WO2021206104A1

WO2021206104A1 - pp65-CONTAINING ARTIFICIAL ADJUVANT VECTOR CELLS USED IN TREATMENT OF CANCER

Info

Publication number: WO2021206104A1
Application number: PCT/JP2021/014686
Authority: WO
Inventors: 恵介大隅; 中村　仁
Original assignee: アステラス製薬株式会社
Priority date: 2020-04-08
Filing date: 2021-04-07
Publication date: 2021-10-14
Also published as: JP2023078487A

Abstract

Summary: A problem addressed by the present invention is to provide aAVC-pp65 cells for use in the treatment of patients having a pp65-expressing cancer.　aAVC are known to activate innate immunity via CD1d/α-GalCer complexes. The present inventors produced aAVC-pp65 cells that expressed pp65, a structural protein of HCMV virus particles, in aAVC and discovered that the aAVC-pp65 cells exhibit a pp65-specific T cell inducing effect and a pp65 protein-specific antitumor effect in mice in vivo.　Specifically, the present invention provides aAVC-pp65 cells, a pharmaceutical composition including said cells, a method for producing said cells, and a method for treating or preventing pp65-expressing cancers by said cells.

Description

Pp65-containing artificial adjuvant vector cells used for cancer treatment

The present invention relates to a pp65-contining artificial adjuvant vector cell: aAVC-pp65 cell used for the treatment or prevention of cancer.

Tumors that develop from glial cells that support brain nerve cells (neurons) are called gliomas. There are various types of glioma such as astrocytoma and oligodendroglioma, which account for about 25% of tumors originating from the brain. The grade is divided into four according to the malignancy, and the grade 4 having the highest malignancy is called glioblastoma multiforme (GBM).

The standard treatment to be performed at the first onset of GBM has been established. After the tumor is removed by surgery, local radiation is given to the tumor remaining around the removed tumor, and chemotherapy with oral temozolomide is given. After that, continue oral administration of temozolomide as maintenance therapy. However, it is difficult to completely remove the tumor, and most patients have a recurrence within 1 year after radiation therapy. Several treatments have been proposed after recurrence, but there is no standardized method, and the average survival time after recurrence is very short, within one year. Against this background, the development of new drugs and new treatments that are effective for GBM is strongly desired, and for the purpose of providing new treatments, EGFRvIII, IL-13Rα2, survivin, MAGEA1, Ephrin A2, HER2, Development of vaccines and T cell therapies targeting antigens such as AIM2, TRP-2, and gp100 is underway (Non-Patent Documents 1 and 2).

Human cytomegalovirus (HMMV) is a double-stranded DNA virus belonging to the genus Cytomegalovirus. HCMV usually infects the human body through secretions such as saliva and urine in early childhood, and then parasitizes the human body by latent / persistent infection for life. It is known that many healthy people are pre-infected with HCMV and most of them are subclinical infections. However, due to a decrease in immune function, HCMV reactivates and proliferates, resulting in organ transplant patients such as kidney transplantation and bone marrow transplantation, and immunodeficiency such as acquired immunodeficiency syndrome (AIDS), malignant tumors, and immunosuppressive therapy. It often results in serious infections in patients with the condition.

In recent years, it has been reported that human cytomegalovirus may also infect tumor cells, causing tumor cells to acquire resistance to tumor immunity and anticancer drugs and increase malignancy (Non-Patent Document 3). In GBM, expression of HCMV-derived antigen is detected in 90% or more of patients (Non-Patent Documents 4 to 6). In addition, HCMV DNA was detected in colon cancer (Non-Patent Document 7), and HCMV-derived DNA and protein were also detected in prostate cancer (Non-Patent Document 8) and breast cancer (Non-Patent Document 9). Relationships with various cancers have been suggested.

pp65 (65KDa phosphoprotein, gene name UL83) is a protein that constitutes a segment between the envelope of HCMV virus particles and nucleocapsid, and is the most abundant protein among the approximately 70 types of proteins that form HCMV virus particles. be. This pp65 protein is not only useful as an antigen for diagnosing viral antigenemia, but is also being studied as a target antigen for a vaccine for treatment or prevention of HCMV virus infection and GBM (Non-Patent Documents). 10 and 11). However, no drug has yet been launched as a vaccine against the HCMV virus.

Fujii et al. Exogenously expressed CD1d and cancer antigens in human-derived cells, and further pulsed α-galactosylceramide (α-GalCer) into the cells as an artificial adjuvant vector cell (artificial adjuvant vector cell: aAVC) was prepared (Patent Documents 1 to 3). The aAVC activates natural killer T (NKT) cells via the CD1d / α-GalCer complex, and the activated NKT cells produce cytokines such as interferon γ (IFN-γ) to produce natural killer (NKT). ) Activate cells and the like. In a mouse model, NK cell-dependent antitumor effect by administration of aAVC has been shown (Patent Documents 1 to 3). In addition, the aAVC administered to the mouse is rapidly killed by the activated NKT cells in vivo, and the fragment of the aAVC is taken up by the dendritic cells. The dendritic cell incorporating the aAVC fragment presents the cancer antigen fragment incorporated into the major histocompatibility complex (MHC) on the cell surface, and produces cancer antigen-specific T cells. Induce. In a mouse model, an antitumor effect mediated by induction of cancer antigen-specific T cells by administration of aAVC has been shown (Patent Documents 1 to 3, Non-Patent Documents 12 and 13). Thus, it has been shown that aAVC can strongly induce two immune mechanisms: innate immune activation by NK cell activation via NKT cell activation and acquired immune induction by antigen-specific T cell induction. .. However, to date, there have been no reports on cells expressing HCMV antigen such as pp65 in aAVC cells, activation of NK / NKT cells using the cells, and GBM antigen-specific immunoinducing action.

WO2007 / 097370 WO2010 / 061930 WO2013 / 018778

An object of the present invention is to provide aAVC-pp65 cells effective for treating or preventing pp65-expressing cancer.

The present inventors prepared aAVC (also referred to as aAVC (3T3) -pp65 cell) in which pp65 was expressed in mouse cells (Example 1-1), and the aAVC (3T3) -pp65 cell was used in mice. Inducing pp65-specific T cells (Example 1-2), pp65 protein-specific antitumor effect (Example 1-) in pp65-expressing B16 melanoma cell-bearing mice and pp65-expressing GL-261 glioma cell-bearing mice. It was found that 3 and 1-4) are shown. Furthermore, based on these findings, the present inventors prepared aAVC (also referred to as aAVC-pp65) in which pp65 was expressed in human cells (Example 2). That is, the present invention provides aAVC-pp65 cells, a pharmaceutical composition containing the cells, a method for producing the cells, and a method for treating or preventing pp65-expressing cancer by the cells carrying a CD1d ligand on the surface of the cells. do.

That is, the present invention may include the following inventions as medically or industrially useful substances or methods.

[1] A human-derived cell into which a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof have been introduced exogenously.
[2] The cell according to [1], wherein the CD1d is a human CD1d.
[3] The cell according to [1] or [2], wherein the human-derived cell is a normal cell.
[4] The cell according to [3], wherein the normal cell is a cell derived from human fetal kidney cell 293 (HEK293) cell.
[5] The cell according to any one of [1] to [4], expressing CD1d and pp65 or a fragment thereof.
[6] The cell according to [5], wherein the CD1d ligand is loaded on the surface of the cell.
[7] The cell according to [6], wherein the CD1d ligand is α-GalCer.
[8] A pharmaceutical composition containing the cells according to [6] or [7].
[9] The pharmaceutical composition according to [8] for use in the treatment of cancer.
[10] A method for treating cancer, which comprises a step of administering the cells according to [6] or [7] to a subject.
[11] The cell according to [6] or [7] for treating cancer.
[12] Use of the cells according to [6] or [7] for producing a pharmaceutical composition used for treating cancer.
[13] A method for producing cells used for treating cancer.
A step of introducing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into human-derived cells, and
A method comprising culturing the cells.
[14] The method according to [13], wherein the CD1d is a human CD1d.
[15] The method according to [13] or [14], wherein the human-derived cell is a normal cell.
[16] The method according to [15], wherein the normal cells are cells derived from human fetal kidney cells 293 (HEK293) cells.
[17] The method according to any one of [13] to [16], further comprising loading a CD1d ligand on the surface of the cell.
[18] The method according to [17], wherein the CD1d ligand is α-GalCer.

The aAVC-pp65 cells of the present invention and the pharmaceutical composition of the present invention are useful in the treatment of cancer.

FIG. 1-1 shows the number of IFN-γ-producing T cells in the spleen cells of aVC (3T3) -pp65 cell-administered mice. BICANATE on the horizontal axis indicates a control group. aVC (3T3) -in the pp65 cell administration group- (minus) indicates that no peptide for stimulating T cells contained in spleen cells was added, and WT1 and pp65 were used as T cell stimulants, respectively. It is shown that the WT1 peptide and the pp65 peptide were used. ^{The vertical axis shows the number of spots per 3 × 10 5} spleen cells when counted by the ELISPOT reader, the horizontal line shows the average value of the number of spots for each individual, and the error bar shows the standard error of the average value. FIG. 1-2 shows the antitumor effect of aAVC (3T3) -pp65 cells in pp65-expressing B16-bearing mice. aAVC (3T3) -WT1 cells were used as a control test aAVC not expressing pp65. BICANATE indicates a control group. The vertical axis shows the tumor volume, and the horizontal axis shows the number of days after the administration of cancer cells. The solid line shows the transition of the average value of the tumor volume of each group, and the error bar shows the standard error of the average value. The significance probability P value (**: P <0.01) was determined by Dunnett's multiple comparison test on the 17th day tumor volume of the BICANATE administration group, aAVC (3T3) -pp65 cells and aAVC (3T3) -WT1 cell administration group. Was calculated by comparing. The number of cases on the 17th day of the ^{5 × 10 2} cells administration group of aAVC (3T3) -pp65 cells is 11. FIG. 1-3 shows the antitumor effect of aAVC (3T3) -pp65 cells in pp65-expressing GL-261 cancer-bearing mice. aAVC (3T3) -WT1 cells were used as a control test aAVC not expressing pp65. The vertical axis shows the survival rate, and the horizontal axis shows the number of days after cancer cell administration. The significance probability P value was determined by comparing the survival time of the BICANATE-administered group with the survival time of the aAVC (3T3) -pp65 cells and aAVC (3T3) -WT1 cell-administered group using the log rank test. ** in the figure indicates a group in which the P value is smaller than 0.01/6 (significance level corrected by the Bonferroni method).

The present invention will be described in detail below, but the present invention is not limited thereto. Unless otherwise defined herein, scientific and technical terms used in the context of the present invention shall have meanings commonly understood by those skilled in the art.

<Artificial adjuvant vector cell>
In the present specification, "artificial adjuvant vector cell" and "aAVC" are synonymous and are used interchangeably with a CD1d polypeptide (hereinafter, also referred to as "CD1d") and any one or more cancers. It means a cell expressing an antigen or a fragment thereof.
In the present specification, "foreign" or "foreign" refers to the introduction of an artificially produced gene or polynucleotide into a target cell by an operation such as gene manipulation or gene transfer, and the target intracellular. It is used interchangeably as a term to refer to the artificial expression of the proteins encoded by them by genes or polynucleotides artificially introduced into the.

<AAVC-pp65 cells of the present invention>
The present invention
Provided are human-derived cells (also referred to herein as "aAVC-pp65 cells") into which a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof have been exogenously introduced.
Furthermore, the present invention
Provided are aAVC-pp65 cells expressing CD1d and pp65 or fragments thereof and carrying a CD1d ligand on the cell surface.

1. 1. Human-derived cells The human-derived cells used in the present invention are normal cells or cells derived from cancer tissue. In one embodiment, the human-derived cells used in the present invention are normal cells.
As normal cells, human-derived cells such as cells isolated and / or purified from any human tissue, cells derived from a specific cell type, established cells derived from human tissue, and cells differentiated from stem cells are used. be able to. As the cells derived from cancer tissue, cells isolated and / or purified from the cancer tissue of a patient, or cell lines derived from human cancer tissue can be used. Here, "isolation" means separation from living tissue. In addition, "purification" means the separation of human-derived cells from one or more of the other components contained in the tissue from which the cells are derived. Cell isolation and purification can be performed by known methods.

Cells isolated and / or purified from any human tissue include, for example, stomach, small intestine, large intestine, lung, pancreas, kidney, liver, thymus, spleen, prostate, ovary, uterus, bone marrow, skin, muscle, peripheral blood. It may be a cell derived from any human tissue such as. In one embodiment, the human-derived cells used in the present invention are non-blood cell cells. In one embodiment, the human-derived cells used in the present invention are kidney-derived cells.

Cells derived from a particular cell type are those of a particular cell type in human tissue (eg, stem cell epithelial cells, endothelial cells, epidermal cells, stromal cells, fibroblasts, fat cells, human embryonic kidney cells, keratinized cells, etc. Mesangial cells, myoblasts, nerve cells, glial cells, squamous cells, hepatocytes, mammary cells, mesangial cells, pancreatic β cells, exocrine epithelial cells, endocrine cells, skeletal muscle cells, smooth muscle cells, myocardial cells, osteoblasts It is a cell derived from a cell, an embryo cell, an immune cell (for example, a dendritic cell, a macrophage, a lymphocyte, a B cell, etc.). In one embodiment, the human-derived cells used in the present invention are human embryonic kidney cells.

Established cells derived from human tissue can be prepared by a method known to those skilled in the art, or can be commercially available. Examples of the cell line include human fetal kidney cell 293 (HEK293) cell (J. Gen. Virol .; 1977; 36: 59-74), WI-38 cell, SC-01 MFP cell, or MRC-5 cell. , Or cells derived from those cells. In one embodiment, the human-derived cell used in the present invention is a cell derived from HEK293 cells. In one embodiment, the cell derived from HEK293 cells is a FreeStyle 293-F cell.

The cells differentiated from stem cells are cells differentiated from human-derived induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). Preparation of iPS cells or ES cells and cells differentiated from the cells can be prepared by a method known to those skilled in the art.

Cells derived from cancer tissue can be obtained by isolation and / or purification from the cancer tissue of a patient by a method known to those skilled in the art. It is also possible to use cell lines derived from human cancer tissues that are generally available to those skilled in the art from institutions such as the American Type Culture Collection (ATCC®).

2. pp65
pp65 is one of the constituent proteins of HCMV virus particles and is encoded by the UL83 gene. The UL83 gene is expressed not only in HCMV but also in viruses of the genus Cytomegalovirus such as Chimpanzee cytomegalovirus.

The pp65 used in the present invention may be a naturally occurring pp65, or a variant thereof as long as it induces pp65-specific T cells when administered as aAVC. In one embodiment, the pp65 used in the present invention is a pp65 derived from a virus belonging to the genus Cytomegalovirus. In one embodiment, the pp65 used in the present invention is HPMV-derived pp65 (hereinafter referred to as "human pp65"). In one embodiment, human pp65 is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, human pp65 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. It is a polypeptide consisting of. In one embodiment, human pp65 has 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, 1 to 20 in the amino acid sequence set forth in SEQ ID NO: 2. A polypeptide consisting of an amino acid sequence in which three or one or two amino acids have been deleted, substituted, inserted and / or added. Further, pp65 used in the present invention may be a fragment of pp65 as long as it induces pp65-specific T cells when administered as aAVC. In one embodiment, the fragment of pp65 is a fragment of human pp65. In one embodiment, the fragment of human pp65 is 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more of the amino acid sequence shown in SEQ ID NO: 2. A polypeptide having a length of 85% or more, 90% or more, or 95% or more. The induction of pp65-specific T cells by administration as aAVC can be evaluated, for example, by the method described in Example 1-2 below. The polynucleotide encoding pp65 or a fragment thereof used in the present invention can be prepared by designing a base sequence from the amino acid sequence of pp65 or a fragment thereof used by a method known in the art. In one embodiment, the polynucleotide encoding pp65 is a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1.

As used herein, the term "identity" means the value of Identity obtained by the parameters provided by default using EMBOSS Needle (Nucleic Acids Res .; 2015; 43: W580-W584). The above parameters are as follows.
Gap Open Penalty = 10
Gap Extend Penalty = 0.5
Matrix = EBLOSUM62
End Gap Penalty = false

3. 3. CD1d
The aAVC-pp65 cells of the present invention have exogenously introduced a polynucleotide encoding CD1d. CD1d is an MHC class I-like glycoprotein expressed on the cell surface, and it is known that CD1d-binding NKT cells are activated by presenting a CD1d ligand, which is a glycolipid antigen, on CD1d (Science). 1997; 278 (5343): 1626-1629). The CD1d used in the present invention may be a naturally occurring CD1d or a variant thereof as long as it has the function of CD1d when expressed in human-derived cells. The function of CD1d can be evaluated by measuring the binding between CD1d and the CD1d ligand (eg, α-GalCer). The evaluation can be easily evaluated by those skilled in the art using known methods. The function of CD1d can also be evaluated using the ability of aAVC to activate human NKT cells as an index. The ability to activate human NKT cells is described, for example, in Fig. It can be evaluated in the in vitro evaluation system described in 1. Simply, the activation of NKT cells can be evaluated by co-culturing CD1d-expressing cells cultured in the presence of α-GalCer with NKT cells and measuring the amount of IFN-γ in the culture medium.

In one embodiment, the CD1d used in the present invention is a CD1d derived from a mammal (eg, human, monkey, mouse, rat, dog, chimpanzee, etc.). In one embodiment, the CD1d is a human CD1d. In one embodiment, human CD1d is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, human CD1d lacks 1-10, 1-7, 1-5, 1-3, or 1-2 amino acids in the amino acid sequence set forth in SEQ ID NO: 4. A polypeptide consisting of a substituted, inserted, and / or added amino acid sequence and having the function of CD1d. In one embodiment, human CD1d is an amino acid having at least 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the polypeptide set forth in SEQ ID NO: 4. It is a polypeptide consisting of a sequence and having the function of CD1d.

The polynucleotide encoding CD1d used in the present invention is an exogenously introduced polynucleotide, and the polynucleotide uses a method known in the art to obtain a base sequence from the amino acid sequence of CD1d used. Can be designed and manufactured. In one embodiment, the polynucleotide encoding CD1d is a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3.

4. Promoter In one embodiment, in the aAVC-pp65 cells of the invention, the promoter is operably linked to the polynucleotide encoding CD1d and / or the polynucleotide encoding pp65 or a fragment thereof. As the promoter, either a promoter that constitutively promotes expression or an inducible promoter can be used. As used herein, "operably linked" means a state in which a promoter is linked to a polynucleotide in order to control expression of the polypeptide in a host cell.

Examples of the "promoter that constantly promotes expression" include virus-derived promoters such as CMV (cytomegalovirus), RSV (respiratory syncytial virus), SV40 (simian virus 40), actin promoter, EF (elongation factor), etc. Can be mentioned.

An "inducible promoter" is a promoter that induces expression, and is an inducing factor that drives the promoter (also referred to herein as an "inducible factor of an inducible promoter" or simply an "inducible factor"). A promoter capable of inducing the expression of a polynucleotide operably linked to the promoter in the presence of the promoter. Examples of the inducible promoter include a thermal inducible promoter (for example, a heat shock promoter) and a drug-inducible promoter. In one embodiment, the inducible promoter is a drug-inducible promoter. As used herein, the term "drug-induced promoter" means a promoter whose expression of a polynucleotide operably linked to the promoter is regulated by a drug that is an inducing factor. Examples of the drug-induced promoter include a Cumate operator, a λ operator (for example, 12 × λOp), an inducible promoter of a tetracycline gene expression-inducing system (hereinafter, referred to as “tetracycline-based inducible promoter”), and the like. The Cumate operator is inactive in the presence of the CymR repressor, but in the presence of the inducing factor Cumate, dissociates from the CymR repressor and induces expression of the polynucleotide operably linked to the operator. .. The λ operator is operably linked to the operator in the presence of an inducer (eg, cumermycin) that dimers an activator (λRep-GyrB-AD) capable of activating transcription by dimerization. Induces the expression of the polynucleotide. A tetracycline-based inducible promoter can be used in the presence of an inducing factor, tetracycline or a derivative thereof (eg, doxycycline, etc.) and a reverse tetracycline-regulated transactivator (rtTA) (eg, Tet-On 3G protein). Induces the expression of operably linked polynucleotides. Examples of the tetracycline-based inducible promoter include the TRE3G promoter. In one embodiment, the drug-induced promoter is a tetracycline-based inducible promoter, and in one embodiment, the tetracycline-based inducible promoter is a TRE3G promoter.

5. CD1d Ligand In one embodiment, the aAVC-pp65 cells of the present invention may carry the CD1d ligand on the cell surface. The loading of the CD1d ligand on the cell surface is carried out by pulsing the CD1d ligand (for example, α-GalCer) to the cells expressing CD1d as described in the section <Method for producing aAVC-pp65 cells of the present invention>. Can be done. As used herein, "pulse the CD1d ligand" refers to contacting the cell with the CD1d ligand in the culture medium. In aAVC pulsed with the CD1d ligand, all or at least some of the CD1d are loaded with the CD1d ligand.

Examples of the CD1d ligand used in the present invention include α-galactosylceramide (α-GalCer), α-C-galactosylceramide (α-C-GalCer), 7DW8-5, and isoglobotolihexosylceramide (iGb3). Can be mentioned. In one embodiment, the CD1d ligand is α-GalCer. The chemical name of α-GalCer is (2S, 3S, 4R) -1-O- (α-D-galactosyl) -N-hexacosanoyl-2-amino-1,3,4-octadecanetriol, and CAS RN: It is a substance (molecular weight: 858.34) registered under 158021-47-7 and represented by the molecular formula: C ₅₀ H ₉₉ NO _9. α-GalCer may be synthesized according to a technique known in the art, or commercially available one (for example, α-Galactosylceramide (Funakoshi, Cat. KRN7000)) may be used.

<Method for producing aAVC-pp65 cells of the present invention>
The aAVC-pp65 cells of the present invention can be prepared by introducing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into human-derived cells. The method for producing aAVC-pp65 cells of the present invention may further include a step of culturing the aAVC-pp65 cells, a step of cloning the cells, and a step of loading the CD1d ligand on the cell surface.

1. 1. Introduction of polynucleotide into human-derived cells The aAVC-pp65 cells of the present invention can be prepared by introducing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into human-derived cells (hereinafter,). , The polynucleotide to be introduced is also referred to as a "target polynucleotide").

For the polynucleotide encoding CD1d and the polynucleotide encoding pp65 or a fragment thereof, for example, a nucleotide sequence encoding the amino acid sequence of CD1d and pp65 is obtained from NCBI RefSeq ID or GenBank's Accession number, and standard molecular biology is performed. And / or can be designed and made using chemical techniques. For example, the polynucleotide can be synthesized based on its base sequence by a phosphoramidite method or the like, or is prepared by combining DNA fragments obtained from a cDNA library using a polymerase chain reaction (PCR). can do.

The target polynucleotide can be introduced into human-derived cells by using either a non-viral vector method such as electroporation, lipofection, or microinjection, or a viral vector method. When a non-viral vector system method is used, the target polynucleotide may be in the form of a plasmid vector, cDNA, etc. or in the form of mRNA.

The plasmid vector or viral vector can be used as an expression vector for exogenously expressing CD1d or pp65 or a fragment thereof in human-derived cells. The expression vector is not particularly limited as long as it can express the target polypeptide, and can be prepared by a method known to those skilled in the art. As the expression vector, for example, pcDNA series (Thermo Fisher Scientific), pALTER (registered trademark) -MAX (Promega), pHEK293 Ultra Expression Vector (Takara Bio Inc.) and the like can be used as the plasmid vector. As the viral vector, for example, lentivirus, adenovirus, retrovirus, and adeno-associated virus can be used. For example, when a lentivirus is used for introducing a target polynucleotide into a human-derived cell, the lentivirus can be prepared using a pLVSIN-CMV / EF1α vector (Takara Bio Inc.) or a pLenti vector (Thermo Fisher Scientific). .. When CD1d and pp65 or a fragment thereof are expressed exogenously in human-derived cells, a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof are introduced into human-derived cells using one expression vector. Alternatively, they may be introduced using separate expression vectors. In addition, the expression vector contains a gene that can be a marker for confirming the expression of the target gene or polynucleotide (for example, a drug resistance gene, a gene encoding a reporter enzyme, a gene encoding a fluorescent protein, etc.). You may. The expression vector may further include start and stop codons, enhancer sequences, untranslated regions, splicing junctions, polyadenylation sites, replicable units, and the like.

In one embodiment, the method for producing aAVC-pp65 cells of the present invention is a step of introducing an expression vector containing a polynucleotide encoding CD1d and an expression vector containing a polynucleotide encoding pp65 or a fragment thereof into human-derived cells. including. In one embodiment, the method for producing aAVC-pp65 cells of the present invention comprises introducing an expression vector containing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into human-derived cells. For these methods, in one embodiment the expression vector is a plasmid vector or a viral vector, in one embodiment the expression vector is a viral vector, and in one embodiment the viral vector is a lentivirus. It is a vector.

In one embodiment, the aAVC-pp65 cells of the invention are made by introducing into human-derived cells an expression vector containing a polynucleotide encoding CD1d and an expression vector containing a polynucleotide encoding pp65 or a fragment thereof. Cell. In one embodiment, the aAVC-pp65 cells of the invention are cells made by introducing into a human-derived cell an expression vector containing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof. .. For these cells, in one embodiment, the expression vector is a plasmid vector or viral vector, in one embodiment, the expression vector is a viral vector, and in one embodiment, the viral vector is lentivirus. It is a vector. In one embodiment, the prepared aAVC-pp65 cells have a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof on extrachromosomal or cell nucleus genomic DNA. In one embodiment, the aAVC-pp65 cells produced have a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof on the genomic DNA of its cell nucleus.

2. Culturing aAVC-pp65 cells The aAVC-pp65 cells produced by the above method can be further grown by culturing. Culture of cells for maintaining or growing aAVC-pp65 cells is performed by a known method. Examples of the basal medium include MEM medium (Science; 1952; 122: 501), DMEM medium (Virology; 1959; 8: 396-397), and RPMI 1640 medium (J. Am. Med. Assoc .; 1967; 199: 519). -524), 199 medium (Proc.Soc.Exp.Biol.Med.; 1950; 73: 1-8), FreeStyle293 Expression Medium (Thermo Fisher Science, Cat.12338022), CD 293 Cat.11913019), Expi293 Expression Medium (Thermo Fisher Scientific, Cat. A1435101) can be used. The culture medium is a basal medium, for example, serum (eg, fetal bovine serum), serum substitute (eg, KnockOut Serum Replacement: KSR), fatty acid or lipid, amino acid, vitamin, growth factor, cytokine, antioxidant, 2 -Mercaptoethanol, pyruvate, buffers, inorganic salts, antibiotics and the like can be further included. In one embodiment, the culture medium is a serum-free medium or a chemically defined medium.

Culture conditions (for example, culture conditions such as culture time, temperature, pH of medium, CO ₂ concentration, etc.) can be appropriately selected by those skilled in the art. The pH of the medium is preferably about 6 to 8, and the culture temperature is not particularly limited, but is, for example, about 30 to 40 ° C, preferably about 37 ° C. The CO ₂ concentration is about 1 to 10%, preferably about 5%. The culturing time is not particularly limited, but is about 15 to 336 hours. Ventilation and agitation can be performed if necessary.

In aAVC-pp65 cells, when an inducible promoter is operably linked to a polynucleotide encoding CD1d and / or a polynucleotide encoding pp65 or a fragment thereof, the aAVC-pp65 cells are cultivated when the aAVC-pp65 cells are cultured. Induction factors for the inducible promoter may be contacted to induce expression of CD1d and / or pp65 or fragments thereof. In one embodiment, the method of producing aAVC-pp65 cells comprises the step of inducing the expression of CD1d and / or pp65 or a fragment thereof by culturing the aAVC-pp65 cells in the presence of an inducer of an inducible promoter. .. In one embodiment, the inducible promoter is a drug-inducible promoter, and the production method comprises culturing aAVC-pp65 cells in the presence of an inducer of the drug-inducible promoter. When a drug-induced promoter is used, cells are cultured in a culture medium containing drugs such as tetracycline, doxycycline, Cumate, and cumermycin, which are inducing factors, and a gene or polynucleotide operably linked to the drug-induced promoter is used. May be induced. The step can be performed according to a gene induction method using a general gene induction system. In one embodiment, the drug-induced promoter is a tetracycline-based inducible promoter, and the production method comprises culturing aAVC-pp65 cells in the presence of tetracycline or a derivative thereof and rtTA. In one embodiment, the tetracycline-based inducible promoter is a TRE3G promoter, the method of preparation comprising culturing aAVC-pp65 cells in the presence of tetracycline or a derivative thereof and a Tet-On 3G protein. The expression level of CD1d and pp65 or a fragment thereof in the obtained aAVC-pp65 cells is determined by a method known to those skilled in the art such as Western Blot method, ELISA method, flow cytometry method or the like using an anti-CD1d antibody or an anti-pp65 antibody. Can be measured.

3. 3. When a polynucleotide is introduced into cloned cells of aAVC-pp65 cells, cells in which the target polynucleotide has been introduced and cells in which the target polynucleotide has not been introduced are mixed. Therefore, when the target polynucleotide is further incorporated into the genomic DNA of the cell nucleus. Since the place of incorporation also differs depending on the cell, it may be a heterogeneous population of cells. In the present specification, the heterogeneous cell population is referred to as a "cell pool". A method of obtaining cells having a single genotype from the cell pool (referred to herein as "cloning") introduced a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof. By acquiring and proliferating one cell (hereinafter referred to as "cloned cell"), a cell population having a single genotype (hereinafter referred to as "cloned cell population") is acquired. be able to. Cloning of aAVC-pp65 cells can be performed using methods well known to those of skill in the art, for example, ultradilution, single cell sorting, or colony pickup methods. In one embodiment, the method for cloning aAVC-pp65 cells can also be a combination of one or more of the ultradilution method, the single cell sorting method, or the colony pickup method. In one embodiment, the cloning method for aAVC-pp65 cells is the ultradilution method.

By measuring the expression level of CD1d and pp65 or a fragment thereof for each cloned aAVC-pp65 cell obtained by the above method, the expression level of CD1d and pp65 or a fragment thereof was cloned to show the desired expression level. A population of aAVC-pp65 cells can be selected. When an inducible promoter is operably linked to a polynucleotide encoding CD1d and / or a polynucleotide encoding pp65 or a fragment thereof, a portion of each cloned aAVC-pp65 cell is said to be inducible. A clone that shows the desired expression level for CD1d and pp65 or a fragment thereof in the presence of the inducer by contacting with an inducer for a promoter and measuring the expression level of CD1d and pp65 or a fragment thereof in the obtained cells. A population of clonized aAVC-pp65 cells may be selected. A frozen population of selected cloned cells can be used as a research cell bank (RCB). RCB cells can be cultured, proliferated, frozen and used as a master cell bank (MCB). MCB cells can be cultured, proliferated, and frozen to be used as a working cell bank (WCB). Cultured WCB cells can be used as a raw material for pharmaceutical products. Each cell bank may contain a cryoprotectant. Each cell bank may be dispensed into a plurality of containers (for example, 2 to 10000 containers, for example, 10 to 1000 containers, for example, 100 to 500 containers) and stored in a frozen state. Thawing, culturing, and contacting inducing factors of cloned aAVC-pp65 cells, and measuring the expression level of CD1d and pp65 or fragments thereof can be performed by methods known to those skilled in the art, respectively, and culturing and inducing. Contact with the factor and measurement of the expression level can be performed, for example, by using the method described in "2. Culture of aAVC-pp65 cells". In one embodiment, culturing cloned aAVC-pp65 cells, contact with inducers, and measurement of expression levels of CD1d and pp65 or fragments thereof can be performed after thawing of RCB and WCB.

4. Loading of CD1d ligand into aAVC-pp65 cells By pulsing the CD1d ligand into aAVC-pp65 cells, aAVC-pp65 cells loaded with the CD1d ligand can be prepared. The conditions for pulsing the CD1d ligand (for example, the timing of adding the CD1d ligand to the cell culture medium, the concentration of the CD1d ligand in the culture medium, the culture time, etc.) are determined in consideration of the cells to be used and various conditions at the time of culture. It can be adjusted appropriately by the vendor. The concentration of the CD1d ligand added to the culture medium of aAVC-pp65 cells is not particularly limited, but can be appropriately selected in the range of, for example, 1 ng / mL to 10000 ng / mL. In one embodiment, the CD1d ligand is α-GalCer. In the case of aAVC-pp65 cells in which an inducible promoter is operably linked to a polynucleotide encoding CD1d and / or a polynucleotide encoding pp65 or a fragment thereof, in one embodiment, CD1d to aAVC-pp65 cells. Ligand loading can occur prior to contact of the cell with the inducer. In one embodiment, loading of the CD1d ligand into aAVC-pp65 cells can occur after contact of the cells with the inducer. In one embodiment, loading of the CD1d ligand into aAVC-pp65 cells can occur at the same time as the contact of the cells with the inducing factor. In these embodiments, the aAVC-pp65 cells are preferably cloned.

In aAVC-pp65 cells expressing the CD1d and pp65 prepared above or fragments thereof and loading the CD1d ligand on the surface of the cells, the proliferation of the cells may be stopped by an artificial method. The method for stopping the proliferation of the cells is not particularly limited, and for example, a method for stopping the cell proliferation by irradiation with radiation (for example, X-ray), a method for adding a drug such as mitomycin C, or the like can be used. In one embodiment, the aAVC-pp65 cells of the present invention are cells in which cell proliferation has stopped. In one embodiment, the method of stopping cell proliferation is irradiation.

<Pharmaceutical composition of the present invention, etc.>
The present invention also provides a pharmaceutical composition containing aAVC-pp65 cells (hereinafter referred to as "CD1d ligand-loaded aAVC-pp65 cells") expressing CD1d and pp65 or fragments thereof and loading the surface of the cells with a CD1d ligand. offer. In one embodiment, the CD1d ligand is α-GalCer. In one embodiment, the pharmaceutical composition is a pharmaceutical composition for use in the treatment of cancer. The pharmaceutical composition can be prepared by a commonly used method using excipients commonly used in the art, such as pharmaceutical excipients and pharmaceutical carriers. In formulating the pharmaceutical composition, excipients, carriers, additives and the like corresponding to these dosage forms can be used within a pharmaceutically acceptable range. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections and infusions. In one embodiment, the pharmaceutical composition of the present invention may comprise a frozen product of CD1d ligand loaded aAVC-pp65 cells. In one embodiment, the pharmaceutical composition of the present invention may comprise a frozen product of CD1d ligand loaded aAVC-pp65 cells and a cryoprotectant. In one embodiment, the pharmaceutical composition of the invention may comprise a suspension of CD1d ligand loaded aAVC-pp65 cells. In one embodiment, the pharmaceutical composition of the present invention may comprise a suspension of CD1d ligand loaded aAVC-pp65 cells and a cryoprotectant.

The present invention also provides CD1d ligand loaded aAVC-pp65 cells for treating cancer. In one embodiment, the CD1d ligand is α-GalCer. The present invention also provides the use of the CD1d ligand loaded aAVC-pp65 cells of the present invention for producing pharmaceutical compositions used in the treatment of cancer. In one embodiment, the CD1d ligand is α-GalCer.

The present invention also provides a method of treating cancer, comprising the step of administering to the subject the CD1d ligand loaded aAVC-pp65 cells of the present invention. In one embodiment, the CD1d ligand is α-GalCer. As used herein, the "subject" is a mammal (eg, human, monkey, mouse, rat, dog, chimpanzee, etc.), and in one embodiment, the subject is a human. As used herein, the term "treatment" is used to include therapeutic and prophylactic treatment. When a CD1d ligand-laden aAVC-pp65 cells are administered to a subject, they can be administered to the subject in the form of a pharmaceutical composition comprising the cells and a pharmaceutically acceptable excipient. The dose and frequency of administration of CD1d ligand-loaded aAVC-pp65 cells to a subject can be appropriately adjusted according to the type, location, severity of cancer, age, body weight and condition of the subject to be treated. The dose of aAVC-pp65 cells loaded with CD1d ligand can be, for example, 1 × 10 ³ cells / kg to 1 × 10 ⁹ cells / kg in a single administration to a subject. As a method of administering the CD1d ligand-loaded aAVC-pp65 cells to the subject, for example, it can be administered by injection or infusion into a vein, intratumor, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial or the like. The treatment method of the present invention can be used in combination with other cancer treatment methods. Other cancer treatment methods include surgery, radiation therapy, hematopoietic stem cell transplantation, or treatment with other anticancer agents.

The cancer to be treated according to the present invention is not particularly limited, but is limited to acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), hodgkin lymphoma, non-hodgkin lymphoma, B-cell lymphoma, and multiple cancers. Hematological cancers such as myeloma and T-cell lymphoma, myelodystrophy syndrome, adenocarcinoma, squamous cell carcinoma, squamous cell carcinoma, undifferentiated cancer, large cell cancer, non-small cell lung cancer, small cell lung cancer , Middle dermatoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, cholecyst cancer, Solid cancers such as bile duct cancer, kidney cancer, pancreatic cancer, lung cancer, colon cancer, colon cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testis cancer, ovarian cancer, and brain cancer , And cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue and hematopoietic tissue, as well as sarcoma such as chondrosarcoma, Ewing sarcoma, malignant vascular endothelial tumor, malignant Schwan tumor, osteosarcoma, and soft tissue sarcoma. , Glioblastoma, polymorphic glioblastoma, hepatic blastoma, medullary blastoma, renal blastoma, neuroblastoma, pancreatic blastoma, pleural lung blastoma, blastoma such as retinal blastoma and the like. In one embodiment, the cancer targeted for the treatment of the present invention is a pp65 positive cancer. In one embodiment, the cancer targeted for the treatment of the present invention is glioblastoma or glioblastoma polymorphism.

<Manufacturing method of cells or pharmaceutical compositions used for cancer treatment>
The present invention also provides a method for producing a cell or pharmaceutical composition used in the treatment of cancer, which comprises the following steps:
A step of introducing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into a human-derived cell, and a step of culturing the cell.

In one embodiment, the CD1d used in the method for producing cells or pharmaceutical compositions used in the treatment of cancer of the present invention is human CD1d.

In one embodiment, the cells used in the treatment of cancer of the present invention or the human-derived cells used in the method for producing a pharmaceutical composition are normal cells. In one embodiment, the cells used in the treatment of cancer or the human-derived cells used in the method for producing a pharmaceutical composition are cells derived from HEK293 cells.

In one embodiment, the method for producing a cell or pharmaceutical composition used in the treatment of cancer of the present invention further comprises loading a CD1d ligand on the cell surface. In one embodiment, the CD1d ligand is α-GalCer.

In one embodiment, the method for producing a cell or pharmaceutical composition used in the treatment of cancer of the present invention encodes a polynucleotide encoding CD1d operably linked with an inducible promoter and / or pp65 or a fragment thereof. Including the step of introducing the polynucleotide to be used into human-derived cells. In one embodiment, the production method induces the expression of CD1d and / or pp65 or a fragment thereof by contacting the cells (aAVC-pp65 cells) obtained in the above step with an inducing factor for the inducible promoter. Further includes steps. In one embodiment, the production method comprises inducing the expression of pp65 or a fragment thereof by culturing the aAVC-pp65 cells in the presence of an inducing factor for the inducible promoter. In one embodiment, the production method further comprises loading the CD1d ligand on the cell surface, and in one embodiment, the CD1d ligand is α-GalCer. In one embodiment, loading of the CD1d ligand on the cell surface may be before, after, or at the same time the contact of the aAVC-pp65 cells with the inducer.

The present invention also provides a method for producing a cell or pharmaceutical composition used for treating cancer, which comprises the step of culturing the aAVC-pp65 cell of the present invention. In one embodiment, the CD1d used for the aAVC-pp65 cells is human CD1d. In one embodiment, the human-derived cells used for the aAVC-pp65 cells are normal cells, and in one embodiment, the human-derived cells are cells derived from HEK293 cells. In one embodiment, the production method further comprises loading a CD1d ligand on the cell surface. In one embodiment, the CD1d ligand is α-GalCer.

In one embodiment, the aAVC-pp65 cells of the invention used in the production method are polynucleotides encoding CD1d to which an inducible promoter is operably linked and / or poly that encodes pp65 or a fragment thereof. Human-derived cells into which nucleotides have been introduced exogenously, in one embodiment, the method involves contacting the aAVC-pp65 cells of the invention with an inducer of an inducible promoter to produce CD1d and / or pp65 or a fragment thereof. It further comprises the step of inducing expression. In one embodiment, the method comprises inducing the expression of CD1d and / or pp65 or a fragment thereof by culturing the aAVC-pp65 cells in the presence of an inducing factor of an inducible promoter. In one embodiment, the production method further comprises loading the CD1d ligand on the cell surface, and in one embodiment, the CD1d ligand is α-GalCer. In one embodiment, loading of the CD1d ligand on the cell surface may be before, after, or at the same time the contact of the aAVC-pp65 cells with the inducer. The present invention also provides the use of the aAVC-pp65 cells of the present invention in the manufacture of pharmaceutical compositions used in the treatment of cancer.

Specific embodiments relating to the cells and steps used in the production method described in this section are described in the above-mentioned <aAVC-pp65 cells of the present invention> and <method for producing aAVC-pp65 cells of the present invention>. It is as follows.

Specific examples referred to herein for further understanding of the invention are provided, but these are for illustrative purposes only and are not intended to limit the invention.

[Example 1: Antitumor effect of mouse-type aAVC (3T3) -pp65 cells]
The antigen-specific antitumor effect of aAVC carrying pp65, which is a cancer antigen, was confirmed by an in vivo evaluation system.

[Example 1-1: Preparation of mouse-type aAVC (3T3) -pp65 cells]
MRNAs of the pp65 gene and the CD1d gene were introduced into mouse NIH / 3T3 cells (ATCC, Cat.CRL-1658) to prepare mouse-type aAVC (also referred to as aAVC (3T3) -pp65).

The pp65 gene (SEQ ID NO: 1) was prepared by artificial gene synthesis based on the amino acid sequence (UniProt: P06725-1) of HCMV pp65 shown in SEQ ID NO: 2. Furthermore, the pp65 gene is a sequence complementary to 16 bases containing a HindIII or EcoRI recognition sequence of pGEM®-4Z plasmid (hereinafter referred to as "pGEM-4Z plasmid") (Promega, Cat. P2161). It was amplified by the PCR method using the primer to which was added. The amplified pp65 gene was inserted into the HindIII-EcoRI site of the pGEM-4Z plasmid using In-Fusion® HD Cloning Kit (Takara Bio Inc., Cat. 639648). The resulting plasmid is referred to as the pGEM-4Z-pp65 plasmid. At the HindIII-BamHI site of the pGEM-4Z plasmid, the mouse CD1d gene consisting of the nucleotide sequence shown in SEQ ID NO: 5 (gene was synthesized by artificial gene synthesis based on the amino acid sequence of UniProt: P11609-1 (SEQ ID NO: 6)). I inserted it. The obtained plasmid is referred to as a pGEM-4Z-mCD1d plasmid. The pGEM-4Z-pp65 plasmid and the pGEM-4Z-mCD1d plasmid were cleaved with EcoRI and BamHI, respectively, and linearized, and used as a template. Pp65 mRNA and CD1d mRNA were prepared. NIH / 3T3 cells were added to Dulbecco's Modified Eagle's medium (Merck, Cat. D6429) containing 10% of fetal bovine serum supplemented with 350 ng / mL of α-GalCer (consigned to Juzen Kagaku Co., Ltd.). After culturing for 2 days, the cells were collected. Pp65 mRNA and CD1d mRNA are added to the collected cell suspension, and electroporation (pouring pulse: voltage 150 V, pulse width 8 ms, pulse interval 50 ms, number of times 2) is performed using a NEPA21 electroporator (Neppagene Co., Ltd.). Times, attenuation rate 10%, polarity +, transfer pulse: voltage 20V, pulse width 50ms, pulse interval 50ms, number of times ± 5 times, attenuation rate 40%, polarity +/-). The cells after electroporation were collected, and the cells were irradiated with 30 Gy of X-rays using an X-ray irradiation device MBR-1520R-3 (Hitachi Power Solutions Co., Ltd.). The cells obtained by the above method are referred to as aAVC (3T3) -pp65 cells.

Control test of aAVC (3T3) -pp65 cells The aAVC (3T3) -WT1 cells used as aAVC were prepared by introducing mRNA of WT1 gene and CD1d gene according to the method for producing aAVC (3T3) -pp65 cells. .. In this step, NIH / 3T3 cells were cultured in Dulvecco's Modified Eagle's medium (Merck, Cat. D6429) containing 10% of fetal bovine serum supplemented with 500 ng / mL of α-GalCer for 2 days. The WT1 gene was prepared from pcDNA3-ATG-WT1 described in WO2013 / 018778.

[Example 1-2: pp65-specific T cell-inducing ability of aAVC (3T3) -pp65 cells in mice]
Five aAVC (3T3) -pp65 cells suspended in 200 μL BICANATE Injection (Otsuka Pharmaceutical Factory, Inc.) were placed in the tail vein of 5 week-old C57BL / 6J female mice (Charles River Laboratories, Japan) in 5 cases in each group. ^{X10 3} cells, 5 × 10 ⁴ cells, and 5 × 10 ⁵ cells were administered at a time. The control group received 200 μL of BICANATE Injection. Spleen cells were harvested from the mice after administration 7 days, attached mouse IFN-γ single-color enzymatic ELISPOT the recovered cells according to the method assay kit (Cellular Technology Limited, Inc., mIFNGp-2M / 5) plates 3 × 10 ⁵ of Cells / wells were sown. In addition, to stimulate pp65-specific T cells, the pp65 peptide, a pp65 protein-specific overlapping peptide (PepTivator CMV pp65-premium grade, human (Miltenyi Biotec, Cat. 130-093-435)), Then, a non-specific overlapping peptide WT1 peptide (PepTivator WT1-premium grade, human (Miltenyi Biotec, Cat. 130-095-918)) was added to the pp65 protein, and the temperature was 37 ° C. and 5% CO ₂ condition. It was cultured underneath for 1 day. The spots were colored according to the attached method, and the number of IFN-γ-producing cells was measured by counting the number of cell spots at the bottom of the well with ELISpot Reader 08 Classic (Autoimmun Diagnostica). In aVC (3T3) -pp65 cell-administered mice, the number of cells producing IFN-γ increased by pp65 peptide stimulation (Fig. 1-1). The difference from the number of spots stimulated by WT1 peptide, which is non-antigen specific, indicates the number of pp65-specific T cells. This result suggests that administration of aAVC (3T3) -pp65 cells induces pp65-specific T cells.

[Example 1-3: Antigen-specific antitumor effect of aAVC (3T3) -pp65 cells in pp65-expressing B16 melanoma cell-bearing mice]
B16-F10 melanoma cells expressing the pp65 protein by introducing a plasmid carrying the pp65 gene (SEQ ID NO: 1) into B16-F10 melanoma cells (ATCC, Cat.CRL-6475) by electroporation and selecting a drug. (Also referred to as B16-pp65 cells) were prepared. ^{Under the skin of a 5-week-old C57BL / 6J female mouse, 1 × 10 5} cells B16-pp65 cells suspended in 50 μL of D-PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. 045-29795) were placed. It was administered. Four days after administration, the mice were divided into seven groups of 12 patients in each group based on the tumor volume, and one day after grouping, aAVC (3T3) -pp65 cells suspended in BICANATE Injection or pp65 non-expressed in the tail vein. As a control test aAVC, aAVC (3T3) -WT1 cells were administered at ^{5 × 10 2} cells, 5 × 10 ³ cells, and 5 × 10 ^{4 cells, respectively.} The control group received 200 μL of BICANATE Injection. The day when the cancer cells were inoculated was set as the 0th day, and the change in tumor volume for 17 days was measured. The growth of B16-pp65 tumors was significantly suppressed in all groups administered with aAVC (3T3) -pp65 cells and ^{in the group administered with 5 × 10 4 cells of aAVC (3T3) -WT1 cells, whereas aAVC (3T3)} ) -The growth of B16-pp65 tumors was not suppressed in the group to which 5 × 10 ² cells and 5 × 10 ^{3 cells were administered to WT1 cells (Fig. 1-2).} The antitumor effect at this dose was not specific to the pp65 protein, as antitumor effects were observed in both the aAVC (3T3) -pp65 cells and aAVC (3T3) -WT1 ^{cells administered with 5 × 10 4 cells.} It is considered that the antitumor effect including the effect is detected. On the other hand, in ^{the aAVC (3T3) -pp65 cell administration group of 5 × 10 2} cells and 5 × 10 ³ cells, a stronger antitumor effect was obtained than in the aAVC (3T3) -WT1 cell administration group. The difference in antitumor activity is considered to be the pp65-specific antitumor effect based on the induction of specific immunity to the pp65 protein. The expression level of pp65 per ^{1 × 10 6} cells of aAVC (3T3) -pp65 was 96.8 μg.

[Example 1-4: Antigen-specific antitumor effect of aAVC (3T3) -pp65 cells in pp65-expressing GL-261 glioma cell-bearing mice]
GL-261 glioma cells (GL) expressing the pp65 protein by introducing a plasmid carrying the pp65 gene (SEQ ID NO: 1) into GL-261 glioma cells (DSMZ, Cat.ACC802) by electroporation and selecting a drug. -261-pp65 cells) were prepared. Twelve 5-week-old C57BL / 6J female mice in each group received 3 μL of 1 × 10 ⁵ cells GL-261-pp65 cells suspended in D-PBS (−). Seven days after administration, aAVC (3T3) -pp65 cells or aAVC (3T3) -WT1 cells suspended in BICANATE Injection were placed in the tail vein of the mouse in 5 × 10 ² cells, 5 × 10 ³ cells, 5 ×, respectively. 10 ⁴ cells were administered at a time. The control group received 200 μL of BICANATE Injection. Survival was observed for 50 days after administration of GL-261-pp65 cells. When the survival time was compared by the log rank test, the survival time of all the aAVC (3T3) -pp65 cell-administered group and the aAVC (3T3) -WT1 cell- ^{administered 5 × 10 4} cells group was significantly higher than that of the control group. In contrast, aAVC (3T3) -WT1 cells were treated with 5 × 10 ² cells and 5 × 10 ³ cells, and the effect of prolonging survival was low or not observed (Fig. 1-3). .. Similar to the results of Example 1-3, the antitumor activity exhibited by administration of aAVC (3T3) -pp65 cells of ^{5 × 10 2} cells and 5 × 10 ^{3 cells was pp65 based on the induction of pp65-specific immunity.} It is considered to have a specific antitumor effect.

From the results of Example 1, it was confirmed that the mouse-type aAVC (3T3) -pp65 cells exhibited a pp65-specific antitumor effect by inducing pp65-specific T cells in the mouse in vivo evaluation system. In humans, human-type aAVC-pp65 cells can be expected to induce innate immunity and exert an antitumor effect, induce antigen-specific acquired immunity, and exert an antitumor effect based on acquired immunity. For the purpose of creating a therapeutic drug showing a pp65-specific antitumor effect on human pp65-expressing cancer, the production of human-type aAVC-pp65 cells was examined below.

[Example 2: Preparation and preparation of human aAVC-pp65 cells]
Human aAVC-pp65 cells were constructed by introducing the pp65, CD1d, and Tet-On 3G genes into FreeStyle 293-F cells (Thermo Fisher Scientific, Cat. R79007).

[Example 2-1: Lentivirus preparation]
(1) Construction of plasmid for lentivirus preparation A lentivirus plasmid constructed based on the nucleotide sequences of pLVSIN-CMV Pur plasmid (Takara Bio Inc., Cat. 6183) and pTRE3G (Takara Bio Inc., Cat. 631173) is pLVsin-TRE3G. Called a plasmid. The pp65 gene (SEQ ID NO: 1) was amplified by the PCR method using a primer in which a sequence complementary to 16 bases including the EcoRI or BamHI recognition sequence of the pLVsyn-TRE3G plasmid was added. The amplified pp65 gene was inserted into the pLVsin-TRE3G plasmid treated with EcoRI and BamHI using the In-Fusion® HD Cloning Kit. The obtained plasmid is referred to as a pLVsyn-TRE3G-pp65 plasmid. The WPRE sequence of the obtained pLVsyn-TRE3G-pp65 plasmid was subjected to Gene Ther. 2009; 16 (5): 605-19 was replaced with the mutant WPRE sequence (mut6). The resulting plasmid is referred to as pLVsyn-TRE3G-pp65-mWPRE (SEQ ID NO: 7).

The CD1d gene (SEQ ID NO: 3) is designed based on the amino acid sequence of human CD1d shown in SEQ ID NO: 4, and is an XhoI recognition sequence on the 5'end side of the gene and a NotI recognition sequence on the 3'end side by artificial gene synthesis. Was added to the gene. The CD1d gene excised with restriction enzymes XhoI and NotI was inserted into the XhoI-NotI site of the pLVsin-CMV plasmid (SEQ ID NO: 8) constructed based on the nucleotide sequence of the pLVSIN-CMV Pur plasmid. The obtained plasmid is referred to as a pLVsyn-CMV-CD1d plasmid. A pLVsyn-CMV-CD1d-mWPRE plasmid was prepared by converting the WPRE sequence into a mutant in the same procedure as for pLVsyn-TRE3G-pp65-mWPRE.

The Tet-On 3G gene is based on the Tet-On 3G gene sequence of the pCMV-Tet3G plasmid (Takara Bio Inc., Cat. 613335), and has an XhoI recognition sequence on the 5'end side of the gene and NotI recognition on the 3'end side. A sequence was added and prepared by artificial gene synthesis. The Tet-On 3G gene excised with restriction enzymes XhoI and NotI was inserted into the XhoI-NotI site of the pLVsin-CMV plasmid. The obtained plasmid is referred to as a pLVsyn-CMV-Tet3G plasmid. A pLVsyn-CMV-Tet3G-mWPRE plasmid was prepared by converting the WPRE sequence into a mutant in the same procedure as for pLVsyn-TRE3G-pp65-mWPRE.

(2) Lentivirus preparation Using the pLVsin-TRE3G-pp65-mWPRE plasmid, pLVsin-CMV-Tet3G-mWPRE plasmid, and pLVsin-CMV-CD1d-mWPRE plasmid prepared in (1), the pp65 gene and Tet-On A lentivirus carrying a 3G gene and a CD1d gene was prepared. The obtained lentiviruses are referred to as TRE3G-pp65-equipped lentivirus, Tet3G-equipped lentivirus, and CD1d-equipped lentivirus, respectively.

30 μg of either the pLVsyn-TRE3G-pp65-mWPRE plasmid or the pLVsin-CMV-Tet3G-mWPRE plasmid and ViraPower Lentiviral Packing Mix (Thermo Fisher Scientific) Mix (Thermo Fisher Scientific) Mix (Thermo Fisher Scientific) Mix (Thermo Fisher Scientific) SFM (Thermo Fisher Scientific, Cat. 12309019) was added and mixed (A). 540 μL of LV-MAX Transfection Reagent (a component of Thermo Fisher Scientific, Cat. A35348) and 4.5 mL of OptiProSFM were mixed and allowed to stand for 1 minute (B). (A) and 1.5 mL (B) were mixed and allowed to stand at room temperature for 10 minutes. Incubate Visual Production Cells (Thermo Fisher Scientific, Cat. A35347) in a 125 mL Erlenmeyer flask (Thermo Fisher Scientific, Cat. 4115-0125) and in culture (1.2 x 10 ⁸ cells / mL). The whole amount of the mixed solution of (A) and (B) was added to carry out gene transfer. The Viral Production Cells were cultured in LV-MAX Production Medium (Thermo Fisher Scientific, Cat. A3583401) _{under the conditions of 37 ° C., 8% CO 2} , and 120 rpm. After the gene transfer, the cells were cultured under the above conditions for 2 days, and the culture supernatant containing the lentivirus carrying the TRE3G-pp65 or Tet3G gene was collected. The culture supernatant was centrifuged at 760 xg at 4 ° C. for 10 minutes. The supernatant is filtered through a 0.45 μm filter (Merck, Cat. SLHV033RS), and PEG-it Virus Precision Solution (5x) (System Biosciences, Cat.LV810A-1) is used in a quarter amount of the supernatant. The mixture was added, mixed, and allowed to stand at 4 ° C. overnight. The supernatant was removed by centrifugation at 1500 xg at 4 ° C. for 30 minutes, and again centrifuged at 1500 xg at 4 ° C. for 5 minutes to completely remove the supernatant. The pellet was suspended in 725 μL of DPBS (Thermo Fisher Scientific, Cat. 14190144) to obtain a TRE3G-pp65-loaded lentivirus and a Tet3G-loaded lentivirus.

18 μg of pLVsyn-CMV-CD1d-mWPRE plasmid and 54 μL of ViraPower Lentivir Packing Mix (Thermo Fisher Scientific, Cat.K497500) were mixed, and 9 mL of OptiPeriSciPr. A). Lipofectamine 2000 CD Transfection Reagent (Thermo Fisher Scientific, Cat. 12566014) 648 μL and OptiProSFM 27 mL were mixed and allowed to stand for 5 minutes (B). (A) and 9 mL (B) were mixed and allowed to stand at room temperature for 20 minutes, and then 5 × 10 ⁶ cells / in Falcon® 100 mm TC-treated Cell Culture Dish (Corning, Cat. 353003) the day before. Gene transfer was performed by adding 3 mL / plate of a mixed solution of (A) and (B) to Lenti-X 293T cells (Takara Bio Inc., Cat. 632180) seeded by plate. Lenti-X 293T cells are DMEM containing 10% amount of fetal bovine serum (SAFC Biosciences, Cat. 12007C (γ-irradiated product)) and 0.1% amount of gentamicin (Thermo Fisher Scientific, Cat. 15750060). The cells were cultured in a medium (Thermo Fisher Scientific, Cat. 10569010) at 37 ° C. _{under 5% CO 2 conditions.} After the gene transfer, the cells were cultured under the same conditions for 2 days, and the culture supernatant containing the lentivirus carrying the CD1d gene was collected. The culture supernatant was centrifuged at 760 xg at 4 ° C. for 10 minutes. The supernatant is filtered through a 0.45 μm filter (Merck, Cat. SLHV033RS), and PEG-it Virus Precision Solution (5x) (System Biosciences, Cat.LV810A-1) is used in a quarter amount of the supernatant. The mixture was added, mixed, and allowed to stand at 4 ° C. overnight. The supernatant was removed by centrifugation at 1500 xg at 4 ° C. for 30 minutes, and again centrifuged at 1500 xg at 4 ° C. for 5 minutes to completely remove the supernatant. The pellet was suspended in 1 mL of DPBS (Thermo Fisher Scientific, Cat. 14190144) to obtain a CD1d-loaded lentivirus.

[Example 2-2: Preparation and cloning of Lentivirus-infected cells]
FreeStyle 293-F cells (Thermo Fisher Scientific, Cat. 510029) were infected with each lentivirus prepared in Example 2-1. A cell population infected with a TRE3G-pp65-equipped lentivirus, a CD1d-equipped lentivirus, and a Tet3G-equipped lentivirus is referred to as a FreeStyle 293F_Tet-on_pp65_CD1d cell pool. Cells were cloned from these cell pools, and multiple clones from each cell pool were obtained.

(1) Preparation of FreeStyle 293F_Tet-on_pp65_CD1d cell pool FreeStyle 293-F cells were ^{prepared at a concentration of 2 × 10 6} cells / mL, and Falcon® cell culture 12-well cell culture multi-well plate flat bottom at 500 μL / well. The cells were seeded with a lid (hereinafter referred to as "12-well plate") (Corning, Cat. 353043), 100 μL of the CD1d-loaded lentivirus obtained in Example 2-1 was added, and 400 μL of the medium was further added. The medium used was FreeStyle 293 Expression Medium (Thermo Fisher Scientific, Cat. 12338018) containing a 0.1% amount of gentamicin (Thermo Fisher Scientific, Cat. 15750-060). After centrifuging at 540 × g at room temperature for 30 minutes, the cells were gently suspended by pipetting and cultured with shaking. After culturing for several days, the 12-well plate was subcultured from the Corning® polycarbonate Erlenmeyer flask vent cap 125 mL (hereinafter referred to as "125 mL Erlenmeyer flask") (Corning, Cat.431143), and further substituting at appropriate intervals. Instead, cell pool A was obtained. Cell pool A ^{was prepared at a concentration of 2 × 10 6} cells / mL, seeded on a 12-well plate at 500 μL / well, and 200 μL of each of the TRE3G-pp65-loaded lentivirus and Tet3G-loaded lentivirus obtained in Example 2-1. / Well or 25 μL / well were added at a time. Further, 100 μL or 450 μL of medium was added, respectively. As the medium, FreeStyle 293 Expression Medium containing 0.1% amount of gentamicin was used. After centrifuging at 540 × g at room temperature for 30 minutes, the cells were gently suspended by pipetting and cultured with shaking. After culturing for several days, the cells were subcultured from a 12-well plate into a 125 mL Erlenmeyer flask and further subcultured at appropriate intervals to obtain a FreeStyle 293F_Tet-on_pp65_CD1d cell pool.

(2) Cloning Single cell cloning was performed from the FreeStyle 293F_Tet-on_pp65_CD1d cell pool prepared in (1), and clones in which the CD1d gene, pp65 gene, and Tet3G gene were stably expressed were selected. The cells were seeded on a 96-well plate so as to be 1 cell / well, and the cells were subcultured according to the growth of the cells to proliferate the cells. The expression levels of the pp65 protein and the CD1d protein in the proliferated cells were measured by the ELISA method for the pp65 protein and the flow cytometry method for the CD1d protein, and clones that stably expressed the pp65 protein and the CD1d protein were selected. For the cells used for measuring the expression level of pp65 protein, doxycycline (Takara Bio Inc., Cat. 631311) having a final concentration of 0.8 to 1 μg / mL was added to the medium to express the expression of pp65 protein via Tet-On System. Evaluation was performed after induction. For measurement by the ELISA method, CMV pp65 (3A12) antibody (Santa Cruz Biotechnology Cat.sc-56973) was used as the primary antibody, and Goat Anti-Mouse IgGFc-HRP antibody (Jackson ImmunoResearch Lab) was used as the secondary antibody. 035-071) was used, and the measurement was performed according to the general measurement method of the ELISA direct method. For the measurement by the flow cytometry method, an APC Mouse Anti-Human CD1d antibody (BD Biosciences, Cat.563505) was used, and the measurement was performed according to a general measurement method using FACSVerse (BD Biosciences). rice field.

The aAVC-pp65 cells of the present invention can be expected to be useful in the treatment of cancer.

In the number heading <223> of the following sequence listing, the description of "Artificial Sequence" is described. The nucleotide sequence shown by SEQ ID NO: 1 in the sequence listing is the nucleotide sequence encoding the human cytomegalovirus pp65 protein, and the nucleotide sequence shown by SEQ ID NO: 2 in the sequence listing is the amino acid sequence encoded by SEQ ID NO: 1. be. The base sequence shown by SEQ ID NO: 3 is a base sequence encoding a human CD1d protein, and the amino acid sequence shown by SEQ ID NO: 4 in the sequence listing is an amino acid sequence encoded by SEQ ID NO: 3. The nucleotide sequence shown by SEQ ID NO: 5 is the nucleotide sequence encoding the mouse CD1d protein, and the amino acid sequence shown by SEQ ID NO: 6 in the sequence listing is the amino acid sequence encoded by SEQ ID NO: 5. The nucleotide sequences shown by SEQ ID NO: 7 and SEQ ID NO: 8 in the Sequence Listing are the nucleotide sequences of the pLVsin-TRE3G-pp65-mWPRE plasmid and the pLVsin-CMV plasmid, respectively.

Claims

A human-derived cell into which a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof have been introduced exogenously.
The cell according to claim 1, wherein the CD1d is a human CD1d.
The cell according to claim 1 or 2, wherein the human-derived cell is a normal cell.
The cell according to claim 3, wherein the normal cell is a cell derived from human fetal kidney cell 293 (HEK293) cell.
The cell according to any one of claims 1 to 4, expressing CD1d and pp65 or a fragment thereof.
The cell according to claim 5, wherein the CD1d ligand is loaded on the surface of the cell.
The cell according to claim 6, wherein the CD1d ligand is α-GalCer.
A pharmaceutical composition comprising the cells according to claim 6 or 7.
The pharmaceutical composition according to claim 8, for use in the treatment of cancer.
A method for treating cancer, which comprises a step of administering the cell according to claim 6 or 7 to a subject.
The cell according to claim 6 or 7, for treating cancer.
Use of the cells according to claim 6 or 7 for producing a pharmaceutical composition used for the treatment of cancer.
A method of producing cells used in the treatment of cancer.
A step of introducing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into human-derived cells, and
A method comprising culturing the cells.
The method according to claim 13, wherein the CD1d is a human CD1d.
The method according to claim 13 or 14, wherein the human-derived cell is a normal cell.
The method according to claim 15, wherein the normal cells are cells derived from human fetal kidney cells 293 (HEK293) cells.
The method according to any one of claims 13 to 16, further comprising a step of loading a CD1d ligand on the surface of a cell.
The method according to claim 17, wherein the CD1d ligand is α-GalCer.