JP2023078487A - pp65-CONTAINING ARTIFICIAL ADJUVANT VECTOR CELLS USED IN TREATMENT OF CANCER - Google Patents

Abstract

To provide aAVC-pp65 cells for use in the treatment of patients having a pp65-expressing cancer.SOLUTION: The present invention provides: a human-derived cell having a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof exogenously introduced thereto; and a method for producing cells for use in the treatment of cancer that includes the steps of introducing, to a human-derived cell, a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof and culturing the cell having the polynucleotides introduced thereto.SELECTED DRAWING: None

Description

The present invention relates to pp65-containing artificial adjuvant vector cells (aAVC-pp65 cells) for use in the treatment or prevention of cancer.

Tumors that develop from glial cells (glia) that support brain nerve cells (neurons) are called gliomas. Glioma includes types such as astrocytoma and oligodendroglioma, and accounts for approximately 25% of tumors originating from the brain. There are four grades according to the degree of malignancy, and grade 4, which has the highest degree of malignancy, is called glioblastoma multiforme (GBM).

A standard of care for the first onset of GBM has been established. After surgical removal of the tumor, local radiation is applied to the tumor remaining around the removal, followed by oral chemotherapy with temozolomide. After that, oral administration of temozolomide is continued as maintenance therapy. However, it is difficult to remove the tumor completely, and recurrence is seen within one year after radiotherapy in most patients. Several treatment methods have been proposed after recurrence, but there is no standardized method, and the average survival period after recurrence is very short, less than 1 year. Against this background, there is a strong demand for the development of new drugs and novel treatments effective for GBM. -13 receptor alpha chain variant 2), survivin, MAGEA1, Ephrin A2, HER2, AIM2, TRP-2, gp100 and other antigen-targeted vaccines and T-cell therapies are being developed (Non-Patent Documents 1 and 2 ).

Human cytomegalovirus (HCMV) is a double-stranded DNA virus belonging to the genus Cytomegalovirus. HCMV usually infects the human body through secretions such as saliva and urine during infancy and childhood, and then remains parasitic on the human body for the rest of one's life through latent and persistent infection. It is known that many healthy individuals are pre-infected with HCMV, most of which are subclinical infections. However, HCMV is reactivated and proliferates due to decreased immune function, and organ transplant patients such as kidney and bone marrow transplants, Acquired Immunodeficiency Syndrome (AIDS), malignant tumors, immunodeficiency such as immunosuppressive therapy. It often causes serious infections in affected patients.

In recent years, it has been reported that human cytomegalovirus also infects tumor cells, and that the tumor cells acquire tumor immunity and resistance to anticancer drugs, thereby increasing the degree of malignancy (Non-Patent Document 3). Expression of HCMV-derived antigens has been detected in more than 90% of patients with GBM (Non-Patent Documents 4-6). In addition, HCMV DNA has been detected in colon cancer (Non-Patent Document 7), and HCMV-derived DNA and proteins have also been detected in prostate cancer (Non-Patent Document 8) and breast cancer (Non-Patent Document 9). A relationship with various cancers has been suggested.

pp65 (65 KDa phosphoprotein, gene name UL83) is a protein that constitutes the tegument between the envelope and nucleocapsid of HCMV virions, and is the most abundant protein among about 70 kinds of proteins that form HCMV virions. 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 therapeutic or preventive vaccines against HCMV viral infection and GBM (Non-Patent Document 10 and 11). However, there is still no drug marketed as a vaccine against the HCMV virus.

Fujii et al. exogenously expressed CD1d and cancer antigens in human-derived cells, and further α-galactosylceramide (α-GalCer) pulsed artificial adjuvant vector cells (artificial adjuvant vector cells: aAVC) was produced (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 become natural killer (NK ) to activate cells. In mouse models, NK cell-dependent antitumor effects by aAVC administration have been demonstrated (Patent Documents 1 to 3). In addition, aAVC administered to mice is rapidly killed by NKT cells activated in vivo, and the aAVC fragment is taken up by dendritic cells. Dendritic cells that have incorporated the aAVC fragment present cancer antigen fragments that have been incorporated into the major histocompatibility complex (MHC) on the cell surface, and generate cancer antigen-specific T cells. Induce. Antitumor effects mediated by induction of cancer antigen-specific T cells by aAVC administration have been shown in mouse models (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 immunity activation by NK cell activation via NKT cell activation and acquired immunity induction by antigen-specific T cell induction. . However, to date, there have been no reports on aAVC cells expressing HCMV antigens such as pp65, activation of NK/NKT cells using such cells, and GBM antigen-specific immunity-inducing effects.

International publication number 2007/097370 International publication number 2010/061930 International publication number 2013/018778

Lim M et al. , Nature reviews. Clinical oncology, (England), 2018; 15(7):422-442. McGranahan T et al. , "Current treatment options in oncology", (USA), 2019; 20(3):24. Michaelis M. et al. , "Neoplasia", (USA), 2009;11(1):1-9 CobbsC. S. et al. , Cancer Research, (USA), 2002;62(12):3347-3350. Mitchell D. A. et al. , Neuro-oncology, (England), 2008; 10(1):10-18. ScheurerM. E. et al. , "Acta Neuropathologicala", (Germany), 2008; 116(1):79-86. Chen H. P. et al. , "Journal of clinical virology", (Netherlands), 2012;54(3):240-244. Samanta M. et al. , "The Journal of urology", (USA), 2003; 170(3):998-1002 Harkins L. E. et al. , "Herpesviridae", (England), 2010;1(1):8 Batich KA et al. , Clinical Cancer Research, (USA), 2017;23(8):1898-1909. Schuessler A et al. , Cancer Research, (USA), 2014;74(13):3466-3476. Shimizu K. et al. , Cancer Research, (USA), 2013;73(1):62-73. Shimizu K. et al. , Cancer Research, (USA), 2016;76(13):3756-3766.

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

The present inventors prepared aAVC (also referred to as aAVC(3T3)-pp65 cells) in which pp65 was expressed in mouse cells (Example 1-1), and the aAVC(3T3)-pp65 cells were pp65-specific T cells were induced (Example 1-2), and pp65 protein-specific antitumor effects (Example 1- 3 and 1-4). 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, pharmaceutical compositions containing the cells, methods for producing the cells, and methods for treating or preventing pp65-expressing cancers using 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 exogenously introduced.
[2] The cell of [1], wherein the CD1d is human CD1d.
[3] The cell of [1] or [2], wherein the human-derived cell is a normal cell.
[4] The cell of [3], wherein the normal cell is derived from human embryonic kidney 293 (HEK293) cells.
[5] The cell of any one of [1] to [4], which expresses CD1d and pp65 or a fragment thereof.
[6] The cell of [5], which loads a CD1d ligand on the surface of the cell.
[7] The cell of [6], wherein the CD1d ligand is α-GalCer.
[8] A pharmaceutical composition comprising the cells of [6] or [7].
[9] The pharmaceutical composition of [8], for use in treating cancer.
[10] A method of treating cancer, comprising the step of administering the cell of [6] or [7] to a subject.
[11] The cell of [6] or [7] for treating cancer.
[12] Use of the cells of [6] or [7] for producing a pharmaceutical composition for cancer treatment.
[13] A method for producing cells for use in cancer treatment, comprising:
introducing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into a human-derived cell;
A method comprising the step of culturing said cells.
[14] The method of [13], wherein the CD1d is human CD1d.
[15] The method of [13] or [14], wherein the human-derived cells are normal cells.
[16] The method of [15], wherein the normal cells are derived from human embryonic kidney 293 (HEK293) cells.
[17] The method of any one of [13] to [16], further comprising the step of loading a CD1d ligand on the surface of the cell.
[18] The method of [17], wherein the CD1d ligand is α-GalCer.

The aAVC-pp65 cells of the invention and the pharmaceutical compositions of the invention are useful for treating cancer.

FIG. 1-1 shows the number of IFN-γ-producing T cells in spleen cells of aAVC(3T3)-pp65 cell-administered mice. BICANATE on the horizontal axis indicates the control group. - (minus) in the aAVC(3T3)-pp65 cell administration group indicates that peptides for stimulating T cells contained in spleen cells were not added, and WT1 and pp65 were used as T cell stimulators, respectively. WT1 peptide and pp65 peptide were used. The vertical axis indicates the number of spots per 3×10 ⁵ spleen cells counted with an ELISPOT reader, the horizontal line indicates the average number of spots for each individual, and the error bars indicate ±standard error of the mean. Figures 1-2 show the antitumor effect of aAVC(3T3)-pp65 cells in pp65-expressing B16 tumor-bearing mice. aAVC(3T3)-WT1 cells were used as control test aAVC without pp65 expression. The vertical axis indicates the tumor volume, and the horizontal axis indicates the number of days after cancer cell administration. The solid line indicates changes in the average tumor volume in each group, and the error bars indicate the standard error of the average. Significance probability P value (**: P < 0.01) is the tumor volume on day 12 of the BICANATE administration group and the aAVC (3T3) -pp65 cell and aAVC (3T3) -WT1 cell administration group by Dunnett's multiple comparison test It was calculated by comparing Figures 1-3 show the anti-tumor effect of aAVC(3T3)-pp65 cells in pp65-expressing GL-261 tumor-bearing mice. aAVC(3T3)-WT1 cells were used as control test aAVC without pp65 expression. The vertical axis indicates the survival rate, and the horizontal axis indicates the number of days after cancer cell administration. The significance probability P value was obtained by comparing the survival period of the BICANATE-administered group and the survival periods of the aAVC(3T3)-pp65 cell and aAVC(3T3)-WT1 cell-administered groups using the log-rank test. ** in the figure indicates a group with a P value smaller than 0.01/6 (significance level corrected by Bonferroni's method).

Although the present invention will be described in detail below, the present invention is not limited to these. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

<Artificial adjuvant vector cells>
As used herein, "artificial adjuvant vector cells" and "aAVC" are synonymous and used interchangeably, CD1d polypeptide (hereinafter also referred to as "CD1d") and any one or more cancer cells A cell that expresses an antigen or a fragment thereof is meant.
As used herein, "exogenous" or "exogenous" refers to introduction of an artificially produced gene or polynucleotide into a target cell by manipulation such as genetic engineering or gene introduction, and The terms are used interchangeably to refer to the artificial expression of the protein they encode by a gene or polynucleotide that has been artificially introduced into a cell.

<aAVC-pp65 cells of the present invention>
The present invention
Human-derived cells (also referred to herein as "aAVC-pp65 cells") exogenously introduced with a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof are provided.
Furthermore, the present invention provides
aAVC-pp65 cells expressing CD1d and pp65 or fragments thereof and loading CD1d ligand on the cell surface are provided.

1. Human-Derived Cells Human-derived cells used in the present invention are cells derived from normal cells or cancer tissues. In one embodiment, the human-derived cells used in the present invention are normal cells.
Normal cells are, for example, cells isolated and/or purified from any human tissue, cells derived from specific cell types, established cell lines derived from human tissue, cells differentiated from stem cells, etc., as long as they have proliferative ability. human-derived cells. As the cancer tissue-derived cells, cells isolated and/or purified from a patient's cancer tissue, or human cancer tissue-derived cell lines can be used. Here, "isolation" means separation from living tissue. In addition, "purification" means separation of human-derived cells from one or more other components contained in the tissue from which the cells were derived. Isolation and purification of cells can be performed by known methods.

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

Cells derived from specific cell types may be derived from specific cell types in human tissue (e.g., epithelial cells, endothelial cells, epidermal cells, stromal cells, fibroblasts, adipocytes, human embryonic kidney cells, keratinocytes, mesocytes). Leaf cells, myoblasts, nerve cells, glial cells, squamous cells, stem cells, mammary cells, mesangial cells, pancreatic β cells, exocrine epithelial cells, endocrine cells, skeletal muscle cells, smooth muscle cells, cardiomyocytes, osteoblasts, Cells derived from embryonic cells, immune cells (eg, dendritic cells, macrophages, lymphocytes, B cells, etc.). In one embodiment, the human-derived cells used in the present invention are human embryonic kidney cells.

Human tissue-derived cell lines can be produced using methods known to those skilled in the art, or can be obtained commercially. Such cell lines include, for example, human embryonic kidney 293 (HEK293) cells (J. Gen. Virol.; 1977; 36: 59-74), WI-38 cells, SC-01MFP cells, or MRC-5 cells. , or cells derived from these cells. In one embodiment, the human-derived cells used in the present invention are cells derived from HEK293 cells. In one embodiment, the cells derived from HEK293 cells are FreeStyle 293-F cells.

Cells differentiated from stem cells are cells differentiated from human-derived induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). Production of iPS cells or ES cells and cells differentiated from the cells can be produced using methods known to those skilled in the art.

Cancer tissue-derived cells can be obtained by isolation and/or purification from cancer tissues of patients by methods known to those skilled in the art. In addition, those skilled in the art can also use established cell lines derived from human cancer tissue that are generally available from organizations such as the American Type Culture Collection (ATCC (registered trademark)).

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 in viruses belonging to the genus Cytomegalovirus, such as Chimpanzee cytomegalovirus, in addition to HCMV.

The pp65 used in the present invention may be naturally occurring pp65 or may be a variant thereof so long as it induces pp65-specific T cells when administered as aAVC. In one embodiment, the pp65 used in the present invention is pp65 derived from a virus belonging to the genus Cytomegalovirus. In one embodiment, the pp65 used in the present invention is HCMV-derived pp65 (hereinafter referred to as “human pp65”). In one embodiment, human pp65 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:2. In one embodiment, human pp65 has at least 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2. A polypeptide consisting of In one embodiment, the human pp65 is 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 shown in SEQ ID NO: 2. It is a polypeptide consisting of an amino acid sequence in which 3 or 1-2 amino acids are deleted, substituted, inserted and/or added. Also, the 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. Induction of pp65-specific T cells by administration as aAVC can be evaluated, for example, by the method described in Example 1-2 below. A polynucleotide encoding pp65 or a fragment thereof used in the present invention can be produced by designing a nucleotide sequence from the amino acid sequence of pp65 or a fragment thereof using methods known in the art. In one embodiment, the polynucleotide encoding pp65 is a polynucleotide consisting of the base sequence shown in SEQ ID NO:1.

The term “identity” as used herein means an identity value obtained by default parameters using EMBOSS Needle (Nucleic Acids Res.; 2015; 43: W580-W584). Said parameters are:
Gap Open Penalty = 10
Gap Extend Penalty = 0.5
Matrix = EBLOSUM62
End Gap Penalty = false

3. CD1d
The aAVC-pp65 cells of the present invention have been exogenously introduced with a polynucleotide encoding CD1d. CD1d is an MHC class I-like glycoprotein expressed on the cell surface, and is known to activate CD1d-restricted NKT cells by presenting a CD1d ligand, which is a glycolipid antigen, on CD1d (Science 1997;278;5343:1626-1629). CD1d used in the present invention may be naturally occurring CD1d or a variant thereof, as long as it has the function of CD1d when expressed in human-derived cells. CD1d function can be assessed by measuring the binding of CD1d to a CD1d ligand (eg, α-GalCer). Such evaluation can be readily 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. This ability to activate human NKT cells is shown in Fig. 1 of Non-Patent Document 12, for example. 1 can be evaluated in the in vitro evaluation system. Briefly, CD1d-expressing cells cultured in the presence of α-GalCer are co-cultured with NKT cells, and the amount of IFN-γ in the culture medium is measured to evaluate activation of NKT cells.

In one embodiment, the CD1d used in the present invention is CD1d from a mammal (eg, human, monkey, mouse, rat, dog, chimpanzee, etc.). In one embodiment, the CD1d is human CD1d. In one embodiment, human CD1d is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:4. In one embodiment, the human CD1d has a deletion of 1-10, 1-7, 1-5, 1-3, or 1-2 amino acids in the amino acid sequence shown in SEQ ID NO:4; It is a polypeptide consisting of a substituted, inserted and/or added amino acid sequence and having the function of CD1d. In one embodiment, the human CD1d has at least 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity to the polypeptide set forth in SEQ ID NO:4. 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. It can be designed and made. In one embodiment, the CD1d-encoding polynucleotide is a polynucleotide consisting of the base sequence shown in SEQ ID NO:3.

4. Promoters In one embodiment, in the aAVC-pp65 cells of the invention, a 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 promotes expression constantly or an inducible promoter can be used. As used herein, "operably linked" means a state in which a promoter is linked to a polynucleotide so that the expression of the polypeptide in the host cell is controlled by the promoter.

Examples of the "promoter that promotes constant expression" include virus-derived promoters such as CMV (cytomegalovirus), RSV (respiratory syncytial virus), SV40 (simian virus 40), actin promoter, EF (elongation factor) 1α promoter, and the like. is mentioned.

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

5. CD1d Ligand In one embodiment, the aAVC-pp65 cells of the invention may be loaded with CD1d ligand on the cell surface. Loading of CD1d ligand on the cell surface can be achieved by pulsing CD1d-expressing cells with CD1d ligand (eg, α-GalCer) as described in the section <Method for producing aAVC-pp65 cells of the present invention>. can do As used herein, "pulsing CD1d ligand" refers to contacting cells with CD1d ligand in culture medium. In CD1d ligand-pulsed aAVC, all or at least some CD1d is loaded with CD1d ligand.

CD1d ligands used in the present invention include, for example, α-galactosylceramide (α-GalCer), α-C-galactosylceramide (α-C-GalCer), 7DW8-5, isoglobotrihexosylceramide (iGb3). is mentioned. In one embodiment, the CD1d ligand is α-GalCer. α-GalCer has the chemical name (2S,3S,4R)-1-O-(α-D-galactosyl)-N-hexacosanoyl-2-amino-1,3,4-octadecanetriol and has the CAS RN: 158021-47-7 and represented by the molecular formula: C ₅₀ H ₉₉ NO ₉ (molecular weight: 858.34). α-GalCer may be synthesized according to techniques known in the art, or commercially available (eg, 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 produced 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 comprise the steps of culturing the aAVC-pp65 cells, cloning the cells, and loading CD1d ligand on the cell surface.

1. Introduction of Polynucleotides 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 (see below). , the introduced polynucleotide is also referred to as the "polynucleotide of interest").

Polynucleotides encoding CD1d and polynucleotides encoding pp65 or a fragment thereof are obtained, for example, from NCBI RefSeq ID or GenBank Accession numbers to obtain nucleotide sequences encoding CD1d and pp65 amino acid sequences, and subject them to standard molecular biology. and/or can be designed and fabricated using chemical methods. For example, the polynucleotide can be synthesized using the phosphoramidite method or the like based on its nucleotide sequence, or can be prepared by combining DNA fragments obtained from a cDNA library using the polymerase chain reaction (PCR). can do.

Introduction of the target polynucleotide into human-derived cells can be carried out using either non-viral vector-based methods such as electroporation, lipofection, microinjection, or viral vector-based methods. When using non-viral vector-based methods, the target polynucleotide may be in the form of a plasmid vector, cDNA, or the like, or in the form of mRNA.

Plasmid or viral vectors can be used as expression vectors to exogenously express CD1d or pp65 or fragments thereof in human-derived cells. The expression vector is not particularly limited as long as it can express the polypeptide of interest, and can be constructed by those skilled in the art using methods known to those skilled in the art. Examples of plasmid vectors that can be used include pcDNA series (Thermo Fisher Scientific), pALTER (registered trademark)-MAX (Promega), pHEK293 Ultra Expression Vector (Takara Bio), and the like. Viral vectors that can be used include, for example, lentiviruses, adenoviruses, retroviruses, and adeno-associated viruses. For example, when using a lentivirus to introduce a target polynucleotide into human-derived cells, the lentivirus can be prepared using pLVSIN-CMV/EF1α vector (Takara Bio Inc.) and pLenti vector (Thermo Fisher Scientific). . When exogenously expressing CD1d and pp65 or a fragment thereof 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 serve as a marker for confirming the expression of the target gene or polynucleotide (for example, a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein, etc.). You can Expression vectors may further include start and stop codons, enhancer sequences, untranslated regions, splice junctions, polyadenylation sites, replicable units, and the like.

In one embodiment, the method for producing aAVC-pp65 cells of the present invention includes the 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 of producing aAVC-pp65 cells of the present invention comprises introducing an expression vector comprising a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into a human-derived cell. 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, in one embodiment the viral vector is a lentivirus. is a vector.

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

2. Culture of aAVC-pp65 Cells The aAVC-pp65 cells prepared by the above method can be further grown by culturing. Cell culture for maintaining or growing aAVC-pp65 cells is performed by known methods. Examples of basal media include MEM medium (Science; 1952; 122: 501), DMEM medium (Virology; 1959; 8: 396-397), RPMI1640 medium (J. Am. Med. Assoc.; 1967; 199: 519 -524), 199 medium (Proc. Soc. Exp. Biol. Med.; 1950; 73: 1-8), FreeStyle 293 Expression Medium (Thermo Fisher Scientific, Cat. 12338022), CD 293 Medium (Thermo F Isher Scientific, Inc. Cat. 11913019), Expi293 Expression Medium (Thermo Fisher Scientific, Cat. A1435101) can be used. The culture medium contains serum (e.g., fetal bovine serum), serum replacement (e.g., KnockOut Serum Replacement: KSR), fatty acids or lipids, amino acids, vitamins, growth factors, cytokines, antioxidants, 2 - can further include mercaptoethanol, pyruvic acid, buffers, inorganic salts, antibiotics, etc.; In one embodiment, the culture medium is serum-free or chemically defined medium.

Culture conditions (eg, culture conditions such as culture time, temperature, medium pH, CO ₂ concentration, etc.) can be appropriately selected by those skilled in the art. The pH of the medium is preferably about 6-8, and the culture temperature is not particularly limited, but is, for example, about 30-40°C, preferably about 37°C. Also, the CO ₂ concentration is about 1-10%, preferably about 5%. Cultivation time is not particularly limited, but is carried out for about 15 to 336 hours. Aeration and stirring can also 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 cultured during the culture of the aAVC-pp65 cells. Expression of CD1d and/or pp65 or a fragment thereof may be induced by contacting with an inducer for the inducible promoter. In one embodiment, the method for producing aAVC-pp65 cells includes the step of inducing expression of CD1d and/or pp65 or a fragment thereof by culturing 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 method of making comprises culturing aAVC-pp65 cells in the presence of an inducer of the drug-inducible promoter. When a drug-inducible promoter is used, cells are cultured in a culture medium containing an inducer such as tetracycline, doxycycline, cumate, coumermycin, etc., and a gene or polynucleotide operably linked to the drug-inducible promoter. expression 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-inducible promoter is a tetracycline-based inducible promoter, and the method of making 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 the TRE3G promoter and the method comprises culturing aAVC-pp65 cells in the presence of tetracycline or a derivative thereof and Tet-On 3G protein. The expression levels of CD1d and pp65 or fragments thereof in the obtained aAVC-pp65 cells can be determined by methods known to those skilled in the art, such as Western Blot method using anti-CD1d antibody or anti-pp65 antibody, ELISA method, and flow cytometry method. can be measured.

3. Cloning of aAVC-pp65 Cells When a polynucleotide is introduced into cells, cells into which the target polynucleotide has been introduced and cells into which the target polynucleotide has not been introduced are mixed. In some cases, a heterogeneous population of cells is formed because the site of integration differs depending on the cell. The heterogeneous population of cells is referred to herein as a "cell pool." A polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof were introduced by a method of obtaining cells having a single genotype from the cell pool (herein referred to as "cloning"). Obtaining a cell population having a single genotype (hereinafter referred to as a "cloned cell population") by obtaining and growing one cell (hereinafter referred to as a "cloned cell") be able to. Cloning of aAVC-pp65 cells can be performed using methods well known to those skilled in the art, for example, limiting dilution, single cell sorting, or colony picking can be used. In one embodiment, the aAVC-pp65 cell cloning method can be one or a combination of two or more of the limiting dilution method, single cell sorting method, or colony picking method. In one embodiment, the method for cloning aAVC-pp65 cells is the limiting dilution method.

By measuring the expression levels of CD1d and pp65 or fragments thereof for each cloned aAVC-pp65 cell obtained by the method, the cloned cells showing the desired expression levels for CD1d and pp65 or fragments thereof A population of aAVC-pp65 cells can be selected. If an inducible promoter is operably linked to the polynucleotide encoding CD1d and/or the polynucleotide encoding pp65 or a fragment thereof, for each cloned aAVC-pp65 cell, a portion of the inducible A clone that exhibits a desired expression level of 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 resulting cells. A population of transformed aAVC-pp65 cells may be selected. A cryopreserved population of selected cloned cells can be used as a research cell bank (RCB). RCB cells can be grown in culture and frozen to serve as a master cell bank (MCB). MCB cells can be grown in culture and frozen to serve as a working cell bank (WCB). Cultured WCB cells can be used as raw materials for pharmaceuticals. Each cell bank may contain a cryoprotectant. Each cell bank may be aliquoted into multiple containers (eg, 2-10,000 containers, such as 10-1000 containers, such as 100-500 containers) and stored frozen. Thawing the cloned aAVC-pp65 cells, culturing them, contacting them with an inducer, and measuring the expression levels of CD1d and pp65 or fragments thereof can be performed by methods known to those skilled in the art. Contact with the factor and measurement of the expression level can be performed using, for example, the method described in "2. Culture of aAVC-pp65 cells". In one embodiment, culturing the cloned aAVC-pp65 cells and contacting with inducers and measuring the expression levels of CD1d and pp65 or fragments thereof can be performed after thawing the RCB and WCB.

4. CD1d Ligand Loading of aAVC-pp65 Cells CD1d ligand-loaded aAVC-pp65 cells can be generated by pulsing aAVC-pp65 cells with CD1d ligand. The conditions for pulsing the CD1d ligand (e.g., 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.) should be determined in consideration of the cells to be used and the culture conditions. It can be adjusted as appropriate by the trader. The concentration of the CD1d ligand to be added to the culture medium of aAVC-pp65 cells is not particularly limited, but can be selected appropriately within the range of, for example, 1 ng/mL to 10000 ng/mL. In one embodiment, the CD1d ligand is α-GalCer. For 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 may occur prior to contacting the cells with the inducer. In one embodiment, loading of aAVC-pp65 cells with a CD1d ligand may be performed after contacting the cells with an inducer. In one embodiment, loading of aAVC-pp65 cells with CD1d ligand can occur simultaneously with contacting the cells with an inducer. In these embodiments, the aAVC-pp65 cells are preferably cloned.

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

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

The invention also provides a method of treating cancer comprising administering the CD1d ligand-loaded aAVC-pp65 cells of the invention to a subject. In one embodiment, the CD1d ligand is α-GalCer. As used herein, a "subject" is a mammal (eg, human, monkey, mouse, rat, dog, chimpanzee, etc.), and in one embodiment, the subject is human. As used herein, the term "treatment" is used to include therapeutic treatment and prophylactic treatment. When administering the CD1d ligand-loaded aAVC-pp65 cells 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 dosage and frequency of administration of the CD1d ligand-loaded aAVC-pp65 cells to the subject can be appropriately adjusted according to the type, location, severity of cancer, age, weight and condition of the subject to be treated, and the like. Dosages of CD1d ligand-loaded aAVC-pp65 cells can be, for example, 1×10 ³ cells/kg to 1×10 ⁹ cells/kg in a single administration to a subject. The CD1d ligand-loaded aAVC-pp65 cells can be administered to a subject by, for example, intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, or intraarterial injection or infusion. 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 anti-cancer agents.

Cancers to be treated by the present invention include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiplex Hematological cancers such as myeloma and T-cell lymphoma, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer , mesothelioma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, gallbladder cancer, Solid cancer such as bile duct cancer, kidney cancer, pancreatic cancer, lung cancer, colon cancer, colon cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, brain tumor, etc. , and cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue, sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma , glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma. In one embodiment, the cancer targeted for treatment of the present invention is a pp65-positive cancer. In one embodiment, the cancer targeted for treatment of the present invention is glioblastoma or glioblastoma multiforme.

<Method for Producing Cells or Pharmaceutical Composition Used for Treatment of Cancer>
The present invention also provides a method for producing a cell or pharmaceutical composition for treating cancer, comprising the steps of:
A step of introducing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into human-derived cells, and culturing the cells.

In one embodiment, the CD1d used in the method for producing a cell or pharmaceutical composition for treating cancer of the present invention is human CD1d.

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

The present invention also provides a method for producing cells or pharmaceutical compositions for treating cancer, comprising culturing the aAVC-pp65 cells of the present invention. In one embodiment, the CD1d used in 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 manufacturing method further comprises the step of loading a CD1d ligand onto the cell surface. In one embodiment, the CD1d ligand is α-GalCer.

In one embodiment, the aAVC-pp65 cells of the present invention used in the production method contain a polynucleotide encoding CD1d and/or a polynucleotide encoding pp65 or a fragment thereof operably linked to an inducible promoter. human-derived cells into which nucleotides have been exogenously introduced; Further comprising the step of inducing expression. In one embodiment, the method comprises inducing 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 manufacturing method further comprises loading a CD1d ligand onto the cell surface, and in one embodiment, the CD1d ligand is α-GalCer. In one embodiment, the loading of the CD1d ligand on the cell surface may be before, after, or simultaneously with the contacting of the aAVC-pp65 cells with the inducer. The present invention also provides use of the aAVC-pp65 cells of the present invention in the manufacture of pharmaceutical compositions for treating cancer.

Specific embodiments of the cells and processes used in the production method described in this section are described in the sections <aAVC-pp65 cells of the present invention> and <Method for producing aAVC-pp65 cells of the present invention>. It is as follows.

Specific examples are provided herein by reference for a further understanding of the present invention, which are intended for purposes of illustration and are not intended to limit the invention.

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

[Example 1-1: Preparation of mouse aAVC (3T3)-pp65 cells]
Mouse NIH/3T3 cells (ATCC, Cat. CRL-1658) were transfected with pp65 gene and CD1d gene mRNA 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 of HCMV pp65 shown in SEQ ID NO: 2 (UniProt: P06725-1). Furthermore, the pp65 gene is a primer with a sequence complementary to 16 bases containing the HindIII or EcoRI recognition sequence of the pGEM (registered trademark)-4Z plasmid (hereinafter referred to as pGEM-4Z plasmid) (Promega, Cat. P2161). was used and amplified by the PCR method. The amplified pp65 gene was inserted into the HindIII-EcoRI site of pGEM-4Z plasmid using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio Inc., Cat. 639648). The resulting plasmid is called pGEM-4Z-pp65 plasmid. A mouse CD1d gene consisting of the base sequence shown in SEQ ID NO: 5 (Gene synthesized by artificial gene synthesis based on the amino acid sequence of UniProt: P11609-1 (SEQ ID NO: 6)) was inserted into the HindIII-BamHI site of the pGEM-4Z plasmid. inserted. The resulting plasmid is called pGEM-4Z-mCD1d plasmid. The pGEM-4Z-pp65 plasmid and pGEM-4Z-mCD1d plasmid were cleaved with EcoRI and BamHI and linearized, respectively. to generate pp65 mRNA and CD1d mRNA. NIH/3T3 cells were placed in Dulbecco's Modified Eagle medium (Merck, Cat. D6429) containing 10% fetal bovine serum supplemented with 350 ng/mL of α-GalCer (synthesized by Juzen Chemical Co., Ltd.). The cells were harvested after 2 days of culture at room temperature. pp65 mRNA and CD1d mRNA were added to the collected cell suspension, and electroporation was performed using a NEPA21 electroporator (Neppa Gene Co., Ltd.) (pouring pulse: voltage 150 V, pulse width 8 ms, pulse interval 50 ms, number of times 2). times, attenuation rate 10%, polarity +, transfer pulse: voltage 20 V, pulse width 50 ms, pulse interval 50 ms, number of times ±5 times, attenuation rate 40%, polarity +/-). After the electroporation, the cells were recovered and irradiated with 30 Gy of X-rays using an X-ray irradiator MBR-1520R-3 (Hitachi Power Solutions Co., Ltd.). The cells obtained by the above method are called aAVC(3T3)-pp65 cells.

aAVC(3T3)-pp65 cell control aAVC(3T3)-WT1 cells used as test aAVC were produced 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 for 2 days in Dulbecco's Modified Eagle medium (Merck, Cat. D6429) containing 10% fetal bovine serum supplemented with 500 ng/mL of α-GalCer. The WT1 gene was prepared from pcDNA3-ATG-WT1 described in WO2013/018778.

[Example 1-2: Ability of aAVC(3T3)-pp65 cells to induce pp65-specific T cells in mice]
Five aAVC(3T3)-pp65 cells suspended in 200 μL of BICANATE Injection (Otsuka Pharmaceutical Factory, Inc.) were injected into the tail veins of five 5-week-old C57BL/6J female mice (Charles River Japan) in each group. ×10 ³ cells, 5×10 ⁴ cells and 5×10 ⁵ cells were administered respectively. 200 μL of BICANATE Injection was administered to the control group. Seven days after administration, spleen cells were collected from the mice, and the cells collected according to the attached method were placed on a plate of mouse IFN-γ single-color enzymatic ELISPOT assay kit (Cellular Technology Limited, mIFNGp-2M/5) at 3×10 ^{5 .} Cells/well were seeded. Furthermore, for stimulating pp65-specific T cells, the pp65 peptide, an overlapping peptide specific for the pp65 protein (PepTivator CMV pp65 - premium grade, human (Miltenyi Biotech, Cat. 130-093-435)), And, WT1 peptide (PepTivator WT1-premium grade, human (Miltenyi Biotech, Cat. 130-095-918)), which is a non-specific overlapping peptide to pp65 protein, was added, 37 ° C., 5% CO ₂ conditions cultured for 1 day under The spots were colored according to the attached method, and the number of IFN-γ-producing cells was counted by counting the number of cell spots on the well bottom with an ELISpot reader 08 classic (Autoimmun Diagnostica). In aAVC(3T3)-pp65 cell-administered mice, pp65 peptide stimulation increased the number of IFN-γ-producing cells (FIG. 1-1). The difference from the number of spots by WT1 peptide stimulation, which is antigen-nonspecific, indicates the number of pp65-specific T cells. This result suggested 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 tumor-bearing mice]
B16-F10 melanoma cells (ATCC, Cat. CRL-6475) were introduced with a plasmid carrying the pp65 gene (SEQ ID NO: 1) by electroporation, and drug selection was performed to obtain B16-F10 melanoma cells that express the pp65 protein. (also called B16-pp65 cells) were generated. 1x suspended in D-PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. 10 ⁵ cells of B16-pp65 cells were administered. Five days after administration, aAVC(3T3)-pp65 cells or aAVC(3T3)-WT1 cells suspended in BICANATE Injection were injected into the tail vein of the mice at 5×10 ² cells, 5×10 3 cells, and 5×10 ³ cells, respectively. 10 ⁴ cells were administered at a time. 200 μL of BICANATE Injection was administered to the control group. The day when cancer cells were inoculated was set as day 0, and changes in tumor volume were measured for 17 days. B16-pp65 tumor growth 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. )-WT1 cells of 5×10 ² cells and 5×10 ³ cells of B16-pp65 tumor growth were not inhibited (FIG. 1-2). In the groups administered 5×10 ⁴ cells of aAVC(3T3)-pp65 cells and aAVC(3T3)-WT1 cells, both antitumor effects were observed, so the antitumor effect at this dose was non-specific to the pp65 protein. It is thought that anti-tumor effects including anti-tumor effects are 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 than the aAVC(3T3)-WT1 cell administration group was obtained. The difference in antitumor activity is considered a pp65-specific antitumor effect based on the induction of specific immunity against 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 tumor-bearing mice]
A plasmid carrying the pp65 gene (SEQ ID NO: 1) was introduced into GL-261 glioma cells (DSMZ, Cat. ACC802) by electroporation, and drug selection was performed to obtain GL-261 glioma cells (GL -261-pp65 cells) were generated. 1 suspended in D-PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. GL-261-pp65 cells were administered at ×10 ⁵ cells. Seven days after administration, aAVC(3T3)-pp65 cells or aAVC(3T3)-WT1 cells suspended in BICANATE Injection were added to the tail vein of the mice at 5×10 ² cells, 5×10 3 cells, and 5×10 ³ cells, respectively. 10 ⁴ cells were administered at a time. 200 μL of BICANATE Injection was administered to the control group. Survival was observed for 50 days after administration of GL-261-pp65 cells. When the survival period was compared by the log-rank test, the survival period of all aAVC(3T3)-pp65 cell administration groups and the group of aAVC(3T3)-WT1 cell administration of 5×10 ⁴ cells was significantly longer than that of the control group. In contrast, no effect of prolonging the survival period was observed in the groups administered 5×10 ² cells and 5×10 ³ cells of aAVC(3T3)-WT1 cells (FIG. 1-3). Similar to the results of Example 1-3, the antitumor activity exhibited by the administration of 5×10 ² cells and 5×10 ³ cells of aAVC(3T3)-pp65 cells was confirmed by the induction of pp65-specific immunity. It is considered as a specific anti-tumor effect.

The results of Example 1 confirmed that mouse aAVC(3T3)-pp65 cells exhibited pp65-specific antitumor effects by inducing pp65-specific T cells in a mouse in vivo evaluation system. Human aAVC-pp65 cells can be expected to induce innate immunity and exhibit antitumor effects in humans, as well as induce antigen-specific acquired immunity and exhibit antitumor effects based on acquired immunity. , for the purpose of creating therapeutic agents that exhibit pp65-specific anti-tumor effects against human pp65-expressing cancers, the preparation of human aAVC-pp65 cells was investigated below.

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

[Example 2-1: Preparation of lentivirus]
(1) Construction of plasmid for producing lentivirus A lentiviral plasmid constructed based on the nucleotide sequences of pLVSIN-CMV Pur plasmid (Takara Bio Inc., Cat.6183) and pTRE3G (Takara Bio Inc., Cat.631173) was constructed as pLVsyn-TRE3G. called a plasmid. The pp65 gene (SEQ ID NO: 1) was amplified by PCR using primers added with sequences complementary to 16 bases containing the EcoRI or BamHI recognition sequence of the pLVsyn-TRE3G plasmid. The amplified pp65 gene was inserted into the EcoRI and BamHI treated pLVsyn-TRE3G plasmid using the In-Fusion® HD Cloning Kit. The resulting plasmid is called pLVsyn-TRE3G-pp65 plasmid. The WPRE sequence of the resulting pLVsyn-TRE3G-pp65 plasmid was cloned using methods well known to those skilled in the art according to Gene Ther. ; 2009; 16(5):605-19. The resulting plasmid is called pLVsyn-TRE3G-pp65-mWPRE (SEQ ID NO:7).

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

The Tet-On 3G gene is based on the Tet-On 3G gene sequence of pCMV-Tet3G plasmid (Takara Bio Inc., Cat.631335). Sequences were added and produced by artificial gene synthesis. The Tet-On 3G gene cut out with restriction enzymes XhoI and NotI was inserted into the XhoI-NotI site of the pLVsyn-CMV plasmid. The resulting plasmid is called pLVsyn-CMV-Tet3G plasmid. A pLVsyn-CMV-Tet3G-mWPRE plasmid was prepared by converting the WPRE sequence into a mutant type by the same procedure as for pLVsyn-TRE3G-pp65-mWPRE.

(2) Lentivirus preparation Using the pLVsyn-TRE3G-pp65-mWPRE plasmid, pLVsyn-CMV-Tet3G-mWPRE plasmid, and pLVsyn-CMV-CD1d-mWPRE plasmid prepared in (1), the pp65 gene, Tet-On A lentivirus carrying the 3G gene and the CD1d gene was prepared. The resulting 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 pLVsyn-CMV-Tet3G-mWPRE plasmid and 45 μL of ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific, Cat. K497500) were mixed, 1.5 mL of OptiPRO SFM (Thermo Fisher Scientific, Cat. 12309019) was added and mixed (A). 540 μL of LV-MAX Transfection Reagent (Component of Thermo Fischer Scientific, Cat. A35348) and 4.5 mL of OptiProSFM were mixed and allowed to stand for 1 minute (B). (A) and 1.5 mL of (B) were mixed and allowed to stand at room temperature for 10 minutes. Viral Production Cells (Thermo Fischer Scientific, Cat. A35347) were cultured in a 125 mL Erlenmeyer flask (Thermo Fischer Scientific, Cat. 4115-0125), and added to the culture solution (1.2×10 ⁸ cells/mL, 27 mL). Gene introduction was carried out by adding the total amount of the mixture of (A) and (B). The Viral Production Cells were cultured in LV-MAX Production Medium (Thermo Fisher Scientific, Cat. A3583401) under conditions of 37° C., 8% CO ₂ and 120 rpm. After the gene introduction, the cells were cultured for 2 days under the above conditions, and the culture supernatant containing the lentivirus carrying the TRE3G-pp65 or Tet3G gene was collected. The culture supernatant was centrifuged at 760 xg for 10 minutes at 4°C. The supernatant was filtered through a 0.45 μm filter (Merck, Cat. SLHV033RS), and PEG-it Virus Precipitation Solution (5x) (System Biosciences, Cat. LV810A-1) was added to ¼ volume of the supernatant. Add, mix and let stand overnight at 4°C. After centrifugation at 1500×g and 4° C. for 30 minutes, the supernatant was removed and centrifuged again at 1500×g and 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 TRE3G-pp65-loaded lentivirus and Tet3G-loaded lentivirus.

18 μg of the pLVsyn-CMV-CD1d-mWPRE plasmid and 54 μL of ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific, Cat. K497500) were mixed and added to 9 mL of OptiPRO SFM (Thermo Fisher Scientific). fic, Cat. 12309019) was added and mixed ( A). 648 μL of Lipofectamine 2000 CD Transfection Reagent (Thermo Fischer Scientific, Cat. 12566014) and 27 mL of OptiProSFM were mixed and allowed to stand for 5 minutes (B). ( ^A ) and 9 mL of (B) were mixed and allowed to stand at room temperature for 20 minutes. A mixed solution of (A) and (B) was added to Lenti-X 293T cells (Takara Bio Inc., Cat. 632180) seeded in a plate at 3 mL/plate for gene transfer. Lenti-X 293T cells were grown in DMEM containing 10% fetal bovine serum (SAFC Biosciences, Cat. 12007C (γ-irradiated)) and 0.1% gentamicin (Thermo Fisher Scientific, Cat. 15750060). Cultivation was performed in a medium (Thermo Fisher Scientific, Cat. 10569010) at 37°C and 5% _CO2 . After the gene transfer, the cells were cultured under the same conditions for 2 days, and the culture supernatant containing the lentivirus loaded with each gene was collected. The culture supernatant was centrifuged at 760 xg for 10 minutes at 4°C. The supernatant was filtered through a 0.45 μm filter (Merck, Cat. SLHV033RS), and PEG-it Virus Precipitation Solution (5x) (System Biosciences, Cat. LV810A-1) was added to ¼ volume of the supernatant. Add, mix and let stand overnight at 4°C. After centrifugation at 1500×g and 4° C. for 30 minutes, the supernatant was removed and centrifuged again at 1500×g and 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 CD1d-loaded lentivirus.

[Example 2-2: Preparation and cloning of Lenti virus-infected cells]
FreeStyle 293-F cells (Thermo Fisher Scientific, Cat. 510029) were infected with each lentivirus prepared in Example 2-1. Cell populations infected with TRE3G-pp65 loaded lentiviruses, CD1d loaded lentiviruses and Tet3G loaded lentiviruses are referred to as FreeStyle 293F_Tet-on_pp65_CD1d cell pools. Cell cloning was performed from these cell pools, and multiple clones derived 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 (registered trademark) cell culture was performed at 500 μL/well. 12-well multiwell plate for cell culture, flat bottom The cells were seeded in a lidded (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 medium was further added. The medium used was FreeStyle 293 Expression Medium (Thermo Fisher Scientific, Cat. 12338018) containing 0.1% gentamicin (Thermo Fisher Scientific, Cat. 15750-060). After centrifugation at 540×g for 30 minutes at room temperature, the cells were gently suspended by pipetting and cultured with shaking. After culturing for several days, the cells were subcultured from the 12-well plate to Corning (registered trademark) polycarbonate Erlenmeyer flask vent cap 125 mL (hereinafter referred to as 125 mL Erlenmeyer flask) (Corning, Cat.431143), and further subcultured at appropriate intervals. Pool A was obtained. Cell pool A was prepared at a concentration of 2×10 ⁶ cells/mL, seeded in a 12-well plate at 500 μL/well, and 200 μL each of TRE3G-pp65-loaded lentivirus and Tet3G-loaded lentivirus obtained in Example 2-1. /well or 25 μL/well was added. Furthermore, 100 μL or 450 μL of medium was added, respectively. FreeStyle 293 Expression Medium containing 0.1% gentamicin was used as the medium. After centrifugation at 540×g for 30 minutes at room temperature, 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 to 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 stably expressing all genes were selected. Cells were seeded in a 96-well plate at 1 cell/well, and subcultured according to cell growth to proliferate the cells. The expression levels of pp65 protein and CD1d protein in the proliferated cells were measured by ELISA for pp65 protein and by flow cytometry for CD1d protein, and clones stably expressing pp65 protein and CD1d protein were selected. Cells used for measuring the expression level of pp65 protein added doxycycline (Takara Bio Inc., Cat.631311) at a final concentration of 0.8 to 1 μg/mL to the medium, and expressed pp65 protein via the Tet-On System. Evaluation was performed after induction. For measurement by ELISA, a CMV pp65 (3A12) antibody (Santa Cruz Biotechnology, Cat. sc-56973) was used as a primary antibody, and a Goat Anti-Mouse IgG Fc-HRP antibody (Jackson Immuno Research Laboratories, Cat. sc-56973) was used as a secondary antibody. 115- 035-071) was used, and the measurement was carried out according to the general measurement method of the ELISA direct method. For measurement by flow cytometry, APC Mouse Anti-Human CD1d antibody (BD Biosciences, Cat.563505) is used, and measurement is performed according to a general measurement method using FACSVerse (BD Biosciences). rice field.

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

Numerical heading <223> in the Sequence Listing below provides a description of "Artificial Sequence". The nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing is the nucleotide sequence encoding the human cytomegalovirus pp65 protein, and the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing is the amino acid sequence encoded by SEQ ID NO: 1. be. The nucleotide sequence shown by SEQ ID NO:3 is the nucleotide sequence encoding the human CD1d protein, and the amino acid sequence shown by SEQ ID NO:4 in the sequence listing is the amino acid sequence encoded by SEQ ID NO:3. The nucleotide sequence shown by SEQ ID NO:5 is a 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 represented by SEQ ID NO: 7 and SEQ ID NO: 8 in the Sequence Listing are the nucleotide sequences of the pLVsyn-TRE3G-pp65-mWPRE plasmid and the pLVsyn-CMV plasmid, respectively.

Claims (18)

A human-derived cell exogenously introduced with a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof. 2. The cell of claim 1, wherein the CD1d is human CD1d. 3. The cell according to claim 1 or 2, wherein the human-derived cell is a normal cell. 4. The cell of claim 3, wherein the normal cell is a cell derived from human embryonic kidney 293 (HEK293) cells. A cell according to any one of claims 1 to 4, which expresses CD1d and pp65 or a fragment thereof. 6. The cell of claim 5, wherein the cell surface is loaded with CD1d ligand. 7. The cell of claim 6, wherein the CD1d ligand is α-GalCer. A pharmaceutical composition comprising the cells of claim 6 or 7. 9. A pharmaceutical composition according to claim 8 for use in treating cancer. A method of treating cancer, comprising administering the cells of claim 6 or 7 to a subject. 8. A cell according to claim 6 or 7 for treating cancer. Use of the cells according to claim 6 or 7 for manufacturing a pharmaceutical composition for treating cancer. A method of producing cells for use in treating cancer, comprising:
introducing a polynucleotide encoding CD1d and a polynucleotide encoding pp65 or a fragment thereof into a human-derived cell;
A method comprising the step of culturing said cells. 14. The method of claim 13, wherein the CD1d is human CD1d. 15. The method of claim 13 or 14, wherein the human-derived cells are normal cells. 16. The method of claim 15, wherein the normal cells are cells derived from human embryonic kidney 293 (HEK293) cells. 17. The method of any of claims 13-16, further comprising the step of loading the surface of the cell with a CD1d ligand. 18. The method of claim 17, wherein the CD1d ligand is α-GalCer.

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