JP6903866B2 - Method for inducing proliferation of blood-derived monocytes - Google Patents

Description

The present invention relates to a method for inducing proliferation of monocytes derived from blood, particularly peripheral blood. The present invention also relates to a method for producing a large amount of monocytes using the method for inducing proliferation. The present invention further relates to a method for providing monocyte-derived dendritic cells that can be used for immunotherapy, and also relates to immunotherapy using those dendritic cells.

Monocytes (Mo) are mononuclear cells that are classified as white blood cells and are immature phagocytes that circulate in the blood. Monocytes play an important role in immunity against infection, and take up foreign substances such as bacteria into cells and digest them. The fragmented foreign substance is bound to the MHC molecule originally contained in the cytoplasm, presented on the cell surface, and the helper T cell recognizes this to initiate an immune reaction. Monocytes are also known to differentiate into macrophages, dendritic cells and osteoclasts.

Macrophages are major foreign body-treating cells in the living body, and have a role of protecting the living body from infectious diseases by phagocytosing infectious microorganisms and the like that have invaded the living body and decomposing them. In addition, a large amount of cells die every day in the living body, and macrophages phagocytose and decompose the debris existing in the tissues in the living body. In addition, macrophages play an essential role in maintaining homeostasis of the living body by phagocytosing and decomposing various metabolites generated in the living body. In addition, macrophages are often infiltrated locally in malignant tumors. Macrophages located locally in the tumor are thought to be both attacking the tumor cells and promoting the growth of the tumor cells. Attempts have also been made to treat malignant tumors by utilizing the ability of macrophages to attack tumor cells.

Dendritic cells (DCs) are cells that strongly stimulate and activate T lymphocytes, and are cells that control the immune response in vivo. Upon invasion of an infectious microorganism into the body, dendritic cells phagocytose the microorganism, present an antigenic substance derived from the microorganism to T lymphocytes, and stimulate and activate antigen-specific T lymphocytes to stimulate an immune response. Induce. Attempts have been made to utilize dendritic cells as a cell vaccine in immunotherapy for cancer and infectious diseases, taking advantage of the ability of dendritic cells to strongly stimulate T lymphocytes.

Vaccine treatment with antigenic peptides is widely used to treat cancer patients. These antigenic peptides are, for example, emulsified with an adjuvant or added to dendritic cells. Over the last two decades, considerable effort has been made to identify cancer antigen-derived CTL epitopes, limited to ^{common alleles of HLA class I, such as HLA-A * 02:01.} Yes, and as a result, a vast amount of information about the epitopes presented by the major HLA class I alleles has been accumulated. On the other hand, relatively few types of epitopes have been identified for low-frequency HLA alleles. Therefore, cancer patients who are negative for a common type of HLA class I are excluded from most current vaccine therapies.

Although HLA-A ^* 02:01 is the most common class I allele in the world, ^{the gene frequency of HLA-A *} 02:01 is at most 30% in most racial populations. Therefore, a significant number of patients do not benefit from vaccine therapy. In addition, HLA-B restrictive epitopes have been poorly identified, probably due to the absence of particularly dominant alleles at the HLA-B locus. However, there should be many useful HLA-B-restrictive epitopes, including known cancer antigens. If HLA-B-restrictive CTL can be stimulated, the effectiveness of anticancer vaccine therapy will be substantially improved.

Gene-based vaccine therapies, such as plasmid DNA vaccines and recombinant virus vaccines, have been considered as possible means to overcome the limitations associated with synthetic peptide-based vaccine therapies. However, plasmid-based DNA vaccines are not sufficiently effective in inducing anticancer immunity. Therapy with recombinant viruses poses a problem with the potential risks associated with administration of the infectious virus to patients. Vaccines using genetically modified dendritic cells that present cancer antigens are more effective and safer than them.

Since dendritic cells are present in very small numbers in human blood, dendritic cells used in chemotherapy are usually made by differentiating monocytes in peripheral blood in vitro. Genetic modification of monocytes using a viral vector has been reported as a means for producing cancer antigen-presenting dendritic cells (Non-Patent Documents 1 to 3). However, monocytes are usually unable to proliferate, and selective proliferation of transgenic cells is not a feasible means. Therefore, a method of proliferating dendritic cells or monocytes which are precursors thereof is desired in order to more effectively produce antigen-presenting dendritic cells.

The inventor has previously reported that transduction of cMYC with BMI1 using a lentivirus can induce the proliferation of ^{CD14 + monocytes.} This result was the first to establish a method for amplifying human monocytes, and the present inventor named the monocyte-derived proliferating cells CD14-ML (Patent Document 1, Non-Patent Document 4). The grown CD14-ML differentiated into functional dendritic cells (CD14-ML-DCs) by the addition of IL-4 to the medium. One drawback of this method is that proliferation induction is highly donor-dependent and CD14 ⁺ monocyte proliferation has been reportedly unsuccessful in 3 of 12 healthy blood donors (about 30%).

WO2012 / 043651

Su, Z., et al. 2003 Cancer Res. 63: 2127-2133. Kyte, J.A., et al. 2006 Cancer Gene Ther. 13: 905-918. Sundarasetty, B.S., et al. 2015 Gene Ther. Doi: 10.1038 / gt.2015.43. Haruta, M., et. Al. 2013. Hum. Immunol. 74: 1400-1408.

An object of the present invention is to provide an improved method of the conventional method reported by the present inventors. More specifically, it is to provide a more efficient and improved method for inducing the proliferation of monocytes derived from peripheral blood. Such a method is, for example, a method for inducing proliferation of monocytes derived from peripheral blood with improved donor dependence.
An object of the present invention is also to provide dendritic cells capable of presenting antigens from proliferative monocytes, and further to provide a drug containing such dendritic cells.

As a result of trying to improve the efficiency and improvement of the conventional method of the present inventors in order to solve the above-mentioned problems, it is very possible to introduce BCL2 or LYL1 into monocytes together with cMYC and BMI1. The present invention was completed because it was found to be advantageous. Furthermore, the present inventors have also succeeded in producing antigen-presenting dendritic cells using a specific antigenic peptide or using a gene modification technique.

That is, the present invention includes the following aspects.
[1] A method for inducing the proliferation of monocytes derived from human peripheral blood.
A method comprising producing a proliferative monocyte by introducing and expressing at least the following three genes: cMYC, BMI1, and BCL2 or LYL1 gene into monocytes.
[2] The method for inducing proliferation of monocytes according to the above [1], wherein the monocytes derived from peripheral blood are fresh monocytes that have not been cryopreserved.
[3] The method for inducing proliferation of monocytes according to the above [1] or [2], wherein the monocytes are monocytes derived from a cancer patient.
[4] A method for producing dendritic cells from monocytes derived from human peripheral blood, wherein the following steps:
(A) The step of producing a monocyte having a proliferative ability by the method for inducing the proliferation of monocytes according to any one of the above [1] to [3], and (b) the monocyte having the proliferative ability (b). The process of inducing differentiation into dendritic cells by culturing (with differentiation-inducing factors),
Method for producing dendritic cells including.
[5] The dendritic cell preparation according to the above [4], which further comprises loading the dendritic cells prepared in the step (b) with an arbitrary antigen to prepare an antigen-presenting dendritic cell. Method.
[6] The method for producing a dendritic cell according to the above [4], further comprising introducing a gene of an arbitrary antigen into the dendritic cell produced in the step (b) to produce an antigen-presenting dendritic cell. ..
[7] A medicine containing monocytes having a proliferative ability, which is produced by the method for inducing the proliferation of monocytes according to any one of the above [1] to [3].
[8] A drug containing dendritic cells prepared by the method according to any one of the above [4] to [6].
[9] The drug according to the above [8], wherein the drug is a drug for use for the treatment or prevention of cancer.
[10] By co-culturing the antigen-presenting dendritic cells prepared in [5] or [6] with CD4 ⁺ T cells or CD8 ⁺ T cells derived from peripheral blood, the antigen-specific CD4 ⁺ T cells are obtained. A method of establishing a line or CD8 ^{+ T cell line.}

[11] A monocyte derived from human peripheral blood having a proliferative ability.
At least the following three exogeneous genes:
cMYC, BMI1, and monocytes having the BCL2 or LYL1 genes and forcibly expressing them.
[12] The monocyte according to the above [11], wherein the monocyte derived from peripheral blood is a fresh monocyte that has not been cryopreserved.
[13] The monocyte according to the above [11] or [12], wherein the monocyte is a monocyte derived from a cancer patient.
[14] A dendritic cell induced to differentiate from the monocyte according to any one of the above [11] to [13].
[15] The dendritic cell according to the above [14], which is loaded with an arbitrary antigen and presents the antigen.
[16] The dendritic cell according to the above [14], which has a gene of an arbitrary antigen and presents the antigen by forcibly expressing the gene.
[17] The medicine containing monocytes according to any one of the above [11] to [13].
[18] The medicament containing the dendritic cell according to any one of the above [14] to [16].
[19] The drug according to the above [18], wherein the drug is a drug for use for the treatment or prevention of cancer.

By using the improved method of the present invention, a large amount of proliferative monocytes (hereinafter, may be referred to as proliferative monocytes) can be stably supplied without physical load on the cell donor. Will be possible. Further, according to the present invention, proliferative monocytes can be obtained from various samples including healthy subjects and cancer patients. In addition, proliferative monocytes established using the methods of the invention (referred to herein as CD14-ML) can differentiate into functionally mature dendritic cells (inducing differentiation of CD14-ML). The prepared dendritic cells are referred to herein as CD14-ML-DC). Therefore, according to the method of the present invention, for example, a sufficient number of monocytes and / or dendritic cells necessary for vaccine treatment can be produced from a small amount of peripheral blood.

Further, some of the proliferative monocytes (CD-14-ML) and dendritic cells (CD-14-ML-DC) produced according to the method of the present invention are not limited to these depending on the purpose. , For example, it can be used as follows. For example, antigen-presenting dendritic cells can be produced by loading an arbitrary antigenic peptide. For example, a cancer antigen-presenting CD14-ML-DC can be prepared by loading a peptide derived from a cancer antigen, and further, when peripheral blood T cells are stimulated in vitro using the CD4 ⁺ cell, the peptide is recognized. Lines and CD8 ⁺ T cell lines can be established. In addition, since CD14-ML proliferates for one month or longer, genetic modification can be easily performed. For example, an antigen-presenting dendritic cell can be prepared by introducing a gene encoding an antigen.
Furthermore, according to the method of some aspects of the present invention, it is possible to produce antigen-presenting dendritic cells without information on T cell epitopes. This means that information about T cell epitopes is no longer needed in vaccine methods using cancer antigen-presenting CD14-ML-DC, and thus anti-anti-therapy using the present invention for all patients regardless of HLA type. Cancer treatment will be useful.

FIG. 1 shows the age, sex, human leukocyte antigen (HLA) serotype, and proliferative CD14 of the donors (10 healthy donors, 2 cancer patient donors) from which the peripheral blood monocytes used in the examples were derived. It is a figure which showed the result of making-ML. "NT" = Not Tested. It is a result of morphological observation of CD14ML and CD14-ML-DC produced by the method of the present invention. A phase-contrast microscope image (A and B, C: left figure) and a May-Grunwald Giemsa stained image (B, C: right figure) are shown. (A): It is a form of CD14-ML produced by the method of the present invention (introduced MYC, BMI1, and LYL1). (B) and (C): A form of CD14-ML produced by the method of the present invention (introduced with MYC, BMI1, and BCL2). (B) Shows the morphology of human monocytes and human monocyte-derived myeloid cell lines (CD14-ML). (C) Shows the morphology of mo-DCs and CD14-ML-DCs stimulated with OK432. It is the result of the production of IL-12p70 by cell surface molecules of CD14-ML-DCs and CD14-ML-DC. (A) It is the result of analyzing the expression of CD40, CD80, CD83, CD86, HLA class I and HLA class II for CD14-ML-DC before and after the treatment with OK432. As a control, the results of analysis of mo-DC (monocyte-derived DC) stimulated with OK432 are shown. The staining profile of a specific mAb (shown by a thick line) and an isotype-matched control mAb (shadow region) is shown. (B) It is a result of measuring the concentration of IL-12p70 in the culture medium supernatant after 60 hours of CD14-ML-DCs and mo-DCs by ELISA. The data are the results of two experiments. It shows T cell stimulation and antigen presentation by CD14-ML-DCs. (A) This is the result of stimulation with CD14-ML (diamond), IL-4 (square) or OK432 (triangle). (B) When the indicated number of CD-14-ML-DCs was loaded with the GAD65 _111-131 peptide (diamond) or not loaded (square), the T cell proliferation reaction was carried out in the last 16 cases. of ^[3 H] in cultures of time - the result of measuring methyl thymidine incorporation. Experiments were performed on CD14-ML from two different donors. (C) This is the result of analyzing the proliferation reaction of T cells when CD14-ML-DCs are pulsed with recombinant GST-fused GAD65 protein or GST protein for 16 hours. Experiments were performed on CD14-ML from two different donors. It is the result of induction of ^{cancer antigen-reactive CD8 +} T cell line by CD14-ML-DCs. (A) The protocol for inducing ^{cancer antigen-specific CD8 +} T cells by CD14-ML-DCs is shown. (B) On day 21, ^{the number of IFN-γ producing CD8 +} T cells was analyzed by the ELISPOT assay (Day 21). Results for T cells prior to stimulation culture are also shown (Day 0). (C) On the 21st day, T cells were collected and stained with anti-CD8 mAb and HLA-A * 24: 02 / CDCA1 _56-64 or HLA-A * 24: 02 / LY6K _{177-186 tetramer.} is there. ^{The numbers in the figure indicate the percentage of CD8 +} T cells stained with the HLA-peptide complex tetramer (Day 21: right figure). The results of T cells before stimulation culture are also shown (Day 0: left figure). It is the result of induction of ^{cancer antigen-reactive CD8 +} T cell line by CD14-ML-DCs. (A) This is the result of analyzing the number of IFN-γ producing ^{CD8 + T cells by the ELISPOT assay.} (B) Collect T cells, anti-CD8 mAb, and HLA-A * 02: 01 / MART1 _26-35 detremer, HLA-A * 02: 01 / CDCA1 _351-359 tetramer, or HLA-A * 02: 01 / IMP3 _{515-523 This} is the result of staining with a tetramer. ^{The numbers in the figure indicate the percentage of CD8 +} T cells stained with the tetramer or tetramer of the HLA-peptide complex (right figure). The results of T cells before stimulation culture are also shown (Day 0: left figure). The result is the induction of cancer antigen-reactive CD4 ^{+ T cell lines by CD14-ML-DCs.} (A) The protocol for inducing ^{cancer antigen-specific CD4 +} T cells by CD14-ML-DCs is shown. (B, C) After stimulation (more than 3 times), ^{the number of CD4 +} T cells reacting with each peptide was analyzed by the IFN-γELISPOT assay (Day 28). The results of T cells before stimulation culture are also shown (Day 0). Dimethyl sulfoxide was used as a control. The results of healthy donor 3 (B) and donor 2 (C) are shown. It is the result of induction of ^{cancer antigen-reactive CD8 +} T cell line by CD14-ML-DCs obtained from HNC patients. (A, C), day 21, ^{the number of CD8 +} T cells reacting with the peptide was analyzed by the IFN-γ ELISPOT assay. The HIV peptide was used as the control peptide. _{(B, D) On day 21, T cells were harvested} , anti-CD8 mAb, and HLA-A * 24: 02 / CDCA1 56-64, HLA-A * 24: 02 / KIF20A _66-75 or HLA-A. * 24: 02 / LY6K1 _{77-186 This} is the result of staining with a tetramer. The numbers in the figure indicate the percentage of ^{CD8 +} cells stained with a tetramer of the HLA-peptide complex. The results of cancer patient 1 (A, B) and cancer patient 2 (C, D) are shown. It is the result of induction of ^{cancer antigen-reactive CD4 +} T cell line by CD14-ML-DCs obtained from HNC patients. ^{The results of analyzing the number of CD4 +} T cells that reacted with each peptide after stimulation (more than 3 times) by the ELISPOT assay are shown. The results of cancer patient 1 (A) and cancer patient 2 (B) are shown. This is the result of induction of ^{cancer antigen-reactive CD8 +} T cell lines by CD14-ML-DCs expressing antigenic proteins. (A) Lentivirus constructs of CMVpp65 (EF-CMV-IP) and MART1 (EF-MART1-IP) are shown. (B) It is a result of analyzing the expression of CMVpp65 by CD14-ML / CMV by flow cytometry. The staining profile of the control mAb (shadow region) whose isotype matches the specific mAb (indicated by the thick line) is shown. (C) On day 9, the results of analyzing the number of T cells reactive with the _{CMVpp65 341-349 peptide by the ELISPOT assay are shown.} The HIV peptide was used as the control peptide. The results of T cells before stimulation culture are also shown (Day 0). (D) On day 9, T cells were collected and stained with anti-CD8 mAb and HLA-A * 24: 02 / CMVpp65 _341-349 complex tetramer. ^{The numbers in the figure indicate the percentage of CD8 +} T cells stained with the HLA-peptide complex tetramer (Day 9: right figure). The results of T cells before stimulation culture are also shown (Day 0: left figure). (E) It is the result of analyzing the number of IFN-γ producing ^{CD8 +} T cells on the 9th day by the ELISPOT assay. The results of T cells before stimulation culture are also shown (Day 0). This is the result of preparation of CD14-ML-DCs presented by MART1 / Melan-A. (A) This is the result of flow cytometric analysis of the expression of MART1 by CD14-ML / MART1. The staining profile of the control mAb (shadow region) whose isotype is matched with the specific mAb (indicated by the thick line) is shown. (B) HLA-A * 02: 01 CD8 ⁺ T cells obtained from a positive healthy donor were co-cultured with CD14-ML-DC-MART1 derived from the same donor, and on the 21st day, MART1 _26-35 was obtained. It is the result of analyzing the frequency of the reacting CD8 ^{+ T cells by the ELISPOT assay.} The HIV peptide was used as the control peptide. The results of T cells before stimulation culture are also shown (Day 0). (C) The results of staining with anti-CD8 mAb and HLA-A * 02: 01 / MART1 _{26-35 extractramer on the 21st day are shown.} ^{The numbers in the figure indicate the percentage of CD8 +} T cells stained with the extractramer of the HLA-peptide complex (Day 21: right figure). The results of T cells before stimulation culture are also shown (Day 0: left figure). (D) ^{The frequency of CD8 +} T cells reactive to MART1 is the result of analysis by ELISPOT assay using CD14-ML-DC and CD14-ML-DC / MART1 as stimulants. The results of T cells before stimulation culture are also shown (Day 0).

Hereinafter, the present invention will be described together with preferred methods and materials that can be used in the practice of the present invention, using exemplary embodiments as examples.
Unless otherwise specified in the text, all technical terms and scientific terms used in the present specification have the same meanings as generally understood by those skilled in the art to which the present invention belongs. Also, any material and method equivalent to or similar to that described herein can be used similarly in the practice of the present invention.
In addition, all publications and patents cited herein in connection with the invention described herein are described herein, for example, as indicating methods, materials, etc. that can be used in the present invention. It constitutes a part.

(Separation of monocytes from human peripheral blood)
In the present invention, monocytes as a raw material are collected from peripheral blood. Methods for separating and preparing monocytes (cells expressing the CD14 molecule in peripheral blood) in human peripheral blood are known, and these methods can be used in the present invention without limitation. Although not limited to this, for example, the following method can be exemplified.

Collect human peripheral blood. As the anticoagulant, heparin, citric acid or the like is used. The collected blood is diluted with an equal volume of physiological saline, phosphate buffer physiological saline, Hanks buffer solution, or the like. Next, the diluted blood is layered on a Ficoll solution (GE Healthcare) that has been previously dispensed into a centrifuge tube (BD-Falcon 352070 or the like). Then, after centrifuging with a centrifugal force of 500 g for 20 minutes using a centrifuge device, mononuclear cell fractions (including lymphocytes and monocytes) existing near the interface are collected.

Monocytes can be separated from mononuclear cells by the magnetic bead method or the like using the expression of the CD14 molecule as an index. For example, it can be separated by using CD14 microbeads (Milteny 130-050-201) or the like. Alternatively, the mononuclear cell fraction is cultured for about 6 to 16 hours in a cell culture vessel that has been surface-treated for cell culture, and the cells attached to the vessel are collected to collect monospheres or macrophages derived from the monospheres. It is also possible to obtain. Usually, 200,000-500,000 monocytes can be recovered from 10 ml of peripheral blood in a healthy adult.
The peripheral blood-derived monocytes used in the present invention are preferably fresh monocytes that have not been cryopreserved.
In addition, monocytes derived from human peripheral blood are commercially available and can be used.

(Granting proliferative ability to peripheral blood-derived monocytes)
The method of the present invention imparts long-term proliferative ability to monocytes by forcibly expressing at least the following three genes: cMYC gene, BMI1 gene, and BCL2 gene or LYL1 gene in peripheral blood monocytes. It is characterized by that. As a method for forcibly expressing the genes, for example, forcible expression of these genes may be carried out by introducing a foreign gene into monocytes. From the viewpoint of achieving efficient and high-level expression of genes and reliably imparting long-term proliferative ability to monocytes, a method of introducing a foreign gene into monocytes using genetic engineering technology is preferable.
In the present invention, the "monocyte having a proliferative ability" means a monocyte to which a long-term proliferative ability is imparted by forcibly expressing the above gene in a monocyte derived from peripheral blood as described above. The "proliferative monocyte" of the present invention proliferates over a longer period of time as compared with a control monocyte (that is, a peripheral blood-derived monocyte used as a starting material) into which the above gene has not been introduced. For example, it is possible to proliferate for 2 weeks or more, preferably 1 month or more from the time when the gene is forcibly expressed (when the gene is introduced into cells).

Specific examples of the cMYC gene include the human cMYC gene (NM_002467). Specific examples of the BMI1 gene include the human BMI1 gene (NM_005180). Specific examples of the BCL2 gene include the human BCL2 gene (NM_000633). Specific examples of the LYL1 gene include the human LYL1 gene (NM_005583). The NCBI accession number is shown in parentheses.

The cMYC gene, BMI1 gene, BCL2 gene, and LYL1 gene are genes commonly present in mammals including humans, and are derived from any mammal in the present invention (for example, derived from mammals such as humans, mice, rats, and monkeys). ) Gene can be used. Also, for wild-type genes, several (for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3). Mutant genes in which bases are substituted, inserted and / or deleted and having the same function as wild-type genes can also be used. Further, as long as it has a function equal to or higher than that of the wild-type gene, the gene may be artificially modified so that the product of the gene is expressed as a fusion protein with another protein or peptide.

The method for introducing the cMYC gene, BMI1 gene, BCL2 gene, and LYL1 gene into peripheral blood-derived monocytes is not particularly limited as long as these introduced genes can be expressed to impart long-term proliferative ability to the monocytes. .. For example, an expression vector containing a transgene can be used to introduce the gene into monocytes. Further, a plurality of genes may be incorporated into one expression vector and the expression vector may be introduced into monocytes, or an expression vector in which each gene is individually incorporated may be prepared and introduced into monocytes. May be good.

The type of expression vector is not particularly limited, and may be a viral vector or a plasmid vector, but is preferably a viral vector, and particularly preferably a viral vector in which the introduced gene is integrated into a monocytic chromosome. Examples of the viral vector that can be used in the present invention include a retrovirus vector, a lentiviral vector, and an adeno-associated virus vector.

The packaging cells used to prepare the recombinant viral vector include cells capable of supplementing the defective protein of the recombinant viral vector plasmid lacking at least one of the genes encoding the proteins required for virus packaging. If so, any cell can be used. For example, HEK293 cells derived from human kidney and packaging cells based on mouse fibroblast NIH3T3 can be used.

Recombinant viral vector A recombinant viral vector can be produced by introducing a plasmid into packaging cells. The method for introducing the viral vector plasmid into the packaging cells is not particularly limited, and can be carried out by a known gene transfer method such as a calcium phosphate method, a lipofection method or an electroporation method. Furthermore, a solution in which the recombinant virus is concentrated can be recovered from the culture supernatant of the packaging cells into which the plasmid has been introduced by a centrifugation method or a concentration method using a column commercially available for virus purification. ..

By adding a solution containing the recombinant virus prepared as described above to peripheral blood monocytes collected from humans in a culture vessel, the virus is infected and the target gene is introduced. ..

(Proliferation method of monocytes with proliferative ability)
The monocytes having the ability to proliferate in vitro prepared as described above can be cultured using a cell culture medium containing M-CSF. The content of macrophage colony stimulating factor (M-CSF) in the culture medium can be in the range of 25 to 100 ng / ml. Alternatively, it is also possible to cause the monocytes themselves to produce M-CSF by introducing the M-CSF gene into the cells themselves using a lentiviral vector or the like. In this case, it is possible to culture and proliferate using a cell culture medium to which M-CSF has not been added.

(Differentiation from monocytes to dendritic cells)
It is possible to produce dendritic cells from monocytes having the ability to proliferate in vitro produced by the method of the present invention. For example, dendritic cells are prepared by culturing, but not limited to, monocytes having the long-term proliferative capacity of the present invention in the presence of M-CSF, GM-CSF and interleukin 4 (IL-4). can do. The content of M-CSF and GM-CSF in the culture solution is not limited to this, for example, in the range of 50 to 200 ng / ml, and the content of IL-4 is not limited to this, for example, 5 to. It can be in the range of 20 ng / ml. Further, dendritic cell maturation can be induced by culturing in the presence of OK432 (penicillin-sterilized Streptococcus pyrogenes), which is a maturation inducer. The content of OK432 in the culture is not limited to this, but can be, for example, in the range of 5 to 50 μ / ml. In the present invention, the "dendritic cell" has properties similar to those of natural (unmodified) monocyte-derived dendritic cells in terms of morphology, cell surface molecules, and T cell stimulating ability. It is a cell. Such a method for producing dendritic cells and dendritic cells produced by the method are also a part of the present invention.

(Preparation of antigen-presenting dendritic cells from monocytes)
By pulsing an arbitrary substance that can be an antigen, for example, a peptide, into a dendritic cell produced by the method of the present invention, a dendritic cell that presents the antigen can be produced. The antigen can be used without particular limitation as long as it can be presented as an antigen. For example, but not limited to, cancer-specific peptides or proteins can be mentioned. Cancer-specific antigen-presenting dendritic cells prepared by pulsing cancer-specific peptides or proteins are useful as anticancer drugs in cancer vaccine therapy.
The method of pulsing the antigen can be carried out according to a conventional method, and can be carried out by culturing CD14-ML-DC prepared by using the method of the present invention together with an antigen candidate substance. As known methods, for example, Haruta, M., et. Al. 2013. Hum. Immunol. 74: 1400-1408 can be referred to, and these known methods can be modified as appropriate. Examples thereof include, but are not limited to, a method of culturing for 3 to 24 hours in a culture solution in which a peptide or protein serving as an antigen is added to a concentration of 1 to 20 μM.

In addition, dendritic cells expressing a specific protein can be produced by introducing a gene expressing a specific protein into monocytes produced by the method of the present invention and culturing the monocytes. A known gene transfer method can be used for gene transfer, and the gene transfer can be performed using, for example, an expression vector containing the transgene.
The type of expression vector is not particularly limited, and may be a viral vector or a plasmid vector, but a viral vector is preferable. Examples of the viral vector that can be used in the present invention include a retrovirus vector, a lentiviral vector, and an adeno-associated virus vector. When a plasmid vector is used, a method with high introduction efficiency should be used, and introduction by electroporation is preferable.
The method for introducing the viral vector and the method for selecting the cell into which the gene has been introduced can be carried out according to a conventional method, and a known method can be used.

(Establishment of antigen-specific CD4 ⁺ T cell line or CD8 ⁺ T cell line)
^{Furthermore, by co-culturing with CD4 +} T cells or CD8 ⁺ T cells derived from the same donor using the antigen-presenting dendritic cells prepared by the method of the present invention, antigen-specific CD4 ⁺ T cell lines or CD8 ⁺ A T cell line can be established.
Many reports have already been made on the procedure for establishing a CD4 ⁺ T cell line or a CD8 ^{+ T cell line, and these methods can be used.} For example, the method described in Harao M., et al., Int J Cancer 2008; 123: 2616-25 and the method described in Tomita., Et al., 2013 Int J Cancer 134: 352-66 can be mentioned. , These methods can be modified as appropriate and used.

(Cell medicine)
The monocytes produced by the method of the present invention have the same properties as macrophages and the like existing in the living body, and can provide a cell medicine for performing immuno-cell therapy for infectious diseases and malignant tumors. The monospheres of the present invention also provide cell medicines for use in the treatment of diseases caused by the accumulation of specific substances in the body, such as Alzheimer's disease, prion disease, amyloidosis, or certain metabolic diseases. You can also do it. In addition, the monocyte-derived dendritic cells having long-term proliferative ability of the present invention can be used as a cell vaccine for use in the treatment of malignant tumors and infectious diseases. Furthermore, the dendritic cells of the present invention can provide a cell medicine for use in suppressive control of immune response for the purpose of treating autoimmune diseases, rejection reactions associated with organ transplantation, and the like.

In the production of the cell medicine of the present invention, an auxiliary agent other than those described above, for example, for the purpose of stably retaining the monocytes having the long-term proliferative ability of the present invention and the dendritic cells derived from the monocytes. , Medium and the like may be used as appropriate.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.

(Materials and methods)
1. 1. Cell samples and donors This study was approved by the Institutional Review Board of the Graduate School of Medical Education, Kumamoto University. Peripheral blood monocytic cell (PBMC) samples were obtained from healthy donors or head and neck cancer (HNC) patients who gave informed consent. CD14 ⁺ monocytes were purchased from Takara Bio and used. In total, monocytes obtained from 10 healthy donors and 2 cancer patients were used. Information about each donor is shown in FIG.

2. Preparation of recombinant lentivirus The cDNA fragment of human cMYC was obtained by PCR and cloned into the pENTR-TOPO vector (Invitrogen, USA). The cDNAs for BMI1, BCL2 and LYL1 were provided by the RIKEN BioResource Center (Tsukuba, Japan). The respective cDNAs of CMVpp65 and MART1 / MelanA were prepared by gene synthesis (GenScript, USA). These cDNAs were introduced into a lentiviral vector, pCSII-EF or pCSIIEF-IRES-PuroR, using the LR Clonase System (Invitrogen). The pCSII-EF and the plasmids of the lentiviral vector package, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp, were provided by Dr. Miyoshi (RIKEN BioResource Center). The plasmid construct was introduced into 293T cells using Lipofectamine 2000, Invitrogen, and after 3 days, recombinant lentivirus was recovered from the centrifuged (50,000 × g, 2h) culture supernatant.

3. 3. Preparation of Proliferative Myeloid Lineages from Monocytes Monocytes were obtained from healthy donors using Ficoll-Plaque (GE Healthcare UK, UK) by positive selection using anti-human CD14 MicroBeads (Miltenyi Biotec, Germany). It was purified from isolated PBMCs and transduced with a lentivirus in the presence of polybrene (8 ng / ml). Cells were cultured in α-MEM containing 20% FCS, 50 ng / ml M-CSF, and 50 ng / ml GM-CSF. Proliferative cells (CD14-ML) appeared after 4-5 weeks of culture. To induce the differentiation of CD14-ML into DC, cells were cultured for 3 days in the presence of M-CSF (50 ng / ml), GM-CSF (50 ng / ml) and IL-4 (20 ng / ml). did. To induce maturation, CD14-ML-DC was stimulated with penicillin-sterilized Streptococcus pyogenes (10 μg / ml) for 2 days.

4. Flow cytometric analysis
The following FITC or PE conjugated mAbs were purchased from BD Pharmingen (USA), eBioscience (USA), R & D Systems (USA), Abcam (UK) or Miltenyi Biotec (Germany): Anti-HLA Class II (clone TU39, mouse) IgG2a), anti-HLA class I (clone G46-2.6, mouse IgG1), anti-CD80 (clone L307.4, mouse IgG1), anti-CD83 (clone HB15e, mouse IgG1), anti-CD86 (clone FUN-1, mouse) IgG1), anti-CD40 (clone 5C3, mouse IgG1), and anti-CD8 (clone T8, mouse IgG1). Mouse IgG2a (clone G155-178), mouse IgG2b (clone 27-35), mouse IgG1 (clone MOPC-21), affinity purified mouse IgM (eBioscience), and rat IgG2a (clone eBR2a) were used as isotype-matched controls. used. Cell samples were treated with Fc Receptor Blocking Reagent (Miltenyi Biotec) for 10 minutes, stained with fluorescent dye conjugate mAb for 30 minutes, and then washed 3 times with PBS containing 2% FCS. Stained cell samples were analyzed by FACScan (BD Biosciences, USA) flow cytometry.

5. Quantification of IL-12p70 Production To analyze the production of IL-12p70 by mo-DCs and CD14-ML-DCs, cells were cultured in 96-well flat bottom culture plates in the presence of OK432 (10 μg / ml) (1 ×). 10 ⁵ cells / 200 [mu] l culture / well). After 60 hours of culturing, the supernatant was collected and the concentration of IL-12p70 was measured using an ELISA kit (eBioscience).

6. T cell proliferation analysis.
T cells were purified from PBMCs by negative selection using Pan T cell isolation kit II (Miltenyi Biotec) and 200 μl AIM supplemented with 5% decomplemented human plasma in 96-well round-bottom culture plates. Cultured in -V (Life Technologies, USA) for 5 days with a gradual number of X-ray (45 Gy) allogeneic stimulating cells (mo-DC, CD14-ML or CD14-ML-DC). .. [ ³ H] -Methylthymidine (247.9 Gbq / mmol) was added to the culture broth to make 0.037 Mbq / well, and the cells were cultured for 16 hours. [ ³ H] -Methylthymidine uptake was measured with a scintillation counter. A gradual number of CD14-ML-DCs was _{pulsed with glutamate decarboxylase 65 (GAD65) 111-131} peptide (LQDVMNILLQYVVKSFDRSTK (SEQ ID NO: 1), 10 μM) for 3 hours, irradiated with X-rays (45 Gy), and then , 96-well flat-bottomed culture plate, GAD65-specific HLA-DR53-restrained human CD4 ⁺ T cells (3 × 10 ⁴ cells /) in 200 μl AIM-V supplemented with 5% immobilized human plasma for 3 days. Well) was cultured together. Proliferative response of T cells, ^[3 H] - were measured in methyl thymidine incorporation. To investigate the natural processing of protein antigens, CD14-ML-DCs (2 × 10 ⁴ cells / well) were fused with recombinant glutathione S-transferase (GST) in 96-well flat-bottomed culture plates for 16 hours. Pulsed with GAD65 protein or GST protein, X-ray irradiation, and then added to GAD65-specific T cells (3 × 10 ⁴ cells / wells) in 200 μl AIM-V supplemented with 5% human complementary plasma. It was. Proliferative response of T cells, ^[3 H] - were measured in methyl thymidine incorporation.

7. Preparation of cancer antigen-reactive CD8 ⁺ T cell line HLA-A, DRB1 and DPB1 gene typing of blood samples was performed at the HLA Laboratory (Japan) based on the PCR-SSOP (sequence-specific oligonucleotide probe) method. .. Induction of antigen-specific CD8 ⁺ T cells was performed with minor modifications to previous reports (Harao M., et al., Int J Cancer 2008; 123: 2616-25). In summary, CD8 ⁺ T cells were purified from PBMCs by positive selection using magnetic microbeads (Miltenyi Biotec, USA). CD14-ML-DC was used as a stimulating cell ^{to induce antigen-specific CD8 +} T cell lines. CDCA1 _351-359 (KLATAQFKI) (SEQ ID NO: 2), KIF20A _809-817 (CIAEQYHTV) (SEQ ID NO: 3), MART1 _26-35 (EAAGIGILTV) (SEQ ID NO: 4) and IMP3 _515-523 (NLSSAEVVV) (SEQ ID NO: 5) ) Was identified as an HLA-A * 02: 01 restrictive epitope. CDCA1 _56-64 (VYGIRLEEHF) (SEQ ID NO: 6), KIF20A _66-75 (KVYLRVRPLL) (SEQ ID NO: 7), LY6K _177-186 (RYCNLEGPPI) (SEQ ID NO: 8) and IMP-3 _508-516 (KTVNELQNL) (SEQ ID NO: 7) Number 9) was identified as an HLA-A * 24: 02 restrictive epitope. T cells - the beginning of the stimulated cultures (day 0), the pretreated CD14-ML-DCs (1 × 10 5 / well) with OK432, 3 hours pulsed with these 9-mer or 10-mer peptides 10μg , X-ray irradiation (45 Gy), then on a 24-well culture plate, 2 ml of AIM supplemented with 5% immobilized human plasma and 10 ng / ml recombinant human IL-7 (rhIL-7). in -V, were both cultured CD8 ⁺ T cells (2 × 10 ⁶ / well). On days 7 and 14, half of the medium was removed from each culture and preloaded with peptide (10 μg / ml) and 10 ng / ml rhIL-7, irradiated (45 Gy) CD14-ML-. Fresh medium (1 ml / well) containing DCs (1 × 10 ^{4 / well not stimulated with OK432) was added.} On day 9, rhIL-2 was added to each well at 20 IU / ml. At the end of the culture, stimulated CD8 ⁺ T cells were analyzed for specificity by IFN-γELISPOT analysis and PE-labeled HLA-A * 02: 01 / CDCA1 _351-359 , HLA-A * 02: 01 / IMP3. _515-523 , HLA-A * 24: 02 / CDCA1 _56-64 or HLA-A * 24: 02 / LY6K _{177-186 Tetramer} (MBL, Japan) of peptide complex, or PE-labeled HLA-A * 24: 01 / MART1 _26-35 peptide complex stained with dexramer (Immunodex, Denmark) and analyzed by flow cytometry in combination with FITC-labeled anti-human CD8 mAb (clon T8; Beckman Coulter, USA).

8. Preparation of antigen-reactive CD4 ⁺ T cell lines Antigen-specific CD4 ⁺ T cell induction was performed with some modifications to the method described in Tomitay., Et al., 2013 Int J Cancer 134: 352-66. It was. CD4 ⁺ T cells were purified from PBMCs obtained by positive selection using magnetic microbeads (Miltenyi Biotec, USA). CD14-ML-DCs were used as APCs to induce ^{antigen-specific CD4 + T cells.} CDCA1 _39-64 (NPKPEVLHMIYMRALQIVYGIRLEHF) (SEQ ID NO: _{10), CDCA1 55-78 (IVYGIRLEHFYMMPVNSEVMYPHL)} ( SEQ ID NO: _{11), KIF20A 60-84 (DSMEKVKVYLRVRPLLPSELERQED)} ( SEQ ID NO: _{12), KIF20A 809-833 (CIAEQYHTVLKLQGQVSAKKRLGTN)} ( SEQ ID NO: 13 ), LY6K _119-142 (KWTEPYCVIAAAVKIFPRPFFMVAKQ) (SEQ ID NO: 14) and LY6K _172-191 (KCCKIRYCNLEGP PINSSVF) (SEQ ID NO: 15) have been identified as epitopes of ^{human CD4 + T cells in previous studies.} On day 0, CD14-ML-DCs (1 × 10 ⁴ cells / well) pretreated with OK432 were pulsed with a mixture of peptides (10 μg / ml each) for 3 hours and irradiated with X-rays (45 Gy). ^{They were then co-cultured with CD4 +} T cells (3 × 10 ⁴ cells / well) in 200 μl AIM-V supplemented with 5% immobilized human plasma on a 96-well flat bottom culture plate. On day 7, half of the medium was removed and stimulated with irradiated (45 Gy) irradiated CD14-ML-DCs (OK432) preloaded with peptides (10 μg / ml) and rhIL-7 (5 ng / ml). No fresh medium (100 μl / well) containing 1 × 10 ^{4 cells / well) was added.} On day 9, rhIL-2 was added to each well (10 IU / ml). ^{On day 14, CD4 +} T cells in each well were analyzed for peptide-specific reactivity based on IFN-γ ELISPOT analysis. T cells show a specific response to cognate peptide, were transferred to 24-well plates, (not stimulated with OK432, 1 × 10 ⁵ cells / well) CD14-ML-DCs that receive a radiation-loaded with peptides Restimulated every other week. Then, a medium supplemented with rhIL-2 (20 IU / ml) and rhIL-7 (5 ng / ml) was added. On day 28, stimulated CD4 ⁺ T cells were collected and washed and the peptide-specific production of IFN-γ was analyzed by ELISPOT analysis. To determine the binding HLA molecule, anti-HLA-DP mAb (B7 / 21, Abcam), anti-HLA-DQ mAb (SPV-L3, Abcam) or anti-HLA-DR mAb (L243, BioLegend), ELISPOPT plate It was added to the stimulating cells inside (PBMCs from the same donor) (all mAbs were concentrated at 5 μg / mL). After 1 hour incubation, peptides and T cells were added. The number of spots was measured after 16 hours of co-culture.

9. Preparation of antigenic protein-presenting CD14-ML-DC CD14-ML was transformed with CMVpp65 or a lentiviral vector encoding MART1 / MelanA in the presence of polybrene (8 ng / ml). Cells were cultured in α-MEM containing 20% FCS, M-CSF (50 ng / ml) and GM-CSF (50 ng / ml). From 2 days after the introduction of lentivirus, cells were cultured in the presence of puromycin (5 μg / ml) for 2 weeks. Presentation of the antigenic protein in the modified CD14-ML was confirmed by flow cytometric analysis. Then, IL-4 (20 ng / ml) was added to the antigen-presenting CD-14-ML (CD14-ML / CMV or CD14-ML / MART1), and DC (CD14-ML-DC / CMV or CD14-ML-DC /). It was differentiated into MART1).

10. Stimulation of CD8 ⁺ T cells with CD14-ML-DC / CMV CD14-ML-DC / CMV was stimulated with TNF-α (5 ng / ml) for 2 days, irradiated and seeded on a 24-well culture plate. There was (1 × 10 ⁵ cells / well). The same donor from peripheral blood CD8 ⁺ T cells were added to the wells (2 × 10 ⁶ cells / well). Cells were cultured in AIM-V medium containing 5% human immobilized plasma and rhIL-7 (10 ng / ml). rhIL-2 (20 U / ml) was added on day 2. On day 9, cells were collected, combined with FITC-labeled anti-human _CD8mAb , and PE-labeled tetramer (MBL, Japan) of the HLA-A * 24: 02 / CMVpp65 341-349 (QYDPVAALF) (SEQ ID NO: 16) complex. ), And analyzed by flow cytometry. Pre-pulse ^{CD8 +} T cells (1 × 10 ⁴ cells / well) with HLA-A * 24: 02 binding CMV pp65 _341-349 peptide to analyze IFN-γ production by CMV-specific T cells. The cells were co-cultured with CD14-ML / CMV or C1R-A24 (HLA-A * 24: 02 presented C1R cells (1 × 10 ⁴ cells / well). 16 hours later, the culture was terminated and IFN-γ-producing cells were used. , ELISPOT analysis was used for counting.

11. Stimulation of CD8 ⁺ T cells with CD14-ML-DC / MART1 CD14-ML-DC / MART1 was stimulated with OK432 and irradiated, followed by a 24-well culture plate with 5% human immobilized plasma. In 2 mL of AIM-V supplemented with rhIL-7 (10 ng / ml), co-cultured with ^{CD8 +} T cells (2 × 10 ^{6 cells / well) derived from the same donor.} CD14-ML-DC-MART1 and rhIL-7 were added on days 7 and 14, and rhIL-2 (20 U / ml) was added on days 9 and 16. On day 21, CD8 ⁺ T cells were collected, combined with FITC-labeled anti-human CD8 mAbs, stained with PE-labeled extruder of HLA-A * 02: 01 / MART1 peptide complex, and then flow cytometry. Was analyzed by. To analyze IFN-γ production by ELISPOT analysis, CD8 ⁺ T cells (1 × 10 ⁴ cells / well) were _{pre-pulsed with MART1 26-35} peptide CD14-ML-DC-MART1 or HLA from the same donor. -A * 02: 01 co-cultured with positive T2 cells (1 × 10 ⁴ cells / well) for 16 hours.

(Example 1)
Improved production method of CD14-ML The inventors have previously ^{reported that the proliferation of CD14 +} monocytes can be induced by introducing cMYC and BMI1 by lentivirus, and produced CD14-ML. Nevertheless, the growth efficiency varied among blood donors, and the growth of monocytes from 3 of the 12 blood donors could not be induced. Therefore, we tried to improve the production efficiency of CD14-ML. For this purpose, in addition to the two genetic factors, other factors were studied. Factors that may aid or promote cell proliferation include, for example, BCL2, LYL1, BCL-XL, MLLT1, MEIS1, HOXXA9, E2F2, E4F1, PHC1, PBX1, CDT1, MLF1, Cyclin D2, MYB, SOX2, CBX5. AURKB and BUB1 were tried. As a result, the introduction of BCL2 or LYL1 in addition to the two previously reported factors cMYC and BMI1 significantly improved the growth induction efficiency.

Cell proliferation was observed 3-5 weeks after the introduction of cMYC, BMI1 and BCL2. Monocyte samples from all donors showed marked proliferation. As the monocytes, newly prepared monocytes were used instead of freeze-thawed monocytes. The samples used in this example include samples from donors whose growth could not be induced by the introduction of cMYC and BMI1 alone in previous reports. Although the duration, rate, and degree of growth still varied among donors, the methods of the invention increased cell numbers by at least 100-fold. The results are shown in FIG. Similarly, the number of cells increased significantly when cMYC, BMI1, and LYL1 were used.
Proliferation of CD14-ML by the method of the present invention (introducing cMYC, BMI1, and BCL2 or LYL1) is similar to CD-14-ML produced by the previous method (introducing cMYC and BMI1). And GM-CSF were both present. The result of morphological observation of the CD14-ML produced by the method of the present invention is shown in FIG. As shown in FIG. 2B, CD14-ML is larger than monocytes and has round nuclei instead of monocyte-like irregular nuclei.

(Example 2)
Differentiation of CD14-ML produced by the modified method (introducing cMYC, BMI1, and BCL2) into DC IL-4 was added to differentiate CD14-ML into DCs. After 3 days, OK432 (penicillin-sterilized Streptococcus pyrogenes), a potent maturation inducer of human monocyte-derived direct current (mo-DC), was added. mo-DC and CD14-ML-DC were stimulated with OK432 for 2 days and then microscopically analyzed. The results are shown in FIG. 2C. The data are the results of two experiments. Like the CD14-ML produced by the previous method, the CD14-ML produced by the method of the present invention also differentiated into CD14-ML-DC with typical DC-like morphology and protrusions ( FIG. 2C).

Next, the production of cell surface molecules of CD14-ML-DCs and IL-12p70 by CD14-ML-DC was confirmed. The expression of CD40, CD80, CD83, CD86, HLA class I and HLA class II was analyzed for CD14-ML-DC before and after treatment with OK432. As a control, mo-DC (monocyte-derived DC) stimulated with OK432 was used. The results are shown in FIG. 3A. Expression of CD40, CD80, CD83, CD86, HLA class I and HLA class II was detected on CD14-ML-DC, and their expression was amplified by stimulation with OK432. The expression levels of these molecules were almost the same as those of mo-DC.
CD14-ML-DCs and mo-DCs were cultured in 96-well flat bottom culture plates (1 × 10 ⁵ cells / 200 μl medium / well) in the presence of OK432 (10 μg / ml). After 60 hours, the concentration of IL-12p70 in the culture medium supernatant was measured by ELISA. The results are shown in FIG. 3B. The data are the results of two experiments. Upon stimulation with OK432, CD14-ML-DC produced more IL-12p70 than mo-DC. Overall, the CD14-ML produced by the method of the invention differentiated into CD14-ML-DC with a fully mature DC phenotype.

(Practical example 3)
T cell stimulation and antigen presentation by CD14-ML-DC Using a primary allogeneic T cell stimulation assay, the ability of CD14-ML-DC to stimulate native T cells was confirmed. Peripheral blood T cells (4 ×) derived from the same donor were irradiated with CD14-ML-DCs or mo-DC (circle) stimulated with CD14-ML (diamond), IL-4 (square) or OK432 (triangle). 10 were co-cultured with ¹⁰⁴ cells / well). Alternatively, only CD14-ML-DCs (circles) were irradiated and cultured. Culturing was carried out on a 96-well round bottom culture plate for 5 days. T cell proliferation, ^[3 H] in culture for the last 16 hours - was measured by methyl thymidine incorporation. Experiments were performed on CD14-ML from two different donors. The results are shown in FIG. 4A. Compared to CD14-ML and mo-DC, CD14-ML-DC induced the proliferation of allogeneic T cells more prominently. Treatment with OK432 further enhanced the T cell stimulating ability of CD14-ML-DC.

Next, it was confirmed whether CD14-ML-DCs could present antigenic peptides in relation to HLA class II. The inventors have previously established clones of _{GAD65 111-131} specific DR53 (DRB4 * 01: 03) restrictive human CD4 ^{+ T cells as responders.} CD14-ML-DCs from DR53 positive donors were pulsed with GAD65 peptide and cultured with their T cell clones. The number of CD-14-ML-DCs shown in the figure _{was loaded with or without the GAD65 111-131} peptide (diamond) (square) and X-rayed, followed by GAD65-specific HLA-DR53. It was co-cultured with a restrained clone of human CD4 ⁺ T cells (3 × 10 ⁴ cells / well) for 3 days. Proliferative response of T cells, ^[3 H] in culture for the last 16 hours - measured in methyl thymidine incorporation. Experiments were performed on CD14-ML from two different donors. The results are shown in FIG. 4B. T cells exhibit a specific proliferative response, indicating that CD14-ML-DCs can present antigenic peptides in relation to HLA class II and stimulate T cells.

Next, the processing ability of the protein antigen was examined. CD14-ML-DC was cultured (pulsed) for 16 hours in the presence of recombinant GST-fused GAD65 protein or GST protein, irradiated with X-rays, and then GAD65-specific T cells were added. The T cell proliferation response was analyzed. Experiments were performed on CD14-ML from two different donors. The results are shown in FIG. 4C. GAD65-specific T cells proliferate in a dose-dependent manner, indicating that CD14-ML-DC can process protein antigens and present antigenic peptides.

(Example 4)
^{Induction of antigen-specific CD8 +} T cells from healthy donors by CD14-ML-DCs.
^{Cancer antigen-specific CD8 +} T cells were primed by in vitro stimulation with CD14-ML-DC loaded with an antigenic peptide according to the culture protocol shown in FIG. 5A. IL-4 was added to CD14-ML to make CD14-ML-DCs. After 3 days, OK432 was added. CD14-ML-DCs were pulsed with peptides for 3 hours, irradiated with X-rays (45 Gy), and then mixed with ^{CD8 + T cells from the same donor.} Cells were cultured with rIL-7 (10 ng / ml) in AIM-V containing 5% immobilized human plasma. On days 7 and 14, it was stimulated with peptide-pulsed CD14-ML-DCs from the same donor, and on days 9 and 16, rIL-2 (20 IU / ml) was added. CD14-ML-DCs were prepared each time and only IL-4 was added (OK432 was not added). IFN-γ ELISPOT analysis and flow cytometric analysis were performed on the 6th or 7th day after repeating the peptide stimulation for 3 cycles.

HLA-A * 02: 01 or HLA-A * 24: 02 Peripheral blood CD8 ⁺ T cells derived from positive healthy donors, 4 types of already identified cancer antigen-derived peptides (HLA-A * 24: 02) CDCA1 _56-64 , KIF20A _66-75 , LY6K _177-186 and IMP-3 _{508-516 ; CDCA1 351-359} , KIF20A _809-817 , MART1 _26-35 and IMP3 _515-523 for HLA-A * 02: 01. ) Was preloaded with CD14-ML-DCs from the same donor. T cell stimulation with peptide-loaded DCs was repeated once a week for 3 weeks. The HIV peptide was used as the control peptide. On day 21, T cells were collected and their reactivity to individual peptides was analyzed by ELISPOT analysis using HLA-A * 24: 02 positive CIR cells or HLA-A * 02: 01 positive T2 cells as stimulants. .. The results are shown in FIGS. 5B and C.
_{T cells reactive to CDCA1 56-64} and LY6K _177-186 were induced from T cells from healthy donors (one HLA-A * 24: 02 positive) by induction culture for 3 weeks (Fig. 5B, FIG. C). HLA-A * 24: 02 / peptide complex staining with tetramer confirmed amplification of specific T cells during 3 weeks of stimulation culture.

Similarly, experiments were performed on cells obtained from HLA-A * 02: 01 positive donor (donor 2). Four peptides (CDCA1 _351-359 , KIF20A _809-817 , MART1 _26-35 , and IMP3 _515-523 ) were used for T cell stimulation. The results are shown in FIG. Similar to the above _{, T cells responsive to CDCA1 351-359} , MART1 _26-35 , and IMP3 _515-523 ^{are from peripheral blood CD8 +} T cells of healthy donor 2 positive for HLA-A * 02: 01. I was induced.
These results, CD14-ML-DCs loaded with HLA class I restricted peptides can stimulate specific CD8 ⁺ T cells in CD8 ⁺ T cell population from the same donor, in the same manner as mo-DC , Suggesting that their amplification can be induced.

(Example 5)
Stimulation of cancer antigen-specific CD4 ⁺ T cells with CD14-ML-DCs Next, ^{the ability of CD14-ML-DC to induce antigen-specific CD4 +} T cells was examined. FIG. 7A shows the culture schedule. HLA-DR 6 type that has already been identified as a restricted epitopes of cancer antigen-derived peptides _{(CDCA1 39-64, CDCA1 55-78, KIF20A} 60-84, KIF20A 809-833, LY6K 119-142 and LY6K _172-191 ) Was used. IL-4 was added to CD14-ML to make CD14-ML-DCs. After 3 days, OK432 was added. The CD14-ML-DCs, 6 one peptide for 3 hours pulsed with a mixture of _{(CDCA1 39-64, CDCA1 55-78, KIF20A} 60-84, KIF20A 809-833, LY6K 119-142, and LY6K _172-191), X-ray irradiation (45 Gy) was then mixed with ^{CD4 +} T cells from the same donor in AIM-V containing 5% immobilized human plasma. On day 7, rIL-7 (5 ng / ml) was added, stimulated with peptide-pulsed CD14-ML-DCs from the same donor. Two days later, rIL-2 (10 IU / ml) was added to the medium. For CD14-ML-DCs, only IL-4 was added (OK432 was not added). On day 14, ^{the properties of stimulated CD4 +} cells in each well were analyzed by the IFN-γELISPOT assay. T cells exhibiting specific reactivity to the cognate peptide were transferred to a 24-well plate and restimulated with peptide-pulsed CD14-ML-DCs from the same donor, followed by rIL-7 (5 ng / ml) and rIL. -2 (20 IU / ml) was added. On day 21, peptide-pulsed CD14-ML-DCs from the same donor were restimulated and rIL-7 and rIL-2 were added. IFN-γ ELISPOT analysis was performed on the 6th or 7th day after repeating the peptide stimulation for 4 cycles.

The results are shown in FIGS. 7B and C. FIG. 7B shows the results of reacting ^{CD4 +} T cell lines obtained from healthy donor 3 _{with CDCA1 39-64} , CDCA1 _55-78 , and LY6K _172-191 in an HLA-DR-dependent manner. , The same result as that of the healthy donor 2 (Fig. 7C) was obtained. These results suggest that peptide-loaded CD14-ML-DCs were able to induce ^{antigen-specific CD4 + T cells in in vitro stimulated culture.}

(Example 6)
T cell stimulation with CD14-ML-DCs established from cancer patients Monocytes obtained from cancer patients are often less likely to survive than monocytes from healthy donors. In such cases, as reported, the production of dendritic cells from monocytes is very difficult. Therefore, it is very useful to be able to establish CD14-ML not only from healthy donors but also from cancer patients. Therefore, we attempted to establish CD14ML from monocytes obtained from HNC patients. The ability of CD14-ML-DCs ^{to induce antigen-specific CD8 +} T cell lines was tested.
^{Peripheral blood CD8 +} T cells obtained from HLA-A * 24: 02 positive HNC patients _{were subjected to} peptide mixture (CDCA1 56-64, KIF20A _66-75 , LY6K) on a schedule similar to that shown in FIG. 5A. _177-186 and IMP-3 _508-516 ) were co-cultured with preloaded CD14-ML-DCs from the same donor to induce T cells reactive to those peptides. On days 7 and 14, CD8 ⁺ T cells were restimulated with peptide-loaded CD14-ML-DCs. ^{On day 21, the number of CD8 +} T cells responding to the peptide was analyzed by the IFN-γ ELISPOT assay. The HIV peptide was used as the control peptide. The results are shown in FIG. 8A (cancer patient 1) and FIG. 8B (cancer patient 2).
_{On day 21, T cells were harvested} and anti-CD8 mAb and HLA-A * 24: 02 / CDCA1 56-64, HLA-A * 24: 02 / KIF20A _66-75 or HLA-A * 24: 02 / It was stained with _{LY6K1 77-186 tetramer.} The results are shown in FIG. 8B (cancer patient 1) and FIG. 8D (cancer patient 2). The numbers in the figure indicate the percentage of ^{CD8 +} T cells stained with the HLA-peptide complex tetramer.
_{Reactivity of} CD8 ⁺ T cells to CDCA1 56-64, LY6K _177-186 , and KIF20A _66-75 was observed in cancer patients. As a result, we succeeded in establishing CD14ML from monocytes obtained from two patients (cancer patients 1 and 2). The obtained CD14ML could be differentiated into CD14-ML-DC by stimulation with IL-4.

Similarly, we attempted to induce ^{cancer antigen-specific CD4 +} T cell lines using CD14-ML-DCs derived from cancer patients.
The CD4 ⁺ T cells isolated from PBMCs of HNC patients, six of the peptides _{(CDCA1 39-64, CDCA1 55-78, KIF20A} 60-84, KIF20A 809-833, LY6K 119-142, and LY6K _172-191 ) Stimulated with pulsed CD14-ML-DCs. After stimulation for more than 3 cycles, ^{the number of CD4 +} T cells that responded to each peptide was analyzed by ELISPOT assay. Since only small amounts of blood samples are available from cancer patients, CD14-ML-DCs were used as stimulants in the assay. The results are shown in FIG. ^{CD4 +} T cell lines from cancer patient 1 collected from the induction culture were HLA-DQ or HLA-DR restrictive _{and reacted with the CDCA1 55-78} and LY6K _172-191 peptides (FIG. 9A). ^{CD4 +} T cell lines from cancer patient 2 collected from induction cultures were HLA-DQ or HLA-DR restrictive _{and reacted with CDCA1 39-764} , CDCA1 _55-78 , and LY6K _172-191 peptides (Figure). 9B).
From the above, the production of CD14-ML-DCs from monocytes of cancer patients was feasible. And CD14-ML-DCs derived from cancer patients had sufficient ability to induce an antigen-specific T cell reaction.

(Example 7)
Preparation of CMVpp65-presented CD14ML-DC by gene recombination of CD14-ML Since the antigen-specific CD4 ⁺ or CD8 ⁺ T cell line could be established by in vitro stimulation using CD14-ML-DCs preloaded with synthetic peptides, the following In addition, we attempted to produce genetically modified CD14-ML-DCs that present antigenic proteins by utilizing the proliferative capacity of CD14-ML.
FIG. 10A shows the lentivirus constructs of CMVpp65 (EF-CMV-IP) and MART1 (EF-MART1-IP). HLA-A * 24: 02 CD14-ML from a positive donor was transformed with a CMVpp65 lentiviral vector with an IRES (internal ribosome entry site) -puromycin acetyltransferase cassette. The cells were cultured in the presence of puromycin (5 μg / ml) for 2 weeks to select and proliferate a cell population carrying the transgene to produce CD14-ML / CMV and CD14-ML / MART1. Then, the CMVpp65 expression of the obtained CD14-ML-DCs (CD14-ML-DC / CMV) was confirmed by flow cytometric analysis (FIG. 10B).

IL-4 was added to the CD14-ML culture and the HLA-A * 24: 02 obtained as a stimulant was derived from a positive healthy donor using CD14-ML-DC / CMV from the same donor. CMV-specific CD8 ⁺ T cell lines were induced from peripheral blood CD8 ^{+ T cells.} To examine CMV-specific reactions, IFN-γ-producing cells were examined after co-culturing overnight with C1R cells pulsed with an HLA-A * 24: 02 restrictive CMVpp65-derived epitope (CMVpp65 _341-349). On day 9, the number of T cells reactive with the _{CMVpp65 341-349 peptide was analyzed by the ELISPOT assay.} The results shown in FIG. 10C indicate _{successful induction of CMVpp65 341-349} reactive CD8 ⁺ T cell lines.

In addition, on day 9, T cells were collected and stained with anti-CD8 mAb and HLA-A * 24: 02 / CMVpp65 _341-349 complex tetramer. As shown in FIG. 10D, CMVpp65 _341-349 reactive CD8 ⁺ T cells were also detected by staining with tetramer.

Next, we attempted to create ^{a CMV-pp65 reactive CD8 +} T cell line from another blood donor (HLA-A * 24: 02 negative healthy donor) homozygous for HLA-A * 26: 01. There was no information on CMV-derived epitopes for this HLA-A allele. Preparation of CMVpp65-presented CD14-ML-DCs ^{and co-culture with CD8 +} T cells derived from the same donor were carried out by the same procedure as described above. In order to detect the reactivity to CMVpp65, the obtained T cells were co-cultured with CD14-ML derived from the same donor in the presence or absence of the transgene of CMVpp65. On day 9, ^{the number of IFN-γ producing CD8 +} T cells was analyzed by ELISPOT assay using CD14-ML and CD14-ML / CMV as stimulants. A reaction to the pp65 protein was observed in this analysis, as shown in FIG. 10E. T cells stimulated with CD14-ML-DC / CMVpp65 were further activated and compared to T cells before stimulation culture. This reaction was blocked by the addition of anti-HLA class I blocking antibody, indicating that the reaction was restricted by HLA class I. Although the epitope of this donor has not been identified, it has been demonstrated that antigen-specific CD8 ⁺ T cell stimulation is possible regardless of the availability of information on the epitope.

(Example 8)
Preparation of MART1 / Melan-A Presented CD14-ML-DCs Next, MART1 / Melan-A presented CD14-ML-DC was prepared by the same method as described above. CD14-ML-DC / MART1 was made from two blood donors. One (healthy donor 2) was positive for HLA-A * 02: 01, and the other (healthy donor 4) was negative for HLA-A * 02: 01. Expression of the MART1 protein in the resulting CD14-ML-DCs (CD14-ML-DC / MART1) was confirmed by staining with specific antibodies followed by flow cytometric analysis. The results are shown in FIG. 11A.

CD14-ML-DC-MART1 was co-cultured with ^{peripheral blood CD8 + T cells from the same donor.} CD8 ⁺ T cells were stimulated with CD14-ML-DC / MART1 once a week and cultured for 3 weeks. On day 21, the reactivity of cultured T cells to MART1 was analyzed. Cultured T cells from HLA-A * 02: 01 positive donors were co-cultured with HLA-A02 *: 01-presented T2 cells loaded with a _{known HLA-A * 02: 01 restrictive epitope (MART1 26-35).} .. The results shown in FIG. 11B show the induction of T cells that are reactive to the HLA-A * 02: 01 restrictive epitope.

On day 21, T cells were harvested, stained with anti-CD8 mAb and HLA-A * 02: 01 / MART1 _26-35 dextramer, and the proportion of ^{CD8 +} T cells stained with the HLA-peptide complex dextramer. Examined. As the results are shown in FIG. 11C, 38.1% of T cells were stained with the extractramer of _{the HLA-A * 02: 01 / MART1 26-35 complex.}

^{The recovered CD8 +} T cells derived from the HLA-A * 02: 01 negative donor were then co-cultured with CD14ML-DC / MART1 from the same donor or CD14-ML-DCs from the same donor. On day 21, ^{the frequency of CD8 +} T cells responsive to MART1 was analyzed by ELISPOT assay using CD14-ML-DC and CD14-ML-DC / MART1 as stimulants. The results shown in FIG. 11D indicate that the T cells responded to MART1 and that the response was HLA-Class I restrictive.

From the above results, the method of the present invention, which is an improvement of the conventional method, was able to induce the proliferation of monocytes from all healthy people and cancer patients. The CD14-ML prepared by this method proliferated more actively than the CD14-ML prepared by our conventional method. The properties of CD14-ML produced by this method are the conventional methods of the present inventors in terms of morphology, cell surface molecules, dependence on GM-CSF and M-CSF, and ability to differentiate into dendritic cells. It was indistinguishable from the CD14-ML produced by.
It was also found that a genetically modified CD14-ML-DC expressing an antigenic protein can be produced by using the method of the present invention. The antigen-expressing CD14-ML-DC produced by the method of the present invention succeeded in inducing ^{a CD8 +} T cell line responsive to CMVpp65 or MART1 / MelanA by in vitro stimulation of peripheral blood T cells using the antigen-expressing CD14-ML-DC. It was shown that it has the ability to induce antigen-specific T cells. These results suggest application to vaccine therapy.
Then, based on the CD14-ML technique of the present invention, functional DCs expressing antigenic proteins can be easily prepared, and more importantly, information on HLA-binding epitopes can be easily produced using antigen-presenting DCs. Is not available, but can induce antigen-specific T cells.

The above detailed description merely describes the object and object of the present invention, and does not limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein, without departing from the appended claims.

According to the method of the present invention, it is possible to efficiently provide a large amount of peripheral blood monocytes having proliferative ability without depending on a donor. In addition, the monocytes provided by the method of the present invention can differentiate into functional dendritic cells. Monospheres can be used as cell therapies for infectious diseases and malignant tumors, as cell medicines for diseases caused by the accumulation of certain substances in the body, such as Alzheimer's disease, amyloidosis, or certain metabolic diseases. The monospheres provided by the methods of the invention can be used for those applications. Furthermore, dendritic cells are used as cell vaccines against malignant tumors, infectious diseases, etc., and the dendritic cells provided by the method of the present invention can be used for those purposes.

Claims (19)

A method for inducing the proliferation of monocytes derived from human peripheral blood.
A method comprising producing proliferative monocytes by introducing and expressing at least the following three genes: cMYC, BMI1, and BCL2 genes into monocytes isolated from human peripheral blood. A method for inducing the proliferation of monocytes derived from human peripheral blood.
A method comprising producing proliferative monocytes by introducing and expressing at least the following three genes: cMYC, BMI1, and LYL1 genes into monocytes isolated from human peripheral blood. The method for inducing proliferation of monocytes according to claim 1 or 2, wherein the monocytes derived from peripheral blood are fresh monocytes that have not been cryopreserved. The method for inducing monocyte proliferation according to any one of claims 1 to 3, wherein the monocyte is a monocyte derived from a cancer patient. The method for inducing monocytes according to any one of claims 1 to 4, wherein the monocytes isolated from the human peripheral blood are monocytes that cannot acquire proliferative ability when only the cMYC and BMI1 genes are introduced. .. A method for producing dendritic cells from monocytes derived from human peripheral blood, wherein the following steps:
(A) A step of producing a monocyte having a proliferative ability by the method for inducing the proliferation of monocytes according to any one of claims 1 to 5, and (b) culturing the monocyte having the proliferative ability. The process of inducing differentiation into dendritic cells,
Method for producing dendritic cells including. The method for producing a dendritic cell according to claim 6, further comprising loading the dendritic cell produced in the step (b) with an arbitrary antigen to produce an antigen-presenting dendritic cell. The method for producing a dendritic cell according to claim 6, further comprising introducing a gene of an arbitrary antigen into the dendritic cell produced in the step (b) to produce an antigen-presenting dendritic cell. A method for establishing an antigen-specific CD4 + T cell line or CD8 + T cell line by co-culturing the antigen-presenting dendritic cells prepared according to claim 7 or 8 with CD4 + T cells or CD8 + T cells derived from peripheral blood. , The antigen-presenting dendritic cell comprises the exogenous genes , cMYC, BMI1, and BCL2 genes, or cMYC, BMI1, and LYL1 genes. Proliferative human peripheral blood-derived monocytes
At least the following three exogeneous genes:
A monocyte that has the cMYC, BMI1, and BCL2 genes and forcibly expresses them. Proliferative human peripheral blood-derived monocytes
At least the following three exogeneous genes:
A monocyte that has the cMYC, BMI1, and LYL1 genes and forcibly expresses them. The monocyte according to claim 10 or 11, wherein the monocyte derived from peripheral blood is a fresh monocyte that has not been cryopreserved. The monocyte according to any one of claims 10 to 12, wherein the monocyte is a monocyte derived from a cancer patient. The dendritic cell derived from the monocyte according to any one of claims 10 to 13, wherein the dendritic cell is an exogenous gene , cMYC, BMI1, and BCL2 gene, or cMYC, BMI1. , And a dendritic cell having the LYL1 gene. The dendritic cell according to claim 14, which is loaded with an arbitrary antigen and presents the antigen. The dendritic cell according to claim 14, which has a gene of an arbitrary antigen and presents the antigen by forcibly expressing the gene. The medicine containing monocytes according to any one of claims 10 to 13. A medicament containing a dendritic cell according to any one of claims 14 to 16. The medicine according to claim 18, wherein the medicine is a medicine for use for treating or preventing cancer.

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