GB2484869A - Pharmaceutical composition containing transiently surviving CTL - Google Patents
Pharmaceutical composition containing transiently surviving CTL Download PDFInfo
- Publication number
- GB2484869A GB2484869A GB1202719.9A GB201202719A GB2484869A GB 2484869 A GB2484869 A GB 2484869A GB 201202719 A GB201202719 A GB 201202719A GB 2484869 A GB2484869 A GB 2484869A
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- cells
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- pharmaceutical composition
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- A—HUMAN NECESSITIES
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- A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
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
Title of the Invention: PHARMACEUTICAL COMPOSITION CONTAINING
TRANSIENTLY SURVIVING CTL
Field of the invention
[0001] The present invention relates to a pharmaceutical composition comprising cells derived from a human hematopoietic stem cell, and more particularly to a pharmaceutical composition comprising cytotoxic T cells derived from umbilical cord blood, wherein the cells can recognize a disease-associated antigen presented by an lILA class I molecule of a patient, and the cells derived from the hematopoietic stem cell do neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
Background of the invention
[0002] Umbilical cord blood hematopoietic stem cell transplantation (hereinafter, referred to as UCBHCT) is an effective therapy for child and adult patients suffering from leukemia such as acute myeloid leukemia; hematopoietic disorder such as aplastic anemia; congenital irnmunodeficiency; inborn error of metabolism; and EBV infection (Non-Patent Document 1).
In many countries, an umbilical cord blood bank has been established, storing frozen umbilical cord bloods with laboratory data of histocompatibility. UCBHCT is thus the currently most popular stem cell transplant therapy. UCBHCT is generally known to succeed in most transplantation cases of child patients having an BLA type in which four to six out of six antigens encoded on HLA-A, HLA-B, and DRB1 loci are matched with an unrelated umbilical cord blood, or zero-to two-antigen mismatch (Non-Patent Documents 2 and 3). Transplanted cells survive permanently in the host patient.
[0003] Adoptive cell transfer (hereinafter, referred to as ACT) currently has attracted attention as the most effective treatment of cancer (Non-Patent Document 4). ACT is a therapeutic method comprising of culturing patient's (autologous) tumor-infiltrating lymphocytes or patient's lymphocytes sensitized in vitro by a disease-associated antigen in the presence of Interleukin-2; and transplanting the lymphocytes to the patient after the patient s lymphocytes are suppressed. For example, objective cancer regression has been reported in approximately 50% of patients with metastatic melanoma by transfusion of autologous lymphocytes using tumor-infiltrating lymphocytes.
Prior Art Document
Non-Patent Document [0004] Non-Patent Document 1: Broxmeyer, H. E. andSmith, F. 0., "Thomas' Hematopoietic Cell Transplantation," 4th ed., Appelbaum, F.R. et al. eds.., Blackwell Publishing, ISBN:978l405153485 (2009), Chapter 39, pp 559-576 Non-Patent Document 2: Gluckrnan, E. and Rocha, V.,, Curr.
Opin. Immunol.,18;565-570 (2006) Non-Patent Document 3: Brunstein, C. G. et al., Br. J. Hasmatol., 137:20-35 (2007) Non-Patent Document 4: Rosenberg, S. A. et al., Nature Reviews Cancer 8, 299-308 (2008)
Summary of the invention
Problem to be solved by the Invention [0005] In some cases a donor umbilical cord blood suitable for transplantation cannot be found for a patient, even though one-or two-antigen mismatch on BLA loci is permissible in UCBHCT.
In other cases, mismatching lILA loci of the donor umbilical cord blood may fall under an incompatible combination of lILA types which is involved in severe GVH diseases and surviving. Further, even if permanent surviving of transplanted cells in the patient is successfully achieved by UCBI-ICT, the patient then may develop a graft-versus-host disease (hereinafter, referred to as "GVH disease"). In addition, UCEHCT has a higher risk of developing leukemia caused by transplanted cells (i.e., donor cell leukemia) than bone-mallow transplantation and peripheral blood stem cell transplantation.
[0006] In ACT, lymphocytes for transplantation must be prepared for individual patient. The procedure is highly labor-intensive and requires a professional skill. Also in ACT, there is a risk of developing leukemia caused by transplanted cells.
[0007] Further, as the cells for transplantation are prepared for a particular patient in UCBHCT and ACT, there is a risk of propagating infections due to transplantation of cells without being able to detect a pathogen such as prion that are hardly
detectable.
[0008] Therefore, there is a need for developing transplantation cells for immunotherapy, that are applicable to a wider range of patients than conventional transplantation cells for immunotherapy, and that can survive transiently and do not develop severe GVH disease.
Means for Solving Problem [0009] The present invention provides a pharmaceutical composition comprising cells derived from a human hematopoietic stem cell. In the pharmaceutical composition of the present invention, one locus of human HLA class I molecule of the cells has at least one antigen match with a patient. Further, at least one locus of human liLA class I and II molecules of the cells has at least one antigen mismatch with the patient, and the cells derived from the human hematopoietic stem cell do neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
[00101? The pharmaceutical composition of the present invention may be administered after the patient's lymphocytes are suppressed, and the pharmaceutical composition may survive transiently in the body of the patient, but may disappear as the patient's lymphocytes repopulate.
[0011] In the pharmaceutical composition of the present invention, the locus of human HLA class I molecule of the cells may be liLA-A or liLA-B; the locus of human liLA class II molecule of the cells may be DRB1; at least one locus of liLA-A, HLA-8, HLA-C and DRB1 may have at least one antigen mismatch with the patient; and the cells derived from the human hematopoie tic stem cell may neither survive permanently nor develop an acute GVF! disease having a severity of III or IV in the body of the patient.
[0012J In the pharmaceutical composition of the present invention, HLA-A, liLA-B, and DRB1 loci of the cells may have five or six antigen mismatch with the patient, and the cells derived from the human hematopoietic stem cell may neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
[0013] In the pharmaceutical conposition of the present invention, the human hematopoietic stem cell maybe an umbilical cord blood stem cell.
[0014) In the pharmaceutical composition of the present invention, the cells derived from the human hematopoietic stem cell may be cytotoxic T cells specific to an epitope restricted by a human HLA class I molecule.
[0015] The pharmaceutical composition of the present invention may be for treating cancer.
[0016] The pharmaceutical composition of the present invention nay be for treating an infectious disease.
[0017] In the pharmaceutical composition for treating cancer of the present invention, the cytotoxic T cells specific to the epitope restricted by the human HLA class I molecule may be amplified without stimulation by the epitope restricted by the human lILA class I molecule.
[0018) In the pharmaceutical composition for treating cancer of the present invention, the HLA-A locus of the cells may have at least one antigen match with the patient, and the cytotoxic T cells may be specific to the Wilrn's tumor gene product (WT1).
[0019] The present invention provides a pharmaceutical composition comprising cytotoxic T cells derived from human umbilical cord blood. In the pharmaceutical composition comprising cytotoxic T cells of the present invention, the cytotoxic T cells are specific to an epitope restricted by a human lILA class I molecule encoded on any one locus of the LiLA-A, lILA-B and HLA-C; the cytotoxic T cells specific to the epitope restricted by the human HLA class I molecule are amplified without stimulation by the epitope restricted by the human ElLA class I molecule; at least one locus of human LiLA class I molecule of the cells has at least one antigenrnatchwithapatient; LiLA-A, lILA-B and DRB1 loci of the cells have five or six antigen mismatch with the patient; and the cells derived from the human hematopoietic stem cell do neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
[0020] The present invention also provides a method for producing the pharmaceutical composition for treating cancer according to the present invention, in which the cytotoxic T cells specific to the epitope restricted by the human HLA class I molecule are amplified without stimulation by the epitope restricted by the human liLA class I molecule. The method for producing the pharmaceutical composition comprises a step of stimulating human umbilical cord blood with C03/CD28 immunobeads.
[0021] The present invention also provides a method for treating an infectious disease or cancer. The method of the present invention comprises steps of preparing cells derived from a human hematopoietic stem cell, and transplanting the prepared cells in a patient, wherein one locus of human HLA class I molecule of the cells has at least one antigen match with a patient, at least one locus of human ElLA class I and II molecules of the cells has at least one antigen mismatch with the patient, and the cells derived from the human hematopoietic stem cell do neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
[0022] The method for treating an infectious disease or cancer of the present invention may further comprise a step of suppressing the patient's lymphocytes before the step of transplanting the cells in the patient, wherein the cells derived from the human hematopoietic stem cell may survive transiently in the body of the patient, but may disappear as the patient's lymphocytes repopulate. 0023]
In the method for treating an infectious disease or cancer of the present invent ion, the locus of human LiLA class I molecule of the cells may be selected from a group consisting of I-ILA--A, lILA-B and HLA-C; the locus of human LiLA class II molecule of the cells may be DRE1; at least one locus of human lILA class I and II molecules of the cells may have at least one antigen mismatch with the patient; and the cells derived from the human hematopoietic stem cell may neither survive permanently nor develop an acute GVH disease having a severity of III or Iv in the body of the patient.
[0024] In the method for treating an infectious disease or cancer of the present invention, lILA-A, lILA-B, and DRB1 loci of the cells may have five or six antigen mismatch with the patient and the cells derived from the human hematopoietic stem cell may neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
[0025 11 In the method for treating an infectious disease or cancer of the present invention, the human hematopoietic stem cell may be an umbilical cord blood stem cell.
[0026) In the method for treating an infectious disease or cancer of the present invention, the cells derived from the human hematopoietic stem cell may be cytotoxic T cells specific to an epitope restricted by a human BLA class I molecule.
[0027) In the method for treating an infectious disease or cancer of the present invention, the step of preparing cells derived from human hematopoietic stem cell comprises a step of amplifying the cytotoxic T cells specific to the epitope restricted by the human liLA class I molecule without stimulation by the epitope restricted by the human HLA class I molecule.
[0028) In the method for treating an infectious disease or cancer of the present invention, the step of amplifying the cytotoxic T cells specific to the epitope restricted by the human HLA class I molecule without stimulation by the epitope restricted by the human liLA class I molecule comprises a step of stimulating human umbilical cord blood with C03/CD28 immunobeads.
[0029) In the method for treating an infectious disease or cancer of the present invention, the liLA-A locus of the cells derived from human henatopoietic stem cell may have at least one antigen match with the patient( and the cytotoxic T cells may be specific to the Wilm's tumor gene product (WT1).
[0030] In the method for treating an infectious disease or cancer of the present invention, the cytotoxic T cells derived from human umbilical cord blood may be specific to an epitope restricted by a human HLA class I molecule encoded on any one locus of the ElLA-A, ElLA-B and lILA-C; the cytotoxic T cells specific to the epitope restricted by the human HLA class I molecule may be amplified without stimulation by the epitope restricted by the human ElLA class I molecule; at least one locus of human HLA class I molecule of the cells may have at least one antigen match with a patient; ElLA-A, HLA-B and DRB1 loci of the cells may have five or six antigen mismatch with the patient; and the cells derived from the human hematopcietic stem cell may neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
[0031] The technical scope of the present invention is defined by the description of appended claims. The present invention can be modified, for example, by adding, deleting, or substituting a constituent element of the present invention without departing from the spirit of the present invention.
[0032] The human hematopoietic stem cells used in the present invention are derived from tissues including but not limited to, peripheral blood after administration of G-CSF as well as umbilical cord blood and bone marrow. A preferred origin of the human hematopoietic stemcells is umbilical cordblood. The hematopoietic stem cells may also be derived from embryonic stem cells, adult stem cells or induced pluripotent stem tiPS) cells.
[0033] The cells derived from the human hematopoietic stem cell of the present invention include T cell, MX cell, dendritic cell, B cell, macrophage, and granulocytes such as neutrophil and eosinophil. The cells derived from the human hematopoietic stem cell of the present invention may be produced through inducing differentiation of hematopoietic stem cells in peripheral blood after administration of G-CSF as well as umbilical cord blood and bone marrow, or directly from embryonic stem cells, adult stem cells or induced pluripotent stem (iPS) cells through inducing differentiation thereof.
[0034] Umbilical cord bloods and other tissues containing hematopoietic stem cells may be cryopreserved after having been determined HLA types of antigens encoded on HLA class I and II loci. Once the patient's BLA types of these antigens are determined, the cells derived from the human hematopoietic stem cell according to the present invention is produced by thawing the tissue which meets the requirements of the present invention; and culturing with proliferation stimulation.
Pdternatively, cells derived from the human hematopoie tic stem cell according to the present invention may have been produced by culturing with proliferation stimulation and cryopreserved beforehand. In the latter case, it is possible to administer cells with confirmed efficacy, as the cells have been amplified in the large scale beforehand. In the latter case, it is also possible to administer cells of uniform quality to one or more patients.
[0035] HLA class I molecules are comprised of three types: lILA-A, LILA-B, and LILA-C. Genes on the LILA class I loci and gene products therefrom each are highly polymorphic. LILA class I molecules present peptide antigens to cytotoxic T cells, and also have a role as the underlying irnmunogen for distinguishing "self" and "non-self" in allograft to induce an J4VG reaction in which host cells attack transplanted cells and a GVH reaction in which the transplanted cells attack the host cells. Viewing HLA class I molecules from the perspective of immunogen involved in histocompatibility in the allograft, on each locus in human, two gene products encoded by two chromosomes derived from a mother and a father respectively are recognized as two different antigens. In other words, each locus of human LILA class I molecule has two antigens and entire loci of human LILA class I molecules have six antigens. For representing the degree of HLA compatibility, a difference of only one antigen on a locus is referred to as "one antigen mismatch," and a difference of one or two antigens on a locus is referred to as "one locus mismatch". That is, the one locus mismatch includes one antigen mismatch and two antigen mismatch. Similarly, "two loci mismatch" includes two to four antigen mismatches. Five or six antigens on three bc! mismatch means all three bc! mismatch.
[003611
In the present specification, the lILA type can be
determined by serological or DNA testing. The DNA testing includes methods of middle resolution such as a fluorescent bead method (PCR-rsso) and of high resolution such as SET. An lILA antigen has an allele name represented by four digits, as well as by six digits for distinguishing base substitutions within the translating region, and by eight digits for distinguishing base substitutions in untranslated regions. An allele name with the six digits or the eight digits represents a polymorphism without change of an amino acid sequence and can be considered to be the immunologically equivalent to an allele name with four digits.
[0037] lILA class II molecules include three heterodimers: lILA-OR, F{LA-DP, and HLA-DQ. Each heterodimer is composed of an alpha and a beta chains. These chains are encoded on adjacent loci on a chromosome. Only the beta chain of lILA-DR has two loci.
There are thus seven loci of lILA class II molecule: DRA, DRB1, DRB2, DPA, DPE, DQA and DQB. HLA class II molecules present peptide antigens to helper T cells, and similarly to lILA class I molecules, also have a role as the underlying imrnunogen for distinguishing "self" and "non-self" in allograft to induce an HVG reaction in which host cells attack transplanted cells and a GVH reaction in which the transplanted cells attack the host cells. Genes on the loci of HLA class II molecule and gene products therefrom respectively are highly polymorphic similar to HLA class I molecules.
[0038] As used herein, the "transiently survive" refers that transplanted cells can be detected in the body of a host, such as in peripheral blood, one month after the transplantation, but cannot be detected after three, six, twelve or eighteen months. The "permanently survive" refers that transplanted cells can be detected in the body of a host as long as the host survives. Detecting of transplanted cells can be carried out by genetic tests such as ECR; chromosomal tests such as FISH and chromosomal analysis; and immunohistochemical tests such as flow cytonetry with anti-HLA. antibody.
[0039] As used herein, the "acute GVH disease having a severity of III or IV" refers that rated to the severest stage or the second severest stage on the severity scale of I to IV proposed by Prezepiorka, D. et al., at Consensus Conference on Acute GVHD Grading (1994) and described in Bone Marrow Transplant., 15:825-855 (2002) . In cases of a patient developing a GVH disease, before transplantation of the pharmaceutical composition of the present invention, blood is taken from the patient and processed by leukapheresis to purify and collect lymphocytes. The lymphocytes are cryopreserved, and when needed, are amplified by stimulation with interleukin 2 and/or CD3/CD28 and returned into the patient for treatment (autologous lymphocyte infusion).
[00401 Mismatching of antigens encoded on ULA class I and II loci are known to have effects at various degrees on transplantation of hematopoietic stem cells depending on the locus and the number of stem cells transplanted or a dose thereof.
Transplantation is generally known to succeed in most cases when child patient's HLA-A, HLA-B and DRB1 loci have four to six antigen match with an unrelated umbilical cord blood, or zero to two antigen mismatch (Japanese Cord Blood Bank Network, Transplantation Data Management Subcommittee, Outcomes of unrelated umbilical cord blood transplantation in Japan result of analyst F. Y. 2007", https://www.j-corchgr.jp/ja/wnew/isyokuseiseki2007.pdf; Gluckman, E. and Rocha, V., Curt. Opin. Immunoi., 18:565-570(2006); Brunstein, C.G. et al., Br. J. Haematol., 137:20-35(2007)). The report of Japanese Cord Blood Bank Network Transplantation Data Management Subcommittee shows data in which young adult ALL patients transplanted with umbilical cord bloods of one to tour antigen mismatch had a rate of 500 days event-free survival (EFS) from the transplantation of 20 to 56%, while two patients transplanted with an umbilical cord blood of five-antigen mismatch had the rate of 0 (Figs.
to 35). These results indicate that transplanted cells having five antigen mismatch with a host do neither survive permanently nor develop an acute GVH disease having a severity of III or IV. There is another report that in a severe neutron-exposed patient, when an umbilical cord blood having only one antigen mismatch on the DRB1 locus was transplanted, monocytes derived from the umbilical cord blood disappeared from peripheral blood and bone marrow 51 days after the transplantation (Nagayama, I-I. et al., Bone Marrow Transplant., 29:197-204 (2002)). The result indicates that transplanted cells having at least one antigen mismatch with a host do neither survive permanently nor develop an acute GVH disease having a severity of III or IV.
[0041] Therefore, for at least one locus of HLA-A, HLA-B and DRB1 of the cells of the present invention having at least one antigen mismatch with a patient, those skilled in the art of umbilical cord blood transplantation can determine conditions that transplanted cells derived from umbilical cord blood do neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient. HLA-A, HLI1⁄2-B, and DRB1 loci of the cells of the present invention preferably have four, five or six antigen mismatch with the patient.
(0042] The pharmaceutical composition of the present invention can be transplanted in a dramatically wider range of patients than ever before, even when it contains cells derived from one donor. In addition, cells with efficacy such as cytotoxic T cells specific for various disease-associated antigens can be prepared from a single umbilical cord blood. Therefore, a smaller library than conventional umbilical cord blood bank and bone marrow bank is enough to treat various diseases with a large number of patients.
(0043] The cells derived from the hematopoietic stem cell of the present invention may be comprised of cell types that can differentiate into a dendritic cell or a cytotoxic T cell.
Examples of the cell type include, but not limited to, common lyinphoid progenitor, dendritic cell, double negative T cell expressing neither CD4 nor 0DB, double positive T cell expressing both CD4 and 008, 0D8-positive T cell, as well as common progenitor for myeloid and lymphoid, common progenitor for rnyeloid and T cell, and common progenitor for macrophage and T cell, which have been proposed by Katsura and Kawamoto.
[0044] For preparing the cells derived from a hematopoietic stem cell of the present invent ion, various procedures known to those skilled in the art can be used. For example, leukocytes can be separated from an umbilical cord blood by Ficoll gradient centrifugation. For separation of T cells from the leukocytes, irnmunomagnetic beads can be used, including, but not limited to Dynal bead (trade mark) from Invitrogen and C1InIMACS (trade mark) fromMilteriyBiotech. The immunomagnetic beads separate and purify cells expressing the cell surface antigen(s) CD3, C014 and/or CD28. For culturing the resultant cell, serum-free media suitable for culturing hematopoietic cells such as lymphocyte, including XVivol5 medium can be used. The culture medium may be supplemented with human AB serum (0 to 15%) available from BioWhittaker or the like or with human donated blood serum albumin (0 to 10%) available from Japanese Red Cross Society. The culture medium may also be supplemented with a cell growth factor such as, but not limited to, interleukin 2 (0 to 10,000 ItJ/mL), interleukin 15 (0 to 100 ng/mL), and/or interleukin 21 (0 to 100 ng/mL), which are available from Peprotech or the like.
[0045] CD3/CD28 beads from Invitrogen or the like may be used as a proliferation stimulator. In this casey the cells derived from human hematopoietic stem cell of the present invention can be stimulated for proliferation without an epitope restricted by a human HLA class I molecule. Alternatively, the cells of the present invention may be stimulated for proliferation with an epitope restricted by a human HLA class I molecule. Examples of the epitope restricted by a human MbA class I molecule include cancer-specific peptides such as an oligopeptide having an amino acid sequence CMTWNQMNL (SEQ. ID NO: 1) (HLAA*24D2restricted WTJ.-derived peptide fragment), an oligopeptide having an amino acid sequence ADVEFCLSL (SEQ. ID NO: 2) or SADVEFCLSL (SEQ. ID NO: 3) (HLAB*4OO2restricted tyrosinase-derived peptide fragment), and an oligopeptide having a sequence from an eighty-first to a hundred-tenth amino acid residues of a cancer antigen NY-ESO---1 (HLAS*35Ol_ or HLA_C*O3Q4restricted peptide fragment) and viral proteins and peptide fragments thereof such as of ES virus, cytomegalovirus, and AIDS virus. The cells derived from human hematopoietic stem cell of the present invention such as cells expressing the cell surface antigen(s) CD3, CD14 and/or C028 purified from leukocytes in an umbilical cord blood can be stimulated for proliferation with the epitope restricted by a human HLA class I molecule through culture in a medium comprising such a protein or peptide. Proliferation stimulation with the epitope restricted by a human LiLA class I molecule can also be performed, but not limited to, through co-culture with dendritic cells derived from an umbilical cord blood, that dendritic cells are co-cultured with a lysate of cancer cells or cells infected with a virus or other pathogen.
[0046] Stimulated cells for proliferation may be cultured at 31°C in the presence of 5 to 7% CO2. Cells may be seeded at a concentration of 1 x 106 cells/mL. A culture medium may be replaced after two to four days from seeding. Cultured cells may again be seeded on a new medium at a concentration of 1 x 106 cells/mL. For mass culture of the total of not less than 1 x 108 cells, apparatuses such as WAVE Bioreactor 2/10 (trade mark) from GE Healthcare may be used.
[0047] The pharmaceutical composition of the present invention may comprise any pharmaceutically acceptable ingredient for cellular preparation to be transplanted in a blood vessel of a patient, as well as the cells derived from a human hematopoietic stem cell. In cases of transporting the pharmaceutical composition of the present invention in a frozen state, the pharmaceutical composition may comprise a liquid for cryopreservation in such amount as that a thawed composition comprises effective cytotoxic T cells in a satisfactory amount as a cellular preparation for transplantation.
[0048] The pharmaceutical composition of the present invention may be transplanted after the patient's lymphocytes are suppressed. The reasons of suppression are, similar to general Adoptive Cell Transfer (ACT): that is, to avoid competition between cells in the pharmaceutical composition and lymphocytes of the patient over cytokines necessary for proliferation (interleukin 7, interleukin 15, and the like); and to prevent reduction of activity of cells in the pharmaceutical composition through cell interactions with lymphocytes of the patient. A method of suppressing lymphocytes from the patient includes, but not limited to, administration of an agent such as cyclophosphamide (e.g., 60mg/kg, for two days), fludarabine (e.g., 25 mg/m2, for five days) and/or radiation (e.g., 2 or 12 Gy), as well as administration of interleukin 2 (7.2 x iü ID/kg every eight hours for two to three days).
[0049] As shown in Examples, the pharmaceutical composition of the present invention comprises cytotoxic T cells specific for a cancer antigen WT1-derived peptide restricted by HLA-A of an umbilical cord blood cell. The pharmaceutical composition thus can attack cancer cells overexpressing WT1 in a transplanted patient having at least one antigen match with the umbilical cord blood on the LiLA-A locus.
[00501 Other than those described above, cytotoxic T cells have been known that cytotoxic T cells specific to other antigens can be amplified in vitro and that the amplified cytotoxic T cells exhibit a clinical efficacy in a transplanted patient.
For example, Godet, Y. et al., produced cytotoxic T cells specific to HLA-B-restricted peptide derived front a melanoma-specific cancer antigen tyrosinase (Cancer Immunol. Immunother., 58: 271-280 (2009)). Straathof, K. C. N. etal., demonstrated clinical effects of CTL lines specific to an epitope restricted by HLA-B of immunogenic EB viral antigen LMP2, an epitope restricted by HLA-B of immunogenic ES viral antigen EBNA1, an epitope restricted by HLA-B of immunogenic ES viral antigen EBNA3, an epitope restricted by HLA-B of immunogenic ES viral antigen BZLF1 by transplanting the CTLs in patients with nasopharyngeal carcinoma associated with ES virus (Blood, 105: 1898-1904 (2005)). Walter, E. A. et al., demonstrated clinical effects of cytomegalovirus (CMV)-specific CTLs by transplanting the CTLs in patients who had reactivated OW/s during administration of an immunosuppressive agent after transplantation of bone marrow from a CMV-positive related donor for leukemia therapy (N. Engl. J. Med., 333: 1038-44 (1995)) [0051] Bioley G. et a!., reported that vaccination with a cancer antigen NY-ESO-l in a patient provided cytotoxic T cells specific to an HLA-B--or HLA-C---restricted peptide derived from the antigen in the patient (Clin. Cancer Res. 15: 299-306 (2009)) . Cytotoxic T cells thus may be specific to an epitope restricted by HLA-B or HLA-C, as well as to an epitope restricted by HLA-P such as a WT1 protein-derived peptide.
[005211 In the pharmaceutical composition of the present invention, one locus of human lILA class I molecule of the cells derived from human hematopoietic stem cell has at least one antigen match with a patient. When a cell of the patient expresses a disease-associated antigen such as a cancer antigen and a pathogen-derived antigen, the disease-associated antigen is presented together with human HLA. class I molecules to the cytotoxic T cells in the pharmaceutical composition. The pharmaceutical composition of the present invention thus specifically attacks the cell of the patient expressing the disease-associated antigen by recognizing the antigen through a context of a common locus allele of the 1-ILA class I molecule.
[0053] Examples of the disease-associated antigen expressed by a cell in the patient include, but not limited to, tumor-associated antigens such as WT1, tyrosinase, NY-ESO-l, CEA, NSE, PSA, gplOO, MART-i, and MAGE-3, antigens (over)expressed in a cancer cell such as LB viral antigens (e.g., LEER and LMP-l), cytomegalovirus-specific antigens such as CMVgp65, AIDS virus-specific antigens such as flIVgpl6O, and other virus-specific antigens expressed in an infected cell.
The pharmaceutical composition of the present invention thus can be used for treating cancer or an infectious disease.
Brief Description of the Drawings
[00541 Fig. 1 is a graph showing results on proliferation conditions for CD3-positive cells derived from urribilical cord blood.
Fig. 2 is a flow cytometry histogram of CD3-positive cells derived from an umbilical cord blood, that are stimulated with immunobeads, cultured for 14 days to be amplified, and double stained with an Aft-labeled anti-human CDB mouse monoclonal antibody and a PE-labeled HLA-At24O2WT1 (wild) CMTWNQMNL-tetramer.
Modes for carrying out the invention [0055] Examples described below are intended only to illustrate and not to limit the scope of the present invention. The scope of the present invention should be limited only by the description of Claims. All Patent Documents and Non-Patent Documents cited herein are entirely incorporated herein by reference.
[0056] Experiments described in Examples below were performed after approval by the Research Ethics Committee of Institute of Medical Science, University of Tokyo.
Example J
[0057] Human umbilical cord blood A human umbilical cord blood sample was taken from a mother who had given written informed consent, the sample being cryopreserved in the liquid nitrogen system of RIKEN BioResource Center. Blood sampling was carried out as described in Rubinstein, P. et al., (Blood, 81:1679-1690 (1993)), and a procedure of cryopreservation was carried out as described inRubinstein, P. etal., (Proc. Natl. Acad. Sci. USA, 92:10119-10122 (1995)).
[0058) Culture medium and reagent * A culture medium used was X-VivolS (trade mark, TaFtaRa Bio, Shiga). In some experiments, to the medium was supplemented with human AB serum (Lonza) at a final concentration of 0 to 5%. A recombinant human interleukin-2 (IL-2) available from Peprotech (Rocky Hill, New Jersey) was added at a final concentration of 0 to 1,500 IU/mL.
[0059] Purification of CD3-positive cells derived from an umbilical cord blood ruL of the umbilical cord blood obtained from RIKEN was thawed in a warm bath of 37°C, layered on Ficoll-Paque (trade mark, GE Healthcare), and centrifuged for 30 minutes at 1,500 rpm in a room temperature. The buffy coat was collected and re-suspended in PBS. The resultant leukocytes were further washed three times with PBS. Then, to a suspension of the leukocytes was added 25 jaL of Dynal (trade mark) immunomagnetic bead CD14 (Invitrogen) per jQl leukocytes, and agitated for 30 minutes or longer on ice or at 4°C. COil-positive cells and monocyte-macrophages phagocytizing the bead were removed with a magnetic particle concentrator (MPC-1, Dynal). The remaining cells were re-suspended in PBS. To this was added 25.iL of Dynal (trade mark) immunomagnetic bead CD3 (Invitrogen) per io cells, and agitated for 30 minutes or longer on ice or at 4°C. CD3-positive cells were separated with a magnetic particle concentrator (MPC-1, Dynal).
[0060] Cultivation of CD3-positive cells derived from the umbilical cord blood The CD3-positive cells were suspended at a concentration of iO cells/mL in an XVivol5 medium supplemented with 5% human KB serum, and cultured in the presence of Dynabeads CD3/CD2S T cell expander (Invitrogen) and recombinant IL-2. On the days 4, 7, 9, 12, and 14 of cultivation, the number of living cells was counted by trypan blue staining. The culture was diluted in an ExVivoiS medium supplemented with 5% human AB serum comprising recombinant IL-2 such that the concentration of cells was set at 106 cells/mL.
[0061] Result Fig. 1 shows results on proliferation conditions for CD3-positive cells derived from the umbilical cord blood. The ordinate of Fig. 1 represents a proliferation rate normalized by the number of the total CD3-positive cells at the start of cultivation, set to I. The abscissa represents number of days from the start of cultivation. CD3/C028 iramunobeads for stimulation were used at about two beads per cell. Regardless of concentrations of the recombinant IL-2 and the human serum added in the medium, under all conditions, these C03-positive cells proliferated at almost same rate for nine days. However at day 12, only cells cultured under the conditions at IL-2 concentration of 1,500 IU/mL and 5% of human serum kept a proliferation rate, but the cells under other conditions showed a decrease in a proliferation rate andlor the number of cells.
At day 14, only cells cultured under the condition of 1,500 IU/mL of IL-2 proliferated at the same rate. As shown above, it is now demonstrated to be feasible to isolate and purify only CD3-positive T cells from leukocytes derived from an umbilical cord blood and to selectively proliferate these cells by mitotic stimulation.
Example 2
[0062] Amplification of cancer antigen-specific cytotoxic T cells CD3-positive cells derived from the umbilical cord blood were stimulated with CD3/0D28 iromunobeads, cultured and amplified in an ExVivolS medium supplemented with 5% human AS serum and 1,500 IU/mL of IL-2, and collected on day 14 of cultivation. The cells were double stained with anAPC-labeled anti-human CDB mouse monoclonaJ. antibody and a PE-labeled HLA_A*2402WTl (wild) CMTWNQMNL-tetramer, and analyzed by flow cytome try.
[0063] Results Fig. 2 shows a flow cytometry histogram of the cells. The ordinate represents an intensity of PE fluorescence resulting from a reaction with the HLAA*2402WT1 (wild) CMTWNQMNL-tetramer, and the abscissa represents an intensity of APC fluorescence resulting from a reaction with the anti-CD8 antibody. As shown in Fig. 2, about 11.6% of 0DB-positive cells or cytotoxic T cells in the amplified 0D3-positive cells derived from the umbilical cord blood specifically recognized a cancer antigen WT1-derived peptide, through a context of FiLA_A*2402 allele. A WT1-specific cytotoxic T cell is known to react with a cell overexpressing WT1 such as a lymphoma cell, but not attack to a normal cell expressing WT1 in a relatively small amount (Gao, L. etal, Blood, 95:2198-2203 (2000), Oka, Y. etal, Curr.
Op. in Immunol., 20:211-220 (2008)).
[0064] The results in Fig. 2 demonstrates that leukocytes derived from umbilical cord blood can be selected based on a specified surface antigen, and stimulated and amplified under appropriate conditions to generate a large amount of cytotoxic T cells specifically recognizing a cancer antigen restricted by a specific HLA-A allele.
[0065] In Example 2, without WT1 peptide for stimulating cytotoxic T cells for proliferation, cytotoxic T cells specifically recognizing a WT1-derived peptide were obtained.
This suggests the possibility that WT1 was expressed in leukocytes in the umbilical cord blood, and lymphocytes used for proliferation included those differentiating into dendritic cell, and hence dendritic cells presented WT1 as an antigen to cytotoxic T cells during cultivation. Umbilical cord blood comprises a population of immature CD4-positivefCD8-positive T cells before thymic selection, and the cell population does not exist in adult peripheral blood.
The cell population is specific to the umbilical cord blood.
To activate naive T cells antigen-specifically (priming), dendritic cells are indispensable. However, in cases of such a self protein expressed also in normal cells as WT1, umbilical cord blood contains immature CD4-positive/CDS-positive autoreactive memory cells before thymic selection, and thus, there is a possibility that WT1-specific cytotoxic T cells are amplified from the cell population. In the adult, the cell population cannot be obtained by amplifying adult peripheral blood, but can be prepared only from the umbilical cord blood, because the cell population has already removed at the stage of thymic selection as auto reactive clones. Experiments of Example 2 demonstrate that the cell population can be prepared only from the umbilical cord blood for the first time in the EFSLIVE-#10278330-vl-Sequence_l i sting_for_fl ii ng. TXT
SEQUENCE LISTING
<110> THE UNIVERSITY OF TOKYO <120> Pharmaceutical Composition Comprising Transiently Surviving CTL <130> TEK/FP6810899 <140> <141> 2010-08-04 <151> 2010-08-04 <150> JP 2009-188251 <151> 2009-08-17 <160> 3 <170> Patentln version 3.3 <210> 1 <211> 9 <212> PRT <213> Artificial <220> <223> HLA_A*2402 restricted peptide epitope derived from human wilm's tumor responsible gene product (wTl) <400> 1 Cys Met Thr Trp Asn Gln Met Asn Leu 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial <220> <223> HLA_B*4002 restricted peptide epitope derived from human tyrosi nase <400> 2 Ala sp val Glu Phe Cys Leu Ser Leu 1 5 <210> 3 <211> 10 <212> PRT <213> Artificial <220> <223> HLA_B*4002 restricted peptide epitope derived from human tyrosinase <400> 3 Ser Ala Asp Val clu Phe Cys Leu Ser Leu 1 5 10
Claims (12)
- Claims 1. A pharmaceutical composition comprising cells derived from a human hematopoietic stem cell, wherein, one locus of human HLA class I molecule of the cells has at least one antigen match with a patient; at least one locus of human HLA class I and II molecules of the cells has at least one antigen mismatch with the patient; and the cells derived from the human hematopoietic stem cell do neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
- 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered after the patient's lymphocytes are suppressed; and the pharmaceutical composition survives transiently in the body of the patient, but disappears as the patient's lymphocytes repopulate.
- 3. The pharmaceutical composition according to claim 1 or 2, wherein the locus of human lILA class I molecule of the cells is selected from a group consisting of lILA-A, lILA-B and lILA-C; the locus of human LILA class II molecule of the cells is DRB1; at least one locus of human LILA class I and II molecules of the cells has at least one antigen mismatch with the patient; and the cells derived from the human hematopoietic stem cell do neither survive permanently nor develop an acute CS/H disease having a severity of III or IV in the body of the patient.
- 4. The pharmaceutical composition according to claim 3, wherein LILA-A, LILA-B, and DRB1 loci of the cells have five or six antigen mismatch with the patient; and the cells derived from the human hematopoietic stem cell do neither survive permanently nor develop an acute GVH disease having a severity of III or IV in the body of the patient.
- 5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the human hematopoietic stem cell is an umbilical cord blood stem cell.
- 6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the cells derived from the human hematopoietic stem cell are cytotoxic T cells specific to an epitope restricted by a human LILA class I molecule.
- 7. The pharmaceutical composition according to any one of claims 1 to 6, which is for treating cancer.
- 8. The pharmaceutical composition according to any one of claims 1 to 6, which is for treating an infectious disease.
- 9. The pharmaceutical composition according to claim 8, wherein the cytotoxic T cells specific to the epitope restricted by the human HLA class I molecule are amplified without stimulation by the epitope restricted by the human ElLA class I molecule.
- 10. The pharmaceutical composition according to claim 9, wherein the HLA-A locus of the cells has at least one antigen match with the patient; and the cytotoxic T cells are specific to the Wilm's tumor gene prcduct (WT1).
- 11. The pharmaceutical composition comprising cytotoxic T cells derived from human umbilical cord blood, wherein the cytotoxic T cells are specific to an epitope restricted by a human HLA class I molecule encoded on any one locus of the HLA-A, ElLA-B and ElLA-C; the cytotoxic T cells specific to the epitope restricted by the human HLA class I molecule are amplified without stimulation by the epitope restricted by the human liLA class I molecule; at least one locus of human liLA class I molecule of the cells has at least one antigen match with a patient; liLA-A, liLA-B and DRB1 loci of the cells have five or six antigen mismatch with the patient; and the cells derived from the human hematopoietic stem cell do neither survive permanently nor develop an acute GVH disease having a severity of III or Iv in the body of the patient.
- 12. A method for producing the pharmaceutical composition according to claim 9, comprising a step of stimulating human umbilical cord blood with CD3/CD28 immunobeads.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009188251 | 2009-08-17 | ||
| PCT/JP2010/063181 WO2011021503A1 (en) | 2009-08-17 | 2010-08-04 | Pharmaceutical composition containing transiently surviving ctl |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB201202719D0 GB201202719D0 (en) | 2012-04-04 |
| GB2484869A true GB2484869A (en) | 2012-04-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB1202719.9A Withdrawn GB2484869A (en) | 2009-08-17 | 2010-08-04 | Pharmaceutical composition containing transiently surviving CTL |
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| Country | Link |
|---|---|
| JP (1) | JPWO2011021503A1 (en) |
| KR (1) | KR20120054018A (en) |
| CN (1) | CN102596209A (en) |
| GB (1) | GB2484869A (en) |
| WO (1) | WO2011021503A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3154567B1 (en) | 2014-06-11 | 2020-11-25 | polybiocept GmbH | Expansion of lymphocytes with a cytokine composition for active cellular immunotherapy |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PE20171135A1 (en) * | 2014-11-05 | 2017-08-09 | Memorial Sloan Kettering Cancer Center | METHODS TO SELECT A LINE OF T-CELLS AND DONOR THEM FOR ADOPTIVE CELLULAR THERAPY |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09510456A (en) * | 1994-03-17 | 1997-10-21 | バクスター、インターナショナル、インコーポレイテッド | Immunotherapy of cancer with allogeneic lymphocytes |
| 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 |
-
2010
- 2010-08-04 WO PCT/JP2010/063181 patent/WO2011021503A1/en active Application Filing
- 2010-08-04 GB GB1202719.9A patent/GB2484869A/en not_active Withdrawn
- 2010-08-04 KR KR1020127004088A patent/KR20120054018A/en not_active Application Discontinuation
- 2010-08-04 CN CN2010800362638A patent/CN102596209A/en active Pending
- 2010-08-04 JP JP2011527629A patent/JPWO2011021503A1/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09510456A (en) * | 1994-03-17 | 1997-10-21 | バクスター、インターナショナル、インコーポレイテッド | Immunotherapy of cancer with allogeneic lymphocytes |
| 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 |
|---|
| Blood, Vol.105, 2005, Straathof, K. C. et al., "Treatment of nasopharyngeal carcinoma...", pp.1898-1904 * |
| Bone Marrow Transplant, Vol.29, 2002, Nagayama, H. et al., "Transient hematopoietic stem cell...", pp.197-204 * |
| Japanese Cord BLood Bank Network, 2007, https://www.j-cord.gr.jp/ja/wnew/isyokuseiseki2007.pdf * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3154567B1 (en) | 2014-06-11 | 2020-11-25 | polybiocept GmbH | Expansion of lymphocytes with a cytokine composition for active cellular immunotherapy |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2011021503A1 (en) | 2011-02-24 |
| GB201202719D0 (en) | 2012-04-04 |
| JPWO2011021503A1 (en) | 2013-01-24 |
| KR20120054018A (en) | 2012-05-29 |
| CN102596209A (en) | 2012-07-18 |
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