vector. (vector name: pLZRS-BMN-Z).

Note that the sequences of the anti-CD19 scFv domain and Truncated EGFR domain include a GM-CSF receptor leader sequence added to the N-terminus.

Test example 2. CAR-T cell growth factor screening
CAR-T cell growth factors were screened by GW (Genome Wide)-CRISPR screening. An overview is shown in Figure 2. Specifically, it was performed as follows.

Blood was collected from three healthy donors, CD8-positive lymphocytes were sorted using immunomagnetic beads (Myltenii), and the CD8-positive cells were stimulated with CD3/CD28 beads to start proliferation (Day 0). On Day 1, a lentivirus expressing GW-gRNA was introduced, and on Day 2, Cas9 protein was introduced by electroporation. On Day 3, a retrovirus packaged with a CAR construct (Test Example 1) consisting of CD19CAR and EGFR (truncated EGFR, tEGFR) lacking an intracellular domain as a gene transfer marker was introduced. On Day 7, tEGFR-positive cells (CD19CAR-positive cells) were purified using biotinylated EGFR antibodies and anti-biotin microbeads using tEGFR as a gene transfer marker. Culture was continued until Day 10, after which stimulation was repeated three times using the Raji cell line (CD19+, Burkitt's lymphoma-derived cell line) that had been irradiated with 100 Gy every 10 days, and the remaining cells were collected on Day 40. The gRNA sequences were determined and quantified using DNA collected from Day 10 CAR-T cells and Day 40 CAR-T cells. The ranking of knocked out genes for each donor was performed using GFOLD analysis. By integrating GFOLD analysis, we extracted six genes among the top 10 genes reported to be expressed in T cells.

Next, a secondary screening was conducted. In place of the GW-gRNA Library, use a lentiviral vector (vector name: pLV-EGFP-U6) containing an expression cassette of one of the two gRNAs targeting each of the six genes extracted above. prepared CAR-T cells according to the scheme in Figure 2. The gRNA target sequence is as follows.
SMCO2 gene (NCBI gene ID: 341346)
gRNA1: SEQ ID NO: 17
gRNA2: SEQ ID NO: 18
TSR2 gene (NCBI gene ID: 90121)
gRNA1: SEQ ID NO: 19
gRNA2: SEQ ID NO: 20
PKM gene (NCBI gene ID: 5315)
gRNA1: SEQ ID NO: 21
gRNA2: SEQ ID NO: 22
CUL5 gene (NCBI gene ID: 8065)
gRNA1 (#4690): SEQ ID NO: 23
gRNA2 (#8377): SEQ ID NO: 24
KLRC1 gene (NCBI gene ID: 3821)
gRNA1: SEQ ID NO: 25
gRNA2: SEQ ID NO: 26
ING3 gene (NCBI gene ID: 54556)
gRNA1: SEQ ID NO: 27
gRNA2: SEQ ID NO: 28

The results are shown in FIGS. 3 and 4. Only when CUL5 was suppressed, the GFP-positive rate increased. The results of measuring the CUL5 expression level on Day 7 by Western blotting are shown in FIG. 5.

Test example 3. Analysis of the effect of CUL5 knockout 1
Except for using cells derived from each of the five donors and using a lentiviral vector containing an expression cassette for one of two types of gRNA (Test Example 2) targeting CUL5 instead of the GW-gRNA Library. CAR-T cells were prepared according to the scheme in Figure 2.

After stimulation with Raji cells irradiated on Day 10, CAR-T cells were stained with CellTrace Violet on the 4th day (14th day in total), and the extent of cell division was measured by flow cytometry 72 hours later (17th day in total). It was measured by diluting CellTrace Violet using The results are shown in FIG. It was found that cell division was accelerated by CUL5 knockout.

Intracellular cytokine production after stimulation was measured by flow cytometry with intracellular cytokine staining. Control sgRNA-introduced CAR-T and CUL5KO CAR-T were stimulated with K562 (CD19 negative) and Raji (CD19 positive), respectively, and intracellular cytokine (IFN-γ) was stained 4 hours later. The results are shown in FIG. It was found that cytokine production was enhanced by CUL5 knockout.

Test example 4. Analysis of the effect of CUL5 knockout 2
Except for using cells from each of the four donors and using a lentiviral vector containing an expression cassette for one of two types of gRNA (Test Example 2) targeting CUL5 instead of the GW-gRNA Library. CAR-T cells were prepared according to the scheme in Figure 2.

After priming CAR-T cells on day 10 with CD3/28 beads and Raji cells, the surface characteristics on day 4 were measured by flow cytometry, and effector memory T cells ( _TEM : CD45RA (-) CCR7 (-)) were detected. and the percentage of central memory T cells (T _CM :CD45RA(-)CCR7(+)) was calculated. The results are shown in FIG. We found that CUL5 knockout increased effector memory T cells.

Test example 5. Analysis of the effect of CUL5 knockout 3
CAR-T cells were prepared according to the scheme in Figure 2, except that instead of the GW-gRNA Library, a lentiviral vector containing an expression cassette for one of the two gRNAs targeting CUL5 (Test Example 2) was used. did.

Raji cells (Raji/Luc cells) that constitutively express firefly luciferase and GFP were intravenously injected into NOD-Scid shi common gamma chain knock out mice (NOG mice) on Day 0, and various T cells were injected on Day 7. (CAR-T cells expressing control gRNA, CAR-T cells expressing CUL5 gRNA, T cells expressing tEGFR) were administered intravenously at 1.0x10e6. After that, luciferin was administered every week, and the luminescence emitted by tumor cells was photographed.

The results are shown in Figures 9 and 10. With CUL5 knockout, CAR-T cells showed lower bioluminescent levels over time and survival was significantly better. The experiment was conducted using T cells generated from 3 different donors: tEGFR-T, n=6; control CAR-T and CUL5KO CAR-T, n=10.

Test example 6. Two-in-one vector design and production 1
From the N-terminal side,
Anti-CD19 scFv domain (amino acid sequence: SEQ ID NO: 1, nucleotide sequence: SEQ ID NO: 9), IgG4 hinge region (amino acid sequence: SEQ ID NO: 2, nucleotide sequence: SEQ ID NO: 10),
CD28 transmembrane domain (amino acid sequence: SEQ ID NO: 3, nucleotide sequence: SEQ ID NO: 11),
CD28 intracellular domain (amino acid sequence: SEQ ID NO: 4, nucleotide sequence: SEQ ID NO: 12),
CD3ζ intracellular domain (amino acid sequence: SEQ ID NO: 5, nucleotide sequence: SEQ ID NO: 13),
5 amino acid spacer,
T2A sequence (amino acid sequence: SEQ ID NO: 6, base sequence: SEQ ID NO: 14),
A CAR expression cassette consisting of Truncated EGFR domains (amino acid sequence: SEQ ID NO: 7, base sequence: SEQ ID NO: 15), and
Each of the expression cassettes for shRNA (nucleotide sequence: SEQ ID NO: 29) against CUL5 was incorporated into a lentiviral vector (vector name: pGreenPuro_shRNA(EF1)) using restriction enzyme 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. A schematic diagram of the obtained CAR construct is shown in FIG. 11. Note that the sequences of the anti-CD19 scFv domain and Truncated EGFR domain include a GM-CSF receptor leader sequence added to the N-terminus.

Test example 7. Analysis of the effect of two-in-one vector
Method 1
Except for using cells derived from three donors and using a lentiviral vector containing an expression cassette of one of two gRNAs targeting CUL5 (Test Example 2) instead of the GW-gRNA Library. CAR-T cells were prepared according to the scheme in Figure 2.

Method 2
On the other hand, CAR-T cells were prepared by changing part of the scheme in FIG. 2 (scheme before the start of stimulation) to the scheme in FIG. 12. Specifically, blood was collected from three healthy donors, CD8-positive lymphocytes were sorted using immunomagnetic beads (Myltenii), and the CD8-positive cells were stimulated with CD3/CD28 beads to begin proliferation (Day 0). ). On Day 1, the lentivirus obtained from the Two-in-one vector (Test Example 6) was introduced. On Day 5, tEGFR-positive cells (CD19CAR-positive cells) were purified using a biotinylated EGFR antibody and anti-biotin microbeads using tEGFR as a gene transfer marker. Culture was continued until Day 10, after which stimulation was repeated three times using the Raji cell line (CD19+, Burkitt's lymphoma-derived cell line) that had been irradiated with 100 Gy every 10 days, and the remaining cells were collected on Day 40.

Method 3
Furthermore, as a control for 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.

For all of the above methods, CAR-T cell production was started from 1x10e6 CD8 positive cells.

The number of CAR-T cells was determined by trypan blue staining. Figure 13 shows the number of CAR-T cells on Day 7. The results of measuring the CUL5 expression level on Day 10 by Western blotting are shown in FIG. 14. Furthermore, the proliferation of CAR-T cells is shown in FIG. 15. By producing CUL5 knockdown CAR-T cells using a polynucleotide containing an expression cassette for a CUL5 gene expression suppression polynucleotide and an expression cassette for a chimeric antigen receptor as in Method 2, the production efficiency of CAR-T cells, It was found that proliferation could be greatly improved.

Test example 8. Analysis of the effect of two-in-one vector 2
CAR-T cells were prepared according to Method 2 and Method 3 of Test Example 7.

NOD-Scid shi common gamma chain knock out mice (NOG mice, n = 8 to 10) were intravenously injected with 2.0x10e6 Raji cells (Raji/Luc cells) that constitutively expressed firefly luciferase and GFP on Day 0. On Day 7, 1.0x10e6 of various T cells were intravenously administered. After that, luciferin was administered every week, and the luminescence emitted by tumor cells was photographed.

The results are shown in FIG. In CAR-T cells in which CUL5 was knocked down using a two-in-one vector, bioluminescent levels decreased over time.

Test example 9. Analysis of the effect of two-in-one vector 3
CAR-T cells were prepared according to Method 2 and Method 3 of Test Example 7.

Raji cells (Raji/Luc cells) that constitutively express firefly luciferase and GFP were intravenously injected into NOD-Scid shi common gamma chain knock out mice (NOG mice) on Day 0, and various T cells were injected on Day 10. was administered intravenously at 1.0x10e6. Thereafter, luciferin was administered every week, and the luminescence emitted by tumor cells was photographed to measure tumor volume. In addition, when the tumor volume exceeded 1500 mm3, euthanasia was performed.

The measurement results of tumor volume are shown in FIG. 17, and the survival rates are shown in FIG. 18. In CAR-T cells in which CUL5 was knocked down using a two-in-one vector, tumor growth was significantly suppressed and survival rate was also greatly improved.

Test example 10. Two-in-one vector design and production 2
An anti-Eva1 scFv domain is used instead of the anti-CD19 scFv domain, and a 4-1BB intracellular domain (amino acid sequence: SEQ ID NO: 30, nucleotide sequence: SEQ ID NO: 33) or CD79a is used instead of the CD28 intracellular domain. Test examples except for adopting the tandem linking domain of intracellular domain (amino acid sequence: SEQ ID NO: 31, nucleotide sequence: SEQ ID NO: 34) and CD40 intracellular domain (amino acid sequence: SEQ ID NO: 32, nucleotide sequence: SEQ ID NO: 35). A lentiviral vector was obtained in the same manner as in 6.

Test example 11. Analysis of the effect of two-in-one vector 4
CAR-T cells were prepared in the same manner as Method 2 and Method 3 of Test Example 7 except that the Two-in-one vector of Test Example 10 was used.

NCI-H1975 (NCI-H1975-firefly luciferase-GFP, cells that constitutively express firefly luciferase and GFP) 1x10e6 were 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) was administered intravenously. Thereafter, luciferin was administered at regular intervals, and the luminescence emitted by tumor cells was photographed to measure tumor volume. In addition, when the tumor volume exceeded 2000 mm3, euthanasia was performed.

The luminescence image is shown in FIG. 19, and the measurement results of the tumor volume are shown in FIG. 20.

Claims (4)

A cell that has been modified to reduce expression of the CUL5 gene and expresses a chimeric antigen receptor. The cell according to claim 1, which is a lymphocyte cell. A pharmaceutical composition containing the cell according to claim 1 or 2. The pharmaceutical composition according to claim 3, which is used for treating, preventing, or improving cancer.

Images