WO2011021503A1 - Pharmaceutical composition containing transiently surviving ctl - Google Patents
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- WO2011021503A1 WO2011021503A1 PCT/JP2010/063181 JP2010063181W WO2011021503A1 WO 2011021503 A1 WO2011021503 A1 WO 2011021503A1 JP 2010063181 W JP2010063181 W JP 2010063181W WO 2011021503 A1 WO2011021503 A1 WO 2011021503A1
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0636—T lymphocytes
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Definitions
- the present invention relates to a pharmaceutical composition comprising cells derived from human hematopoietic stem cells, and more specifically, a pharmaceutical composition comprising cytotoxic T cells derived from umbilical cord blood, presented by a patient's HLA class I molecule.
- the present invention relates to a pharmaceutical composition capable of recognizing a disease-related antigen, and wherein the hematopoietic stem cell-derived cell does not cause permanent engraftment or acute GVH disease of severity III or IV in the patient.
- Umbilical cord blood hematopoietic stem cell transplantation (Umbilical Cord Blood Hematopoietic Cell Transplantation, hereinafter referred to as “UCBHCT”) is acute myeloid leukemia and other leukemia, aplastic anemia and other hematopoietic disorders, congenital immunodeficiency, congenital This is an effective treatment for children and adults such as metabolic disorders and EBV infection (Non-patent Document 1).
- UCBHCT has become the most popular stem cell transplantation therapy today because the cord blood bank system that is cryopreserved after obtaining histocompatibility test data is widespread in each country.
- UCBHCT In UCBHCT, in general, for HLA-A, HLA-B, and DRB1 loci, 4 to 6 of 6 antigens whose matching type of pediatric patients undergoing transplantation matches that of unrelated cord blood are matched. It is known that transplantation is usually successful when six are included, that is, when the patient's matched type contains 0 to 2 antigens that do not match the matched type of cord blood (Non-patent Documents 2 and 3). The transplanted cells are permanently engrafted in the patient's host body.
- ACT adoptive immunotherapy
- Non-patent Document 4 Non-patent Document 4
- ACT removes lymphocytes by mass-culturing patients 'own lymphocytes that have infiltrated the tumor tissue or patients' lymphocytes sensitized in vitro with a disease-related antigen in the presence of interleukin-2. It is a treatment method that is transplanted to a given patient. For example, in metastatic melanoma, objective tumor regression has been reported in about 50% of cases in which autologous lymphocyte infusion using tumor-infiltrating lymphocytes has been performed.
- UCBHCT accepts one or two mismatched antigens, but donor umbilical cord blood suitable for transplantation may not be found depending on the compatible type of the patient's HLA locus.
- donor umbilical cord blood may not be suitable for transplantation because it corresponds to an incompatible combination of HLA types involved in severe GVH disease and survival.
- GVH disease graft-versus-host disease
- the risk of developing transplanted cell-derived leukemia is higher in UCBHCT than in bone marrow transplantation or peripheral blood stem cell transplantation.
- ACT needs to prepare lymphocytes for transplantation for each patient, and this operation is quite labor intensive and requires specialized skills. Also in ACT, there is a risk of developing leukemia derived from transplanted cells.
- transplant cells for immunotherapy that can target a wider range of patients than conventional transplant cells for immunotherapy and that do not cause severe GVH disease. is there.
- the present invention provides a pharmaceutical composition comprising cells derived from human hematopoietic stem cells.
- any one locus of the human HLA class I molecule of the cell contains at least one antigen whose matched type of the cell matches that of the patient.
- the human HLA class I and II molecular loci of the cells contain at least one antigen whose compatible type of the cells does not match that of the patient, and the cells derived from the hematopoietic stem cells are in the body of the patient.
- neither permanent engraftment nor acute GVH disease of severity III or IV occurs.
- the pharmaceutical composition of the present invention is administered after the patient's lymphocytes have been removed and is engrafted transiently in the patient's body, but the patient's lymphocytes may re-grow and disappear. .
- the human HLA class I molecule locus of the cell is HLA-A and HLA-B
- the human HLA class II molecule locus of the cell is DRB1
- the HLA-A, HLA-B, HLA-C and DRB1 antigen loci comprise at least one antigen whose compatible type of cells does not match that of the patient, and the hematopoietic stem cell-derived cells are In the body, there may be no permanent engraftment or acute GVH disease of severity III or IV.
- the HLA-A, HLA-B, and DRB1 antigen loci of the cells contain 5 to 6 antigens whose matched type of the cells does not match that of the patient, and Cells derived from hematopoietic stem cells may not cause permanent engraftment in the patient's body or acute GVH disease of severity III or IV.
- the human hematopoietic stem cell may be a cord blood stem cell.
- the human hematopoietic stem cell-derived cell may be a cytotoxic T cell specific for a human HLA class I molecule-restricted epitope.
- the pharmaceutical composition of the present invention may be used for cancer treatment.
- the pharmaceutical composition of the present invention may be used for treating infectious diseases.
- the cytotoxic T cells specific to the human HLA class I molecule-restricted epitope are amplified without being stimulated by the human HLA class I molecule-restricted epitope There is.
- the HLA-A locus of the cell contains at least one antigen whose matched type of the cell matches that of the patient, and the cytotoxic T cell is a Wilms tumor. It may be specific for the causative gene product (WT1).
- the present invention provides a pharmaceutical composition comprising cytotoxic T cells derived from human umbilical cord blood.
- the cytotoxic T cell is restricted to HLA class I molecules at any one locus of HLA-A, HLA-B and HLA-C.
- a cytotoxic T cell that is specific for an epitope and specific for said human HLA class I molecule restricted epitope is amplified without being stimulated by said human HLA class I molecule restricted epitope, and any one of said cells
- the HLA class I molecule of the type of locus contains at least one antigen whose matched type of the cell matches that of the patient, and the HLA-A, HLA-B and DRB1 loci of the cell And 5 to 6 antigens that do not match the patient's compatible type, and the hematopoietic stem cell-derived cells are present in the patient's body, with permanent engraftment, severity II Or it does not cause even acute GVH disease IV.
- the present invention is the pharmaceutical composition for cancer treatment of the present invention, wherein the cytotoxic T cells specific for the human HLA class I molecule-restricted epitope are stimulated by the human HLA class I molecule-restricted epitope.
- the present invention provides a method for producing a pharmaceutical composition for cancer treatment that is amplified without any problem.
- the manufacturing method includes stimulating human umbilical cord blood with CD3 / CD28 immunobeads.
- the present invention provides a method for treating infectious diseases or cancer.
- the method for treating infection or cancer of the present invention comprises the steps of preparing cells derived from human hematopoietic stem cells, and transplanting the cells derived from human hematopoietic stem cells to a patient, and human HLA class I molecules of the cells Any one of the loci comprises at least one antigen whose matched type of the cell matches that of the patient, and the antigen of the human HLA class I and II molecule locus of the cell.
- the cell type contains at least one antigen that does not match the patient's type, and the hematopoietic stem cell-derived cell is in the patient's body, either permanently engrafted, or with an acute GVH disease of severity III or IV It does not wake up.
- the method for treating infection or cancer of the present invention comprises the step of removing lymphocytes of the patient before the step of transplanting the cells into the patient, and the cells derived from the human hematopoietic stem cells It may be characterized by the fact that the patient's lymphocytes re-proliferate and disappear while transiently taking in the body.
- any one locus of human HLA class I molecules of the hematopoietic stem cell-derived cell is selected from the group consisting of HLA-A, HLA-B and HLA-C,
- the locus of the human HLA class II molecule is DRB1
- the antigen of the human HLA class I and II molecule locus is at least one antigen whose matched cell type does not match that of the patient.
- the hematopoietic stem cell-derived cell may be characterized in that it does not cause permanent engraftment or an acute GVH disease of severity III or IV in the patient.
- the antigens at the HLA-A, HLA-B and DRB1 loci of the cells include 5 to 6 antigens in which the compatible type of the cells does not match that of the patient.
- the cells derived from the hematopoietic stem cells may be characterized in that they do not cause permanent engraftment or acute GVH disease of severity III or IV in the patient.
- the human hematopoietic stem cells may be umbilical cord blood stem cells.
- the human hematopoietic stem cell-derived cell may be a cytotoxic T cell specific for a human HLA class I molecule-restricted epitope.
- the step of preparing a cell derived from the human hematopoietic stem cell comprises the step of preparing the human HLA class I molecule-restricted epitope without being stimulated by the human HLA class I molecule-restricted epitope.
- Amplifying specific cytotoxic T cells may be included.
- the step of amplifying cytotoxic T cells specific to the human HLA class I molecule-restricted epitope without being stimulated by the human HLA class I molecule-restricted epitope Stimulating human umbilical cord blood with CD3 / CD28 immunobeads may be included.
- the compatible type of at least one antigen of the HLA-A locus of the hematopoietic stem cell matches the compatible type of the patient, and the cytotoxic T cell is a Wilms tumor causative gene. It may be specific for the product (WT1).
- the human umbilical cord blood-derived cytotoxic T cell is an HLA class I molecule at any one locus of HLA-A, HLA-B and HLA-C.
- Cytotoxic T cells that are specific for a restricted epitope and specific for the human HLA class I molecule restricted epitope are amplified without being stimulated by the human HLA class I molecule restricted epitope,
- the HLA class I molecule at any one locus contains at least one antigen whose cell type matches that of the patient, and the cell's HLA-A, HLA-B and DRB1 loci are 5 to 6 antigens that do not match the patient's compatible type, and the cells derived from the hematopoietic stem cells are in the patient's body, permanent engraftment, It may not occur even acute GVH disease in degrees III or IV.
- the human hematopoietic stem cells of the present invention may be derived from tissues including, but not limited to, cord blood, bone marrow, and peripheral blood after administration of G-CSF.
- a preferred source of human hematopoietic stem cells is cord blood.
- hematopoietic stem cells generated from embryonic stem cells, adult stem cells, and induced pluripotent stem (iPS) cells may also be used.
- the human hematopoietic stem cell-derived cells of the present invention include T cells, NK cells, dendritic cells, B cells, macrophages, neutrophils, eosinophils and other granulocytes.
- the human hematopoietic stem cell-derived cells of the present invention may be generated by inducing differentiation of hematopoietic stem cells contained in umbilical cord blood, bone marrow, and peripheral blood after administration of G-CSF. In some cases, it is generated by directly inducing differentiation from adult stem cells and induced pluripotent stem (iPS) cells.
- iPS induced pluripotent stem
- the human hematopoietic stem cell-derived cells of the present invention are prepared by determining the HLA class I and II molecular locus conformation antigens and cryopreserving tissues containing cord blood and other hematopoietic stem cells. It may be generated by thawing the tissue having an adaptive form that meets the requirements of the present invention and culturing it with growth stimulation after the compatible form of the antigen at the locus of the I and II molecules has been determined .
- the cells derived from the human hematopoietic stem cells of the present invention may be generated by culturing in advance with growth stimulation, amplified, and cryopreserved. In the latter case, since a large amount has been amplified in advance, cells whose drug efficacy has been confirmed in advance can be administered to the patient. In addition, cells of uniform quality can be administered to one or more patients.
- HLA class I molecules There are three types of HLA class I molecules: HLA-A, HLA-B, and HLA-C. There are numerous polymorphisms of genes at each locus of HLA class I molecules and their products. While HLA class I molecules have the function of presenting peptide antigens to cytotoxic T cells, host cells are transplanted as immunogens that serve as a basis for self-non-self discrimination during tissue transplantation between allogeneic species. It also plays a role in inducing an HVG reaction that attacks the cells and a GVH reaction in which the transplanted cells attack the host cells.
- each locus of HLA class I molecules has 2 antigens, and the locus of HLA class I molecules has a total of 6 antigens. Therefore, when expressing the fitness of HLA, the case where only one antigen's fitness type at one locus is different is “one antigen mismatch”, and one or two antigen's fitness types at one locus are different. The case is expressed as “one-seat mismatch”. That is, “one locus mismatch” includes the case of one antigen mismatch and two antigen mismatch. Similarly, “bidentate mismatch” includes the case where 2 to 4 antigens do not match. Also, when 5 or 6 antigens do not match for 3 loci, they also do not match.
- the conforming type may be determined by serological examination or DNA examination.
- DNA tests There are two types of DNA tests: those with medium resolution such as the fluorescent bead method (PCR-rsso method) and those with high resolution such as the SBT method.
- Suitable types include a so-called 4-digit allele name, a 6-digit allele considering base substitution in the translation region, and an 8-digit allele considering base substitution in the untranslated region. Since the 6-digit or 8-digit allele is a polymorphism without amino acid change, it can be treated immunologically in the same manner as the 4-digit allele.
- HLA-DR There are three types of heterodimers in HLA class II molecules: HLA-DR, HLA-DP and HLA-DQ.
- the alpha and beta chains that make up each heterodimer are encoded at adjacent loci on the chromosome, but there are only two beta chains of HLA-DR. Therefore, there are seven loci of HLA class II molecules: DRA, DRB1, DRB2, DPA, DPB, DQA and DQB.
- HLA class II molecules have the function of presenting peptide antigens to helper T cells, like HLA class I molecules, they serve as immunogens that serve as the basis for self-nonself discrimination during allogeneic tissue transplantation.
- HLA class II the host cell attacks the transplanted cell
- GVGH a GVGH reaction in which the transplanted cell attacks the host cell.
- HLA class I molecules there are numerous polymorphisms in genes at the loci of HLA class II molecules and their products.
- transient engraftment means that the transplanted cells can be detected in the host body, for example, in the peripheral blood even after the lapse of 1 month, but after 3 months, 6 months, 12 months. Or, it means that it can no longer be detected after 18 months.
- Permanent engraftment means that cells transplanted in the host can be detected throughout the life of the host.
- genetic tests such as PCR, chromosome tests such as FISH and chromosome analysis, and cell immunohistochemical tests such as flow cytometry using anti-HLA antibodies. it can.
- the matched type of the pediatric patient undergoing transplantation contains 4 to 6 out of 6 antigens that match the matched type of unrelated cord blood
- transplantation is usually successful if the patient's fitness type contains 0 or 2 antigens that do not match the cord blood adaptation type (Nippon Umbilical Blood Bank Network Committee, “Results of unrelated umbilical cord blood transplantation in Japan (results of analysis in 2007)”, https://www.j-cord.gr.jp/ja/wnew/isyokuseiseki2007.pdf, Gluckman, E. and Rocha, V., Curr. Opin.
- the HLA-A, HLA-B and DRB1 loci of the cells of the present invention if the cell's compatible type contains at least one antigen that does not match the patient's compatible type, specialized in the field of cord blood transplantation
- the home can determine conditions in which cord blood-derived cells do not cause permanent engraftment or severity III or IV acute GVH disease in the patient's body.
- the HLA-A, HLA-B and DRB1 loci of the cells of the present invention comprise 4, 5 or 6 antigens whose matched type of the cell does not match that of the patient.
- the pharmaceutical composition of the present invention can be transplanted to a much wider range of patients than conventional ones even from cells derived from one donor.
- medicinal cells eg, cytotoxic T cells specific for various disease-related antigens
- the cells derived from hematopoietic stem cells of the present invention include any cell type that can differentiate into dendritic cells and cytotoxic T cells. That is, common lymphoid progenitor cells, dendritic cells, so-called double negative T cells that do not express both CD4 and CD8, so-called double positive T cells that express both CD4 and CD8, and CD8 positive T cells.
- the bone marrow-lymphoid common progenitor cells, the bone marrow-T cell common progenitor cells, and the macrophage-T cell common progenitor cells proposed by Katsura and Kawamoto are not limited thereto.
- hematopoietic stem cell-derived cells of the present invention various procedures known to experts in this technical field can be used. For example, when separating leukocytes from cord blood, Ficoll specific gravity centrifugation can be used. When separating T cells from the leukocytes, cell surface antigen CD3 is obtained using immunomagnetic beads including, but not limited to, Dynavial Dynal beads (trademark) manufactured by Invitrogen and CliniMACS (trademark) manufactured by Miltenyi Biotech. , CD14 and / or CD28 expressing cells can be isolated and purified. As a medium for culturing the obtained cells, an XVivo15 medium or other serum-free medium suitable for the growth of blood cells such as lymphocytes can be used.
- a medium for culturing the obtained cells an XVivo15 medium or other serum-free medium suitable for the growth of blood cells such as lymphocytes can be used.
- This medium may be supplemented with human AB serum (0-15%) available from BioWhittaker et al. Or blood donated human serum albumin (0-10%) available from the Japanese Red Cross. Also included are interleukin 2 (0 to 10,000 IU / mL), interleukin 15 (0 to 100 ng / mL) and / or interleukin 21 (0 to 100 ng / mL) available from Peprotech and others. Non-limiting cell growth factors may be added.
- the growth stimulator may be Invitrogen or other CD3 / CD28 beads.
- the cells derived from the human hematopoietic stem cell of the present invention can undergo growth stimulation regardless of the HLA class I molecule-restricted epitope.
- the cells derived from the human hematopoietic stem cells of the present invention can be subjected to growth stimulation by HLA class I molecule-restricted epitopes.
- an oligopeptide consisting of the amino acid sequence of CMTWNQMNL (SEQ ID NO: 1) (HLA-A * 2402-restricted WT1-derived peptide fragment)
- an oligopeptide consisting of the amino acid sequence of ADVEFCSLL (SEQ ID NO: 2) or SADVEFCSLL (SEQ ID NO: 3) ( HLA-B * 4002 restricted tyrosinase-derived peptide fragment)
- oligopeptide consisting of amino acid sequences 81 to 110 of cancer antigen NY-ESO-1 (HLA-B * 3501 or HLA-C * 0304 restricted peptide fragment)
- Cancer-specific peptides such as EB virus, cytomegalovirus, AIDS virus and other viral proteins or peptide fragments thereof are known as HLA class I molecule-restricted epitopes.
- cells derived from the human hematopoietic stem cells of the present invention for example, cells expressing cell surface antigens CD3, CD14 and / or CD28 purified from leukocytes of umbilical cord blood in a medium to which such protein or peptide is added.
- Growth stimulation by HLA class I molecule restricted epitopes can be performed.
- Growth stimulation by HLA class I molecule-restricted epitopes can also be carried out by co-culture with cord blood-derived dendritic cells co-cultured with lysates of cancer cells or cells infected with viruses or other pathogens. However, it is not limited to these.
- Growth-stimulated cells may be cultured at 37 ° C. in the presence of 5-7% CO 2 .
- the cells may be seeded at a concentration of 1 ⁇ 10 6 cells / mL, the medium may be changed after 2 to 4 days, and seeded in a new medium at a concentration of 1 ⁇ 10 6 cells / mL.
- an apparatus such as WAVE Bioreactor 2/10 (trademark) manufactured by GE Healthcare may be used.
- the pharmaceutical composition of the present invention may contain any component that is pharmaceutically acceptable as a cell preparation for transplantation into the blood vessel of a patient, in addition to the cells derived from human hematopoietic stem cells.
- the pharmaceutical composition of the present invention When transported in a cryopreserved state, it may contain a cryopreservation solution that can provide a sufficient amount of effective cytotoxic T cells as a cell preparation for transplantation upon thawing. .
- the pharmaceutical composition of the present invention may be transplanted after the patient's lymphocytes have been removed. This is because, like general adoptive immunotherapy (Adoptive Cell Transfer, ACT), a patient's lymphocytes are used for cytokines (interleukin 7, interleukin 15, etc.) necessary for cell proliferation contained in the pharmaceutical composition of the present invention. In order to avoid competing with cells, and to avoid reducing the activity of cells contained in the pharmaceutical composition of the present invention due to cell-cell interaction with patient lymphocytes.
- cytokines interleukin 7, interleukin 15, etc.
- Methods for removing patient lymphocytes include drug administration such as cyclophosphamide (eg, 60 mg / kg, 2 days), fludarabine (eg, 25 mg / m 2 , 5 days) and / or radiation (eg, 2 or 12 Gy) and interleukin 2 administration (7.2 ⁇ 10 5 IU / kg every 8 hours, 2 to 3 days), but is not limited thereto.
- drug administration such as cyclophosphamide (eg, 60 mg / kg, 2 days), fludarabine (eg, 25 mg / m 2 , 5 days) and / or radiation (eg, 2 or 12 Gy) and interleukin 2 administration (7.2 ⁇ 10 5 IU / kg every 8 hours, 2 to 3 days), but is not limited thereto.
- the pharmaceutical composition of the present invention comprises cytotoxic T cells specific for the HLA-A-restricted cancer antigen WT1 peptide of cord blood cells. Accordingly, the pharmaceutical composition of the present invention may attack cancer cells of a patient overexpressing WT1 when transplanted into a patient having at least one antigen of the same type as cord blood for the HLA-A locus. it can.
- cytotoxic T cells specific for other antigens can be amplified in vitro and that clinical effects can be obtained by transplanting such cytotoxic T cells into patients.
- Godet, Y. et al. (Cancer Immunol. Immunother., 58: 271-280 (2009)), cytotoxic T cells specific for HLA-B-restricted peptides derived from the melanoma-specific cancer antigen tyrosinase were obtained.
- CTLs specific for B-restricted epitopes, HLA-B-restricted epitopes of EB virus-related antigen BZLF1, etc. were transplanted into EB virus-related nasopharyngeal cancer patients to obtain clinical effects. Walter, E .; A. (N. Engl. J.
- CMV cytomegalovirus
- Bioley G. (Clin. Cancer Res. 15: 299-306 (2009)) are specific for HLA-B or HLA-C restricted peptides derived from the antigen from patients vaccinated with the cancer antigen NY-ESO-1. Cytotoxic T cells were obtained. Thus, cytotoxic T cells are specific not only for HLA-A restricted epitopes such as peptides derived from the WT1 protein, but also for HLA-B or HLA-C restricted epitopes. Also good.
- any one locus of human HLA class I molecules of cells derived from human hematopoietic stem cells contains at least one antigen whose cell compatible type matches that of the patient. Therefore, when a patient cell expresses a disease-related antigen, such as a cancer antigen or a pathogen-derived antigen, the disease-related antigen is presented to the cytotoxic T cells of the pharmaceutical composition of the present invention together with human HLA class I molecules. . That is, the pharmaceutical composition of the present invention recognizes the disease-related antigen in the context of the allele of the HLA class I molecular locus common to the patient and specifically attacks the patient's cells that express the disease-related antigen.
- a disease-related antigen such as a cancer antigen or a pathogen-derived antigen
- Disease-related antigens expressed by patient cells include, but are not limited to, WT1, tyrosinase, NY-ESO-1, CEA, NSE, PSA, gp100, MART-1 and MAGE-3, Examples include antigens that are (over-expressed) in cancer cells such as EB virus-related antigens such as EBER and LMP-1, cytomegalovirus-specific antigens such as CMVgp65, and AIDS virus-specific antigens such as HIV gp160. There are cases where the antigen is expressed in infected cells, such as a virus-specific antigen that is not limited. Therefore, the pharmaceutical composition of the present invention may be used for cancer treatment or infectious disease treatment.
- CD14 positive cells and monocyte macrophages phagocytosed by beads were removed using a magnetic particle separator (MPC-1, Dynal).
- the remaining cells were resuspended in PBS and 25 ⁇ L Dynal TM immunomagnetic beads CD3 (Invitrogen) per 10 7 cells were added and stirred for 30 min or longer on ice or at 4 ° C.
- CD3-positive cells were separated using a magnetic particle separator (MPC-1, Dynal).
- the CD3 positive cells are suspended in XVivo15 medium supplemented with 5% human AB type serum at a concentration of 10 6 cells / mL, and recombined with Dynabeads CD3 / CD28 T cell expander (Invitrogen). Incubated with IL-2. Viable cell numbers were measured using trypan blue staining on days 4, 7, 9, 12 and 14 of culture. The cells were diluted with ExVivo15 medium supplemented with 5% human AB type serum containing recombinant IL-2 to a cell concentration of 10 6 cells / mL.
- FIG. 1 shows the results of examining the growth conditions of cord blood-derived CD3 positive cells.
- the vertical axis in FIG. 1 represents the multiplication factor with the total number of CD3 positive cells at the start of culture being 1, and the horizontal axis represents the number of days after the start of culture.
- CD3 / CD28 immunobeads used for stimulation were used at a rate of about 2 beads per cell. Regardless of the concentrations of recombinant IL-2 and human serum added to the medium, umbilical cord blood-derived CD3-positive cells grew at almost the same rate until day 9 of culture under any culture condition.
- FIG. 2 shows the results of the flow cytometry analysis.
- the vertical axis in the figure represents the fluorescence intensity of PE and represents the reaction with HLA-A * 2402WT1 (wild) CMTWNQMNL-tetramer
- the horizontal axis represents the fluorescence intensity of APC and represents the reaction with anti-CD8 antibody.
- CD8 positive cells ie, cytotoxic T cells among the amplified cord blood-derived CD3 positive cells
- WT1 which is a cancer antigen in the context of HLA-A * 2402 allele.
- the derived peptide was specifically recognized.
- WT1-specific cytotoxic T cells are known to react with lymphomas and other malignant cells that overexpress WT1, but do not attack normal cells that express only a relatively small amount of WT1 (Gao, L Et al., Blood, 95: 2198-2203 (2000), Oka, Y. et al., Curr. Op. In Immunol., 20: 211-220 (2008)).
- the WT1 peptide was not used for stimulation of proliferation of cytotoxic T cells, cytotoxic T cells specifically recognizing WT1-derived peptides were obtained. Since lymphocytes expressed in umbilical cord blood leukocytes include those that differentiate into dendritic cells, WT1 was presented as an antigen from dendritic cells to cytotoxic T cells under culture there is a possibility. In cord blood, there is a weak CD4 positive CD8 positive T cell population before undergoing thymic selection that is not present in adult peripheral blood, which is a cell population specific for cord blood.
- Dendritic cells are essential for antigen-specific activation (priming) of naive T cells, but in the case of self-proteins that are also expressed in normal cells such as WT1, juveniles already undergoing thymic selection Since CD4 positive CD8 positive self-reactive memory cells are present in umbilical cord blood, WT1-specific cytotoxic T cells may be amplified from these cell populations. In adults, these cell populations have already been removed as autoreactive clones at the stage of thymus selection and cannot be obtained by amplification of adult peripheral blood, and can only be adjusted from umbilical cord blood. The experiments of this example show for the first time in the world that this cell population can be adjusted only from umbilical cord blood.
Abstract
Disclosed are transplantation cells for an immunotherapy, which are applicable to a wider variety of patients compared to conventional transplantation cells for immunotherapies, and which can survive transiently so that severe GVH disease cannot be induced.
Specifically disclosed is a pharmaceutical composition containing cells derived from human hematopoietic stem cells. In the pharmaceutical composition, the genetic locus of at least one human HLA class I molecule in the cells contains at least one antigen for which the type of matching of the cell is identical to that of a patient. The genetic locus of each of the human HLA class I and II molecules in the cells contains at least one antigen for which the type of matching of the cell is not identical to that of a patient, and the cells derived from the hematopoietic stem cells do not cause acute GVH disease having a severity of level III or IV in the body of a patient even when the cells survive in the patient permanently.
Description
本発明は、ヒト造血幹細胞由来の細胞を含む医薬品組成物に関し、より具体的には、臍帯血由来の細胞傷害性Ｔ細胞を含む医薬品組成物であって、患者のＨＬＡクラスＩ分子が提示する疾患関連抗原を認識でき、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさない、医薬品組成物に関する。
The present invention relates to a pharmaceutical composition comprising cells derived from human hematopoietic stem cells, and more specifically, a pharmaceutical composition comprising cytotoxic T cells derived from umbilical cord blood, presented by a patient's HLA class I molecule. The present invention relates to a pharmaceutical composition capable of recognizing a disease-related antigen, and wherein the hematopoietic stem cell-derived cell does not cause permanent engraftment or acute GVH disease of severity III or IV in the patient.
臍帯血造血幹細胞移植（Ｕｍｂｉｌｉｃａｌ Ｃｏｒｄ Ｂｌｏｏｄ Ｈｅｍａｔｏｐｏｉｅｔｉｃ Ｃｅｌｌ Ｔｒａｎｓｐｌａｎｔａｔｉｏｎ、以下、「ＵＣＢＨＣＴ」という。）は、急性骨髄性白血病その他の白血病や、再生不良性貧血その他の造血障害や、先天性免疫不全症、先天性代謝異常、ＥＢＶ感染症等の小児及び成人に有効な治療法である（非特許文献１）。組織適合性の検査データを得たうえで凍結保存する臍帯血バンク制度が各国で普及しているので、ＵＣＢＨＣＴは今日最も普及した幹細胞移植療法となっている。ＵＣＢＨＣＴでは、一般的に、ＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１遺伝子座について、移植を受ける小児患者の適合型が非血縁の臍帯血の適合型と一致する抗原を６個の抗原のうち４ないし６個含む場合、すなわち、患者の適合型が臍帯血の適合型と一致しない抗原を０ないし２個含む場合にたいてい移植が成功することが知られている（非特許文献２及び３）。移植された細胞は永続的に宿主である患者の体内に生着する。
Umbilical cord blood hematopoietic stem cell transplantation (Umbilical Cord Blood Hematopoietic Cell Transplantation, hereinafter referred to as “UCBHCT”) is acute myeloid leukemia and other leukemia, aplastic anemia and other hematopoietic disorders, congenital immunodeficiency, congenital This is an effective treatment for children and adults such as metabolic disorders and EBV infection (Non-patent Document 1). UCBHCT has become the most popular stem cell transplantation therapy today because the cord blood bank system that is cryopreserved after obtaining histocompatibility test data is widespread in each country. In UCBHCT, in general, for HLA-A, HLA-B, and DRB1 loci, 4 to 6 of 6 antigens whose matching type of pediatric patients undergoing transplantation matches that of unrelated cord blood are matched. It is known that transplantation is usually successful when six are included, that is, when the patient's matched type contains 0 to 2 antigens that do not match the matched type of cord blood (Non-patent Documents 2 and 3). The transplanted cells are permanently engrafted in the patient's host body.
一方、養子免疫療法（Ａｄｏｐｔｉｖｅ Ｃｅｌｌ Ｔｒａｎｓｆｅｒ、以下、「ＡＣＴ」という。）は、最も治療効果が高い癌の治療法として最近注目を集めている（非特許文献４）。ＡＣＴは、腫瘍組織に浸潤した患者自身（自家）のリンパ球や、疾患関連抗原に試験管内で感作された患者自身のリンパ球を、インターロイキン２存在下で大量培養し、リンパ球除去を施された患者に移植する治療法である。例えば転移性メラノーマでは、腫瘍浸潤リンパ球を用いた自家リンパ球輸注が施された約５０％の症例で客観的な腫瘍の退縮が報告されている。
On the other hand, adoptive immunotherapy (Adoptive Cell Transfer, hereinafter referred to as “ACT”) has recently attracted attention as a cancer treatment method having the highest therapeutic effect (Non-patent Document 4). ACT removes lymphocytes by mass-culturing patients 'own lymphocytes that have infiltrated the tumor tissue or patients' lymphocytes sensitized in vitro with a disease-related antigen in the presence of interleukin-2. It is a treatment method that is transplanted to a given patient. For example, in metastatic melanoma, objective tumor regression has been reported in about 50% of cases in which autologous lymphocyte infusion using tumor-infiltrating lymphocytes has been performed.
ＵＣＢＨＣＴは不一致抗原が１個又は２個許容されるが、患者のＨＬＡ遺伝子座の適合型によっては移植に適するドナー臍帯血が見つからないことがある。また、ドナー臍帯血が重症ＧＶＨ病と生存に関与する不適合なＨＬＡ型の組合せに該当するために移植に適さない場合もある。さらに、ＵＣＢＨＣＴで患者に永続的に生着することに成功しても、今度は、移植片対宿主病（以下、「ＧＶＨ病」という。）を発症する場合がある。また、移植された細胞由来の白血病（いわゆるドナー細胞白血病）を発症する危険が、骨髄移植や末梢血幹細胞移植の場合に比べてＵＣＢＨＣＴの場合は高い。
UCBHCT accepts one or two mismatched antigens, but donor umbilical cord blood suitable for transplantation may not be found depending on the compatible type of the patient's HLA locus. In addition, donor umbilical cord blood may not be suitable for transplantation because it corresponds to an incompatible combination of HLA types involved in severe GVH disease and survival. Further, even if the patient is successfully engrafted in the patient with UCBHCT, graft-versus-host disease (hereinafter referred to as “GVH disease”) may be developed. In addition, the risk of developing transplanted cell-derived leukemia (so-called donor cell leukemia) is higher in UCBHCT than in bone marrow transplantation or peripheral blood stem cell transplantation.
ＡＣＴは、患者ごとに移植用のリンパ球を調製する必要があり、この作業は、かなり労働集約的で、専門的な技能を必要とする。またＡＣＴにおいても、移植された細胞由来の白血病を発症する危険がある。
ACT needs to prepare lymphocytes for transplantation for each patient, and this operation is quite labor intensive and requires specialized skills. Also in ACT, there is a risk of developing leukemia derived from transplanted cells.
さらに、ＵＣＢＨＣＴでもＡＣＴでも、特定の患者ごとに移植細胞を調製するため、プリオンのように検出が困難な病原体を検出できないまま移植してしまうことによる、感染症伝播のおそれがある。
Furthermore, in both UCBHCT and ACT, since transplanted cells are prepared for each specific patient, there is a risk of infectious disease transmission due to transplantation without detection of pathogens that are difficult to detect, such as prions.
そこで、従来の免疫療法用移植細胞よりも広範囲の患者を対象とすることができ、かつ、一過性に生着して重症のＧＶＨ病も起こさない、免疫療法用移植細胞を開発する必要がある。
Therefore, there is a need to develop transplant cells for immunotherapy that can target a wider range of patients than conventional transplant cells for immunotherapy and that do not cause severe GVH disease. is there.
本発明はヒト造血幹細胞由来の細胞を含む医薬品組成物を提供する。本発明の医薬品組成物において、前記細胞のヒトＨＬＡクラスＩ分子のいずれか１つの遺伝子座は、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含む。また、前記細胞のヒトＨＬＡクラスＩ及びＩＩ分子の遺伝子座は、前記細胞の適合型が患者の適合型と一致しない抗原を少なくとも１個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさない。
The present invention provides a pharmaceutical composition comprising cells derived from human hematopoietic stem cells. In the pharmaceutical composition of the present invention, any one locus of the human HLA class I molecule of the cell contains at least one antigen whose matched type of the cell matches that of the patient. Further, the human HLA class I and II molecular loci of the cells contain at least one antigen whose compatible type of the cells does not match that of the patient, and the cells derived from the hematopoietic stem cells are in the body of the patient. Thus, neither permanent engraftment nor acute GVH disease of severity III or IV occurs.
本発明の医薬品組成物は、前記患者のリンパ球が除去された後に投与され、前記患者の体内で一過的に生着するけれども、前記患者のリンパ球が再増殖するとともに消失する場合がある。
The pharmaceutical composition of the present invention is administered after the patient's lymphocytes have been removed and is engrafted transiently in the patient's body, but the patient's lymphocytes may re-grow and disappear. .
本発明の医薬品組成物において、前記細胞のヒトＨＬＡクラスＩ分子の遺伝子座は、ＨＬＡ－Ａ及びＨＬＡ－Ｂであり、前記細胞のヒトＨＬＡクラスＩＩ分子の遺伝子座はＤＲＢ１であり、前記細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ、ＨＬＡ－Ｃ及びＤＲＢ１抗原遺伝子座は、前記細胞の適合型が患者の適合型と一致しない抗原を少なくとも１個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさない場合がある。
In the pharmaceutical composition of the present invention, the human HLA class I molecule locus of the cell is HLA-A and HLA-B, the human HLA class II molecule locus of the cell is DRB1, and the cell The HLA-A, HLA-B, HLA-C and DRB1 antigen loci comprise at least one antigen whose compatible type of cells does not match that of the patient, and the hematopoietic stem cell-derived cells are In the body, there may be no permanent engraftment or acute GVH disease of severity III or IV.
本発明の医薬品組成物において、前記細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１抗原遺伝子座は、前記細胞の適合型が患者の適合型と一致しない抗原を５個ないし６個含み、かつ、前記造血幹細胞由来の細胞は、前記患者の体内で永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさない場合がある。
In the pharmaceutical composition of the present invention, the HLA-A, HLA-B, and DRB1 antigen loci of the cells contain 5 to 6 antigens whose matched type of the cells does not match that of the patient, and Cells derived from hematopoietic stem cells may not cause permanent engraftment in the patient's body or acute GVH disease of severity III or IV.
本発明の医薬品組成物において、前記ヒト造血幹細胞は臍帯血幹細胞の場合がある。
In the pharmaceutical composition of the present invention, the human hematopoietic stem cell may be a cord blood stem cell.
本発明の医薬品組成物において、前記ヒト造血幹細胞由来の細胞はヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞の場合がある。
In the pharmaceutical composition of the present invention, the human hematopoietic stem cell-derived cell may be a cytotoxic T cell specific for a human HLA class I molecule-restricted epitope.
本発明の医薬品組成物は癌治療用の場合がある。
The pharmaceutical composition of the present invention may be used for cancer treatment.
本発明の医薬品組成物は感染症治療用の場合がある。
The pharmaceutical composition of the present invention may be used for treating infectious diseases.
本発明の癌治療用医薬品組成物において、前記ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞は、前記ヒトＨＬＡクラスＩ分子拘束性エピトープによる刺激を受けることなく増幅される場合がある。
In the pharmaceutical composition for cancer treatment of the present invention, the cytotoxic T cells specific to the human HLA class I molecule-restricted epitope are amplified without being stimulated by the human HLA class I molecule-restricted epitope There is.
本発明の癌治療用医薬品組成物において、前記細胞のＨＬＡ－Ａ遺伝子座は、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含み、前記細胞傷害性Ｔ細胞はウィルムス腫瘍原因遺伝子産物（ＷＴ１）に特異的である場合がある。
In the pharmaceutical composition for cancer treatment of the present invention, the HLA-A locus of the cell contains at least one antigen whose matched type of the cell matches that of the patient, and the cytotoxic T cell is a Wilms tumor. It may be specific for the causative gene product (WT1).
本発明はヒト臍帯血由来の細胞傷害性Ｔ細胞を含む医薬品組成物を提供する。本発明の細胞傷害性Ｔ細胞を含む医薬品組成物において、前記細胞傷害性Ｔ細胞は、ＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＨＬＡ－Ｃのうちいずれか１種類の遺伝子座のＨＬＡクラスＩ分子拘束性エピトープに特異的であり、前記ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞は前記ヒトＨＬＡクラスＩ分子拘束性エピトープによる刺激を受けることなく増幅され、前記細胞のいずれか１種類の遺伝子座のＨＬＡクラスＩ分子は、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含み、前記細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１の遺伝子座は、前記細胞の適合型が患者の適合型と一致しない抗原を５個ないし６個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさない。
The present invention provides a pharmaceutical composition comprising cytotoxic T cells derived from human umbilical cord blood. In the pharmaceutical composition comprising the cytotoxic T cell of the present invention, the cytotoxic T cell is restricted to HLA class I molecules at any one locus of HLA-A, HLA-B and HLA-C. A cytotoxic T cell that is specific for an epitope and specific for said human HLA class I molecule restricted epitope is amplified without being stimulated by said human HLA class I molecule restricted epitope, and any one of said cells The HLA class I molecule of the type of locus contains at least one antigen whose matched type of the cell matches that of the patient, and the HLA-A, HLA-B and DRB1 loci of the cell And 5 to 6 antigens that do not match the patient's compatible type, and the hematopoietic stem cell-derived cells are present in the patient's body, with permanent engraftment, severity II Or it does not cause even acute GVH disease IV.
本発明は、本発明の癌治療用医薬品組成物であって、前記ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞は前記ヒトＨＬＡクラスＩ分子拘束性エピトープによる刺激を受けることなく増幅される、癌治療用医薬品組成物の製造方法を提供する。前記製造方法は、ヒト臍帯血をＣＤ３／ＣＤ２８免疫ビーズで刺激するステップを含む。
The present invention is the pharmaceutical composition for cancer treatment of the present invention, wherein the cytotoxic T cells specific for the human HLA class I molecule-restricted epitope are stimulated by the human HLA class I molecule-restricted epitope. The present invention provides a method for producing a pharmaceutical composition for cancer treatment that is amplified without any problem. The manufacturing method includes stimulating human umbilical cord blood with CD3 / CD28 immunobeads.
本発明は感染症又は癌の治療方法を提供する。本発明の感染症又は癌の治療方法は、ヒト造血幹細胞由来の細胞を用意するステップと、前記ヒト造血幹細胞由来の細胞を患者に移植するステップとを含むこと、前記細胞のヒトＨＬＡクラスＩ分子のいずれか１つの遺伝子座は、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含むこと、及び、前記細胞のヒトＨＬＡクラスＩ及びＩＩ分子の遺伝子座の抗原は、前記細胞の適合型が患者の適合型と一致しない抗原を少なくとも１個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことを特徴とする。
The present invention provides a method for treating infectious diseases or cancer. The method for treating infection or cancer of the present invention comprises the steps of preparing cells derived from human hematopoietic stem cells, and transplanting the cells derived from human hematopoietic stem cells to a patient, and human HLA class I molecules of the cells Any one of the loci comprises at least one antigen whose matched type of the cell matches that of the patient, and the antigen of the human HLA class I and II molecule locus of the cell The cell type contains at least one antigen that does not match the patient's type, and the hematopoietic stem cell-derived cell is in the patient's body, either permanently engrafted, or with an acute GVH disease of severity III or IV It does not wake up.
本発明の感染症又は癌の治療方法において、前記細胞を患者に移植するステップの前に、前記患者のリンパ球を除去するステップを含むこと、及び、前記ヒト造血幹細胞由来の細胞は前記患者の体内で一過的に生着するが前記患者のリンパ球が再増殖するとともに消失することを特徴とする場合がある。
The method for treating infection or cancer of the present invention comprises the step of removing lymphocytes of the patient before the step of transplanting the cells into the patient, and the cells derived from the human hematopoietic stem cells It may be characterized by the fact that the patient's lymphocytes re-proliferate and disappear while transiently taking in the body.
本発明の感染症又は癌の治療方法において、前記造血幹細胞由来の細胞のヒトＨＬＡクラスＩ分子のいずれか１つの遺伝子座はＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＨＬＡ－Ｃからなるグループから選択され、前記ヒトＨＬＡクラスＩＩ分子の遺伝子座はＤＲＢ１であること、及び、前記ヒトＨＬＡクラスＩ及びＩＩ分子の遺伝子座の抗原は、前記細胞の適合型が患者の適合型と一致しない抗原を少なくとも１個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことを特徴とする場合がある。
In the method for treating infection or cancer according to the present invention, any one locus of human HLA class I molecules of the hematopoietic stem cell-derived cell is selected from the group consisting of HLA-A, HLA-B and HLA-C, The locus of the human HLA class II molecule is DRB1, and the antigen of the human HLA class I and II molecule locus is at least one antigen whose matched cell type does not match that of the patient. And the hematopoietic stem cell-derived cell may be characterized in that it does not cause permanent engraftment or an acute GVH disease of severity III or IV in the patient.
本発明の感染症又は癌の治療方法において、前記細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１の遺伝子座の抗原は、前記細胞の適合型が患者の適合型と一致しない抗原を５個ないし６個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことを特徴とする場合がある。
In the method for treating infection or cancer according to the present invention, the antigens at the HLA-A, HLA-B and DRB1 loci of the cells include 5 to 6 antigens in which the compatible type of the cells does not match that of the patient. And the cells derived from the hematopoietic stem cells may be characterized in that they do not cause permanent engraftment or acute GVH disease of severity III or IV in the patient.
本発明の感染症又は癌の治療方法において、前記ヒト造血幹細胞は臍帯血幹細胞の場合がある。
In the infectious disease or cancer treatment method of the present invention, the human hematopoietic stem cells may be umbilical cord blood stem cells.
本発明の感染症又は癌の治療方法において、前記ヒト造血幹細胞由来の細胞は、ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞の場合がある。
In the infectious disease or cancer treatment method of the present invention, the human hematopoietic stem cell-derived cell may be a cytotoxic T cell specific for a human HLA class I molecule-restricted epitope.
本発明の感染症又は癌の治療方法において、前記ヒト造血幹細胞由来の細胞を用意するステップは、前記ヒトＨＬＡクラスＩ分子拘束性エピトープによる刺激を受けることなく該ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞を増幅するステップを含む場合がある。
In the infectious disease or cancer treatment method of the present invention, the step of preparing a cell derived from the human hematopoietic stem cell comprises the step of preparing the human HLA class I molecule-restricted epitope without being stimulated by the human HLA class I molecule-restricted epitope. Amplifying specific cytotoxic T cells may be included.
本発明の感染症又は癌の治療方法において、前記ヒトＨＬＡクラスＩ分子拘束性エピトープによる刺激を受けることなく該ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞を増幅するステップは、ヒト臍帯血をＣＤ３／ＣＤ２８免疫ビーズで刺激するステップを含む場合がある。
In the method for treating infection or cancer of the present invention, the step of amplifying cytotoxic T cells specific to the human HLA class I molecule-restricted epitope without being stimulated by the human HLA class I molecule-restricted epitope Stimulating human umbilical cord blood with CD3 / CD28 immunobeads may be included.
本発明の感染症又は癌の治療方法において、前記造血幹細胞のＨＬＡ－Ａ遺伝子座の少なくとも１個の抗原の適合型は患者の適合型と一致し、前記細胞傷害性Ｔ細胞はウィルムス腫瘍原因遺伝子産物（ＷＴ１）に特異的な場合がある。
In the method for treating infection or cancer according to the present invention, the compatible type of at least one antigen of the HLA-A locus of the hematopoietic stem cell matches the compatible type of the patient, and the cytotoxic T cell is a Wilms tumor causative gene. It may be specific for the product (WT1).
本発明の感染症又は癌の治療方法において、前記ヒト臍帯血由来の細胞傷害性Ｔ細胞は、ＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＨＬＡ－Ｃのうちいずれか１種類の遺伝子座のＨＬＡクラスＩ分子拘束性エピトープに特異的であり、前記ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞は、前記ヒトＨＬＡクラスＩ分子拘束性エピトープによる刺激を受けることなく増幅され、前記細胞のいずれか１種類の遺伝子座のＨＬＡクラスＩ分子は、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含み、前記細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１の遺伝子座は、前記細胞の適合型が患者の適合型と一致しない抗原を５個ないし６個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさない場合がある。
In the method for treating infection or cancer of the present invention, the human umbilical cord blood-derived cytotoxic T cell is an HLA class I molecule at any one locus of HLA-A, HLA-B and HLA-C. Cytotoxic T cells that are specific for a restricted epitope and specific for the human HLA class I molecule restricted epitope are amplified without being stimulated by the human HLA class I molecule restricted epitope, The HLA class I molecule at any one locus contains at least one antigen whose cell type matches that of the patient, and the cell's HLA-A, HLA-B and DRB1 loci are 5 to 6 antigens that do not match the patient's compatible type, and the cells derived from the hematopoietic stem cells are in the patient's body, permanent engraftment, It may not occur even acute GVH disease in degrees III or IV.
本発明の技術的範囲は、添付する特許請求の範囲の記載に基づいて定められる。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。
The technical scope of the present invention is determined based on the description of the appended claims. Modifications of the present invention, for example, addition, deletion, and replacement of the configuration requirements of the present invention can be made on the condition that the gist of the present invention is not deviated.
本発明のヒト造血幹細胞は、臍帯血と、骨髄と、Ｇ－ＣＳＦを投与後の末梢血とを含むがこれらに限定されない組織に由来する場合がある。好ましいヒト造血幹細胞の出所は臍帯血である。しかし、胚性幹細胞、成体幹細胞及び人工多能性幹（ｉＰＳ）細胞から生成される造血幹細胞であってもかまわない。
The human hematopoietic stem cells of the present invention may be derived from tissues including, but not limited to, cord blood, bone marrow, and peripheral blood after administration of G-CSF. A preferred source of human hematopoietic stem cells is cord blood. However, hematopoietic stem cells generated from embryonic stem cells, adult stem cells, and induced pluripotent stem (iPS) cells may also be used.
本発明のヒト造血幹細胞由来の細胞は、Ｔ細胞、ＮＫ細胞、樹状細胞、Ｂ細胞、マクロファージと、好中球、好酸球その他の顆粒球とを含む。本発明のヒト造血幹細胞由来の細胞は、臍帯血と、骨髄と、Ｇ－ＣＳＦを投与後の末梢血とに含まれる造血幹細胞を分化誘導して生成される場合があるが、胚性幹細胞、成体幹細胞及び人工多能性幹（ｉＰＳ）細胞から直接分化誘導して生成される場合もある。
The human hematopoietic stem cell-derived cells of the present invention include T cells, NK cells, dendritic cells, B cells, macrophages, neutrophils, eosinophils and other granulocytes. The human hematopoietic stem cell-derived cells of the present invention may be generated by inducing differentiation of hematopoietic stem cells contained in umbilical cord blood, bone marrow, and peripheral blood after administration of G-CSF. In some cases, it is generated by directly inducing differentiation from adult stem cells and induced pluripotent stem (iPS) cells.
本発明のヒト造血幹細胞由来の細胞は、ＨＬＡクラスＩ及びＩＩ分子の遺伝子座の抗原の適合型を決定したうえで臍帯血その他の造血幹細胞を含む組織を凍結保存しておき、患者のＨＬＡクラスＩ及びＩＩ分子の遺伝子座の抗原の適合型が決定されてから、本発明の要件を満たす適合型を有する前記組織を解凍して、増殖刺激を与えて培養することにより生成される場合がある。代替策として、本発明のヒト造血幹細胞由来の細胞は、予め増殖刺激を与えて培養することにより生成され、増幅された後に凍結保存される場合がある。後者の場合には、予め大量増幅されているので、あらかじめ薬効が確認できた細胞を患者に投与できる。また、均一な品質の細胞を１人以上の患者に投与することができる。
The human hematopoietic stem cell-derived cells of the present invention are prepared by determining the HLA class I and II molecular locus conformation antigens and cryopreserving tissues containing cord blood and other hematopoietic stem cells. It may be generated by thawing the tissue having an adaptive form that meets the requirements of the present invention and culturing it with growth stimulation after the compatible form of the antigen at the locus of the I and II molecules has been determined . As an alternative, the cells derived from the human hematopoietic stem cells of the present invention may be generated by culturing in advance with growth stimulation, amplified, and cryopreserved. In the latter case, since a large amount has been amplified in advance, cells whose drug efficacy has been confirmed in advance can be administered to the patient. In addition, cells of uniform quality can be administered to one or more patients.
ＨＬＡクラスＩ分子には、ＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＨＬＡ－Ｃの３種類がある。ＨＬＡクラスＩ分子のそれぞれの遺伝子座の遺伝子及びその産物は非常に多くの多型（ｐｏｌｙｍｏｒｐｈｉｓｍ）が存在する。ＨＬＡクラスＩ分子はペプチド抗原を細胞傷害性Ｔ細胞に提示する機能を有する一方で、同種間での組織移植の際の自己－非自己の識別の基礎となる免疫原として、宿主細胞が移植された細胞を攻撃するＨＶＧ反応と、移植された細胞が宿主細胞を攻撃するＧＶＨ反応とを惹起する役割も果たす。かかる組織適合性に関する免疫原としての観点からみると、ヒトのそれぞれの遺伝子座について母親と父親とに由来する２本の染色体のそれぞれにエンコードされる２個の遺伝子産物が別々の抗原として区別される。すなわち、ＨＬＡクラスＩ分子のそれぞれの遺伝子座は２個の抗原を有し、ＨＬＡクラスＩ分子の遺伝子座は全部で６個の抗原を有する。そこで、ＨＬＡの適合度を表すときには、１つの遺伝子座の１個の抗原の適合型のみが異なる場合を「１抗原不一致」、１つの遺伝子座の１個又は２個の抗原の適合型が異なる場合を「１座不一致」と表現する。すなわち「１座不一致」は、１抗原不一致及び２抗原不一致の場合を含む。同様に「２座不一致」は、２個ないし４個の抗原が不一致の場合を含む。また、３つの遺伝子座について５個又は６個の抗原が不一致のとき、３座とも不一致である。
There are three types of HLA class I molecules: HLA-A, HLA-B, and HLA-C. There are numerous polymorphisms of genes at each locus of HLA class I molecules and their products. While HLA class I molecules have the function of presenting peptide antigens to cytotoxic T cells, host cells are transplanted as immunogens that serve as a basis for self-non-self discrimination during tissue transplantation between allogeneic species. It also plays a role in inducing an HVG reaction that attacks the cells and a GVH reaction in which the transplanted cells attack the host cells. From the viewpoint of such immunocompatibility regarding histocompatibility, two gene products encoded on each of the two chromosomes derived from the mother and father for each human locus are distinguished as separate antigens. The That is, each locus of HLA class I molecules has 2 antigens, and the locus of HLA class I molecules has a total of 6 antigens. Therefore, when expressing the fitness of HLA, the case where only one antigen's fitness type at one locus is different is “one antigen mismatch”, and one or two antigen's fitness types at one locus are different. The case is expressed as “one-seat mismatch”. That is, “one locus mismatch” includes the case of one antigen mismatch and two antigen mismatch. Similarly, “bidentate mismatch” includes the case where 2 to 4 antigens do not match. Also, when 5 or 6 antigens do not match for 3 loci, they also do not match.
本明細書において、適合型とは、血清学的検査又はＤＮＡ検査によって決定される場合がある。ＤＮＡ検査には、蛍光ビーズ法（ＰＣＲ－ｒｓｓｏ法）のように中程度の分解能のものと、ＳＢＴ法のように高分解能のものとがある。適合型には、いわゆる４桁アリル名の他、翻訳領域での塩基置換を考慮した６桁アリルや、非翻訳領域での塩基置換を考慮した８桁アリルがある。６桁又は８桁アリルはアミノ酸の変化を伴わない多型なので、免疫学的には４桁アリルと同じに扱うことでよい。
In this specification, the conforming type may be determined by serological examination or DNA examination. There are two types of DNA tests: those with medium resolution such as the fluorescent bead method (PCR-rsso method) and those with high resolution such as the SBT method. Suitable types include a so-called 4-digit allele name, a 6-digit allele considering base substitution in the translation region, and an 8-digit allele considering base substitution in the untranslated region. Since the 6-digit or 8-digit allele is a polymorphism without amino acid change, it can be treated immunologically in the same manner as the 4-digit allele.
ＨＬＡクラスＩＩ分子には、ＨＬＡ－ＤＲ、ＨＬＡ－ＤＰ及びＨＬＡ－ＤＱという３種類のヘテロダイマーがある。それぞれのヘテロダイマーを構成するアルファ鎖及びベータ鎖は染色体上で隣接する遺伝子座にエンコードされるが、ＨＬＡ－ＤＲのベータ鎖のみ２本存在する。そこで、ＨＬＡクラスＩＩ分子の遺伝子座は、ＤＲＡ、ＤＲＢ１、ＤＲＢ２、ＤＰＡ、ＤＰＢ、ＤＱＡ及びＤＱＢの７座がある。ＨＬＡクラスＩＩ分子はペプチド抗原をヘルパーＴ細胞に提示する機能を有する一方で、ＨＬＡクラスＩ分子と同様に、同種間での組織移植の際の自己－非自己の識別の基礎となる免疫原として、宿主細胞が移植された細胞を攻撃するＨＶＧ反応と、移植された細胞が宿主細胞を攻撃するＧＶＨ反応とを惹起する役割も果たす。ＨＬＡクラスＩＩ分子の遺伝子座の遺伝子及びその産物にもＨＬＡクラスＩ分子と同様に非常に多くの多型（ｐｏｌｙｍｏｒｐｈｉｓｍ）が存在する。
There are three types of heterodimers in HLA class II molecules: HLA-DR, HLA-DP and HLA-DQ. The alpha and beta chains that make up each heterodimer are encoded at adjacent loci on the chromosome, but there are only two beta chains of HLA-DR. Therefore, there are seven loci of HLA class II molecules: DRA, DRB1, DRB2, DPA, DPB, DQA and DQB. While HLA class II molecules have the function of presenting peptide antigens to helper T cells, like HLA class I molecules, they serve as immunogens that serve as the basis for self-nonself discrimination during allogeneic tissue transplantation. Also, it plays a role in inducing an HVG reaction in which the host cell attacks the transplanted cell and a GVGH reaction in which the transplanted cell attacks the host cell. As with HLA class I molecules, there are numerous polymorphisms in genes at the loci of HLA class II molecules and their products.
本明細書において、一過性生着とは、移植から１ヶ月経過後も移植された細胞が宿主体内、例えば、末梢血中で検出できているが、３ヶ月後、６ヶ月後、１２ヶ月、あるいは、１８ヶ月後には検出できなくなることをいう。永続的生着とは、宿主が生きている間ずっと宿主体内で移植された細胞が検出できることをいう。ここで、移植された細胞の検出には、ＰＣＲ法などの遺伝子検査、ＦＩＳＨ法や染色体分析などの染色体検査、抗ＨＬＡ抗体を用いたフローサイトメトリーなどの細胞免疫組織化学検査法を用いることができる。
In the present specification, transient engraftment means that the transplanted cells can be detected in the host body, for example, in the peripheral blood even after the lapse of 1 month, but after 3 months, 6 months, 12 months. Or, it means that it can no longer be detected after 18 months. Permanent engraftment means that cells transplanted in the host can be detected throughout the life of the host. Here, for the detection of transplanted cells, it is possible to use genetic tests such as PCR, chromosome tests such as FISH and chromosome analysis, and cell immunohistochemical tests such as flow cytometry using anti-HLA antibodies. it can.
本明細書において、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病とは、１９９４年開催の急性ＧＶＨ病の重症度に関する会議で提案され、Ｐｒｅｚｅｐｉｏｒｋａ， Ｄ．ら（Ｂｏｎｅ Ｍａｒｒｏｗ Ｔｒａｎｓｐｌａｎｔ．、１５：８２５－８５５（２００２））に記載される重症度の分類の４段階のグレードの最も重篤な段階とこれに続く段階とをいう。万一ＧＶＨ病を発症する場合には、本発明の医薬品組成物を移植する前に患者から採血して白血球アフェレーシスにより精製分取して凍結保存されたリンパ球を凍結保存しておき、これをインターロイキン２及び／又はＣＤ３／ＣＤ２８で刺激増幅して患者に戻すこと（自己リンパ球輸注）によって治療することができる。
In this specification, acute GVH disease of severity III or IV was proposed at a conference on the severity of acute GVH disease held in 1994, and Prezepiorka, D. et al. (Bone Marrow Transplant., 15: 825-855 (2002)), the most severe stage of the four grades of severity classification and the subsequent stage. In the event of developing GVH disease, blood is collected from a patient before transplantation of the pharmaceutical composition of the present invention, purified and collected by leukocyte apheresis, and cryopreserved lymphocytes are stored frozen. It can be treated by stimulation amplification with interleukin 2 and / or CD3 / CD28 and return to the patient (autologous lymphocyte infusion).
ＨＬＡクラスＩ分子及びクラスＩＩ分子の遺伝子座の抗原の不一致が造血幹細胞移植に及ぼす影響は、遺伝子座によって異なることや、移植される幹細胞の細胞数、すなわち、投与量によっても異なることが知られている。一般的に、ＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１遺伝子座について、移植を受ける小児患者の適合型が非血縁の臍帯血の適合型と一致する抗原を６個の抗原のうち４ないし６個含む場合、すなわち、患者の適合型が臍帯血の適合型と一致しない抗原を０ないし２個含む場合には、たいてい移植が成功することが知られている（日本さい帯血バンクネットワーク・移植データ管理小委員会、「わが国における非血縁者間さい帯血移植の成績（２００７年度解析結果）」、ｈｔｔｐｓ：／／ｗｗｗ．ｊ－ｃｏｒｄ．ｇｒ．ｊｐ／ｊａ／ｗｎｅｗ／ｉｓｙｏｋｕｓｅｉｓｅｋｉ２００７．ｐｄｆ、Ｇｌｕｃｋｍａｎ，Ｅ．及びＲｏｃｈａ，Ｖ．、Ｃｕｒｒ． Ｏｐｉｎ． Ｉｍｍｕｎｏｌ．，１８：５６５－５７０（２００６）、Ｂｒｕｎｓｔｅｉｎ，Ｃ．Ｇ．ら、Ｂｒ． Ｊ． Ｈａｅｍａｔｏｌ．、１３７：２０－３５（２００７））。前記日本さい帯血バンクネットワーク・移植データ管理小委員会の報告書には、若年成人ＡＬＬ患者に１個ないし４個の抗原が不一致の臍帯血移植を行ったところ術後５００日の無イベント生存率（ＥＦＳ）が２０ないし５６％であったが、５個の抗原が不一致の臍帯血移植を行った２名の患者の術後５００日の無イベント生存率は０であったとのデータが記載される（図５－３５）。この結果から、不一致抗原が５個ある場合には永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことが示唆される。一方で、重篤な中性子被爆患者に臍帯血を移植したときに、ＤＲＢ１遺伝子座の１個の抗原のみが不一致の臍帯血を移植しても、移植後５１日目には末梢血からも骨髄からも移植された臍帯血由来の単核球が消失したとの報告（Ｎａｇａｙａｍａ，Ｈ．ら、Ｂｏｎｅ Ｍａｒｒｏｗ Ｔｒａｎｓｐｌａｎｔ．、２９：１９７－２０４（２００２））がある。この結果から、不一致抗原が少なくとも１個ある場合には永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことが示された。
It is known that the effects of antigen mismatches at HLA class I and class II molecule loci on hematopoietic stem cell transplantation differ depending on the locus and also on the number of stem cells to be transplanted, that is, the dose. ing. In general, for HLA-A, HLA-B and DRB1 loci, the matched type of the pediatric patient undergoing transplantation contains 4 to 6 out of 6 antigens that match the matched type of unrelated cord blood In other words, it is known that transplantation is usually successful if the patient's fitness type contains 0 or 2 antigens that do not match the cord blood adaptation type (Nippon Umbilical Blood Bank Network Committee, “Results of unrelated umbilical cord blood transplantation in Japan (results of analysis in 2007)”, https://www.j-cord.gr.jp/ja/wnew/isyokuseiseki2007.pdf, Gluckman, E. and Rocha, V., Curr. Opin. Immunol., 18: 565-570 (2006), Brunstei , C.G et al., Br J. Haematol, 137:... 20-35 (2007)). According to the report of the Japan Umbilical Cord Blood Bank Network / Transplant Data Management Subcommittee, one to four antigen-mismatched cord blood transplants were performed in young adult ALL patients, and the event-free survival rate was 500 days after surgery. (EFS) was 20 to 56%, but the data showed that the event-free survival rate at 500 days after surgery was 0 for two patients who had 5 cord mismatched cord blood transplants. (Figure 5-35). This result suggests that 5 mismatched antigens do not cause permanent engraftment or acute GVH disease of severity III or IV. On the other hand, when umbilical cord blood is transplanted into a serious neutron-exposed patient, even if umbilical cord blood in which only one antigen at the DRB1 locus does not match is transplanted, peripheral blood is also bone marrow on the 51st day after transplantation. Also reported that the transplanted cord blood-derived mononuclear cells disappeared (Nagayama, H. et al., Bone Marrow Transplant., 29: 197-204 (2002)). This result showed that there was no permanent engraftment or acute GVH disease of severity III or IV when there was at least one mismatched antigen.
したがって、本発明の細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１遺伝子座について、該細胞の適合型が患者の適合型と一致しない抗原を少なくとも１個含む場合には、臍帯血移植の分野の専門家は、臍帯血由来の細胞が前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさない条件を決定することができる。本発明の細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１遺伝子座について、該細胞の適合型が患者の適合型と一致しない抗原を、４、５又は６個含むことがより好ましい。
Therefore, for the HLA-A, HLA-B and DRB1 loci of the cells of the present invention, if the cell's compatible type contains at least one antigen that does not match the patient's compatible type, specialized in the field of cord blood transplantation The home can determine conditions in which cord blood-derived cells do not cause permanent engraftment or severity III or IV acute GVH disease in the patient's body. More preferably, the HLA-A, HLA-B and DRB1 loci of the cells of the present invention comprise 4, 5 or 6 antigens whose matched type of the cell does not match that of the patient.
本発明の医薬品組成物は、１人のドナー由来の細胞であっても、従来より格段に広範囲の患者に移植することができる。また、薬効のある細胞、例えば、さまざまな疾患関連抗原に特異的な細胞傷害性Ｔ細胞を単一の臍帯血から調製することができる。そのため、従来の臍帯血バンクや骨髄バンクと比較して小規模のライブラリを用意することによって、多くの異なる患者のさまざまな疾患に適用することができる。
The pharmaceutical composition of the present invention can be transplanted to a much wider range of patients than conventional ones even from cells derived from one donor. Also, medicinal cells, eg, cytotoxic T cells specific for various disease-related antigens, can be prepared from a single cord blood. Therefore, by preparing a small-scale library as compared with the conventional cord blood bank and bone marrow bank, it can be applied to various diseases of many different patients.
本発明の造血幹細胞由来の細胞は、樹状細胞及び細胞傷害性Ｔ細胞に分化することができるいずれかの細胞タイプを含む。すなわち、リンパ系共通前駆細胞と、樹状細胞と、ＣＤ４及びＣＤ８の両方とも発現しないいわゆる二重陰性Ｔ細胞と、ＣＤ４及びＣＤ８の両方とも発現するいわゆる二重陽性Ｔ細胞と、ＣＤ８陽性Ｔ細胞との他、桂及び河本により提唱された、骨髄－リンパ系共通前駆細胞、骨髄－Ｔ細胞共通前駆細胞及びマクロファージ－Ｔ細胞共通前駆細胞とを含むが、これらに限られない。
The cells derived from hematopoietic stem cells of the present invention include any cell type that can differentiate into dendritic cells and cytotoxic T cells. That is, common lymphoid progenitor cells, dendritic cells, so-called double negative T cells that do not express both CD4 and CD8, so-called double positive T cells that express both CD4 and CD8, and CD8 positive T cells In addition, the bone marrow-lymphoid common progenitor cells, the bone marrow-T cell common progenitor cells, and the macrophage-T cell common progenitor cells proposed by Katsura and Kawamoto are not limited thereto.
本発明の造血幹細胞由来の細胞を用意するためには、本技術分野の専門家に知られたさまざまな手順を用いることができる。例えば臍帯血から白血球を分離する際には、Ｆｉｃｏｌｌ比重遠心法を用いることができる。前記白血球からＴ細胞を分離する際には、Ｉｎｖｉｔｒｏｇｅｎ社のＤｙｎａｌビーズ（商標）や、ミルテニーバイオテック社のＣｌｉｎｉＭＡＣＳ（商標）を含むがこれらに限定されない免疫磁気ビーズを用いて、細胞表面抗原ＣＤ３、ＣＤ１４及び／又はＣＤ２８を発現する細胞を分離精製することができる。得られた細胞を培養するための培地としては、ＸＶｉｖｏ１５培地その他のリンパ球等の血液系細胞の増殖に適する無血清培地を用いることができる。この培地には、ＢｉｏＷｈｉｔｔａｋｅｒ社その他から入手可能なヒトＡＢ型血清（０ないし１５％）や、日本赤十字社から入手可能な献血ヒト血清アルブミン（０ないし１０％）が添加される場合がある。また、ペプロテック社その他から入手可能なインターロイキン２（０ないし１０，０００ＩＵ／ｍＬ）、インターロイキン１５（０ないし１００ｎｇ／ｍＬ）及び／又はインターロイキン２１（０ないし１００ｎｇ／ｍＬ）を含むがこれらに限定されない細胞増殖因子が添加される場合がある。
In order to prepare the hematopoietic stem cell-derived cells of the present invention, various procedures known to experts in this technical field can be used. For example, when separating leukocytes from cord blood, Ficoll specific gravity centrifugation can be used. When separating T cells from the leukocytes, cell surface antigen CD3 is obtained using immunomagnetic beads including, but not limited to, Dynavial Dynal beads (trademark) manufactured by Invitrogen and CliniMACS (trademark) manufactured by Miltenyi Biotech. , CD14 and / or CD28 expressing cells can be isolated and purified. As a medium for culturing the obtained cells, an XVivo15 medium or other serum-free medium suitable for the growth of blood cells such as lymphocytes can be used. This medium may be supplemented with human AB serum (0-15%) available from BioWhittaker et al. Or blood donated human serum albumin (0-10%) available from the Japanese Red Cross. Also included are interleukin 2 (0 to 10,000 IU / mL), interleukin 15 (0 to 100 ng / mL) and / or interleukin 21 (0 to 100 ng / mL) available from Peprotech and others. Non-limiting cell growth factors may be added.
増殖刺激剤は、Ｉｎｖｉｔｒｏｇｅｎ社その他のＣＤ３／ＣＤ２８ビーズの場合がある。この場合には、本発明のヒト造血幹細胞由来の細胞は、ＨＬＡクラスＩ分子拘束性エピトープによらずに増殖刺激を受けることができる。代替策として、本発明のヒト造血幹細胞由来の細胞は、ＨＬＡクラスＩ分子拘束性エピトープによる増殖刺激を受けることができる。例えば、ＣＭＴＷＮＱＭＮＬ（配列番号１）のアミノ酸配列からなるオリゴペプチド（ＨＬＡ－Ａ*２４０２拘束性ＷＴ１由来ペプチド断片）、ＡＤＶＥＦＣＬＳＬ（配列番号２）又はＳＡＤＶＥＦＣＬＳＬ（配列番号３）のアミノ酸配列からなるオリゴペプチド（ＨＬＡ－Ｂ*４００２拘束性チロシナーゼ由来ペプチド断片）、癌抗原ＮＹ－ＥＳＯ－１の第８１ないし１１０番目のアミノ酸配列からなるオリゴペプチド（ＨＬＡ－Ｂ*３５０１又はＨＬＡ－Ｃ*０３０４拘束性ペプチド断片）のような癌特異的ペプチドや、ＥＢウイルス、サイトメガロウイルス、エイズウイルスその他のウイルスのタンパク質又はそのペプチド断片がＨＬＡクラスＩ分子拘束性エピトープとして知られている。かかるタンパク質又はペプチドを添加した培地中で、本発明のヒト造血幹細胞由来の細胞、例えば、臍帯血の白血球から精製された細胞表面抗原ＣＤ３、ＣＤ１４及び／又はＣＤ２８を発現する細胞を培養することによりＨＬＡクラスＩ分子拘束性エピトープによる増殖刺激を行うことができる。ＨＬＡクラスＩ分子拘束性エピトープによる増殖刺激は、癌細胞や、ウイルスその他の病原体に感染した細胞の溶解物と共培養された臍帯血由来樹状細胞との共培養によっても実施することができるが、これらに限定されない。
The growth stimulator may be Invitrogen or other CD3 / CD28 beads. In this case, the cells derived from the human hematopoietic stem cell of the present invention can undergo growth stimulation regardless of the HLA class I molecule-restricted epitope. As an alternative, the cells derived from the human hematopoietic stem cells of the present invention can be subjected to growth stimulation by HLA class I molecule-restricted epitopes. For example, an oligopeptide consisting of the amino acid sequence of CMTWNQMNL (SEQ ID NO: 1) (HLA-A * 2402-restricted WT1-derived peptide fragment), an oligopeptide consisting of the amino acid sequence of ADVEFCSLL (SEQ ID NO: 2) or SADVEFCSLL (SEQ ID NO: 3) ( HLA-B * 4002 restricted tyrosinase-derived peptide fragment), oligopeptide consisting of amino acid sequences 81 to 110 of cancer antigen NY-ESO-1 (HLA-B * 3501 or HLA-C * 0304 restricted peptide fragment) Cancer-specific peptides such as EB virus, cytomegalovirus, AIDS virus and other viral proteins or peptide fragments thereof are known as HLA class I molecule-restricted epitopes. By culturing cells derived from the human hematopoietic stem cells of the present invention, for example, cells expressing cell surface antigens CD3, CD14 and / or CD28 purified from leukocytes of umbilical cord blood in a medium to which such protein or peptide is added. Growth stimulation by HLA class I molecule restricted epitopes can be performed. Growth stimulation by HLA class I molecule-restricted epitopes can also be carried out by co-culture with cord blood-derived dendritic cells co-cultured with lysates of cancer cells or cells infected with viruses or other pathogens. However, it is not limited to these.
増殖刺激された細胞は５ないし７％ＣＯ2存在下３７°Ｃで培養される場合がある。細胞は１×１０6個／ｍＬの濃度で播種され、２ないし４日後に培地交換され、新たな培地に１×１０6個／ｍＬの濃度で播種される場合がある。総数１×１０8個以上の大量培養の場合には、ＧＥ Ｈｅａｌｔｈｃａｒｅ社のＷＡＶＥ Ｂｉｏｒｅａｃｔｏｒ ２／１０（商標）のような装置が用いられる場合がある。
Growth-stimulated cells may be cultured at 37 ° C. in the presence of 5-7% CO 2 . The cells may be seeded at a concentration of 1 × 10 6 cells / mL, the medium may be changed after 2 to 4 days, and seeded in a new medium at a concentration of 1 × 10 6 cells / mL. In the case of mass culture of 1 × 10 8 or more in total, an apparatus such as WAVE Bioreactor 2/10 (trademark) manufactured by GE Healthcare may be used.
本発明の医薬品組成物は、前記ヒト造血幹細胞由来の細胞の他、患者の血管内に移植するための細胞製剤として薬学的に許容されるいかなる成分を含むものであってもよい。本発明の医薬品組成物が、凍結保存状態で輸送される場合には、解凍時に移植用細胞製剤として十分な量の有効な細胞傷害性Ｔ細胞が得られるような凍結保存液を含む場合がある。
The pharmaceutical composition of the present invention may contain any component that is pharmaceutically acceptable as a cell preparation for transplantation into the blood vessel of a patient, in addition to the cells derived from human hematopoietic stem cells. When the pharmaceutical composition of the present invention is transported in a cryopreserved state, it may contain a cryopreservation solution that can provide a sufficient amount of effective cytotoxic T cells as a cell preparation for transplantation upon thawing. .
本発明の医薬品組成物は、患者のリンパ球が除去された後に移植される場合がある。これは、一般の養子免疫療法（Ａｄｏｐｔｉｖｅ Ｃｅｌｌ Ｔｒａｎｓｆｅｒ、ＡＣＴ）と同様に、本発明の医薬品組成物に含まれる細胞の増殖に必要なサイトカイン（インターロイキン７、インターロイキン１５等）をめぐって患者のリンパ球と競合することを避けるため、及び、患者のリンパ球との間の細胞間相互作用によって本発明の医薬品組成物に含まれる細胞の活性が低減することを避けるためである。患者のリンパ球を除去する方法には、シクロホスファミド（例えば、６０ｍｇ／ｋｇ、２日間）、フルダラビン（例えば、２５ｍｇ／ｍ2、５日間）等の薬剤投与及び／又は放射線照射（例えば、２又は１２Ｇｙ）と、インターロイキン２投与（８時間ごとに７．２×１０5ＩＵ／ｋｇ、２ないし３日間）とを含むがこれらに限定されない。
The pharmaceutical composition of the present invention may be transplanted after the patient's lymphocytes have been removed. This is because, like general adoptive immunotherapy (Adoptive Cell Transfer, ACT), a patient's lymphocytes are used for cytokines (interleukin 7, interleukin 15, etc.) necessary for cell proliferation contained in the pharmaceutical composition of the present invention. In order to avoid competing with cells, and to avoid reducing the activity of cells contained in the pharmaceutical composition of the present invention due to cell-cell interaction with patient lymphocytes. Methods for removing patient lymphocytes include drug administration such as cyclophosphamide (eg, 60 mg / kg, 2 days), fludarabine (eg, 25 mg / m 2 , 5 days) and / or radiation (eg, 2 or 12 Gy) and interleukin 2 administration (7.2 × 10 5 IU / kg every 8 hours, 2 to 3 days), but is not limited thereto.
以下の実施例に示すとおり、本発明の医薬品組成物は、臍帯血細胞のＨＬＡ－Ａ拘束性の癌抗原ＷＴ１ペプチドに特異的な細胞傷害性Ｔ細胞を含む。したがって、本発明の医薬品組成物は、ＨＬＡ－Ａ遺伝子座について臍帯血と同じ適合型の抗原を少なくとも１個有する患者に移植されるとき、ＷＴ１を過剰発現する患者の癌細胞を攻撃することができる。
As shown in the following examples, the pharmaceutical composition of the present invention comprises cytotoxic T cells specific for the HLA-A-restricted cancer antigen WT1 peptide of cord blood cells. Accordingly, the pharmaceutical composition of the present invention may attack cancer cells of a patient overexpressing WT1 when transplanted into a patient having at least one antigen of the same type as cord blood for the HLA-A locus. it can.
これ以外にも、試験管内でその他の抗原に特異的な細胞傷害性Ｔ細胞を増幅できること、及び、かかる細胞傷害性Ｔ細胞を患者に移植すると臨床効果が得られることは従来から知られている。例えば、Ｇｏｄｅｔ、Ｙ．ら、（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ． Ｉｍｍｕｎｏｔｈｅｒ．、５８： ２７１－２８０（２００９））メラノーマに特異的な癌抗原チロシナーゼ由来のＨＬＡ－Ｂ拘束性ペプチドに特異的な細胞傷害性Ｔ細胞を得た。Ｓｔｒａａｔｈｏｆ、Ｋ． Ｃ． Ｍ．ら、（Ｂｌｏｏｄ、１０５： １８９８－１９０４（２００５））は、ＥＢウイルス関連抗原ＬＭＰ２のＨＬＡ－Ｂ拘束性エピトープ、ＥＢウイルス関連抗原ＥＢＮＡ１のＨＬＡ－Ｂ拘束性エピトープ、ＥＢウイルス関連抗原ＥＢＮＡ３のＨＬＡ－Ｂ拘束性エピトープ、ＥＢウイルス関連抗原ＢＺＬＦ１のＨＬＡ－Ｂ拘束性エピトープなどに特異的なＣＴＬを、ＥＢウイルス関連鼻咽頭癌患者に移植して臨床効果を得た。Ｗａｌｔｅｒ、Ｅ． Ａ． ら、（Ｎ． Ｅｎｇｌ． Ｊ． Ｍｅｄ．、３３３： １０３８－４４（１９９５））はサイトメガロウイルス（ＣＭＶ）特異的ＣＴＬを、白血病治療のためにＣＭＶ陽性血縁者から骨髄移植を受けた後、免疫抑制剤を投与されている間にＣＭＶが再活性化した患者に移植して臨床効果を得た。
In addition to this, it has been conventionally known that cytotoxic T cells specific for other antigens can be amplified in vitro and that clinical effects can be obtained by transplanting such cytotoxic T cells into patients. . For example, Godet, Y. et al. (Cancer Immunol. Immunother., 58: 271-280 (2009)), cytotoxic T cells specific for HLA-B-restricted peptides derived from the melanoma-specific cancer antigen tyrosinase were obtained. Straathof, K.M. C. M. (Blood, 105: 1898-1904 (2005)) are HLA-B restricted epitopes of the EB virus-related antigen LMP2, HLA-B restricted epitopes of the EB virus-related antigen EBNA1, and HLA-of the EB virus-related antigen EBNA3. CTLs specific for B-restricted epitopes, HLA-B-restricted epitopes of EB virus-related antigen BZLF1, etc. were transplanted into EB virus-related nasopharyngeal cancer patients to obtain clinical effects. Walter, E .; A. (N. Engl. J. Med., 333: 1038-44 (1995)) received cytomegalovirus (CMV) -specific CTL after receiving bone marrow transplantation from a CMV-positive relative for leukemia treatment. A clinical effect was obtained by transplanting into a patient in which CMV was reactivated while being administered an immunosuppressant.
なお、Ｂｉｏｌｅｙ Ｇ．ら（Ｃｌｉｎ． Ｃａｎｃｅｒ Ｒｅｓ． １５： ２９９－３０６（２００９））によると、癌抗原ＮＹ－ＥＳＯ－１をワクチン接種した患者から当該抗原由来のＨＬＡ－Ｂ又はＨＬＡ－Ｃ拘束性ペプチドに特異的な細胞傷害性Ｔ細胞が得られた。そこで、細胞傷害性Ｔ細胞は、ＷＴ１タンパク由来のペプチドのようなＨＬＡ－Ａ拘束性エピトープに特異的なものだけでなく、ＨＬＡ－Ｂ又はＨＬＡ－Ｃ拘束性エピトープに特異的なものであってもよい。
In addition, Bioley G. (Clin. Cancer Res. 15: 299-306 (2009)) are specific for HLA-B or HLA-C restricted peptides derived from the antigen from patients vaccinated with the cancer antigen NY-ESO-1. Cytotoxic T cells were obtained. Thus, cytotoxic T cells are specific not only for HLA-A restricted epitopes such as peptides derived from the WT1 protein, but also for HLA-B or HLA-C restricted epitopes. Also good.
本発明の医薬品組成物においては、ヒト造血幹細胞由来の細胞のヒトＨＬＡクラスＩ分子のいずれか１つの遺伝子座が、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含む。そのため、患者の細胞が疾患関連抗原、例えば、癌抗原や病原体由来抗原を発現するときには、該疾患関連抗原がヒトＨＬＡクラスＩ分子とともに本発明の医薬品組成物の細胞傷害性Ｔ細胞に提示される。すなわち本発明の医薬品組成物は、患者と共通のＨＬＡクラスＩ分子遺伝子座アリルのコンテキストで前記疾患関連抗原を認識して、該疾患関連抗原を発現する患者の細胞を特異的に攻撃する。
In the pharmaceutical composition of the present invention, any one locus of human HLA class I molecules of cells derived from human hematopoietic stem cells contains at least one antigen whose cell compatible type matches that of the patient. Therefore, when a patient cell expresses a disease-related antigen, such as a cancer antigen or a pathogen-derived antigen, the disease-related antigen is presented to the cytotoxic T cells of the pharmaceutical composition of the present invention together with human HLA class I molecules. . That is, the pharmaceutical composition of the present invention recognizes the disease-related antigen in the context of the allele of the HLA class I molecular locus common to the patient and specifically attacks the patient's cells that express the disease-related antigen.
患者の細胞が発現する疾患関連抗原は、ＷＴ１、チロシナーゼ、ＮＹ－ＥＳＯ－１、ＣＥＡ，ＮＳＥ、ＰＳＡ、ｇｐ１００、ＭＡＲＴ－１及びＭＡＧＥ－３を含むが、これらに限られない腫瘍関連抗原と、ＥＢＥＲ，ＬＭＰ－１等のＥＢウイルス関連抗原のように癌細胞で（過剰に）発現する抗原の場合と、ＣＭＶｇｐ６５等のサイトメガロウイルス特異抗原、ＨＩＶｇｐ１６０等のエイズウイルス特異抗原を含むが、これらに限られないウイルス特異抗原のように感染細胞で発現する抗原の場合とがある。したがって本発明の医薬品組成物は、癌治療に用いられる場合と、感染症治療に用いられる場合とがある。
Disease-related antigens expressed by patient cells include, but are not limited to, WT1, tyrosinase, NY-ESO-1, CEA, NSE, PSA, gp100, MART-1 and MAGE-3, Examples include antigens that are (over-expressed) in cancer cells such as EB virus-related antigens such as EBER and LMP-1, cytomegalovirus-specific antigens such as CMVgp65, and AIDS virus-specific antigens such as HIV gp160. There are cases where the antigen is expressed in infected cells, such as a virus-specific antigen that is not limited. Therefore, the pharmaceutical composition of the present invention may be used for cancer treatment or infectious disease treatment.
以下に説明する本発明の実施例は、例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本明細書で言及されるすべての特許文献及び非特許文献はそれらの全体が引用によって本明細書に取り込まれる。
The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims. All patent and non-patent documents referred to herein are hereby incorporated by reference in their entirety.
以下の実施例で説明する実験は、東京大学医科学研究所の倫理審査委員会によって承認された後に実施された。
The experiments described in the following examples were conducted after approval by the Ethics Review Committee of the Institute of Medical Science, the University of Tokyo.
ヒト臍帯血
ヒト臍帯血試料は、母親のインフォームド・コンセント署名を得た後に採血され、理化学研究所バイオリソースセンター内の液体窒素システムで凍結保存された。採血方法はＲｕｂｉｎｓｔｅｉｎ，Ｐ．ら（Ｂｌｏｏｄ，８１：１６７９－１６９０（１９９３））に説明され、凍結保存方法はＲｕｂｉｎｓｔｅｉｎ，Ｐ．ら（Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ， ９２：１０１１９－１０１２２（１９９５））に説明される。 Human umbilical cord blood Human umbilical cord blood samples were collected after obtaining the informed consent signature of the mother and stored frozen in a liquid nitrogen system in the RIKEN BioResource Center. Blood collection methods are described in Rubinstein, P. et al. (Blood, 81: 1679-1690 (1993)) and the cryopreservation method is described in Rubinstein, P. et al. (Proc. Natl. Acad. Sci. USA, 92: 10119-10122 (1995)).
ヒト臍帯血試料は、母親のインフォームド・コンセント署名を得た後に採血され、理化学研究所バイオリソースセンター内の液体窒素システムで凍結保存された。採血方法はＲｕｂｉｎｓｔｅｉｎ，Ｐ．ら（Ｂｌｏｏｄ，８１：１６７９－１６９０（１９９３））に説明され、凍結保存方法はＲｕｂｉｎｓｔｅｉｎ，Ｐ．ら（Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ， ９２：１０１１９－１０１２２（１９９５））に説明される。 Human umbilical cord blood Human umbilical cord blood samples were collected after obtaining the informed consent signature of the mother and stored frozen in a liquid nitrogen system in the RIKEN BioResource Center. Blood collection methods are described in Rubinstein, P. et al. (Blood, 81: 1679-1690 (1993)) and the cryopreservation method is described in Rubinstein, P. et al. (Proc. Natl. Acad. Sci. USA, 92: 10119-10122 (1995)).
培地及び試薬
培地としてＸ－Ｖｉｖｏ１５（商標、ＴａＫａＲａ Ｂｉｏ、滋賀）が使用された。一部の実験では最終濃度０ないし５％のヒトＡＢ型血清（Ｌｏｎｚａ）が添加された。Ｐｅｐｒｏｔｅｃｈ（Ｒｏｃｋｙ Ｈｉｌｌ，ニュージャージー州）製の組換えヒトインターロイキン－２（ＩＬ－２）が最終濃度０ないし１，５００ＩＵ／ｍＬ添加された。 Medium and Reagent X-Vivo15 (Trademark, TaKaRa Bio, Shiga) was used as the medium. In some experiments, a final concentration of 0-5% human AB serum (Lonza) was added. Recombinant human interleukin-2 (IL-2) from Peprotech (Rocky Hill, NJ) was added at a final concentration of 0 to 1,500 IU / mL.
培地としてＸ－Ｖｉｖｏ１５（商標、ＴａＫａＲａ Ｂｉｏ、滋賀）が使用された。一部の実験では最終濃度０ないし５％のヒトＡＢ型血清（Ｌｏｎｚａ）が添加された。Ｐｅｐｒｏｔｅｃｈ（Ｒｏｃｋｙ Ｈｉｌｌ，ニュージャージー州）製の組換えヒトインターロイキン－２（ＩＬ－２）が最終濃度０ないし１，５００ＩＵ／ｍＬ添加された。 Medium and Reagent X-Vivo15 (Trademark, TaKaRa Bio, Shiga) was used as the medium. In some experiments, a final concentration of 0-5% human AB serum (Lonza) was added. Recombinant human interleukin-2 (IL-2) from Peprotech (Rocky Hill, NJ) was added at a final concentration of 0 to 1,500 IU / mL.
臍帯血由来ＣＤ３陽性細胞の精製
理化学研究所から入手した臍帯血２５ｍＬは３７°Ｃの温水浴で解凍され、Ｆｉｃｏｌｌ－Ｐａｑｕｅ（商標、ＧＥ Ｈｅａｌｔｈｃａｒｅ）上に重層され、室温にて１，５００ｒｐｍ、３０分間遠心された。バフィーコートが回収され、ＰＢＳ中に再浮遊された、得られた白血球はＰＢＳでさらに３回洗浄された。その後、前記白血球懸濁液に細胞１０7個あたり２５μＬのＤｙｎａｌ（商標）免疫磁気ビーズＣＤ１４（Ｉｎｖｉｔｒｏｇｅｎ）が添加され、氷上又は４°Ｃで３０分以上攪拌された。ＣＤ１４陽性細胞と、ビーズを貪食した単球マクロファージとが磁気粒子分離器（ＭＰＣ－１、Ｄｙｎａｌ）を用いて除去された。残りの細胞はＰＢＳ中に再浮遊され、細胞１０7個あたり２５μＬのＤｙｎａｌ（商標）免疫磁気ビーズＣＤ３（Ｉｎｖｉｔｒｏｇｅｎ）が添加され、氷上又は４°Ｃで３０分以上攪拌された。ＣＤ３陽性細胞が磁気粒子分離器（ＭＰＣ－１、Ｄｙｎａｌ）を用いて分離された。 Purification of umbilical cord blood-derived CD3 positive cells 25 mL of umbilical cord blood obtained from RIKEN was thawed in a 37 ° C warm water bath, layered on Ficoll-Paque (trademark, GE Healthcare), and 1,500 rpm, 30 at room temperature. Centrifuged for minutes. The buffy coat was collected and resuspended in PBS. The resulting leukocytes were washed three more times with PBS. Thereafter, 25 μL of Dynal ™ immunomagnetic beads CD14 (Invitrogen) per 10 7 cells was added to the leukocyte suspension and stirred on ice or at 4 ° C. for 30 minutes or more. CD14 positive cells and monocyte macrophages phagocytosed by beads were removed using a magnetic particle separator (MPC-1, Dynal). The remaining cells were resuspended in PBS and 25 μL Dynal ™ immunomagnetic beads CD3 (Invitrogen) per 10 7 cells were added and stirred for 30 min or longer on ice or at 4 ° C. CD3-positive cells were separated using a magnetic particle separator (MPC-1, Dynal).
理化学研究所から入手した臍帯血２５ｍＬは３７°Ｃの温水浴で解凍され、Ｆｉｃｏｌｌ－Ｐａｑｕｅ（商標、ＧＥ Ｈｅａｌｔｈｃａｒｅ）上に重層され、室温にて１，５００ｒｐｍ、３０分間遠心された。バフィーコートが回収され、ＰＢＳ中に再浮遊された、得られた白血球はＰＢＳでさらに３回洗浄された。その後、前記白血球懸濁液に細胞１０7個あたり２５μＬのＤｙｎａｌ（商標）免疫磁気ビーズＣＤ１４（Ｉｎｖｉｔｒｏｇｅｎ）が添加され、氷上又は４°Ｃで３０分以上攪拌された。ＣＤ１４陽性細胞と、ビーズを貪食した単球マクロファージとが磁気粒子分離器（ＭＰＣ－１、Ｄｙｎａｌ）を用いて除去された。残りの細胞はＰＢＳ中に再浮遊され、細胞１０7個あたり２５μＬのＤｙｎａｌ（商標）免疫磁気ビーズＣＤ３（Ｉｎｖｉｔｒｏｇｅｎ）が添加され、氷上又は４°Ｃで３０分以上攪拌された。ＣＤ３陽性細胞が磁気粒子分離器（ＭＰＣ－１、Ｄｙｎａｌ）を用いて分離された。 Purification of umbilical cord blood-derived CD3 positive cells 25 mL of umbilical cord blood obtained from RIKEN was thawed in a 37 ° C warm water bath, layered on Ficoll-Paque (trademark, GE Healthcare), and 1,500 rpm, 30 at room temperature. Centrifuged for minutes. The buffy coat was collected and resuspended in PBS. The resulting leukocytes were washed three more times with PBS. Thereafter, 25 μL of Dynal ™ immunomagnetic beads CD14 (Invitrogen) per 10 7 cells was added to the leukocyte suspension and stirred on ice or at 4 ° C. for 30 minutes or more. CD14 positive cells and monocyte macrophages phagocytosed by beads were removed using a magnetic particle separator (MPC-1, Dynal). The remaining cells were resuspended in PBS and 25 μL Dynal ™ immunomagnetic beads CD3 (Invitrogen) per 10 7 cells were added and stirred for 30 min or longer on ice or at 4 ° C. CD3-positive cells were separated using a magnetic particle separator (MPC-1, Dynal).
臍帯血由来ＣＤ３陽性細胞の培養
前記ＣＤ３陽性細胞は５％ヒトＡＢ型血清を添加したＸＶｉｖｏ１５培地に１０6個／ｍＬの濃度で浮遊され、Ｄｙｎａｂｅａｄｓ ＣＤ３／ＣＤ２８ Ｔ ｃｅｌｌ ｅｘｐａｎｄｅｒ（Ｉｎｖｉｔｒｏｇｅｎ）と、組換えＩＬ－２とを添加して培養された。培養４、７、９、１２及び１４日目にトリパンブルー染色を用いて生細胞数が測定された。組換えＩＬ－２を含む５％ヒトＡＢ型血清を添加したＥｘＶｉｖｏ１５培地で、１０6個／ｍＬの細胞濃度になるように希釈された。 Culture of umbilical cord blood-derived CD3 positive cells The CD3 positive cells are suspended in XVivo15 medium supplemented with 5% human AB type serum at a concentration of 10 6 cells / mL, and recombined with Dynabeads CD3 / CD28 T cell expander (Invitrogen). Incubated with IL-2. Viable cell numbers were measured using trypan blue staining on days 4, 7, 9, 12 and 14 of culture. The cells were diluted with ExVivo15 medium supplemented with 5% human AB type serum containing recombinant IL-2 to a cell concentration of 10 6 cells / mL.
前記ＣＤ３陽性細胞は５％ヒトＡＢ型血清を添加したＸＶｉｖｏ１５培地に１０6個／ｍＬの濃度で浮遊され、Ｄｙｎａｂｅａｄｓ ＣＤ３／ＣＤ２８ Ｔ ｃｅｌｌ ｅｘｐａｎｄｅｒ（Ｉｎｖｉｔｒｏｇｅｎ）と、組換えＩＬ－２とを添加して培養された。培養４、７、９、１２及び１４日目にトリパンブルー染色を用いて生細胞数が測定された。組換えＩＬ－２を含む５％ヒトＡＢ型血清を添加したＥｘＶｉｖｏ１５培地で、１０6個／ｍＬの細胞濃度になるように希釈された。 Culture of umbilical cord blood-derived CD3 positive cells The CD3 positive cells are suspended in XVivo15 medium supplemented with 5% human AB type serum at a concentration of 10 6 cells / mL, and recombined with Dynabeads CD3 / CD28 T cell expander (Invitrogen). Incubated with IL-2. Viable cell numbers were measured using trypan blue staining on
結果
図１に臍帯血由来ＣＤ３陽性細胞の増殖条件の検討結果を示す。図１の縦軸は培養開始時のＣＤ３陽性細胞の総数を１とする増殖倍率を表し、横軸は培養開始後の日数を表す。刺激に用いるＣＤ３／ＣＤ２８免疫ビーズは細胞１個あたりビーズ約２個の割合で用いられた。培地に添加される組換えＩＬ－２及びヒト血清の濃度とに関係なく、いずれの培養条件でも臍帯血由来ＣＤ３陽性細胞は培養９日目までほぼ同様の速度で増殖した。しかし、培養１２日目になると、ＩＬ－２濃度が１，５００ＩＵ／ｍＬで、かつ、ヒト血清が５％の培養条件の細胞だけが増殖速度を維持し、他の培養条件の細胞は増殖速度の低下や細胞数の減少がみられた。培養１４日目になると、ＩＬ－２濃度が１，５００ＩＵ／ｍＬの条件で培養された細胞のみが同じ速度で増殖した。以上に示したとおり、臍帯血由来の白血球から、ＣＤ３陽性のＴ細胞のみを精製し、これに分裂刺激を与えて選択的に増殖することが可能になった。 Results FIG. 1 shows the results of examining the growth conditions of cord blood-derived CD3 positive cells. The vertical axis in FIG. 1 represents the multiplication factor with the total number of CD3 positive cells at the start of culture being 1, and the horizontal axis represents the number of days after the start of culture. CD3 / CD28 immunobeads used for stimulation were used at a rate of about 2 beads per cell. Regardless of the concentrations of recombinant IL-2 and human serum added to the medium, umbilical cord blood-derived CD3-positive cells grew at almost the same rate untilday 9 of culture under any culture condition. However, on the 12th day of culture, only cells under culture conditions with an IL-2 concentration of 1,500 IU / mL and 5% human serum maintain the growth rate, while cells under other culture conditions grow. Decreased and the number of cells decreased. On the 14th day of culture, only cells cultured under conditions of IL-2 concentration of 1,500 IU / mL grew at the same rate. As shown above, only CD3-positive T cells were purified from leukocytes derived from umbilical cord blood, and it was possible to selectively proliferate by applying division stimulation thereto.
図１に臍帯血由来ＣＤ３陽性細胞の増殖条件の検討結果を示す。図１の縦軸は培養開始時のＣＤ３陽性細胞の総数を１とする増殖倍率を表し、横軸は培養開始後の日数を表す。刺激に用いるＣＤ３／ＣＤ２８免疫ビーズは細胞１個あたりビーズ約２個の割合で用いられた。培地に添加される組換えＩＬ－２及びヒト血清の濃度とに関係なく、いずれの培養条件でも臍帯血由来ＣＤ３陽性細胞は培養９日目までほぼ同様の速度で増殖した。しかし、培養１２日目になると、ＩＬ－２濃度が１，５００ＩＵ／ｍＬで、かつ、ヒト血清が５％の培養条件の細胞だけが増殖速度を維持し、他の培養条件の細胞は増殖速度の低下や細胞数の減少がみられた。培養１４日目になると、ＩＬ－２濃度が１，５００ＩＵ／ｍＬの条件で培養された細胞のみが同じ速度で増殖した。以上に示したとおり、臍帯血由来の白血球から、ＣＤ３陽性のＴ細胞のみを精製し、これに分裂刺激を与えて選択的に増殖することが可能になった。 Results FIG. 1 shows the results of examining the growth conditions of cord blood-derived CD3 positive cells. The vertical axis in FIG. 1 represents the multiplication factor with the total number of CD3 positive cells at the start of culture being 1, and the horizontal axis represents the number of days after the start of culture. CD3 / CD28 immunobeads used for stimulation were used at a rate of about 2 beads per cell. Regardless of the concentrations of recombinant IL-2 and human serum added to the medium, umbilical cord blood-derived CD3-positive cells grew at almost the same rate until
癌抗原特異的細胞傷害性Ｔ細胞の増幅
ＣＤ３／ＣＤ２８免疫ビーズで刺激され、５％ヒトＡＢ型血清及び１，５００ＩＵ／ｍＬのＩＬ－２を添加したＥｘＶｉｖｏ１５培地で培養増幅された臍帯血由来ＣＤ３陽性細胞を培養１４日目に回収して、ＡＰＣ標識抗ヒトＣＤ８マウスモノクローナル抗体と、ＰＥ標識ＨＬＡ－Ａ*２４０２ＷＴ１（ｗｉｌｄ）ＣＭＴＷＮＱＭＮＬ－テトラマーとで二重染色し、フロー・サイトメトリー解析を行った。 Amplification of cancer antigen-specific cytotoxic T-cells Umbilical cord blood-derived CD3 stimulated with CD3 / CD28 immunobeads and cultured in ExVivo15 medium supplemented with 5% human AB serum and 1,500 IU / mL IL-2 Positive cells were collected on the 14th day of culture and double-stained with APC-labeled anti-human CD8 mouse monoclonal antibody and PE-labeled HLA-A * 2402WT1 (wild) CMTWNQMNL-tetramer, and flow cytometry analysis was performed. .
ＣＤ３／ＣＤ２８免疫ビーズで刺激され、５％ヒトＡＢ型血清及び１，５００ＩＵ／ｍＬのＩＬ－２を添加したＥｘＶｉｖｏ１５培地で培養増幅された臍帯血由来ＣＤ３陽性細胞を培養１４日目に回収して、ＡＰＣ標識抗ヒトＣＤ８マウスモノクローナル抗体と、ＰＥ標識ＨＬＡ－Ａ*２４０２ＷＴ１（ｗｉｌｄ）ＣＭＴＷＮＱＭＮＬ－テトラマーとで二重染色し、フロー・サイトメトリー解析を行った。 Amplification of cancer antigen-specific cytotoxic T-cells Umbilical cord blood-derived CD3 stimulated with CD3 / CD28 immunobeads and cultured in ExVivo15 medium supplemented with 5% human AB serum and 1,500 IU / mL IL-2 Positive cells were collected on the 14th day of culture and double-stained with APC-labeled anti-human CD8 mouse monoclonal antibody and PE-labeled HLA-A * 2402WT1 (wild) CMTWNQMNL-tetramer, and flow cytometry analysis was performed. .
結果
図２に前記フロー・サイトメトリー解析結果を示す。図の縦軸はＰＥの蛍光強度で、ＨＬＡ－Ａ*２４０２ＷＴ１（ｗｉｌｄ）ＣＭＴＷＮＱＭＮＬ－テトラマーとの反応を表し、横軸はＡＰＣの蛍光強度で、抗ＣＤ８抗体との反応を表す。図２に示すとおり、増幅された臍帯血由来ＣＤ３陽性細胞のうちＣＤ８陽性細胞、すなわち、細胞傷害性Ｔ細胞の約１１．６％は、ＨＬＡ－Ａ*２４０２アリルのコンテキストで癌抗原であるＷＴ１由来ペプチドを特異的に認識した。ＷＴ１特異的な細胞傷害性Ｔ細胞は、リンパ腫その他のＷＴ１を過剰発現する悪性細胞とは反応するが、ＷＴ１を比較的少量しか発現しない正常細胞を攻撃しないことが知られている（Ｇａｏ，Ｌ．ら、Ｂｌｏｏｄ、９５：２１９８－２２０３（２０００）、Ｏｋａ、Ｙ．ら、Ｃｕｒｒ． Ｏｐ． ｉｎ Ｉｍｍｕｎｏｌ．、２０：２１１－２２０（２００８））。 Results FIG. 2 shows the results of the flow cytometry analysis. The vertical axis in the figure represents the fluorescence intensity of PE and represents the reaction with HLA-A * 2402WT1 (wild) CMTWNQMNL-tetramer, and the horizontal axis represents the fluorescence intensity of APC and represents the reaction with anti-CD8 antibody. As shown in FIG. 2, about 11.6% of CD8 positive cells, ie, cytotoxic T cells among the amplified cord blood-derived CD3 positive cells, are WT1 which is a cancer antigen in the context of HLA-A * 2402 allele. The derived peptide was specifically recognized. WT1-specific cytotoxic T cells are known to react with lymphomas and other malignant cells that overexpress WT1, but do not attack normal cells that express only a relatively small amount of WT1 (Gao, L Et al., Blood, 95: 2198-2203 (2000), Oka, Y. et al., Curr. Op. In Immunol., 20: 211-220 (2008)).
図２に前記フロー・サイトメトリー解析結果を示す。図の縦軸はＰＥの蛍光強度で、ＨＬＡ－Ａ*２４０２ＷＴ１（ｗｉｌｄ）ＣＭＴＷＮＱＭＮＬ－テトラマーとの反応を表し、横軸はＡＰＣの蛍光強度で、抗ＣＤ８抗体との反応を表す。図２に示すとおり、増幅された臍帯血由来ＣＤ３陽性細胞のうちＣＤ８陽性細胞、すなわち、細胞傷害性Ｔ細胞の約１１．６％は、ＨＬＡ－Ａ*２４０２アリルのコンテキストで癌抗原であるＷＴ１由来ペプチドを特異的に認識した。ＷＴ１特異的な細胞傷害性Ｔ細胞は、リンパ腫その他のＷＴ１を過剰発現する悪性細胞とは反応するが、ＷＴ１を比較的少量しか発現しない正常細胞を攻撃しないことが知られている（Ｇａｏ，Ｌ．ら、Ｂｌｏｏｄ、９５：２１９８－２２０３（２０００）、Ｏｋａ、Ｙ．ら、Ｃｕｒｒ． Ｏｐ． ｉｎ Ｉｍｍｕｎｏｌ．、２０：２１１－２２０（２００８））。 Results FIG. 2 shows the results of the flow cytometry analysis. The vertical axis in the figure represents the fluorescence intensity of PE and represents the reaction with HLA-A * 2402WT1 (wild) CMTWNQMNL-tetramer, and the horizontal axis represents the fluorescence intensity of APC and represents the reaction with anti-CD8 antibody. As shown in FIG. 2, about 11.6% of CD8 positive cells, ie, cytotoxic T cells among the amplified cord blood-derived CD3 positive cells, are WT1 which is a cancer antigen in the context of HLA-
図２の結果から、臍帯血由来の白血球を特定の表面抗原に基づいて選別し、適切な条件で刺激及び増幅を行うと、特定アリルのＨＬＡ－Ａ拘束性で癌抗原を特異的に認識する細胞傷害性Ｔ細胞が大量に得られることが証明された。
From the results shown in FIG. 2, when leukocytes derived from umbilical cord blood are selected based on a specific surface antigen, and stimulated and amplified under appropriate conditions, the cancer antigen is specifically recognized by HLA-A restriction of the specific allele. It has been demonstrated that cytotoxic T cells can be obtained in large quantities.
本実施例では、ＷＴ１ペプチドは細胞傷害性Ｔ細胞の増殖刺激には用いられなかったのに、ＷＴ１由来ペプチドを特異的に認識する細胞傷害性Ｔ細胞が得られたが、これは、ＷＴ１は臍帯血白血球で発現しており、培養に用いられたリンパ球には樹状細胞に分化するものが含まれるため、培養下で樹状細胞から細胞傷害性Ｔ細胞にＷＴ１が抗原として提示された可能性がある。臍帯血では成人末梢血には存在しない胸腺選択を受ける前の幼弱なＣＤ４陽性ＣＤ８陽性Ｔ細胞集団が存在するが、これは臍帯血に特異的な細胞集団である。ナイーブＴ細胞を抗原特異的に活性化する（プライミング）には樹状細胞が必須であるが、ＷＴ１のような正常細胞でも発現している自己蛋白の場合、既に胸腺選択を受ける前の幼弱なＣＤ４陽性ＣＤ８陽性の自己反応性メモリー細胞が臍帯血中に存在しているため、これらの細胞集団からＷＴ１特異的細胞傷害性Ｔ細胞が増幅されている可能性がある。成人ではこれらの細胞集団は自己反応性クローンとして既に胸腺選択の段階で取り除かれているため、成人末梢血の増幅では得ることが出来ず、臍帯血からのみ調整可能である。本実施例の実験は、臍帯血からのみこの細胞集団が調整可能であることを世界で初めて示したものである。
In this example, although the WT1 peptide was not used for stimulation of proliferation of cytotoxic T cells, cytotoxic T cells specifically recognizing WT1-derived peptides were obtained. Since lymphocytes expressed in umbilical cord blood leukocytes include those that differentiate into dendritic cells, WT1 was presented as an antigen from dendritic cells to cytotoxic T cells under culture there is a possibility. In cord blood, there is a weak CD4 positive CD8 positive T cell population before undergoing thymic selection that is not present in adult peripheral blood, which is a cell population specific for cord blood. Dendritic cells are essential for antigen-specific activation (priming) of naive T cells, but in the case of self-proteins that are also expressed in normal cells such as WT1, juveniles already undergoing thymic selection Since CD4 positive CD8 positive self-reactive memory cells are present in umbilical cord blood, WT1-specific cytotoxic T cells may be amplified from these cell populations. In adults, these cell populations have already been removed as autoreactive clones at the stage of thymus selection and cannot be obtained by amplification of adult peripheral blood, and can only be adjusted from umbilical cord blood. The experiments of this example show for the first time in the world that this cell population can be adjusted only from umbilical cord blood.
Claims (12)
- ヒト造血幹細胞由来の細胞を含む医薬品組成物であって、
前記細胞のヒトＨＬＡクラスＩ分子のいずれか１つの遺伝子座は、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含むこと、及び
前記細胞のヒトＨＬＡクラスＩ及びＩＩ分子の遺伝子座の抗原は、前記細胞の適合型が患者の適合型と一致しない抗原を少なくとも１個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことを特徴とする、医薬品組成物。 A pharmaceutical composition comprising cells derived from human hematopoietic stem cells,
Any one locus of the human HLA class I molecule of the cell contains at least one antigen whose matched type of the cell matches that of the patient, and of the human HLA class I and II molecules of the cell The antigen at the locus comprises at least one antigen whose matched type of the cell does not match that of the patient, and the hematopoietic stem cell-derived cell is in the patient's body, whether it is a permanent engraftment or a severity III. Or the pharmaceutical composition characterized by not causing the acute GVH disease of IV. - 前記医薬品組成物は前記患者のリンパ球が除去された後に投与され、
該医薬品組成物は前記患者の体内で一過的に生着するが前記患者のリンパ球が再増殖するとともに消失することを特徴とする、請求項１に記載の医薬品組成物。 The pharmaceutical composition is administered after the patient's lymphocytes have been removed;
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is temporarily engrafted in the body of the patient but disappears as the lymphocytes of the patient re-grow. - 前記細胞のヒトＨＬＡクラスＩ分子のいずれか１つの遺伝子座はＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＨＬＡ－Ｃからなるグループから選択され、前記細胞のヒトＨＬＡクラスＩＩ分子の遺伝子座はＤＲＢ１であること、及び、
前記細胞のヒトＨＬＡクラスＩ及びＩＩ分子の遺伝子座の抗原は、前記細胞の適合型が患者の適合型と一致しない抗原を少なくとも１個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことを特徴とする、請求項１又は２に記載の医薬品組成物。 The locus of any one of the human HLA class I molecules of the cell is selected from the group consisting of HLA-A, HLA-B and HLA-C, and the locus of the human HLA class II molecule of the cell is DRB1 ,as well as,
The antigen of the human HLA class I and II molecular locus of the cell contains at least one antigen whose compatible type of the cell does not match that of the patient, and the hematopoietic stem cell-derived cell is in the body of the patient The pharmaceutical composition according to claim 1 or 2, wherein neither permanent engraftment nor acute GVH disease of severity III or IV occurs. - 前記細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１の遺伝子座の抗原は、前記細胞の適合型が患者の適合型と一致しない抗原を５個ないし６個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことを特徴とする、請求項３に記載の医薬品組成物。 The antigens at the HLA-A, HLA-B and DRB1 loci of the cells include 5 to 6 antigens whose compatible types of cells do not match those of patients, and the cells derived from the hematopoietic stem cells are 4. A pharmaceutical composition according to claim 3, characterized in that it does not cause permanent engraftment or acute GVH disease of severity III or IV in the body of the patient.
- 前記ヒト造血幹細胞は臍帯血幹細胞であることを特徴とする、請求項１ないし４のいずれか１つに記載の医薬品組成物。 The pharmaceutical composition according to any one of claims 1 to 4, wherein the human hematopoietic stem cells are umbilical cord blood stem cells.
- 前記ヒト造血幹細胞由来の細胞は、ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞であることを特徴とする、請求項１ないし５のいずれか１つに記載の医薬品組成物。 The pharmaceutical composition according to any one of claims 1 to 5, wherein the cells derived from human hematopoietic stem cells are cytotoxic T cells specific for human HLA class I molecule-restricted epitopes. .
- 癌治療用であることを特徴とする、請求項１ないし６のいずれか１つに記載の医薬品組成物。 The pharmaceutical composition according to any one of claims 1 to 6, which is used for cancer treatment.
- 感染症治療用であることを特徴とする、請求項１ないし６のいずれか１つに記載の医薬品組成物。 The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition is used for treating infectious diseases.
- 前記ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞は前記ヒトＨＬＡクラスＩ分子拘束性エピトープによる刺激を受けることなく増幅されることを特徴とする、請求項８に記載の医薬品組成物。 The pharmaceutical product according to claim 8, wherein the cytotoxic T cells specific to the human HLA class I molecule-restricted epitope are amplified without being stimulated by the human HLA class I molecule-restricted epitope. Composition.
- 前記細胞のＨＬＡ－Ａ遺伝子座は、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含むこと、及び、
前記細胞傷害性Ｔ細胞はウィルムス腫瘍原因遺伝子産物（ＷＴ１）に特異的であることを特徴とする、請求項９に記載の医薬品組成物。 The HLA-A locus of the cell comprises at least one antigen whose compatible type of cell matches that of the patient; and
The pharmaceutical composition according to claim 9, characterized in that said cytotoxic T cells are specific for Wilms tumor causative gene product (WT1). - ヒト臍帯血由来の細胞傷害性Ｔ細胞を含む医薬品組成物であって、
前記細胞傷害性Ｔ細胞は、ＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＨＬＡ－Ｃのうちいずれか１種類の遺伝子座のＨＬＡクラスＩ分子拘束性エピトープに特異的であること、
前記ヒトＨＬＡクラスＩ分子拘束性エピトープに特異的な細胞傷害性Ｔ細胞は前記ヒトＨＬＡクラスＩ分子拘束性エピトープによる刺激を受けることなく増幅されること、
前記細胞のいずれか１種類の遺伝子座のＨＬＡクラスＩ分子は、前記細胞の適合型が患者の適合型と一致する抗原を少なくとも１個含むこと、及び、
前記細胞のＨＬＡ－Ａ、ＨＬＡ－Ｂ及びＤＲＢ１の遺伝子座は、前記細胞の適合型が患者の適合型と一致しない抗原を５個ないし６個含み、かつ、前記造血幹細胞由来の細胞は前記患者の体内で、永続的生着も、重症度ＩＩＩ又はＩＶの急性ＧＶＨ病も起こさないことを特徴とする、医薬品組成物。 A pharmaceutical composition comprising cytotoxic T cells derived from human umbilical cord blood,
The cytotoxic T cell is specific for an HLA class I molecule-restricted epitope at any one locus of HLA-A, HLA-B and HLA-C;
Cytotoxic T cells specific for the human HLA class I molecule restricted epitope are amplified without being stimulated by the human HLA class I molecule restricted epitope;
The HLA class I molecule at any one locus of the cell comprises at least one antigen whose matched type of the cell matches that of the patient; and
The HLA-A, HLA-B and DRB1 loci of the cells contain 5 to 6 antigens whose matched type of the cells does not match that of the patient, and the hematopoietic stem cell-derived cells are the patient A pharmaceutical composition characterized in that neither permanent engraftment nor acute GVH disease of severity III or IV occurs in the body. - ヒト臍帯血をＣＤ３／ＣＤ２８免疫ビーズで刺激するステップを含むことを特徴とする、請求項９に記載の医薬品組成物の製造方法。 The method for producing a pharmaceutical composition according to claim 9, comprising a step of stimulating human umbilical cord blood with CD3 / CD28 immunobeads.
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| GB1202719.9A GB2484869A (en) | 2009-08-17 | 2010-08-04 | Pharmaceutical composition containing transiently surviving CTL |
| CN2010800362638A CN102596209A (en) | 2009-08-17 | 2010-08-04 | Pharmaceutical composition containing transiently surviving CTL |
| JP2011527629A JPWO2011021503A1 (en) | 2009-08-17 | 2010-08-04 | Pharmaceutical composition comprising transient engraftment CTL |
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| JP2009-188251 | 2009-08-17 | ||
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| KR (1) | KR20120054018A (en) |
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| JP7413639B2 (en) | 2014-06-11 | 2024-01-16 | ポリバイオセプト ゲーエムベーハー | Lymphocyte expansion using cytokine compositions for active cellular immunotherapy |
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| JP7018316B2 (en) * | 2014-11-05 | 2022-02-10 | メモリアル スローン ケタリング キャンサー センター | How to Select T Cell Lines and Their Donors for Adoptive Cell Therapy |
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| WO2003106682A1 (en) * | 2002-06-12 | 2003-12-24 | 中外製薬株式会社 | Hla-a24-restricted cancer antigen peptide |
| JP2004002312A (en) * | 2002-04-08 | 2004-01-08 | Lymphotec:Kk | Hla matching donor-originating activated lymphocyte to be used in prevention/treatment of tumor/infectious disease and autoimmune disease and preparation mainly composed of the lymphocyte, as well as method for manufacturing the preparation, and kit for preparing the preparation |
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| IL112969A (en) * | 1994-03-17 | 2001-05-20 | Baxter Int | Pharmaceutical compositions for the treatment of cancer comprising allogenic lymphocytes or their combination with a t-cell activator |
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| JP2004002312A (en) * | 2002-04-08 | 2004-01-08 | Lymphotec:Kk | Hla matching donor-originating activated lymphocyte to be used in prevention/treatment of tumor/infectious disease and autoimmune disease and preparation mainly composed of the lymphocyte, as well as method for manufacturing the preparation, and kit for preparing the preparation |
| WO2003106682A1 (en) * | 2002-06-12 | 2003-12-24 | 中外製薬株式会社 | Hla-a24-restricted cancer antigen peptide |
Non-Patent Citations (3)
| Title |
|---|
| "Japanese Cord Blood Bank Network Ishoku Data Kanri Shoiinkai", WAGAKUNI NI OKERU HI KETSUENSHA KAN SAITAIKETSU ISHOKU NO SEISEKI (2007 NENDO KAISEKI KEKKA), 7 September 2010 (2010-09-07), Retrieved from the Internet <URL:https://www.j-cord.gr.jp/ja/wnew/isyokuseiseki2007.pdf> * |
| NAGAYAMA, H. ET AL.: "Transient hematopoietic stem cell rescue using umbilical cord blood for a lethally irradiated nuclear accident victim", BONE MARROW TRANSPLANT, vol. 29, no. 3, 2002, pages 197 - 204 * |
| STRAATHOF, K.C. ET AL.: "Treatment of nasopharyngeal carcinoma with Epstein-Barr virus--specific T lymphocytes", BLOOD, vol. 105, no. 5, 2005, pages 1898 - 1904, XP002717844 * |
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| JP7413639B2 (en) | 2014-06-11 | 2024-01-16 | ポリバイオセプト ゲーエムベーハー | Lymphocyte expansion using cytokine compositions for active cellular immunotherapy |
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| JPWO2011021503A1 (en) | 2013-01-24 |
| GB2484869A (en) | 2012-04-25 |
| CN102596209A (en) | 2012-07-18 |
| GB201202719D0 (en) | 2012-04-04 |
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