Method for producing t cells for transplantation, and t cells

Abstract

To provide a method for producing T cells for transplantation, by which T cells having an improved duration in vivo can be produced, to provide T cells having an improved duration in vivo, and to provide a pharmaceutical composition containing the T cells.SOLUTION: A method for producing a T cell for transplantation, comprising a step (A) of increasing an expression level of a nitric oxide synthase interacting protein in a T cell. There is also provided a T cell into which a nucleic acid having a function of improving an expression level of a nitric oxide synthase interacting protein is introduced.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing T cells for transplantation and to T cells. [Background technology]

[0002] In recent years, attention has been focused on transplanting immune cells with enhanced immune activity against diseased cells, such as cancer cells, into patients with cancer and other diseases. Therapeutic immune cells include T cells genetically modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR) that specifically recognizes diseased cells. T cells genetically modified to express a diseased-cell-specific TCR are called TCR-T. T cells genetically modified to express a diseased-cell-specific CAR are called CAR-T.

[0003] For example, Patent Document 1 describes TCR gene-modified T cells that target cancer cells that present hTERT-derived peptides. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2024-051333 Summary of the Invention [Problem to be solved by the invention]

[0005] In T cell therapy using TCR-T, CAR-T, etc., a frequent problem is that the administered T cells cannot persist in vivo for a long period of time. To enhance the effect of T cell therapy, it is preferable that the administered T cells can persist in vivo for a long period of time.

[0006] Therefore, an object of the present invention is to provide a method for producing T cells for transplantation, which can produce T cells with an improved in vivo persistence period, as well as T cells with an improved in vivo persistence period and a pharmaceutical composition containing the T cells. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] A method for producing transplantable T cells, comprising step (A) of increasing the expression level of nitric oxide synthase-interacting protein in T cells. [2] The method for producing transplantable T cells described in [1], wherein the step (A) comprises introducing into the T cells a nucleic acid having the function of increasing the expression level of the nitric oxide synthase-interacting protein. [3] The method for producing transplantable T cells according to [2], wherein the nucleic acid is a nucleic acid encoding the nitric oxide synthase-interacting protein. [4] The method for producing T cells for transplantation described in any one of [1] to [3], wherein the T cells for transplantation have a longer duration in vivo compared to T cells in which the expression level of the nitric oxide synthase-interacting protein is not improved. [5] The method for producing T cells for transplantation according to any one of [1] to [4], wherein the T cells for transplantation are CD8-positive T cells. [6] T cells into which a nucleic acid having the function of increasing the expression level of a nitric oxide synthase-interacting protein has been introduced. [7] The T cell described in [6], wherein the nucleic acid is a nucleic acid encoding the nitric oxide synthase-interacting protein. [8] The T cells according to [6] or [7], which persist in vivo for a longer period of time than T cells to which the nucleic acid has not been introduced. [9] The T cells according to any one of [6] to [8], which are CD8-positive T cells.

[10] A pharmaceutical composition comprising the T cells according to any one of [6] to [9] and a pharmaceutically acceptable carrier. [Effects of the Invention]

[0008] According to the present invention, there are provided a method for producing T cells for transplantation, which can produce T cells with an improved in vivo persistence period, T cells with an improved in vivo persistence period, and a pharmaceutical composition containing the T cells. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the protocol of the mouse test carried out in the Examples. [Figure 2] 1 shows the flow of flow cytometry analysis of lymphocytes obtained from mice transplanted with transduced P14 cells in a mouse test performed in the Examples. [Figure 3] The results of an analysis of the VEX+ cell population obtained from the peripheral blood (PB) of mice transplanted with transduced P14 cells are shown. [Figure 4A] Based on the results of Figure 3, a graph is shown in which the proportions of empty vector-transfected cells and NOSIP vector-transfected cells in the VEX+ cell population are quantified. [Figure 4B] The figures show the results of calculating the number of empty vector-transduced cells and NOSIP vector-transduced cells per 1 × 106 peripheral blood mononuclear cells (PBMCs) for the VEX+ cell population obtained from the peripheral blood (PB) of mice transplanted with transduced P14 cells. [Figure 5A] 1 shows the results of an analysis of the VEX+ cell population obtained from the spleen of mice transplanted with transduced P14 cells. [Figure 5B] The figures show the results of calculating the numbers of empty vector-introduced cells and NOSIP vector-introduced cells per 1 x 106 lymphocytes in a VEX+ cell population obtained from the spleen of a mouse transplanted with transduced P14 cells. [Figure 6A] 1 shows the results of an analysis of the VEX+ cell population obtained from the liver of mice transplanted with transduced P14 cells. [Figure 6B] The figures show the results of calculating the numbers of empty vector-introduced cells and NOSIP vector-introduced cells per 1 x 106 lymphocytes for a VEX+ cell population obtained from the liver of a mouse transplanted with transduced P14 cells. [Figure 7]The results of an analysis of cell surface antigens of VEX+ cell populations obtained from the peripheral blood (PB) of mice transplanted with transduced P14 cells are shown. [Figure 8] The results of an analysis of cell surface antigens of VEX+ cell populations obtained from the peripheral blood (PB) of mice transplanted with transduced P14 cells are shown. [Figure 9] 10 shows a graph quantifying the expression levels of CX3CR1 in empty vector-introduced cells and NOSIP vector-introduced cells obtained from peripheral blood on day 10 of the test in mice transplanted with transduced P14 cells. [Figure 10] The figure shows the results of analyzing the expression level of CX3CR1 in empty vector-introduced cells and NOSIP vector-introduced cells obtained from peripheral blood on day 10 of the test in mice transplanted with transduced P14 cells. [Figure 11] FIG. 11 shows the results of analyzing the expression levels of PD-1, TIGIT, and LAG-3 in the CX3CR1(hi) subset, CX3CR1(int) subset, and CX3CR1(neg) subset grouped in FIG. [Figure 12] The figures show the results of calculating the proportions of CX3CR1(hi), CX3CR1(int), and CX3CR1(neg) subsets in empty vector-transfected cells and NOSIP vector-transfected cells obtained from the peripheral blood of mice transplanted with transfected P14 cells on day 10 of the study. [Figure 13] 1 shows the results of analyzing the expression of intracellular cytokines in empty vector-introduced cells and NOSIP vector-introduced cells obtained from the spleens of mice transplanted with transduced P14 cells. [Figure 14] The results of Western blotting performed on empty vector-transfected cells and NOSIP vector-transfected cells are shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] A numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0011] CD8 positive cells are CD8 + These may be referred to as "CD8-positive cells." Cells positive for other proteins may also be referred to in a similar manner. "CD8-positive cells" refer to cells that express CD8 to an extent that they can be separated using a flow cytometer. The same applies to cells positive for other proteins.

[0012] "Donor" refers to an individual who provides T cells in a T cell transplant. "Recipient" refers to the individual into whom T cells are transplanted in a T cell transplant.

[0013] The "in vivo duration" of transplanted T cells includes not only the period during which T cells identical to the transplanted T cells survive in the recipient's body, but also the period during which T cells derived from the transplanted T cells survive in the recipient's body. "T cells derived from transplanted T cells" include T cells generated by proliferation of the transplanted T cells and T cells generated by differentiation of the transplanted T cells.

[0014] [Method of producing T cells for transplantation] A first aspect of the present disclosure is a method for producing transplantable T cells, which includes a step (A) of increasing the expression level of a nitric oxide synthase-interacting protein in T cells.

[0015] <Process (A)> (T cells) The T cells may be mature T cells or precursor T cells. The T cells may be naive T cells or effector T cells. The T cells may be αβ T cells or γδ T cells. Examples of T cells include cytotoxic T cells, helper T cells, memory T cells, natural killer T cells, and their precursor cells. Examples of T cells include cells expressing at least one selected from the group consisting of CD8 and CD4. Cytotoxic T cells are CD8-positive T cells that express CD8. Helper T cells are CD4-positive T cells that express CD4. In one embodiment, the T cells are CD8-positive T cells.

[0016] The species from which T cells are derived is not particularly limited. Examples of T cells include mammalian T cells. Mammals include, but are not limited to, Primates (humans, gorillas, chimpanzees, monkeys, marmosets, etc.), Rodents (mice, guinea pigs, rats, hamsters, etc.), Carnivores (dogs, cats, bears, weasels, etc.), Artiodactyla (cattle, goats, deer, camels, etc.), and Perissodactyla (horses, donkeys, rhinoceroses, tapirs, etc.). T cells can be from the same species as the recipient into which the T cells are to be transplanted. T cells may be autologous or allogeneic. Autologous T cells are T cells collected from a donor who is the same individual as the recipient into whom the T cells are to be transplanted. Allogeneic T cells are T cells collected from a donor who is a different individual from the recipient.

[0017] The T cells may be genetically modified T cells. The T cells may be T cells (TCR-T) into which a gene encoding a T cell receptor (TCR) that specifically recognizes diseased cells has been introduced. The T cells may be T cells (CAR-T) into which a gene encoding a chimeric antigen receptor (CAR) that specifically recognizes diseased cells has been introduced. Examples of the diseased cells include cancer cells and virus-infected cells.

[0018] (Nitric oxide synthase-interacting protein (NOSIP)) Nitric oxide synthase-interacting protein (NOSIP) affects nitric oxide production by affecting the activity and localization of nitric oxide synthases. The nitric oxide synthases that interact with NOSIP include endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS). NOSIP binds to these nitric oxide synthases and inhibits nitric oxide production by nitric oxide synthases. This allows NOSIP to affect nitric oxide signaling and immune cell function. NOSIP functions as an E3 ubiquitin ligase and is thought to regulate the activity of protein phosphatase 2 phosphatase activator (PP2A). PP2A plays an important role in regulating cell proliferation and signal transduction.

[0019] The biological species from which NOSIP is derived is not particularly limited. NOSIP from the same biological species as that from which T cells are derived can be used. For example, when T cells are human T cells, human NOSIP can be used. Alternatively, NOSIP from the same biological species as the recipient can be used. For example, when the recipient is human, human NOSIP can be used. Examples of the amino acid sequence of human NOSIP (Gene ID: 51070) include GeneBank accession numbers NP_001257889.1 (SEQ ID NO: 1), NP_001350578.1 (SEQ ID NO: 2), and NP_057037.1 (SEQ ID NO: 3). Examples of cDNA encoding NOSIP consisting of the amino acid sequence set forth in SEQ ID NO: 1 include GeneBank accession number NM_001270960.2. Examples of cDNA encoding NOSIP consisting of the amino acid sequence set forth in SEQ ID NO: 2 include GeneBank accession number NM_001363649.2. An example of a cDNA encoding NOSIP consisting of the amino acid sequence set forth in SEQ ID NO: 3 is GeneBank Accession No. NM_015953.5. The amino acid sequence of mouse NOSIP (Gene ID: 66394) is available under GeneBank accession number NP_079809.1. A cDNA encoding the amino acid sequence of NP_079809.1 is available under GeneBank accession number NM_025533.3. Sequence information of NOSIPs in other organisms can also be obtained from publicly known databases such as GenBank.

[0020] NOSIP may be wild-type NOSIP or modified NOSIP. "Wild-type NOSIP" refers to NOSIP that has not been artificially modified. Wild-type NOSIP may be NOSIP found in organisms in nature. Examples of the amino acid sequence of human wild-type NOSIP include the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3. Modified NOSIP is NOSIP that has been artificially modified from wild-type NOSIP. Examples of modified NOSIP include the following proteins:

[0021] (a) A protein comprising an amino acid sequence in which one or more amino acids have been mutated in the amino acid sequence of wild-type NOSIP, and which has a function equivalent to that of wild-type NOSIP. (b) A protein comprising an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of wild-type NOSIP and that has a function equivalent to that of wild-type NOSIP.

[0022] In the above (a), the "mutation" may be any of deletion, substitution, addition, and insertion, or a combination of two or more of these. In the above (a), the term "plurality" is not particularly limited, as long as the resulting protein has a function equivalent to that of NOSIP. Examples of "plurality" include 2 to 50, and may be 2 to 30, 2 to 20, 2 to 10, 2 to 5, 2 to 3, or 2.

[0023] In the above (b), the sequence identity is preferably 85% or more, and may be 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The sequence identity between amino acid sequences or nucleotide sequences is determined by aligning two amino acid sequences or nucleotide sequences, with gaps inserted and deleted to maximize the number of corresponding amino acids or nucleotides, and calculating the percentage of matching amino acids or nucleotides relative to the entire amino acid sequence or the entire nucleotide sequence excluding gaps in the resulting alignment. The sequence identity between amino acid sequences or nucleotide sequences can be determined using various homology search software known in the art. For example, the sequence identity of amino acid sequences can be calculated based on the alignment obtained using the known homology search software BLASTP. For example, the sequence identity of nucleotide sequences can be calculated based on the alignment obtained using the known homology search software BLASTN.

[0024] In the above (a) and (b), "having functions equivalent to those of wild-type NOSIP" means having one or more of the functions of wild-type NOSIP, such as interacting with nitric oxide synthase, suppressing the expression of CX3CR1 (C-X3-C motif chemokine receptor 1), and improving the persistence of T cells in vivo.

[0025] (Method for increasing NOSIP expression level) Known methods can be used to increase the expression level of NOSIP in T cells. Examples of methods for increasing the expression level of NOSIP include introducing a nucleic acid capable of increasing the expression level of NOSIP into T cells. Examples of nucleic acids capable of increasing the expression level of NOSIP include (1) a nucleic acid encoding NOSIP, and (2) a nucleic acid capable of increasing the expression level of the endogenous NOSIP gene in T cells.

[0026] (1) a nucleic acid encoding NOSIP: A "nucleic acid encoding NOSIP" (hereinafter also referred to as a "NOSIP-encoding nucleic acid") is a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of NOSIP (hereinafter also referred to as a "NOSIP-encoding sequence"). A NOSIP-encoding nucleic acid may be DNA or RNA. Examples of NOSIP-encoding nucleic acids include a NOSIP expression vector containing a NOSIP gene and NOSIP mRNA. A "NOSIP gene" refers to a nucleic acid containing an open reading frame (ORF) encoding NOSIP. A NOSIP gene may contain only exons, or may contain exons and introns. A NOSIP gene may contain a 5'UTR, a 3'UTR, and the like in addition to the ORF encoding NOSIP. A NOSIP gene may be a NOSIP cDNA.

[0027] The NOSIP coding sequence may be any of a variety of degenerate codons, as long as it encodes NOSIP. The NOSIP coding sequence may be codon-optimized for the species from which the T cells are derived. "Codon optimization" refers to replacing at least one codon in the original nucleotide sequence with a codon more frequently used in the target species while maintaining the original amino acid sequence. By codon-optimizing the NOSIP coding sequence for the species from which the T cells are derived, the translation efficiency of NOSIP in the T cells can be increased.

[0028] <NOSIP expression vector> Methods for introducing a NOSIP-encoding nucleic acid into T cells include introducing a NOSIP expression vector containing the NOSIP gene into T cells. A NOSIP expression vector refers to a vector equipped with a system that enables the NOSIP gene to be expressed in the cells into which the vector is introduced. A NOSIP expression vector includes, for example, a promoter that can function in T cells and a NOSIP gene operably linked to the promoter. "The promoter is operable" means that the NOSIP gene operably linked to the promoter can be expressed in T cells. "Operatively linked to the promoter" means that the NOSIP gene is linked to the promoter so that the NOSIP gene is expressed in T cells under the control of the promoter.

[0029] The promoter is not particularly limited as long as it has the function of expressing the NOSIP gene in T cells. Examples of promoters include pol II promoters. Examples of pol II promoters include, but are not limited to, the EF1α promoter, CMV promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, and CBh promoter. The promoter may be a constitutive promoter or an inducible promoter. An inducible promoter is a promoter that induces gene expression under specific conditions. The use of a constitutive promoter allows stable expression of the NOSIP gene in T cells. The use of an inducible promoter allows control of NOSIP gene expression in T cells.

[0030] The NOSIP expression vector may contain other components in addition to the NOSIP gene and promoter. These components include a terminator, an enhancer, a poly(A) addition signal, a marker gene, a replication origin, and a gene encoding a protein that binds to the replication origin and controls replication. The terminator is linked to the 3' end of the NOSIP gene. Terminators commonly used in the species from which T cells are derived can be used. The term "marker gene" refers to a gene that enables cell sorting or selection by introducing the marker gene into cells. Examples of marker genes include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. Examples of drug resistance genes include puromycin resistance genes, geneticin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, hygromycin resistance genes, and chloramphenicol resistance genes. Examples of fluorescent protein genes include VEX (violet-excited fluorescent protein), green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, red fluorescent protein (RFP) gene, etc. Examples of luminescent enzyme genes include luciferase gene, etc. Examples of chromogenic enzyme genes include β-galactosidase gene, β-glucuronidase gene, alkaline phosphatase gene, etc.

[0031] The type of expression vector is not particularly limited, and any known expression vector can be used, including, for example, a plasmid vector and a viral vector.

[0032] The plasmid vector is not particularly limited as long as it can be expressed in the target cells. For example, when the target cells are animal cells, a plasmid vector commonly used for expression in animal cells can be used. Examples of plasmid vectors for expression in animal cells include, but are not limited to, pX459, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0033] Examples of viral vectors include, but are not limited to, lymphocytic choriomeningitis virus (LCMV) vectors, retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors, herpes viral vectors, vaccinia viral vectors, pox viral vectors, polio viral vectors, Silvis viral vectors, rhabdoviral vectors, paramyxoviral vectors, and orthomyxoviral vectors.

[0034] The NOSIP expression vector can be introduced into T cells by known methods, such as lipofection, microinjection, DEAE-dextran, gene gun, electroporation, and calcium phosphate. When the NOSIP expression vector is a viral vector, the vector can be transfected into cells (e.g., polybrene).

[0035] <NOSIP mRNA> The NOSIP-encoding nucleic acid may be a NOSIP mRNA. The NOSIP mRNA may contain other components in addition to the NOSIP coding sequence, such as a 5' Cap, a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), and a poly(A) tail.

[0036] NOSIP mRNA may contain a 5' Cap at the 5' end. Examples of 5' Cap structures include Cap0, Cap1, and Cap2 structures. The cap structure is typically a 7-methylguanine ribonucleotide, attached via a 5'-triphosphate to the 5' position of the first nucleotide in the 5'-3' direction of the mRNA, i.e., the first cap-proximal nucleotide. In the Cap0 structure, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain 2'-hydroxyl. In the Cap1 structure, the riboses of the first cap-proximal nucleotide of the mRNA contain 2'-methoxy, and the riboses of the second nucleotide contain 2'-hydroxyl. In the Cap2 structure, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain 2'-methoxy.

[0037] A cap structure can be incorporated into the 5' end of mRNA during transcription by known methods. For example, a cap structure can be incorporated into mRNA by co-transcription using a commercially available capping kit. A cap structure may also be added to mRNA after transcription or to chemically synthesized RNA using a capping enzyme.

[0038] The NOSIP mRNA may contain either or both of the 5'UTR and the 3'UTR. The 5'UTR and the 3'UTR may be those of the wild-type NOSIP mRNA or those of a different mRNA.

[0039] NOSIP mRNA may contain a Kozak sequence. The Kozak sequence can influence translation initiation and the total amount of protein translated from the mRNA. The Kozak sequence contains a methionine codon that can function as an initiation codon. The minimal Kozak sequence is NNNRUGN (N is any nucleotide residue, and R is a purine residue (A or G)). In the formula, the first N is preferably A or G, and the second N is preferably G. In one embodiment, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG.

[0040] The NOSIP mRNA may contain a poly(A) tail at the 3' end. The poly(A) tail may contain at least eight consecutive adenine nucleotides, but may also contain one or more non-adenine nucleotide residues (e.g., G, C, U). The length of the poly(A) tail may be, for example, 10 to 500 nucleotides, 30 to 300 nucleotides, or 60 to 250 nucleotides.

[0041] NOSIP mRNA can be introduced into T cells by known methods, such as those using commercially available RNA transfection reagents such as Lipofectamine (registered trademark) MessengerMAX (Life Technologies).

[0042] (2) A nucleic acid having the function of increasing the expression level of the endogenous NOSIP gene in T cells: Examples of nucleic acids capable of increasing the expression level of the endogenous NOSIP gene in T cells include promoter nucleic acids capable of increasing the expression level of the endogenous NOSIP gene in T cells. Examples of such promoters include promoters of genes that are expressed in higher amounts in T cells than the endogenous NOSIP gene. Alternatively, known promoters commonly used in transduction of animal cells may be used. Examples of such promoters include those described above.

[0043] The promoter may be a constitutive promoter or an inducible promoter. When a constitutive promoter is used, it is preferable to use a promoter of a constitutively expressed gene that is expressed at a higher level than the endogenous NOSIP promoter in T cells. When an inducible promoter is used, it is preferable to use a promoter of an inducible gene that is expressed at a higher level than the endogenous NOSIP promoter in T cells under conditions that induce gene expression.

[0044] The promoter is introduced into T cells so that it is functionally linked to the endogenous NOSIP gene of the T cells. Examples of methods for site-specifically introducing the promoter include methods using a genome editing system such as the CRISPR / Cas system.

[0045] <Other processes> The production method of this embodiment may include other steps in addition to the above step (A), such as a step of selecting T cells with increased NOSIP expression (selection step) and a step of activating T cells (activation step).

[0046] (Selection process) The production method of this embodiment may include, after step (A), a step of selecting T cells in which the expression level of NOSIP is increased. When a nucleic acid capable of increasing the expression level of NOSIP is introduced into T cells together with a marker gene in step (A), T cells into which the nucleic acid capable of increasing the expression level of NOSIP has been introduced can be selected using the expression of the marker gene as an indicator. When the marker gene is a fluorescent protein gene, cells expressing the marker gene can be selected using flow cytometry or the like. When the marker gene is an antibiotic resistance gene, cells expressing the marker gene can be selected by culturing the cells in a medium containing the antibiotic.

[0047] (activation process) The production method of this embodiment may include a step of activating T cells before or after step (A). T cell activation can be performed by known methods. Examples of methods for activating T cells include contacting T cells with antigen-presenting cells and introducing nucleic acids encoding TCRs or CARs that specifically recognize disease cells. Examples of the disease cells include cancer cells and virus-infected cells.

[0048] T cells may be activated by culturing them in a medium containing IL-2 on a plate precoated with CD3ε antibody and CD28 antibody.

[0049] T cells obtained by the production method of this embodiment persist in vivo for a longer period of time than T cells in which the expression level of nitric oxide synthase-interacting protein is not improved. Therefore, when transplanted into a living body, they can persist for a long period of time (e.g., one month or more) in the recipient's body. This may be because high expression of NOSIP suppresses CX3CR1 expression, maintaining a state capable of self-renewal. Because T cells obtained by the production method of this embodiment can persist in vivo for a long period of time, when transplanted into a living body, they can effectively exert an immune effect against disease cells. Therefore, T cells obtained by the production method of this embodiment are useful as T cells for transplantation.

[0050] [T cells] A second aspect of the present disclosure is a T cell into which a nucleic acid capable of increasing the expression level of NOSIP has been introduced.

[0051] The nucleic acid having the function of increasing the expression level of NOSIP is the same as that described above in [Method for producing T cells for transplantation]. Examples of types of T cells include those similar to those listed above in [Method for producing T cells for transplantation]. The T cells of this embodiment can be produced by the production method according to the first aspect. The T cells of this embodiment may contain, for example, at least one selected from the group consisting of an exogenous NOSIP-encoding nucleic acid and an exogenous promoter operably linked to endogenous NOSIP.

[0052] The T cells of this embodiment persist in vivo for a longer period than T cells that are not transfected with a nucleic acid that has the function of increasing the expression level of NOSIP, and are therefore useful as T cells for transplantation.

[0053] [Pharmaceutical composition] A third aspect of the present disclosure is a pharmaceutical composition. The pharmaceutical composition comprises the T cells according to the second aspect and a pharmaceutically acceptable carrier. The pharmaceutical composition of this embodiment comprises the T cells according to the second aspect as an active ingredient.

[0054] The term "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of an active ingredient and is not substantially toxic to a recipient. "Not substantially toxic" means that the ingredient is not toxic to a recipient at a commonly used dose. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the immune function of T cells according to the second aspect and is not substantially toxic to a recipient. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredient that is typically considered an inactive ingredient. Pharmaceutically acceptable carriers include, but are not limited to, solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, buffers, thickeners, fillers, etc. Pharmaceutically acceptable carriers may be, for example, physiological saline, phosphate-buffered saline, glucose solutions, buffered saline, etc. These may also be used with the addition of salts, sugars, sugar alcohols, etc. One type of pharmaceutically acceptable carrier may be used alone, or two or more types may be used in combination.

[0055] The pharmaceutical composition may contain other ingredients in addition to the above-mentioned ingredients. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Examples of the other ingredients include pharmaceutical additives other than those mentioned above. Examples of pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, colorants, etc. The pharmaceutical composition may also contain adjuvants, immune checkpoint inhibitors, etc. One type of other ingredient may be used alone, or two or more types may be used in combination.

[0056] The dosage form of the pharmaceutical composition is not particularly limited and may be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment is preferably a parenteral formulation, preferably a liquid formulation. The pharmaceutical composition of this embodiment is preferably administered by injection, infusion, or the like.

[0057] The pharmaceutical composition of this embodiment is preferably administered parenterally. Examples of parenteral administration routes include intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and enteral administration, with intravenous administration being preferred.

[0058] The pharmaceutical composition can be administered in a therapeutically effective amount of the T cells according to the second aspect. "Therapeutically effective amount" refers to the amount of a drug effective for treating or preventing a target disease. For example, the therapeutically effective amount of the T cells can be an amount effective for the T cells to exert an immune effect against disease cells. The therapeutically effective amount may be determined appropriately depending on the symptoms, weight, age, sex, etc. of the patient, as well as the dosage form and administration method of the pharmaceutical composition. For example, the pharmaceutical composition can be administered in a single dose of about 1 x 10 6 ~Approx. 1×10 12 Approximately 1 x 10 8 ~Approx. 1×10 11 It may be one.

[0059] The pharmaceutical composition may be administered in a single dose or multiple doses. In the case of multiple doses, the administration interval may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., as well as the dosage form of the pharmaceutical composition and the administration method, etc. The administration interval may be, for example, every few hours, 2 to 3 times a day, once a day, once every 2 to 3 days, once a week, once a month, once every few months, etc.

[0060] The disease to which the pharmaceutical composition of this embodiment is applied is not particularly limited as long as it is a disease caused by the presence of diseased cells, and examples of such diseases include cancer and viral infections. [Example]

[0061] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0062] [method] <Mouse> Wild-type (WT) C57BL / 6 mice expressing CD45.1 were purchased from the Jackson Laboratory. CD45.2 + P14 mice of + have a TCR specific for the H-2Db GP33-41 epitope of LCMV introduced into their genes and were provided through the kindness of Dr. Ruka Setoguchi of RCAI, RIKEN, Japan. From these, WT P14 mice expressing both CD45.1 and CD45.2, or WT P14 mice expressing only CD45.1 were generated. WT C57BL / 6 mice expressing CD45.2 were purchased from Sankyo Labo Service Corporation. Male mice aged 6 - 10 weeks were used for the experiments. All animal experiments were conducted in accordance with the guidelines approved by the Animal Experiment Committee of Kanazawa University.

[0063] <Virus Infection> According to the protocol described in the literature (Kurachi, M., Barnitz, R., Yosef, N. et al. Nat Immunol 15, 373 - 383 (2014).), mice were infected with Lymphocytic Choriomeningitis Virus (LCMV). Two strains of LCMV were used: LCMV Armstrong (Arm), which is known as an acute infection, and LCMV Clone 13 (Cl13), a mutant strain that induces chronic infection, to establish an acute infection model and a chronic infection model, respectively. For infection, LCMV Arm was administered intraperitoneally (i.p.) at a dose of 2×10 5 plaque-forming units (PFU), and LCMV Cl13 was administered intravenously (i.v.) at a dose of 4×10 6 PFU.

[0064] <Construction of Retroviral Plasmids Using the Gateway (Registered Trademark) System> Plasmid vectors were constructed using Gateway technology according to the method described previously (Johnson, JL, Georgakilas, G., Petrovic, J., Kurachi, M., Cai, S., Harly, C., Pear, WS, Bhandoola, A., Wherry, EJ, Vahedi, G. Immunity 48(2), 243-257.e10 (2018).). The retroviral vectors used were murine stem cell virus (MSCV)-ccdB-VEX (violet-excited fluorescent protein) and an empty vector control (MSCV-VEX). The cDNA encoding nitric oxide synthase interacting protein (NOSIP) was cloned into the MSCV-ccdB-VEX plasmid to generate MSCV-NOSIP-VEX. The NOSIP cDNA (Origene, MR204220) was first obtained and its sequence confirmed by DNA sequencing. Primers containing Gateway reaction sequences (attB1 and attB2) were designed for PCR amplification of the insert. An entry plasmid was then constructed using the BP clonase reaction, and the NOSIP expression plasmid (MSCV-NOSIP-VEX) was constructed using the LR clonase reaction. Additionally, an empty vector (MSCV-Empty-VEX) was used as a control.

[0065] <Retroviral transduction> Retroviral supernatant was prepared by transfecting 293T cells with the retroviral expression plasmid and pCL-Eco packaging plasmid using the Lipofectamine 3000 transfection kit (Invitrogen, L3000015). Prior to retroviral transfection, cell culture plates were coated with anti-mouse CD3ε antibody (Clone: ​​145-2C11, BioLegend, 100359) and anti-mouse CD28 antibody (Clone: ​​37.51, BioLegend, 102121) at concentrations of 5.0 μg / ml and 2.0 μg / ml, respectively, for 3 hours (Kurachi, M., Kurachi, J., Chen, Z. et al. Nat Protoc 12, 1980-1998 (2017)). Bulk CD8+ T cell isolates were then isolated from the spleens of WT P14 mice using the EasySep® Mouse CD8+ T cell isolation Kit (STEMCELL Technologies, 19853A). + T cells (hereinafter also referred to as "P14 cells") were isolated to ensure the purity of the cell population. The isolated P14 cells were then stimulated for 24 hours on plates precoated with CD3ε and CD28 antibodies in cell culture medium containing 100 U / ml of recombinant human IL-2 to induce T cell activation. After activation, P14 cells were transduced with a retrovirus using spin-infection (2000 g, 30°C, 60 minutes) in the presence of polybrene (final concentration: 4 μg / ml). The NOSIP vector (MSCV-NOSIP-VEX) was transduced with CD8 T cells derived from WT-P14 mice expressing only CD45.1. + T cells (CD45.1 + ) (hereinafter referred to as “P14 cells (CD45.1 + The empty vector (MSCV-Empty-VEX) was transduced into CD8 T cells derived from WT-P14 mice that express both CD45.1 and CD45.2. + T cells (CD45.1 + CD45.2 + ) (hereinafter referred to as “P14 cells (CD45.1 + CD45.2 +) was transduced into the

[0066] <Cell sorting> Twenty-four hours after retroviral transduction, P14 cells were stained with CD45.2-BUV395, CD45.1-BUV805, and CD8-PE, and then sorted using a FACS Aria II (BD Biosciences) to isolate VEX+ cells, which represent successfully transduced cells.

[0067] Adoptive transfer of transduced P14 cells P14 cells (CD45.1 + CD45.2 + ) and NOSIP vector-transduced P14 cells (CD45.1 + ) in a 1:1 ratio, for a total of 1x10 5 The cells were infected with LCMV virus 48 hours prior to the initiation of the transfection. + The cells were administered intravenously to recipient mice, and adoptive immune transfer was performed.

[0068] <Mouse test> The mouse study was performed according to the following protocol (see Figure 1). Day 0: CD45.1 + CD45.2 + TCR transgenic P14 mice or CD45.1 + P14 cells were isolated from the spleens of TCR transgenic P14 mice. They were treated with a standard T cell activation protocol using anti-CD3ε / CD28 antibodies and IL-2. On the same day, naive CD45.2 + Recipient mice (5 mice) were infected with the LCMV clone 13 (Cl13) strain. Day 1: Activated P14 cells were transduced with the empty vector (MSCV-Empty-VEX) and the NOSIP vector (MSCV-NOSIP-VEX). The empty vector transduced P14 cells (CD45.1 + CD45.2 +The NOSIP vector was transfected into P14 cells (CD45.1 + ) was introduced. Day 2: 24 hours after transduction, transduced P14 cells were sorted and transplanted into recipient mice. Days 10, 17, and 24: CD8 cells were collected from mice by blood collection from the retro-orbital plexus under anesthesia. + Cells were harvested and analyzed using flow cytometry. + CD8 + The cells were analyzed. Day 31: Mice were euthanized and CD8 cells were isolated from peripheral blood, spleen, and liver. + Cells were harvested and analyzed using flow cytometry. + CD8 + The cells were analyzed.

[0069] <Flow cytometry> Flow cytometry analysis was performed using a FACSymphony A5 flow cytometer (BD Biosciences). Data were analyzed using FlowJo v10 software. Lymphocytes were obtained from peripheral blood, spleen, and liver for analysis. For spleen and liver, tissue was collected and homogenized using a 70 μm cell strainer to collect immune cells. Liver immune cells were further purified by Percoll density gradient centrifugation. For peripheral blood, red blood cells were lysed using RBC lysis buffer.

[0070] For cell surface staining, 2x10 6 Cells were seeded in 96-well plates and incubated with BD Horizon TMThe cells were suspended in PBS supplemented with Fixable Viability Stain 440UV (BD Biosciences, 566332) and incubated at 4°C for 30 minutes. Subsequently, the cells were incubated with antibodies in staining buffer (PBS containing 2% fetal bovine serum (FBS) and EDTA) for 30 minutes at 4°C. The antibodies used were anti-CD45.1, anti-CD62L, anti-CD27, anti-CD127, anti-CD8, anti-TIGIT, anti-CD69, anti-CX3CR1, anti-TIM3, anti-CD44, anti-PD-1, anti-LAG-3, anti-KLRG1, anti-LY108, and anti-CD45.2.

[0071] For intracellular cytokine staining, cells were incubated with 1 μg / ml gp33 peptide in the presence of 0.1% brefeldin A and 0.1% monensin for 4 hours at 37°C. After surface marker staining, cells were fixed with 2% paraformaldehyde (PFA) for 20 minutes at 4°C to preserve the VEX signal. Cytokine staining was then performed using the cytofix / cytoperm kit (BD Biosciences, 554722). The antibodies used were anti-CD45.1, anti-CD8, anti-CD45.2, anti-CD62L, anti-CD44, anti-CD127, anti-TNFα, anti-GzmB, anti-IFNγ, and anti-IL-2.

[0072] Figure 2 shows the flow of flow cytometry analysis. Lymphocyte population, single cell population, live cell population, CD8 + Cell populations and VEX + Gating was performed in the order of cell populations, and VEX + Cell populations were obtained. VEX + The cell populations were distinguished into empty vector-transfected cell populations and NOSIP vector-transfected cell populations based on the expression status of CD45.1 and CD45.2.

[0073] <Antibody> The antibodies used in flow cytometry analysis are shown in Table 1. Antibodies other than FVS440UV were added to the reaction solution at an antibody:reaction solution ratio of 1:200. FVS440UV was added to the reaction solution at an antibody:reaction solution ratio of 1:2000.

[0074] [Table 1]

[0075] <Western blotting> Proteins were extracted from P14 cells transfected with the empty vector (MSCV-Empty-VEX) or the NOSIP vector (MSCV-NOSIP-VEX) using RIPA Lysis Buffer (Millipore, 20-188) supplemented with a protease inhibitor cocktail (Roche, 04693159001) and phosphatase inhibitor (Roche, 04906837001). Protein concentrations were measured using a BCA assay, and equal amounts of protein were loaded onto a 5-20% gradient SDS-PAGE gel (Wako, 197-15011). After electrophoresis, proteins were transferred from the gel to a PVDF membrane (Millipore, IPVH00010, Immobilon-P). After transfer, the PVDF membrane was blocked for 1 hour in TBST (Tris-buffered saline with Tween-20) containing 5% skim milk (Wako, 190-12865). The PVDF membrane was then incubated overnight at 4°C with a rabbit polyclonal antibody against NOSIP (Abcam, ab102087) diluted 1:1000 as the primary antibody. β-actin was used as an internal control (Cell Signaling, 4967L). The next day, the PVDF membrane was washed and incubated with an anti-rabbit IgG, HRP-conjugated antibody (Cell Signaling, 7074) as the secondary antibody for 30 minutes at room temperature. The PVDF membrane was then washed three times with TBST. Finally, protein bands were visualized using the Clarity Western ECL Substrate kit (Bio-RAD, 1705061).

[0076] <Statistical analysis> Student's t-test and two-way ANOVA were performed using GraphPad Prism 8.0 software. Results were considered significant at a P value of <0.05 for all data. *p<0.05, **p<0.01, ***p<0.0001.

[0077] [result] <Evaluation of persistence of transduced P14 cells> Figure 3 shows VEX obtained from peripheral blood (PB) of mice transplanted with transduced P14 cells. + The results of cell population analysis are shown below. The number of days in the figure indicates the number of days from Cl13 infection (see Figure 1, same below). The results on day 2 show the proportion of empty vector-transfected cells and NOSIP vector-transfected cells among the transplanted P14 cells. The proportion of NOSIP vector-transfected cells increased with the passage of time after transplantation. By day 31, the proportion of NOSIP vector-transfected cells had reached approximately 90%.

[0078] Figure 4A shows the VEX + 1 is a graph quantifying the proportion of empty vector-transfected cells and NOSIP vector-transfected cells in a cell population. It was confirmed that the proportion of NOSIP vector-transfected cells increased with the passage of days.

[0079] Figure 4B shows the results of a 1×10 peripheral blood mononuclear cell (PBMC) 6 The results of calculating the number of empty vector-transfected cells and NOSIP vector-transfected cells per cell are shown. The number of NOSIP vector-transfected cells was greater than that of empty vector-transfected cells at all times. The number of empty vector-transfected cells decreased on day 24 and was close to zero on day 31. On the other hand, the number of NOSIP vector-transfected cells remained at approximately 2,000 even on day 31.

[0080] Figure 5A shows VEX obtained from the spleen of a mouse transplanted with transduced P14 cells. + The results of cell population analysis are shown in Figure 5B.6 The results of calculating the number of empty vector-transduced cells and NOSIP vector-transduced cells per recipient are shown in Figure 5B. The results for five recipient mice are plotted. VEX isolated from the spleen + In cells, as in peripheral blood, the proportion of NOSIP vector-transduced cells was higher than that of empty vector-transduced cells.

[0081] Figure 6A shows VEX obtained from the liver of a mouse transplanted with transduced P14 cells. + The results of cell population analysis are shown in Figure 6B. 6 The results of calculating the number of empty vector-transduced cells and NOSIP vector-transduced cells per recipient are shown in Figure 6B. The results for five recipient mice are plotted. VEX isolated from the liver + In cells, as in peripheral blood and spleen, the proportion of NOSIP vector-transduced cells was higher than that of empty vector-transduced cells.

[0082] From the above results, CD8 + It was confirmed that the in vivo duration could be extended by introducing the NOSIP gene into cells.

[0083] <Cell surface antigen expression analysis> Figures 7 and 8 show VEX obtained from the peripheral blood of mice transplanted with transduced P14 cells. + The results of analyzing cell surface antigens of the cell populations are shown. It was confirmed that there was a difference in the expression level of CX3CR1 between the empty vector-transfected cells and the NOSIP vector-transfected cells.

[0084] 9 is a graph quantifying the expression levels of CX3CR1 in empty vector-introduced cells and NOSIP vector-introduced cells obtained from peripheral blood of mice transplanted with transduced P14 cells on day 10. It was confirmed that the expression level of CX3CR1 was lower in the NOSIP vector-introduced cells than in the empty vector-introduced cells.

[0085] Figure 10 shows the results of analyzing CX3CR1 expression levels in Empty vector-transfected and NOSIP vector-transfected cells obtained from peripheral blood on day 10 of the study in mice transplanted with transfected P14 cells. The cells were grouped into subsets with high CX3CR1 expression (CX3CR1(hi)), intermediate CX3CR1 expression (CX3CR1(int)), and low CX3CR1 expression (CX3CR1(neg)), and the proportion of each subset was calculated. As a result, it was confirmed that the proportion of the CX3CR1(neg) subset was higher in NOSIP vector-transfected cells. On the other hand, the proportion of the CX3CR1(int) subset was higher in Empty vector-transfected cells.

[0086] Figure 11 shows the results of analyzing the expression levels of PD-1, TIGIT, and LAG-3 in the CX3CR1(hi) subset, CX3CR1(int) subset, and CX3CR1(neg) subset grouped in Figure 10. The CX3CR1(neg) subset had a higher proportion of cell populations expressing high levels of PD-1, TIGIT, and LAG-3 than the CX3CR1(hi) subset and CX3CR1(int) subset.

[0087] Figure 12 shows the results of calculating the percentages of the CX3CR1(hi), CX3CR1(int), and CX3CR1(neg) subsets in Empty vector-transfected cells and NOSIP vector-transfected cells obtained from peripheral blood on day 10 of the study in mice transplanted with transfected P14 cells. Similar to the results in Figure 10, the percentage of the CX3CR1(neg) subset was the highest in the NOSIP vector-transfected cells, and the percentage of the CX3CR1(hi) subset was low. On the other hand, the percentage of the CX3CR1(int) subset was the highest in the Empty vector-transfected cells, and the percentage of the CX3CR1(hi) subset was over 10%.

[0088] These results confirmed that the proportion of the CX3CR1(neg) subset was increased in NOSIP vector-transfected cells. CX3CR1 (C-X3-C motif chemokine receptor 1) is a seven-transmembrane domain G protein-coupled receptor. CD8 + Cells are thought to differentiate from the CX3CR1(neg) subset to the CX3CR1(int) subset and from the CX3CR1(int) subset to the CX3CR1(hi) subset (Yamauchi et al., JCI Insight. 2020 Apr 23; 5(8)). The CX3CR1(neg) and CX3CR1(int) subsets are thought to be self-renewing, and differentiation from the CX3CR1(neg) subset to the CX3CR1(int) subset is thought to be reversible. On the other hand, the CX3CR1(hi) subset does not self-renew, and differentiation from the CX3CR1(int) subset to the CX3CR1(hi) subset is reported to be irreversible (Gordon et al., Cell Rep. 2018 Apr 17; 23(3): 768-782.). In NOSIP vector-transfected cells, the increased proportion of the CX3CR1(neg) subset may be involved in the long-term persistence in vivo.

[0089] <Intracellular cytokine expression analysis> Figure 13 shows the results of analyzing intracellular cytokine expression in empty vector-transfected cells and NOSIP vector-transfected cells obtained from the spleen of a mouse transplanted with transfected P14 cells. "FMO" stands for "Fluorescence Minus One." FMO was used as a negative control, in which only one fluorescent dye of interest was added and the remaining dyes were added. There was no significant difference in the expression levels of the cytokines analyzed between the empty vector-transfected cells and the NOSIP vector-transfected cells.

[0090] <Western blotting> The results of Western blotting performed on the empty vector-transfected cells and the NOSIP vector-transfected cells are shown in Figure 14. It was confirmed that the amount of NOSIP expression was increased in the NOSIP vector-transfected cells compared to the empty vector-transfected cells. These results confirmed that the NOSIP vector expresses NOSIP in the cells into which it is introduced. [Industrial Applicability]

[0091] According to the present invention, there are provided a method for producing T cells for transplantation, which can produce T cells with an improved in vivo persistence period, T cells with an improved in vivo persistence period, and a pharmaceutical composition containing the T cells.

Claims

1. A method for producing transplantable T cells, comprising step (A) of increasing the expression level of a nitric oxide synthase-interacting protein in T cells.

2. the step (A) comprises introducing into the T cell a nucleic acid having a function of increasing the expression amount of the nitric oxide synthase-interacting protein; The method for producing transplantable T cells according to claim 1.

3. the nucleic acid is a nucleic acid encoding the nitric oxide synthase-interacting protein; The method for producing transplantable T cells according to claim 2.

4. The T cells for transplantation have a longer duration in vivo compared to T cells in which the expression level of the nitric oxide synthase-interacting protein is not improved. The method for producing the transplantable T cells according to claim 1 or 2.

5. The method for producing T cells for transplantation according to claim 1 or 2, wherein the T cells for transplantation are CD8-positive T cells.

6. A T cell into which a nucleic acid having the function of increasing the expression level of a nitric oxide synthase-interacting protein has been introduced.

7. The T cell of claim 6 , wherein the nucleic acid is a nucleic acid encoding the nitric oxide synthase-interacting protein.

8. The nucleic acid has a longer duration in vivo than T cells into which the nucleic acid has not been introduced. A T cell according to claim 6 or 7.

9. The T cell according to claim 6 or 7, which is a CD8-positive T cell.

10. A pharmaceutical composition comprising the T cells of claim 6 or 7 and a pharmaceutically acceptable carrier.

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