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WO2011068491A1 - Vaccination tumorale combinée à une transplantation de cellules hématopoïétiques (hct) pour cancérothérapie - Google Patents

Vaccination tumorale combinée à une transplantation de cellules hématopoïétiques (hct) pour cancérothérapie Download PDF

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WO2011068491A1
WO2011068491A1 PCT/US2009/006342 US2009006342W WO2011068491A1 WO 2011068491 A1 WO2011068491 A1 WO 2011068491A1 US 2009006342 W US2009006342 W US 2009006342W WO 2011068491 A1 WO2011068491 A1 WO 2011068491A1
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tumor
cells
cancer
purified
subject
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PCT/US2009/006342
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English (en)
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Samuel Strober
Alexander Filatenkov
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The Board Of Trustees Of The Leland Stanford Junior University
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Publication of WO2011068491A1 publication Critical patent/WO2011068491A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • 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
    • C12N5/0693—Tumour cells; Cancer cells
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/50—Colon
    • 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

Definitions

  • Cancer also known as malignant neoplasm, is characterized by an abnormal growth of cells that display uncontrolled cell division, invasion and destruction of adjacent tissues, and sometimes metastasis to other locations in the body.
  • cancer includes breast cancer, skin cancer, lung cancer, colon cancer, prostate cancer, and lymphoma. Cancer is the second leading cause of death in America and it causes about 13% of all deaths. Cancer may affect people at all ages, even fetuses, but the risk for most types of cancer increases with age. Cancers can affect all animals.
  • Chemotherapy has become the standard of care for many cancers. Chemotherapy refers to antineoplastic drugs used to treat cancer or the combination of these drugs into a cytotoxic standardized treatment regimen. Most commonly, chemotherapy acts by killing cells that divide rapidly, one of the main properties of cancer cells. This means that it also harms cells that divide rapidly under normal circumstances: cells in the bone marrow, digestive tract and hair follicles; this results in the most common side effects of chemotherapy— myelosuppression (decreased production of blood cells), mucositis (inflammation of the lining of the digestive tract) and alopecia (hair loss). Newer anticancer drugs act directly against abnormal proteins in cancer cells; this is termed targeted therapy.
  • the present invention provides a method for treating cancer comprising:
  • the tumor cells are purified from a tumor tissue in a tumor-bearing subject.
  • the tumor cells are purified away from stromal cells.
  • the tumor cells are purified away from immunosuppressive cells.
  • the purified tumor cells are irradiated and stimulated prior to vaccination.
  • the purified tumor cells are combined with an adjuvant.
  • the adjuvant can be CpG or GM-CSF or other immunostimulants.
  • the donor subject is tumor-bearing.
  • the immune cells contain T cells. In some embodiments, the immune cells are added to hematopoietic progenitor cells, for example, CD34 + cells. In some embodiments, the
  • hematopoietic cells are mobilized in the vaccinated subject and enriched prior to transplantation.
  • the recipient receives a single or several doses of total body irradiation prior to transplantation with or without local irradiation of the tumor.
  • the subject is a patient diagnosed with cancer.
  • the transplantation is an autologous transplantation of T cells and hematopoietic progenitor cells.
  • the subject method further comprises vaccinating the subject with irradiated tumor cells and adjuvant before the transplantation of immune and hematopoietic cells.
  • the cancer is a solid tumor.
  • the tumor cells are from a primary or metastatic tumor.
  • the cancer is primary or metastatic.
  • the present invention provides a method for purifying tumor cells from vaccination comprising: (a) obtaining a tumor tissue from a subject; (b) making cell suspension of the tumor tissue; (c) separating tumor cells from the cell suspension; and (d) obtaining purified tumor cells with a purity of at least 30%.
  • the tumor is a primary tumor or a metastatic tumor.
  • the tumor is a solid tumor.
  • the solid tumor can be a colorectal tumor, breast tumor, lung tumor, liver tumor, pancreatic tumor, prostate tumor, or ovarian tumor.
  • the subject is a patient diagnosed with cancer.
  • the tumor cells are separated from other components in the cell suspension.
  • the tumor cells are purified from immunosuppressive cells and factors present in the cell suspension. In some embodiments, the tumor cell purity is greater than 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the tumor cell purity is greater than 90%. In some embodiments, the purified tumor cells are subsequently used for vaccinating the subject from whom the tumor tissue is originally obtained. In some embodiments, the purified tumor cells are irradiated and stimulated with an adjuvant. The adjuvant can be CpG or GM-CSF or other immunostimulants.
  • the adjuvant can be CpG or GM-CSF or other immunostimulants.
  • the present invention provides a composition comprising purified and irradiated tumor cells from a subject.
  • the tumor cells are purified from stromal cells.
  • the tumor cells are purified from immunosuppressive cells.
  • the purified and irradiated tumor cells are stimulated prior to vaccination.
  • the purified tumor cells are combined with an adjuvant.
  • the adjuvant can be CpG or GM-CSF or other immunostimulant.
  • the subject is a patient diagnosed with cancer.
  • the purified and irradiated tumor cells are used to vaccinate the subject.
  • the purified and irradiated tumor cells are from a solid tumor.
  • the solid tumor includes but is not limited to colorectal tumor, lung tumor, breast tumor, pancreatic tumor, liver tumor, prostate tumor, and ovarian tumor.
  • the purified and irradiated tumor cells are from a primary or metastatic tumor.
  • the purified tumor cells have a purity greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • FIG. 1A shows the experimental scheme, which uses HCT from tumor- vaccinated donors to treat CT26 colon tumors in syngeneic mice.
  • normal BALB/c donor mice were vaccinated subcutaneously (s.c.) with 10 6 irradiated CT26 tumor cells mixed with 30 ⁇ g CpG, an adjuvant that stimulates antigen presenting cell via TLR-9 (12,13).
  • spleen and bone marrow cells were harvested, and transplanted intravenously (i.v.) into tumor-bearing BALB/c host mice following a single dose of total body irradiation (TBI). Seven days prior to TBI, hosts had been given live tumor cells via s.c.
  • Figure IB shows the progressive growth of s.c. tumors in all untreated mice.
  • tumor bearing recipients of 50 xl0 6 bone marrow cells and 60 x 10 6 spleen cells from unvaccinated donors had uniformly progressive tumors (Figure 1C).
  • Figure ID shows a steady regression of tumor volume over a 100 day observation period
  • Figure 2 shows that "cured" animals are protected from tumor challenge and can serve as donors of immune cells for HCT into syngeneic tumor-bearing hosts.
  • Figure 3 shows requirements for host irradiation and donor T cells in transplants.
  • LPS lipopolysaccharide
  • FIG. 3A illustrates the significant role of the host conditioning regimen in our vaccine strategy.
  • All tumor bearing recipients of bone marrow and splenocytes from vaccinated donors were cured when conditioned with myeloablative TBI (800cGy).
  • myeloablative TBI 800cGy
  • 450 cGy non-myeloablative radiation dose
  • p ⁇ 0.05 non-myeloablative radiation dose
  • p ⁇ 0.0001 causes lymphodepletion which might deplete host regulatory T cells that suppress anti-tumor immune responses.
  • we studied unirradiated tumor-bearing RAG2 -7- recipients that lack T cells we studied unirradiated tumor-bearing RAG2 -7- recipients that lack T cells.
  • HCT from vaccinated donors was further validated in studies of another colon cancer, MC38, which grows only in C57BL/6 (H-2 b ) mice (8).
  • donor mice were vaccinated with lxlO 6 MC38 tumor cells ( Figure 3F).
  • Figure 3F syngeneic recipients of grafts from vaccinated donors were cured, recipients of transplants from unvaccinated donors did not survive beyond 35 days (p ⁇ 0.0001).
  • Figure 4 shows that donor T cells accumulate in host tumors after transplantation.
  • Figure 4A shows the experimental scheme.
  • Figure 4B shows the Thy 1.1 and Thy 2.2 analysis of single cell suspensions of the tumors and spleens obtained on day 28 from tumor-bearing hosts given unvaccinated or vaccinated donor transplants with or without TBI.
  • Figure 5 shows that the effector/regulatory T cell ratios changes in favor of CD8 effector memory cells as a result of vaccination and HCT.
  • Figure 5A shows the experimental scheme.
  • Thy 1.1 donors were vaccinated with 10 6 irradiated CT26 cells and CpG. Bone marrow and splenocytes were transplanted into lethally irradiated Thy 1.2 hosts with s.c. tumors established for 14 days. Tumor-infiltrating cells and host spleens were analyzed d 14 after HCT (28 days after tumors were induced). Control tumor-bearing animals did not receive irradiation and HCT.
  • regulatory T cells of donor or host origin may be capable of infiltrating tumors when wild-type hosts are used.
  • Host and donor T cell subsets infiltrating CT26 subcutaneous tumor nodules were examined in wild-type BALB/c mice before and after HCT, and in controls without HCT as shown in the experimental scheme in Figure 5A.
  • Control Thyl .2 mice given CT26 cells subcutaneously were euthanized 14 or 28 days later, and single cell suspensions from tumors were analyzed for tumor infiltrating T lymphocytes (TIL) subsets.
  • TIL tumor infiltrating T lymphocytes
  • mice were lethally irradiated and given HCT from vaccinated Thy 1.1 donors after 14 days of tumor growth, and tumor cell suspension were analyzed 14 days after HCT.
  • Figure 5B shows the representative staining patterns for CD4 + and CD8 + T cells in cell suspensions using gated Thyl .2 + T cells from control mice and gated Thyl .1 + from mice given HCT at 28 days after the subcutaneous injection of tumor cells (14 days after HCT).
  • CD8 + and CD4 + cells accounted for about 90% and 5% respectively of Thyl .1 + cells in mice given HCT
  • the CD8 + and CD4 + cells accounted for 30% and 28% respectively of Thyl .2 + cells in mice without HCT.
  • Almost all of the CD8 + and CD4 + T cells in mice given HCT were CD62L 10 ( Figure 5B). The staining pattern indicates that few naive or central memory cells were found in these tumors, almost all were effector memory cells, since the CD8 + and CD4 + cells were almost all CD44 hi .
  • the gated CD8 + and CD4 + cells from tumors in control mice contained discrete subsets of both CD62 L low and CD62 L hi cells.
  • the CD62 L hi cells accounted for 26% of CD8 + cells and 58% of CD4 + cells (Figure 5 B).
  • Staining of gated CD4 + tumor cells from control mice and those given HCT for CD4 versus CD25 showed that about 16% of CD4 + cells were CD25 + in controls, and 32% were CD25 + in those given HCT at the day 28 time point (Figure 5C).
  • Figure 5C At day 14, 22% of CD4 + cells were CD25 + .
  • the results of additional staining for intracellular FoxP3 + showed that the mean percentage of CD4 + CD25 + FoxP3 + Treg cells among gated CD4+ cells in the tumors of all 3 groups of mice varied from about 15% to 25% (Figure 5D).
  • Figure 6 shows that tumor vaccination without HCT is not effective, but vaccination combined with HCT is highly effective, even in the presence of growing tumor.
  • Figure 6A shows the experimental scheme. Tumors were induced subcutaneously at day 0. Animals were
  • Figure 6D shows the survival of vaccinated mice after tumor challenge.
  • Figure 6E shows the experimental scheme. Mice with subcutaneous tumors were vaccinated at day 7 after tumor was induced. Tumors were resected at day 21.
  • Figure 6G shows the donors were injected with tumor cells on day 0, vaccinated on day 7 and splenectomized on day 14. Splenectomized donors had their abdominal incisions closed with surgical sutures before receiving a single dose of 800 cGy TBI. Within 6 hours of TBI, the donors were given an autotransplant of all harvested spleen cells injected intravenously.
  • Figure 6A shows the experimental scheme used to determine the effect of vaccination alone on survival of tumor-bearing mice.
  • Figure 6B shows that the survival of vaccinated, but not HCT treated, animals with 7-day tumors did not improve as compared to unvaccinated tumor-bearing animals and all animals died by day 40.
  • Figure 6C when vaccinated non tumor-bearing animals were challenged with as few as 2.5x10 4 CT26 cells 16 and 50 days after vaccination (Figure 6C), only 20% of mice survived 100 days ( Figure 6D).
  • FIG. 6G A model of autolous HCT was studied as shown in Figure 6G.
  • a group of donors was vaccinated 7 days after live tumor cell injection, splenectomized 7 days later,
  • the present invention provides methods for treating cancer comprising the steps of a) obtaining purified tumor cells; b) vaccinating a donor subject with the purified tumor cells; c) collecting immune cells from the vaccinated donor; and d) transplanting the collected hematopoietic cells and immune cells from the donor into a tumor-bearing recipient following total body irradiation of the recipient.
  • Also provided by the present invention is a method for purifying tumor cells from vaccination comprising: a) obtaining a tumor tissue from a subject; b) making cell suspension of the tumor tissue; and c) separating tumor cells from the cell suspension; and d) obtaining purified tumor cells with a purity of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, . 90%, 95%, 96%, 97%, 98%, 99% or more.
  • the tumor cells are purified from a tumor tissue in a tumor-bearing subject.
  • the tumor cells are purified by density gradients using Ficoll or Percoll - followed by centrifugation.
  • the tumor cells are purified by staining for cell surface markers that recognize tumor cells, and subsequent separation of positive staining cells. Following purification, tumor cells are irradiated and then stimulated with an adjuvant.
  • adjuvant examples include but are not limited to various toll-like receptor (TLR) stimulants such as CpG, Lipopolysaccharide (LPS), poly-IC, and cytokines such as granulocyte-macrophage colony-stimulating factor (GM- CSF).
  • TLR toll-like receptor
  • CpG CpG
  • LPS Lipopolysaccharide
  • poly-IC poly-IC
  • cytokines such as granulocyte-macrophage colony-stimulating factor (GM- CSF).
  • the present invention provides a method of treating cancer comprising obtaining purified tumor cells from a cancer patient; vaccinating the patient with the tumor cells mixed with an adjuvant; collecting hematopoietic cells and immune cells from the vaccinated patient; and performing autologous transplantation of the collected hematopoietic cells and immune cells back into the patient following total body irradiation of the patient.
  • the immune cells are T cells.
  • the hematopoietic cells are CD34 + progenitor cells.
  • cancer examples include but are not limited to solid tumors such as colorectal cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, and ovarian cancer.
  • the tumor can be primary or metastatic.
  • the present invention discloses a method for treating cancer via tumor vaccination followed by autologous hematopoietic and immune cell transplantation.
  • the method comprises a) obtaining purified tumor cells; b) vaccinating a donor subject with the purified tumor cells; c) collecting immune cells from the vaccinated donor; and d) transplanting the collected immune cells from the donor into a tumor-bearing recipient following total body irradiation of the recipient.
  • Cancer immunotherapy is the use of the immune system to reject cancer.
  • the main premise is stimulating the patient's immune system to attack the malignant tumor cells that are responsible for the disease. This can be either through immunization of the patient, in which case the patient's own immune system is trained to recognize tumor cells as targets to be destroyed, or through the administration of therapeutic antibodies as drugs, in which case the patient's immune system is recruited to destroy tumor cells by the therapeutic antibodies.
  • tumor cells Since the immune system responds to the environmental factors it encounters on the basis of discrimination between self and non-self, many kinds of tumor cells that arise as a result of the onset of cancer are more or less tolerated by the patient's own immune system since the tumor cells are essentially the patient's own cells that are growing, dividing and spreading without proper regulatory control. In spite of this fact, however, many kinds of tumor cells display unusual antigens that are either inappropriate for the cell type and/or its environment, or are only normally present during the organisms' development (e.g. fetal antigens).
  • antigens include but are not limited to the glycosphingolipid GD2, a disialoganglioside that is normally only expressed at a significant level on the outer surface membranes of neuronal cells, where its exposure to the immune system is limited by the blood-brain barrier.
  • GD2 is expressed on the surfaces of a wide range of tumor cells including neuroblastoma, medulloblastomas, astrocytomas, melanomas, small-cell lung cancer, osteosarcomas and other soft tissue sarcomas.
  • Other kinds of tumor cells display cell surface receptors that are rare or absent on the surfaces of healthy cells, and which are responsible for activating cellular signal transduction pathways that cause the unregulated growth and division of the tumor cell. Examples include ErbB2, a constitutively active cell surface receptor that is produced at abnormally high levels on the surface of breast cancer tumor cells.
  • Vaccines have been tested to be able to induce integrated immune responses composed of target-specific antibodies and CD4+ and CD8+ T lymphocytes, all of which are held to be essential for effective long-term control of cancer. Insights from these studies have generated a strong framework for the selection of components that will likely comprise an ideal therapeutic cancer vaccine, including: multiple cancer-antigens in various forms delivered with potent adjuvants and all administered in a prime-boost setting in conjunction with a modulator of cancer immunosuppression.
  • a skin cancer patient has been treated using immune cells cloned from his own immune system, i.e. autologous cells, which were then re-injected into the patient.
  • autologous in the context of transplantation typically refers to the situation in which the donor and recipient are the same person.
  • An autologous graft is providing a graft, for example of skin, to the same person from which the graft is obtained.
  • the patient, who was suffering from advanced skin cancer, was free from tumors within eight weeks of being injected with autologous immune cells. This result implicates that autologous transplantation can be an effective treatment of cancer in general.
  • OncoVEX GM-CSF is a version of ⁇ herpes simplex virus which has been engineered to replicate selectively in tumor tissue and also to express the immune stimulatory protein GM-CSF. This enhances the anti-tumor immune response to tumor antigens released following lytic virus replication providing an in situ, patient specific antitumor vaccine as a result.
  • Effective cancer vaccines seek to target an antigen specific to the tumor and distinct from self-proteins.
  • the effective vaccine also should seek to provide long term memory to prevent tumor recurrence.
  • both the innate and adaptive immune systems should be activated (Pejawar-Gaddy S, Finn O. (2008) Critical Reviews in Oncology Hematology.67: 93-102).
  • Tumor antigens have been divided into two broad categories: shared tumor antigens; and unique tumor antigens. Shared antigens are expressed by many tumors. Unique tumor antigens result from mutations induced through physical or chemical carcinogens; they are therefore expressed only by individual tumors.
  • vaccines contain whole tumor cells, though these vaccines have been less effective in eliciting immune responses in spontaneous cancer models.
  • tumor antigens decrease the risk of autoimmunity but because the immune response is directed to a single epitope, tumors can evade destruction through antigen loss variance.
  • a process called "epitope spreading" or “provoked immunity” may mitigate this weakness, as sometimes an immune response to a single antigen will lead to development of immunity against other antigens on the same tumor.
  • Most of the cancer vaccines in development are addressing specific cancer types and are therapeutic vaccines.
  • Several cancer vaccines are currently in development by companies such as Antigenics Inc.
  • TILs tumor-infiltrating lymphocytes
  • Bone marrow transplantation has become well established in the treatment of malignant disorders.
  • High-dose chemotherapy with hematopoietic stem cell support is widely used for most hematological malignancies, as well as for some solid tumors.
  • the availability of large numbers of blood stem cells, mobilized by granulocyte colony-stimulating factor and collected by leukapheresis it is possible to overcome histocompatibility barriers in HLA-mismatched patients.
  • the present invention provides a method for treating cancer comprising: a) obtaining purified tumor cells; b) vaccinating a donor subject with the purified tumor cells; c) collecting immune cells from the vaccinated donor; and d) transplanting the collected immune cells from the donor into a tumor-bearing recipient following total body irradiation of the recipient.
  • the method of the present invention combines vaccinating the donor with stimulated tumor cells and transplantation of hematopoietic and immune cells collected from the donor to a tumor-bearing recipient.
  • the subject method comprises autologous tumor vaccination followed by autologous transplantation of hematopoietic and immune cells, wherein the subject is vaccinated with its own tumor cells, for example, tumor cells stimulated with one or more adjuvants, and then transplanted with its own hematopoietic and immune cells collected after tumor vaccination.
  • the subject typically receives total body irradiation before transplantation of hematopoietic and immune cells.
  • the present invention shows that mice that have developed disseminated tumors or bulky primary tumors established for 2 weeks following inoculation with the CT26 colon carcinoma cells can be cured, when treated with a combination of tumor vaccination and
  • HCT hematopoetic cell transplantation
  • the present invention provides a method for treating cancer comprising: a) obtaining purified tumor cells; b) vaccinating a donor subject with the purified tumor cells; c) collecting immune cells from the vaccinated donor; and d) transplanting the collected immune cells from the donor into a tumor-bearing recipient following total body irradiation of the recipient.
  • the present invention provides a method for purifying tumor cells from vaccination comprising: a) obtaining a tumor tissue from a subject; b) making cell suspension of the tumor tissue; c) separating tumor cells from the cell suspension; and d) obtaining purified tumor cells with a purity of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
  • tumor cells are purified by Ficoll gradient.
  • Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions.
  • Ficoll radii range from 2-7 nm. It is prepared by reaction of the polysaccharide with epichlorohydrin. Ficoll is part of Ficoll-Paque which is used in biology laboratories to separate blood to its components (erythrocytes, leukocytes etc.) Ficoll-Paque is normally placed at the bottom of a conical tube, and blood is then slowly layered above Ficoll-Paque. After being centrifuged, the following layers will be visible in the conical tube, from top to bottom: plasma and other
  • PBMC/MNC buffy coat
  • Ficoll-Paque Ficoll-Paque
  • erythrocytes & granulocytes which should be present in pellet form. This separation allows easy harvest of PBMC's. Note that some red blood cell trapping (presence of erythrocytes &
  • granulocytes may occur in the PBMC or Ficoll-Paque layer.
  • Major blood clotting may sometimes occur in the PBMC layer.
  • Ethylene diamine tetra-acetate (EDTA) and heparin are commonly used in conjunction with Ficoll-Paque(TM) to prevent clotting.
  • Ficoll can also be used to separate islets of Langerhans from pancreatic tissue. The separated islets can then be used for transplantation into patients with type 1 diabetes.
  • Separation of viable from non-viable human tumor cells can be performed by differential flotation on Ficoll-Hypaque specific density solution.
  • Ficoll-Hypaque is used to separate tumor cells from necrotic tissue.
  • Ficoll gradient or filtration can also be used to separate tumor cells from normal blood cells.
  • tumor cells are purified by Percoll.
  • Percoll is a tool for efficient density separation. It is used for the isolation of cells, organelles, and/or viruses by density centrifugation.
  • Percoll consists of colloidal silica particles of 15-30 nm diameter (23% w/w in water) which have been coated with polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • tumor cells are purified from other components in the tumor tissue suspension based on their cell surface markers.
  • Tumor markers are substances that can be found in the body when cancer is present. They are most often found in the blood or urine, but they can also be found in tumors and other tissue. They can be products of the cancer cells themselves, or made by the body in response to cancer or other conditions. Most tumor markers are proteins. There are many different tumor markers. Some are seen only in a single type of cancer, while others can be found in many types of cancer.
  • tumor cell surface markers examples include but are not limited to prostate specific membrane antigen (PSMA) on prostate cancer cells, MAGE, GAGE and BAGE in ovarian cancer and melanoma tissues, PLAC1 (PLACenta- specific 1) in human hepatocellular cancer (HCC) tissues, and epithelial tumor antigen (ETA), e.g. MUC1 on breast cancer cells.
  • PSMA prostate specific membrane antigen
  • MAGE MAGE
  • GAGE and BAGE in ovarian cancer and melanoma tissues
  • PLAC1 PLAC1 (PLACenta- specific 1) in human hepatocellular cancer (HCC) tissues
  • ETA epithelial tumor antigen
  • the purity of tumor cells purified from the tumor tissue and suspension by the subject methods of the present invention is at least 70%, 75%, 80%o, 85%, 86%>, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
  • the other components that are removed from the tumor cells during the purification process of the present invention may include but are not limited to stromal cells, immunoregulatory cells such as regulatory T cells, myeloid suppressor cells, and immunosuppressive cytokines.
  • the purified tumor cells are irradiated prior to vaccination.
  • the tumor cells can be washed and irradiated at 5,000 -20,000 rads.
  • the purified irradiated tumor cells are stimulated with an adjuvant to increase immunogenecity.
  • An adjuvant is an agent that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself.
  • An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens, and thus providing increased immunity to a particular disease.
  • PAMPs accomplish this task by mimicking specific sets of evolutionarily conserved molecules, so called PAMPs, which include liposomes, lipopolysaccharide (LPS), molecular cages for antigen, components of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA (Gavin A, et al. (2006) Science 314 (5807): 1936-8).
  • PAMPs liposomes
  • LPS lipopolysaccharide
  • dsRNA double-stranded RNA
  • ssDNA single-stranded DNA
  • CpG dinucleotide-containing DNA unmethylated CpG dinucleotide-containing DNA
  • ligand - either in the form of adjuvant used in vaccinations or in the form of invasive moieties during times of natural infection - to the TLR marks the key molecular events that ultimately lead to innate immune responses and the development of antigen- specific acquired immunity (Medzhitov R, Preston-Hurlburt P, Janeway C (1997) Nature 388 (6640): 394-7).
  • adjuvant that can be used in the subject methods of the present invention include but are not limited to CpG, LPS, polylC, imiquimod, and GM-CSF. Stimulation of tumor cells in the presence of an adjuvant is well known to one skilled in the art.
  • purified tumor cells prior to, during, or after use of the cells for vaccination.
  • the purified tumor cells can be stored upon acquisition to facilitate transport, or to wait for the results of other analyses.
  • purified tumor cells are provided to physicians for appropriate treatment of cancer.
  • purified tumor cells are stored while awaiting instructions from a physician or other medical professional.
  • a portion of the purified tumor cells are stored while another portion of the purified tumor cells is further manipulated. Such manipulations can include but are not limited, to molecular profiling, cytological staining, gene or gene expression product extraction, fixation, and
  • the purified tumor cells may be placed in a suitable medium, excipient, solution, or container for short term or long term storage. Said storage may require keeping the cells in a refrigerated, or frozen environment.
  • the tumor cells may be quickly frozen prior to storage in a frozen environment.
  • the frozen sample may be contacted with a suitable cryopreservation medium or compound including but not limited to: glycerol, ethylene glycol, sucrose, or glucose.
  • a suitable medium, excipient, or solution may include but is not limited to: hanks salt solution, saline, cellular growth medium, or water.
  • the medium, excipient, or solution may or may not be sterile.
  • the medium, excipient, or solution may contain preservative agents to maintain the sample in an adequate state for subsequent diagnostics or manipulation, or to prevent coagulation.
  • Said preservatives may include citrate, ethylene diamine tetraacetic acid, sodium azide, or thimersol.
  • the sample may be fixed prior to or during storage by any method known to the art such as using glutaraldehyde, formaldehyde, or methanol.
  • the container may be any container suitable for storage and or transport of the biological sample including but not limited to: a cup, a cup with a lid, a tube, a sterile tube, a vacuum tube, a syringe, a bottle, a microscope slide, or any other suitable container.
  • the container may or may not be sterile.
  • the sample may be stored in a commercial preparation suitable for storage of cells for subsequent cytological analysis such as but not limited to Cytyc ThinPrep, SurePath, or Monoprep.
  • Also provided by the present invention is a business method of providing purified tumor cells of the present invention to a third party.
  • customer or potential customer refers to individuals or entities that may utilize methods or services of the tumor cell purification business.
  • Potential customers for the tumor cell purification methods and services described herein include for example, patients, subjects, physicians, cytological labs, health care providers, researchers, insurance companies, government entities such as Medicaid, employers, or any other entity interested in achieving more economical or effective system for diagnosing, monitoring and treating cancer.
  • Such parties can utilize the purified tumor cells obtained from the tumor cell purification method of the present invention, for example, to vaccinate patients having such cancer for treatment.
  • the purified irradiated tumor cells are mixed with adjuvant and injected into a subject for vaccination.
  • the subject is bearing a tumor.
  • the vaccination is autologous tumor vaccination wherein the tumor cells are injected into the subject from whom the tumor cells were originally obtained.
  • the tumor cells can be obtained from a primary tumor, a disseminated tumor, or a metastatic tumor.
  • the tumor is a solid tumor.
  • tumor cells are administered to the subject parenterally, including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, intrasplenic, subcutaneous, and intravenous administration.
  • the adjuvant is injected directly into the tumor.
  • peripheral blood immune cells are collected.
  • the immune cells are T lymphocytes, i.e. T cells.
  • the immune cells can be collected several weeks after tumor cell vaccination, for example, peripheral blood T cells can be collected 6 weeks after tumor vaccination.
  • hematopoietic progenitor cells such as CD34 + cells, are mobilized with granulocyte colony- stimulating factor (G-CSF).
  • G-CSF is a colony-stimulating factor hormone. It is a glycoprotein, growth factor or cytokine produced by a number of different tissues to stimulate the bone marrow to produce granulocytes and stem cells.
  • G-CSF then stimulates the bone marrow to release them into the blood.
  • G-CSF is also a potent inducer of HSCs mobilization from the bone marrow into the bloodstream, although it has been shown that it does not directly affect the hematopoietic
  • G-CSF is used to increase the number of hematopoietic stem cells in the blood of the donor before collection by leukapheresis for use in hematopoietic stem cell transplantation. It may also be given to the receiver, to compensate for conditioning regimens.
  • CD34 + hematopoietic progenitor cells are enriched.
  • CD34 molecule is a cluster of differentiation molecule present on certain cells within the human body. It is a cell surface glycoprotein and functions as a cell-cell adhesion factor. It may also mediate the attachment of stem cells to bone marrow extracellular matrix or directly to stromal cells.
  • Known methods in the art can be used to enrich CD34 + hematopoietic progenitor cells.
  • CD34+ cells may be isolated from blood samples using immunomagnetic or immunofluorescent methods.
  • Antibodies are used to quantify and purify hematopoietic progenitor stem cells for research and for clinical bone marrow transplantation.
  • iso-osmolar Percoll density gradient is used to enrich CD34 + cells.
  • an immunomagnetic separation technique using anti-CD34 antibody or magnetic beads coated with anti-CD34 antibody is used to enrich CD34 + cells.
  • both CD34 + progenitor cells and peripheral T cells are collected from the vaccinated subject for subsequent transplantation into the tumor-bearing recipient.
  • transplantation is autologous wherein the CD34 + progenitor cells and peripheral immune cells are collected from a vaccinated cancer patient and subsequently injected back into the same patient following total body irradiation of the patient. Transplantation is typically performed parenterally, for example, via intravenous infusion.
  • the recipient receives a total body irradiation (TBI) prior to receiving hematopoietic and immune cell transplantation.
  • TBI total body irradiation
  • the patient receives TBI before autologous transplantation.
  • Total body irradiation (TBI) is a form of radiotherapy used primarily as part of the preparative regimen for hematopoietic stem cell (or bone marrow) transplantation.
  • TBI involves irradiation of the entire body, though in modern practice the lungs are often partially shielded to lower the risk of radiation-induced lung injury.
  • Total body irradiation in the setting of bone marrow transplantation serves to destroy or suppress the recipient's immune system, preventing immunologic rejection of transplanted donor bone marrow or blood stem cells. Additionally, high doses of total body irradiation can eradicate residual cancer cells in the transplant recipient, increasing the likelihood that the transplant will be successful.
  • Doses of total body irradiation used in bone marrow transplantation typically range from 10 to >12 Gy. For reference, a dose of 4.5 Gy is fatal in 50% of exposed individuals without aggressive medical care. At these doses, total body irradiation both destroys the patient's bone marrow
  • Non-myeloablative bone marrow transplantation uses lower doses of total body irradiation, typically about 2 Gy, which do not destroy the host bone marrow but do suppress the host immune system sufficiently to promote donor engraftment.
  • total body irradiation is fractionated. That is, the radiation is delivered in multiple small doses rather than one large dose. It has been demonstrated that delivering TBI through multiple smaller doses resulted in lower toxicity and better outcomes than delivering a single, large dose (Thomas ED, Buckner CD, Clift RA, et al. (1979) N. Engl. J. Med. 301 (1 1): 597- 9). In other embodiments, TBI is delivered in one single dose.
  • Hematopoietic stem cell transplantation is the transplantation of blood stem cells derived from the bone marrow or blood.
  • Stem cell transplantation is a medical procedure in the fields of hematology and oncology, most often performed for people with diseases of the blood, bone marrow, or certain types of cancer.
  • stem cell growth factors GM-CSF and G-CSF are now performed using stem cells collected from the peripheral blood, rather than from the bone marrow.
  • Autologous HSCT requires the extraction (apheresis) of hematopoietic stem cells (HSC) from the patient and storage of the harvested cells in a freezer.
  • HSC hematopoietic stem cells
  • the patient is typically treated with high-dose chemotherapy with or without radiotherapy with the intention of eradicating the patient's malignant cell population at the cost of partial or complete bone marrow ablation (destruction of patient's bone marrow function to grow new blood cells).
  • the patient's own stored stem cells are then returned to his her body, where they replace destroyed tissue and resume the patient's normal blood cell production.
  • Autologous transplants have the advantage of lower risk of infection during the immune-compromised portion of the treatment since the recovery of immune function is rapid.
  • the subject method further comprises vaccinating the recipient with the purified tumor cells of the present invention after the transplantation.
  • the recipient can be vaccinated with the purified tumor cells one more time after having received the
  • Tumor growth and disease progression is monitored during and after treatment of cancer via the subject methods of the present invention.
  • Clinical efficacy can be measured by any method known in the art.
  • clinical efficacy of the subject treatment method is determined by measuring the clinical benefit rate (CBR).
  • the clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy.
  • CBR for the subject treatment method is at least about 50%. In some embodiments, CBR for the subject treatment method is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.
  • the preclinical data show for the first time that it is possible to eradicate primary, metastatic, or disseminated solid tumors by treating tumor bearing hosts with HCT containing sensitized T cells from vaccinated donors. While there is growing evidence that hematologic cancers can be effectively treated with a combination of tumor vaccination and HCT (15,16), the effect of such treatment on solid tumors has not been tested.
  • Our outcome measure for tumor immunity in the present invention was eradication of the CT26 or MC38 colon tumors.
  • GVHD graft versus host disease
  • a robust anti-tumor immune response can be transferred to tumor bearing mice without ex-vivo T cell expansion or treatment of the mice with cytokines.
  • CD4 + and CD8 + T cells needed to be included in the transplant to achieve cures indicates that effective vaccination requires epitopes recognized by both types of T cells.
  • Such epitopes were lacking in a vaccine consisting of the immunodominant AH-1 peptide and CpG, which may explain why this vaccine was ineffective, in contrast to vaccines containing whole tumor cells, which are a source of multiple CD4 and CD8 epitopes.
  • CD4 T cells provide help to memory CD8 + T cells by enhancing their immune potency, expansion, and persistence after exposure to antigen (24).
  • the subject methods indicate that irradiation of tumor bearing hosts was also required for tumor cures, and markedly augmented the expansion of transplanted T cells in the spleen and their infiltration into tumors. Since lethal irradiation was considerably more effective than sublethal irradiation, hematopoetic stem cells had to be included in the transplants to rescue hosts from marrow aplasia in the present invention. Previous studies indicate that the hematopoetic stem cells injected into irradiated mice not only prevented marrow aplasia, but also facilitated the expansion of CD8 + T cells directed to melanoma tumor antigens by enhancing IL-7 and IL-15 production (22).
  • irradiation and HCT altered the balance of T cell subsets infiltrating the tumors rather than simply depleting T regulatory cells at the tumor site. Since CD4 + CD25 + FoxP3 + Treg cells can suppress tumor immunity (14), and CD8 + effector memory T cells can mediate tumor cell killing, the balance of the subsets was determined in tumor bearing mice with or without HCT. Mice given irradiation and HCT had a 10 fold higher ratio of CD8 + effector memeory T cells:Treg cells in the tumors as compared to control mice without HCT.
  • the HCT procedure not only increases the absolute number of T cells that infiltrated the tumors, but also favors the T cell subsets that kill tumor cells versus the subset that suppresses tumor immunity.
  • Tumor vaccination without HCT was not effective against established tumors.
  • the subject methods demonstrate that vaccination of tumor-bearing animals provided long-term, transferable immunity, which can be enhanced by HCT.
  • One aspect of the present invention relates to a method for treating cancer comprising: a) obtaining purified tumor cells; b) vaccinating a donor subject with the purified tumor cells; c) collecting immune cells from the vaccinated donor; and d) transplanting the collected immune cells from the donor into a tumor-bearing recipient following total body irradiation of the recipient.
  • subject as used herein includes humans as well as other mammals.
  • treating as used herein includes achieving a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant eradication or amelioration of the cancer.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with cancer such that an improvement is observed in the animal subject, notwithstanding the fact that the animal subject may still be afflicted with that cancer.
  • the types of cancer that can be treated using the subject methods of the present invention include but are not limited to adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin' s lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g.
  • Ewing's sarcoma eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin' s lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplasia syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancre
  • uterine sarcoma transitional cell carcinoma
  • vaginal cancer vulvar cancer
  • mesothelioma squamous cell or epidermoid carcinoma
  • bronchial adenoma choriocarinoma
  • head and neck cancers teratocarcinoma, or
  • the subject method is used to treat a solid tumor, for example, colorectal cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer or pancreatic cancer.
  • a solid tumor for example, colorectal cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer or pancreatic cancer.
  • the subject method further comprises administering to a subject in need thereof an anti-tumor agent, or a pharmaceutically acceptable salt or prodrug thereof.
  • the anti-tumor agents include but are not limited to antitumor alkylating agents, antitumor antimetabolites, antitumor antibiotics, plant-derived antitumor agents, antitumor organoplatinum compounds, antitumor campthotecin derivatives, antitumor tyrosine kinase inhibitors, monoclonal antibodies, interferons, biological response modifiers, and other agents having antitumor activities, or a pharmaceutically acceptable salt thereof.
  • the subject method further comprises treating a subject in need thereof one or more of the following therapies in combination with the subject method disclosed herein.
  • Suitable antineoplastic anti -tumor agents to be used in the present invention include, but are not limited to, alkylating agents, antimetabolites, natural antineoplastic agents, hormonal
  • antineoplastic agents angiogenesis inhibitors, differentiating reagents, RNA inhibitors, antibodies or immunotherapeutic agents, gene therapy agents, small molecule enzymatic inhibitors, biological response modifiers, and anti-metastatic agents.
  • Alkylating agents are known to act through the alkylation of macromolecules such as the DNA of cancer cells, and are usually strong electrophiles. This activity can disrupt DNA synthesis and cell division.
  • alkylating reagents suitable for use herein include nitrogen mustards and their analogues and derivatives including, cyclophosphamide, ifosfamide, chlorambucil, estramustine, mechlorethamine hydrochloride, melphalan, and uracil mustard.
  • alkylating agents include alkyl sulfonates (e.g. busulfan), nitrosoureas (e.g.
  • alkylating agent group includes the alkylating-like platinum-containing drugs comprising carboplatin, cisplatin, and oxaliplatin.
  • Antimetabolic antineoplastic agents structurally resemble natural metabolites, and are involved in normal metabolic processes of cancer cells such as the synthesis of nucleic acids and proteins. They differ enough from the natural metabolites so that they interfere with the metabolic processes of cancer cells.
  • Suitable antimetabolic antineoplastic agents to be used in the present invention can be classified according to the metabolic process they affect, and can include, but are not limited to, analogues and derivatives of folic acid, pyrimidines, purines, and cytidine.
  • Members of the folic acid group of agents suitable for use herein include, but are not limited to, methotrexate (amethopterin), pemetrexed and their analogues and derivatives.
  • Pyrimidine agents suitable for use herein include, but are not limited to, cytarabine, floxuridine, fluorouracil (5-fluorouracil), capecitabine, gemcitabine, and their analogues and derivatives.
  • Purine agents suitable for use herein include, but are not limited to, mercaptopurine (6-mercaptopurine), pentostatin, thioguanine, cladribine, and their analogues and derivatives.
  • Cytidine agents suitable for use herein include, but are not limited to, cytarabine (cytosine arabinodside), azacitidine (5-azacytidine) and their analogues and derivatives.
  • Natural antineoplastic agents comprise antimitotic agents, antibiotic antineoplastic agents, camptothecin analogues, and enzymes;
  • Antimitotic agents suitable for use herein include, but are not limited to, vinca alkaloids like vinblastine, vincristine, vindesine, vinorelbine, and their analogues and derivatives. They are derived from the Madagascar periwinkle plant and are usually cell cycle-specific for the M phase, binding to tubulin in the microtubules of cancer cells.
  • Other antimitotic agents suitable for use herein are the podophyllotoxins, which include, but are not limited to etoposide, teniposide, and their analogues and derivatives.
  • antibiotic antineoplastic agents are antimicrobial drugs that have anti-tumor properties usually through interacting with cancer cell DNA.
  • Antibiotic antineoplastic agents suitable for use herein include, but are not limited to, belomycin, dactinomycin, doxorubicin, idarubicin, epirubicin, mitomycin, mitoxantrone, pentostatin, plicamycin, and their analogues and derivatives.
  • the natural antineoplastic agent classification also includes camptothecin analogues and derivatives which are suitable for use herein and include camptothecin, topotecan, and irinotecan. These agents act primarily by targeting the nuclear enzyme topoisomerase I.
  • camptothecin analogues and derivatives which are suitable for use herein and include camptothecin, topotecan, and irinotecan. These agents act primarily by targeting the nuclear enzyme topoisomerase I.
  • Another subclass under the natural antineoplastic agents is the enzyme, L-asparaginase and its variants. L-asparaginase acts by depriving some cancer cells of L-asparagine by catalyzing the hydrolysis of circulating asparagine to aspartic acid and ammonia.
  • Hormonal antineoplastic agents act predominantly on hormone-dependent cancer cells associated with prostate tissue, breast tissue, endometrial tissue, ovarian tissue, lymphoma, and leukemia. Such tissues may be responsive to and dependent upon such classes of agents as glucocorticoids, progestins, estrogens, and androgens. Both analogues and derivatives that are agonists or antagonists are suitable for use in the present invention to treat tumors. Examples of glucocorticoid agonists/antagonists suitable for use herein are dexamethasone, Cortisol,
  • the progestin agonist/antagonist subclass of agents suitable for use herein includes, but is not limited to, hydroxyprogesterone, medroxyprogesterone, megestrol acetate, mifepristone (RU486), ZK98299, their analogues and derivatives.
  • Examples from the estrogen agonist/antagonist subclass of agents suitable for use herein include, but are not limited to, estrogen, tamoxifen, toremifene, RU58668, SR16234, ZD164384, ZK191703, fulvestrant, their analogues and derivatives.
  • aromatase inhibitors suitable for use herein which inhibit estrogen production
  • examples of aromatase inhibitors suitable for use herein, which inhibit estrogen production include, but are not limited to, androstenedione, formestane, exemestane, aminoglutethimide, anastrozole, letrozole, their analogues and derivatives.
  • Examples from the androgen agonist/antagonist subclass of agents suitable for use herein include, but are not limited to, testosterone, dihydrotestosterone,
  • fluoxymesterone e.g. leuprolide, goserelin, triptorelin, buserelin), diethylstilbestrol, abarelix, cyproterone, flutamide, nilutamide, bicalutamide, their analogues and derivatives.
  • gonadotropin- releasing hormone agonists/antagonists e.g. leuprolide, goserelin, triptorelin, buserelin
  • diethylstilbestrol abarelix
  • cyproterone flutamide, nilutamide, bicalutamide, their analogues and derivatives.
  • Angiogenesis inhibitors work by inhibiting the vascularization of tumors.
  • Angiogenesis inhibitors encompass a wide variety of agents including small molecule agents, antibody agents, and agents that target RNA function.
  • Examples of angiogenesis inhibitors suitable for use herein include, but are not limited to, ranibizumab, bevacizumab, SU11248, PTK787, ZK222584, CEP- 7055, angiozyme, dalteparin, thalidomide, suramin, CC-5013, combretastatin A4 Phosphate, LY317615, soy isoflavones, AE-941, interferon alpha, PTK787/ZK 222584, ZD6474, EMD 121974, ZD6474, BAY 543-9006, celecoxib, halofuginone hydrobromide, bevacizumab, their analogues, variants, or derivatives.
  • Differentiating agents inhibit tumor growth through mechanisms that induce cancer cells to differentiate.
  • One such subclass of these agents suitable for use herein includes, but is not limited to, vitamin A analogues or retinoids, and peroxisome proliferator-activated receptor agonists (PPARs).
  • Retinoids suitable for use herein include, but are not limited to, vitamin A, vitamin A aldehyde (retinal), retinoic acid, fenretinide, 9-c/s-retinoid acid, 13-ci ' s-retinoid acid, all-tra «5-retinoic acid, isotretinoin, tretinoin, retinyl palmitate, their analogues and derivatives.
  • Agonists of PPARs suitable for use herein include, but are not limited to, troglitazone, ciglitazone, tesaglitazar, their analogues and derivatives.
  • Antibody agents bind targets selectively expressed in cancer cells and can either utilize a conjugate to kill the cell associated with the target, or elicit the body's immune response to destroy the cancer cells.
  • Immunotherapeutic agents can either be comprised of polyclonal or monoclonal antibodies.
  • the antibodies may be comprised of non-human animal (e.g. mouse) and human components, or be comprised of entirely human components ("humanized antibodies").
  • monoclonal immunotherapeutic agents suitable for use herein include, but are not limited to, rituximab, tosibtumomab, ibritumomab which target the CD-20 protein.
  • trastuzumab examples suitable for use herein include trastuzumab, edrecolomab, bevacizumab, cetuximab, carcinoembryonic antigen antibodies, gemtuzumab, alemtuzumab, mapatumumab, panitumumab, EMD 72000, TheraCIM hR3, 2C4, HGS-TR2J, and HGS-ETR2.
  • Gene therapy agents insert copies of genes into a specific set of a patient's cells, and can target both cancer and non-cancer cells.
  • the goal of gene therapy can be to replace altered genes with functional genes, to stimulate a patient's immune response to cancer, to make cancer cells more sensitive to chemotherapy, to place "suicide" genes into cancer cells, or to inhibit angiogenesis.
  • Genes may be delivered to target cells using viruses, liposomes, or other carriers or vectors. This may be done by injecting the gene-carrier composition into the patient directly, or ex vivo, with infected cells being introduced back into a patient. Such compositions are suitable for use in the present invention.
  • Nanometer-sized particles have novel optical, electronic, and structural properties that are not available from either individual molecules or bulk solids. When linked with tumor-targeting moieties, such as tumor-specific ligands or monoclonal antibodies, these nanoparticles can be used to target cancer-specific receptors, tumor antigens (biomarkers), and tumor vasculatures with high affinity and precision.
  • tumor-targeting moieties such as tumor-specific ligands or monoclonal antibodies
  • tumor antigens biomarkers
  • the formuation and manufacturing process for cancer nanotherapy is disclosed in patent US7179484, and article M. N. Khalid, P. Simard, D. Hoarau, A. Dragomir, J. Leroux, Long Circulating Poly(Ethylene Glycol)Decorated Lipid Nanocapsules Deliver Docetaxel to Solid Tumors, Pharmaceutical Research, 23(4), 2006, all of which are herein incorporated by reference in their entireties.
  • RNA including but not limited to siRNA, shRNA, microRNA may be used to modulate gene expression and treat cancers.
  • Double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotide sequences where the oligonucleotide sequence of one strand is
  • double stranded oligonucleotides are generally assembled from two separate oligonucleotides (e.g., siRNA), or from a single molecule that folds on itself to form a double stranded structure (e.g., shRNA or short hairpin RNA).
  • siRNA oligonucleotide
  • shRNA short hairpin RNA
  • duplex has a distinct nucleotide sequence, wherein only one nucleotide sequence region (guide sequence or the antisense sequence) has complementarity to a target nucleic acid sequence and the other strand (sense sequence) comprises nucleotide sequence that is homologous to the target nucleic acid sequence.
  • MicroRNAs are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression.
  • mRNA messenger RNA
  • RNA inhibiting agents may be utilized to inhibit the expression or translation of messenger RNA (“mRNA”) that is associated with a cancer phenotype.
  • mRNA messenger RNA
  • agents suitable for use herein include, but are not limited to, short interfering RNA (“siRNA”), ribozymes, and antisense oligonucleotides.
  • siRNA short interfering RNA
  • ribozymes ribozymes
  • antisense oligonucleotides include, but are not limited to, Cand5, Sirna-027, fomivirsen, and angiozyme.
  • Certain small molecule therapeutic agents are able to target the tyrosine kinase enzymatic activity or downstream signal transduction signals of certain cell receptors such as epidermal growth factor receptor ("EGFR") or vascular endothelial growth factor receptor (“VEGFR”). Such targeting by small molecule therapeutics can result in anti-cancer effects.
  • EGFR epidermal growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • agents suitable for use herein include, but are not limited to, imatinib, gefitinib, erlotinib, lapatinib, canertinib, ZD6474, sorafenib (BAY 43-9006), ERB-569, and their analogues and derivatives.
  • Certain protein or small molecule agents can be used in anti-cancer therapy through either direct anti-tumor effects or through indirect effects.
  • Examples of direct-acting agents suitable for use herein include, but are not limited to, differentiating reagents such as retinoids and retinoid derivatives.
  • Indirect-acting agents suitable for use herein include, but are not limited to, agents that modify or enhance the immune or other systems such as interferons, interleukins, hematopoietic growth factors (e.g. erythropoietin), and antibodies (monoclonal and polyclonal).
  • Anti-Metastatic Agents include, but are not limited to, agents that modify or enhance the immune or other systems such as interferons, interleukins, hematopoietic growth factors (e.g. erythropoietin), and antibodies (monoclonal and polyclonal).
  • cancer metastasis The process whereby cancer cells spread from the site of the original tumor to other locations around the body is termed cancer metastasis.
  • Certain agents have anti-metastatic properties, designed to inhibit the spread of cancer cells. Examples of such agents suitable for use herein include, but are not limited to, marimastat, bevacizumab, trastuzumab, rituximab, erlotinib, MMI- 166, GRN163L, hunter-killer peptides, tissue inhibitors of metalloproteinases (TIMPs), their analogues, derivatives and variants.
  • marimastat marimastat
  • bevacizumab trastuzumab
  • rituximab rituximab
  • erlotinib MMI- 166
  • GRN163L hunter-killer peptides
  • TRIPs tissue inhibitors of metalloproteinases
  • Certain pharmaceutical agents can be used to prevent initial occurrences of cancer, or to prevent recurrence or metastasis.
  • treatment of cancer with the subject methods is accompanied with the use of chemopreventative agents.
  • chemopreventative agents suitable for use herein include, but are not limited to, tamoxifen, raloxifene, tibolone, bisphosphonate, ibandronate, estrogen receptor modulators, aromatase inhibitors (letrozole, anastrozole), luteinizing hormone-releasing hormone agonists, goserelin, vitamin A, retinal, retinoic acid, fenretinide, 9-c/s-retinoid acid, 13-m-retinoid acid, all-trans-retinoic acid, isotretinoin, tretinoid, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, cyclooxygenase inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs),
  • NSAIDs
  • treatment of cancer with the subject methods is accompanied by administration of pharmaceutical agents that can alleviate the side effects produced by the antineoplastic agents.
  • agents suitable for use herein include, but are not limited to, antiemetics, anti-mucositis agents, pain management agents, infection control agents, and anti- anemia/anti-thrombocytopenia agents.
  • anti-emetics suitable for use herein include, but are not limited to, 5-hydroxytryptamine 3 receptor antagonists, metoclopramide, steroids, lorazepam, ondansetron, cannabinoids, their analogues and derivatives.
  • anti-mucositis agents suitable for use herein include, but are not limited to, palifermin (keratinocyte growth factor), glucagon-like peptide-2, teduglutide, L-glutamine, amifostin, and fibroblast growth factor 20.
  • pain management agents suitable for use herein include, but are not limited to, opioids, opiates, and non-steroidal anti-inflammatory compounds.
  • agents used for control of infection suitable for use herein include, but are not limited to, antibacterials such as
  • agents that can treat anemia or thrombocytopenia associated with chemotherapy include, but are not limited to, erythropoietin, and thrombopoietin.
  • Wild-type male BALB/c (H-2 d ) mice, male BALB/c Rag2 -A mice, wild-type male DBA2/J (H-2 d ) mice, and wild-type female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were 5-8 weeks old. The Stanford University Committee on Animal Welfare (Administration Panel of Laboratory Animal Care) approved all mouse protocols used in this study.
  • the CT26 cell line (an N-nitro-N-methylurethane-induced BALB/c murine colon carcinoma) was purchased from ATCC (Manassas, VA).
  • the MC38 cell line (dimethyl-hydrazine induced C57BL/6 colon adenocarcinoma) was kindly provided by Dr. David Bartlett of the
  • Lyophilized peptide was diluted in DMSO and stored at -20°C until use.
  • Oligonucleotide containing unmethylated CG motifs (CpG) (TCCATGACGTTCCTGACGTT) was synthesized and
  • oligonucleotide was reconstituted in sterile pyrogen- free water and then diluted in PBS for in vivo injections. 3( ⁇ g of ultrapure LPS (Invivogen) was used in some experiments instead of CpG.
  • Irradiation was performed with a Philips X-ray unit (200 kV, 10 mA; Philips Electronic Instruments Inc., Rahway, NJ, USA) at a rate of 84 cGy/min with a 0.5 mm Cu filter.
  • Philips X-ray unit 200 kV, 10 mA; Philips Electronic Instruments Inc., Rahway, NJ, USA
  • Enriched cells were stained with anti-TCR-allophycocyanin (APC) and anti- CD4 or anti-CD8-fluorescein isothiocyanate (FITC) mAbs to check for purity, and preparations were uniformly at least 95% pure.
  • APC anti-TCR-allophycocyanin
  • FITC anti-CD8-fluorescein isothiocyanate
  • HSCs were obtained by modification of the methods described by Spangrude et all (10). Thy-llolin-/loSca-l+ c-Kit+ cells were sorted on a dual laser F ACS (Becton Dickinson, Mountain View, CA) made available through the FACS shared-user group at Stanford University using FlowJo software (TreeStar, Ashland, OR) for data analysis. After sorting cells were checked by FACS reanalysis and determined to be >99% pure.
  • Kaplan-Meier survival curves were generated using Prism software (SAS Institute Inc., Cary, NC, USA), and statistical differences were analyzed using the log-rank (Mantel-Cox) test. Statistical significance in differences between mean percentage of donor cells in host spleens and tumors was analyzed using the two-tailed Student's t-test of means. For all tests, P ⁇ 0.05 was considered significant.
  • HCT Hematopoietic cell transplantation
  • FIG. 1A shows the experimental scheme, which uses HCT from tumor-vaccinated donors to treat CT26 colon tumors in syngeneic mice.
  • normal BALB/c donor mice were vaccinated subcutaneously (s.c.) with 10 6 irradiated CT26 tumor cells mixed with 30 ⁇ g CpG, an adjuvant that stimulates antigen presenting cell via TLR-9 (12,13).
  • spleen and bone marrow cells were harvested, and transplanted intravenously (i.v.) into tumor-bearing BALB/c host mice following a single dose of total body irradiation (TBI). Seven days prior to TBI, hosts had been given live tumor cells via s.c.
  • Figure IB shows the progressive growth of s.c. tumors in all untreated mice.
  • tumor bearing recipients of 50 xl0 6 bone marrow cells and 60 x 10 6 spleen cells from unvaccinated donors had uniformly progressive tumors (Figure 1C).
  • Figure ID shows a steady regression of tumor volume over a 100 day observation period
  • Vaccination and HCT induces long-term anti-tumor immunity.
  • FIG. 3 A illustrates the significant role of the host conditioning regimen in our vaccine strategy. All tumor bearing recipients of bone marrow and splenocytes from vaccinated donors were cured when conditioned with myeloablative TBI (800cGy). In contrast, only 60% of hosts survived 100 days with a non-myeloablative radiation dose (450 cGy) (p ⁇ 0.05), while none survived more than 40 days without irradiation (p ⁇ 0.0001). Radiation causes lymphodepletion which might deplete host regulatory T cells that suppress anti-tumor immune responses.
  • HCT from vaccinated donors was further validated in studies of another colon cancer, MC38, which grows only in C57BL/6 (H-2 b ) mice (8).
  • donor mice were vaccinated with lxlO 6 MC38 tumor cells ( Figure 3F).
  • HCT alters the balance between regulatory and effector cells at the, tumor site
  • regulatory T cells of donor or host origin may be capable of infiltrating tumors when wild-type hosts are used.
  • Host and donor T cell subsets infiltrating CT26 subcutaneous tumor nodules were examined in wild-type BALB/c mice before and after HCT, and in controls without HCT as shown in the experimental scheme in Figure 5A.
  • Control Thy 1.2 mice given CT26 cells subcutaneously were euthanized 14 or 28 days later, and single cell suspensions from tumors were analyzed for tumor infiltrating T lymphocytes (TIL) subsets.
  • TIL tumor infiltrating T lymphocytes
  • mice were lethally irradiated and given HCT from vaccinated Thy 1.1 donors after 14 days of tumor growth, and tumor cell suspension were analyzed 14 days after HCT.
  • Figure 5B shows the representative staining patterns for CD4 + and CD8 + T cells in cell suspensions using gated Thyl .2 + T cells from control mice and gated Thyl .l + from mice given HCT at 28 days after the subcutaneous injection of tumor cells (14 days after HCT).
  • CD8 + and CD4 + cells accounted for about 90% and 5% respectively of Thyl.l + cells in mice given HCT
  • the CD8 + and CD4 + cells accounted for 30% and 28% respectively of Thy 1.2 cells in mice without HCT.
  • Almost all of the CD8 and CD4 T cells in mice given HCT were CD62L 10 ( Figure 5B). The staining pattern indicates that few naive or central memory cells were found in these tumors, almost all were effector memory cells, since the CD8 and CD4 + cells were almost all CD44 hi .
  • the gated CD8 + and CD4 + cells from tumors in control mice contained discrete subsets of both CD62 L low and CD62 L hi cells.
  • the CD62 L hi cells accounted for 26% of CD8 + cells and 58% of CD4 + cells (Figure 5B).
  • Staining of gated CD4 + tumor cells from control mice and those given HCT for CD4 versus CD25 showed that about 16% of CD4 + cells were CD25 + in controls, and 32% were CD25 + in those given HCT at the day 28 time point (Figure 5C).
  • Figure 5C At day 14, 22% of CD4 + cells were CD25 + .
  • the results of additional staining for intracellular FoxP3 + showed that the mean percentage of CD4 + CD25 + FoxP3 + Treg cells among gated CD4+ cells in the tumors of all 3 groups of mice varied from about 15% to 25% (Figure 5D).
  • Example 6 Tumor vaccination becomes effective when combined with HCT and vaccine induced anti-tumor immunity is not prevented by the presence of growing tumors
  • Figure 6A shows the experimental scheme used to determine the effect of vaccination alone on survival of tumor-bearing mice.
  • Figure 6B shows that the survival of vaccinated, but not HCT treated, animals with 7-day tumors did not improve as compared to unvaccinated tumor- bearing animals and all animals died by day 40.
  • vaccinated non tumor-bearing animals were challenged with as few as 2.5xl0 4 CT26 cells 16 and 50 days after vaccination
  • FIG. 6G A model of autolous HCT was studied as shown in Figure 6G.
  • a group of donors was vaccinated 7 days after live tumor cell injection, splenectomized 7 days later, conditioned with TBI immediately after recovery from surgery, and spleen cells were injected intravenously within 6 hours after TBI. Bone marrow cells were not required for rescue of these myeloablated hosts, since mouse spleen cells contain both immune cells and HSCs.
  • Donors that received the autologous transplants had significantly improved survival as compared to those without transplants, and about 40% survived at least 100 days with complete tumor regression (pO.001) (Figure 6H). Thus, large 14 day tumors were either cured or their growth significantly delayed after autologous HCT.
  • Example 8 Example 8:
  • CRC colorectal cancer
  • bevacizumab for first-line therapy of metastatic CRC is standard of care.
  • FOLFOX4 As the standard of care chemotherapy regimen in metastatic CRC when they demonstrated its superiority over two older regimens, IFL (bolus 5-FU/leucovorin/irinotecan) and IROX (irinotecan/oxaliplatin), in terms of prolonging median overall survival (OS), progression-free survival (PFS), and increased response (26).
  • capecitabine an oral pro-drug of 5-FU. It has significant advantages over infusional 5-FU including ease of administration with its oral formulation, lack of infusion-related toxicities, and decreased duration of hospitalization and clinic time. Multiple trials have pitted capecitabine-based therapies against infusional 5-FU regimens and have shown comparable efficacy (27-32). Overall toxicity profiles are also comparable between the two regimens with the exception of less myelosuppression and more hand-foot syndrome with capecitabine compared to the infusional-5-FU-based regimens. Thus, in clinical practice, CAPOX (capecitabine-oxaliplatin) is largely considered to be a comparable regimen to FOLFOX, with significantly more convenient administration.
  • VEGF vascular endothelial growth factor
  • EGFR endothelial growth factor receptor
  • Cetuximab a mouse/human chimeric monoclonal antibody to EGFR, has also shown promise in metastatic CRC (35, 36).
  • K-RAS mutations When using cetuximab, the presence of K-RAS mutations must be considered. Activating mutations in the K-RAS gene are present in 40-45% of colorectal cancer patients (40). The presence of these mutations correlates with a worse outcome and a lack of response to cetuximab in patients with advanced chemotherapy-refractory CRC (41, 42).
  • HCT from in v vo-immunized syngeneic donors can cure solid tumors in mice, most likely due to peripheral memory T cell response against the tumor antigen. Specific timing and priming conditions are critical, while the effect appears to be independent from allo-immune reactivity.
  • complete response was achieved in 60-100% of mice. The response percentage depended on tumor burden.
  • the preclinical studies discovery can be translated to humans with metastatic CRC using the patients own tumor cells, processing the tumor cells to increase antigenicity, and by combining this vaccine and autologous hematopoietic and immune cell rescue with standard of care resection and, when needed, chemotherapy.
  • Vaccines are prepared and formulated as described in section 4.1.5 using validated methodologies.
  • CpG denotes regions of DNA where a cytosine nucleotide exists next to a guanine nucleotide.
  • a phosphate (p) separates the two nucleotides.
  • ODNs oligodeoxynucleotides
  • the immune system utilizes germline-encoded receptors to detect infection via recognition of conserved molecular patterns associated with microbial pathogens (60).
  • TLRs Toll-like receptors
  • Bacterial CpG DNA is the natural ligand for TLR-9.
  • synthetic CpG ODN's are an established method to study innate immunity. When TLR-9 recognizes this immunostimulatory DNA containing unmethylated CpG motifs, innate immune responses are activated that subsequently amplify the adaptive-immune response (61 ).
  • CpG ODNs can trigger predominantly Th-1 type immune responses (60).
  • Th 1 immune activation is desired as it involves activation of NK cells and cytotoxic T lymphocytes (CTLs) that can kill tumor cells, much like they lyse cells infected by viruses and bacteria (62).
  • CTLs cytotoxic T lymphocytes
  • Numerous animal models have demonstrated that synthetic CpG is an effective adjuvant that enhances both humoral and cellular immune responses in diverse indications, ranging from infectious disease to cancer and allergy, and to date, clinical testing has largely affirmed the potency and safety of ISS-adjuvanted vaccines (61).
  • CpG has been studied as an adjuvant to treatment in a variety of human cancers including lymphoma and non-small cell lung cancer.
  • Various lymphoma and viral vaccine studies incorporating CpG as an adjuvant including doses of 6mg and greater have shown mimimal toxicity (61, 64-67).
  • Interleukin-7 IL-7
  • Interleukin-15 IL-15
  • IL-7 a cytokine produced by the bone marrow and thymic stroma
  • IL-7 is a non-redundant cytokine essential for thymopoiesis as well as T cell survival, proliferation, and cytotoxic function in the peripheral circulation (76).
  • Bolotin and colleagues also demonstrated that administration of IL-7 significantly enhanced immune recovery after T cell-depleted bone marrow transplantation (BMT) in a murine model (77).
  • BMT T cell-depleted bone marrow transplantation
  • SCID severe combined immune deficiency
  • ALL acute lymphoblastic leukemia
  • IL-7 a likely regulator of de novo production of T lymphocytes after BMT.
  • R IL-7 receptor
  • IL-7 gene expression by stromal cells is up-regulated by any external stimuli, but it may be negatively regulated by TGF- ⁇ , IL-1 , and IFN- ⁇ (78, 79).
  • TGF- ⁇ TGF- ⁇
  • IL-1 IFN- ⁇
  • IFN- ⁇ IFN- ⁇
  • IL-15 is an important cytokine in the proliferation and survival of naive, effector, and memory T cells. While enhancing the homeostatic proliferation of naive T cells that require IL-7 and TCR interaction, crucial components of its receptor are not expressed until after naive T cells begin homeostatic proliferation in response to IL-7 (81). In addition to promoting the maturation and survival of naive cells, a considerable body of work suggests that IL-15 's primary role is regulation of the CD8 + T cell compartment as it induces proliferation of memory cells with cytolytic function (81 , 82).
  • Cytokines that signal through receptors containing the common ⁇ -chain (yc) such as 11-7 and IL-15 are crucial for T-cell development, survival, expansion, and activation (81). Furthermore, IL-7 and IL-15 are vital to .the homeostatic response to T cell depletion in the setting of
  • IL-7 appears to be more essential for survival of memory T cells while IL-15 promotes proliferation of those same cells (82).
  • serum levels of these cytokines will be measured at serial times before and after hematopoietic and immune cell rescue.
  • Ki-67 is a well established intracellular method for detection of T cell proliferation in peripheral blood and will be performed serially before and after hematopoietic and immune cell rescue (82, 83).
  • TCR T-cell-receptor
  • CML Cell-mediated lympholysis
  • CD8 + T cells In most preclinical models of hematopoietic cell transplantation, eradication of tumor is mediated by CD8 + T cells using a direct cytolytic pathway. Using a standard CML assay, CD8 + and CD4 + cytolytic function will be measured using the patient's purified blood T cells pre- and post- rescue.
  • CTLs cytotoxic T lymphocytes
  • Purified total T cells or purified CD4 + and purified CD8 + T cells (and their memory subsets) will be used as responder cells (50 x 10 3 /well).
  • Stimulator cells will be irradiated (5,000 cGy in vitro) single cell suspensions of tumor cells (50 x 10 3 ) or purified dendritic cells (86) pulsed with the tumor cell lysate obtained by sonication and freeze/thaw.
  • Measurements of responses will include proliferation ( 3 H-thymidine incorporation) and production of the cytokines IL-2, IFN- ⁇ , TNF-a, and IL-10 assayed by intracellular staining or in triplicate culture well supernatants by ELISA (86).
  • autoimmune reactions have been 'seen with immunomodulatory agents such as IFN-a, IL-2, and anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) including thyroiditis, inflammatory bowel disease and enteritis, hepatitis, vitiligo, dermatitis, arthritis, vasculitis, hypophysitis, panhypopituitarism, and anti-phospholipid syndrome (87-90).
  • immunomodulatory agents such as IFN-a, IL-2, and anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4)
  • CTLA-4 anti-cytotoxic T-lymphocyte antigen 4
  • these autoimmune responses often appear to be associated with antitumor responses (88, 90).
  • Gogas and colleagues demonstrated that autoimmunity was an independent prognostic marker for improved relapse-free survival and overall survival in patients with melanoma who received adjuvant IFN-a- 2 ⁇ .
  • Serum will be assessed serially before and after hematopoietic and immune cell rescue for increased levels of autoantibodies including antithyroid antibodies (anti-thyroglobulin, anti-microsomal antibodies), antinuclear antibodies, anti-double stranded DNA antibodies, and anti-cardiolipin antibodies.
  • autoantibodies including antithyroid antibodies (anti-thyroglobulin, anti-microsomal antibodies), antinuclear antibodies, anti-double stranded DNA antibodies, and anti-cardiolipin antibodies.
  • Rheumatoid factor, thyroid stimulating hormone, thyroxine, and triiodothyronine levels will also be measured. Investigators will assess for symptoms of other autoimmune disorders during the history that might not be captured on these laboratories, and patients will be examined for the clinical manifestations such as vitiligo on a regular basis.
  • Primary may be in place Age 18-70
  • Patients may be transfused or receive epoetin alfa to maintain or exceed this level up to the hemoglobin level recommended on the current label for epoetin alfa.
  • hemoglobin levels greater than the level recommended by the current labeling have been associated with the potential increased risk of thrombotic events and increased mortality.
  • a rapid increase in hemoglobin may exacerbate hypertension (a concern in patients with pre-existing hypertension and if bevacizumab is administered).
  • prostate cancer post-prostatectomy within 5 years prior to Day 1 may be discussed with the Medical Monitor.
  • Urine protein creatinine (UPC) ratio > 1.0 at screening OR
  • Urine dipstick for proteinuria > 2+ (patients discovered to have >2+ proteinuria on dipstick urinalysis at baseline should undergo a 24 hour urine collection and must demonstrate ⁇ 1 g of protein in 24 hours to be eligible).
  • examination performed including demographics, vital signs, height, and weight.
  • baseline labs including a CBC with differential, complete metabolic panel, CEA, CA 19-9, autoimmunity panel, thyroid stimulating hormone, thyroxine, and triiodothyronine levels will be obtained.
  • hepatitis panel HepBsAgA, HepB total core Ab, Hep total Ab, Hep C Ab, qualitative Hep C PCR
  • HIV-1 Ag HIV 1 &2 antibody
  • HIV PCR HSV- 1 & -2 Ab
  • HTLV-1 & -2 Ab HSV- 1 & -2 Ab
  • RPR VZV Ab
  • CMV IgG & IgM baseline autoimmunity screen
  • ANA anti-double stranded DNA, anti-microsomal antibodies, anti-thyroglobulin, anti-cardiolipin, rheumatoid factor
  • apheresis Prior to metastectomy and if needed, primary tumor resection, patients will undergo a first . apheresis to establish baseline immune markers. This apheresis will be performed in the blood bank in the standard manner. The goal of this apheresis is twofold: (1) to obtain >10 8 PBMCs and lymphocytes which will be frozen in aliquots of 1 to 10 million, and (2) to collect lOOmL of autologous plasma for use in tumor vaccine preparation and cryopreservation to avoid the use of allogeneic human serum. The apheresis will take approximately 1 hour and approximately 120 mL of blood will be removed which is roughly equivalent to 8 tablespoons.
  • Surgical Pathology service will aseptically collect freshly resected colon cancer tissue for vaccine preparation.
  • Tumor specimens will be placed in cold (2-8°C) medium consisting of RPMI supplemented with 10% autologous plasma. In general, up to 5 g of tumor will be collected for vaccine preparation.
  • Tumor samples will be maintained cold and transferred to the Stanford Blood & Marrow Transplant (BMT) Laboratory for processing.
  • BMT Stanford Blood & Marrow Transplant
  • Freshly resected tumors will be dissociated under aseptic conditions into single-cell suspensions by mechanically mincing tumor into small pieces of approximately 5 mm , followed by enzymatic digestion in Dulbecco's phosphate- buffered saline (DPBS) with an enzyme mixture (Liberase, Roche, Indianapolis, IN) containing collagenase types I and II, and deoxyribonuclease (Pulmozyme, Genentech, South San Francisco, CA). The digestion will be performed at room temperature with gentle agitation until dissociation is complete. The resulting cell suspension will be filtered through nylon mesh (Nytek; TETKO Inc, Briarcliff Manor, NY). After washing with DPBS (Ca 2+ - and Mg 2+ -free), the cells will be resuspended in autologous plasma. Samples will be removed for cell count and viability
  • the tumor cell suspension will be concentrated to yield aliquots of 2x10 7 cells in 90% autologous plasma plus 10% dimethylsulfoxide (DMSO Protide Pharmaceuticals, Lake Zurich, IL) for cryopreservation.
  • DMSO Protide Pharmaceuticals Lake Zurich, IL
  • Vaccine aliquots will be frozen and stored in vapor phase liquid nitrogen at or below -155°C until released for immunization and immunologic assay (see Section 5.2.1 for release testing criteria). Storage freezers are continuously monitored and equipped with remote alarms.
  • cryopreserved tumor cells will be thawed and washed twice in DPBS. Ten to twenty million viable tumor cells in 1 ml will be transferred to the Stanford Blood Center and irradiated to a dose of 25 Gy. The cells will be returned to the BMT Laboratory and resuspended in 0.5-2ml of DPBS containing 6mg CpG to complete the vaccine formulation. Approximately 10% of the volume will be removed for look-back sterility assessment of the vaccine. A dose of 1 xlO 7 viable cells in up to 2 ml final volume will be loaded into a 2 cc syringe and released for vaccination as described below.
  • All injections will be carried out at the GCRC or in the Stanford Cancer Center oncology clinic. Vaccinations will occur at weeks 1 and 2 after surgery (minimum of 7 days after surgery and 7 days between injections) and at a minimum of 7 days after autologous hematopoietic and immune cell rescue. Patients will be vaccinated subcutaneously at one site as per the vaccination schedule. Each dose of vaccine will consists of 1 xlO 7 autologous irradiated tumor cells and 6 mg CpG in DPBS.
  • Appropriate sites of vaccination include: the outer upper arm, abdomen, buttock, or outer thigh.
  • the site of injection will be identified and should be free of skin irritation.
  • the area will be cleansed with an alcohol or betadine swab.
  • approximately 1-2 inches (2.5-5 cm) of skin will be held taut and the vaccine will be injected at a 45 degree angle using a 25-27 gauge 5/8" needle and a 2 mL syringe.
  • pressure will be held over the site with sterile gauze until hemostasis is achieved.
  • the site of injection will be marked and the location will be recorded in the source documentation.
  • PBMCs will be cryopreserved in autologous plasma with 10% DMSO at a dose of at least 2 x 10 8 /kg for immune cell rescue.
  • An additional 10 or more vials containing 1-10 million PBMCs will be cryopreserved for use in for future ex vivo experiments.
  • Apheresis 3 (for hematopoietic cell rescue)
  • the patients will be assessed for response to the chemotherapy with a repeat contrast CT scan of the chest, abdomen, and pelvis during the rest week of cycle 3.
  • FTBI dose will be determined using a 3+3 dose escalation scheme with the following dose levels:
  • Dose level #1 400 cGy (administered as 200 cGy once daily for two days)
  • Dose level #2 600 cGy (administered as 200 cGy once daily for three days)
  • Dose level #3 800 cGy (administered as 200 cGy once daily for four days)
  • the conditioning regimen schedule would be:
  • fludarabine would start 1 to 2 days earlier depending on the number of days of fTBI.
  • Patients will be hydrated prior to, during, or after fTBI as clinically indicated.
  • Furosemide may be utilized to maintain the patient's weight at or near the admission baseline weight.
  • acyclovir 400 mg orally twice daily will be given starting the first day of fTBI until 1 year after the day of rescue (Day 0).
  • the frozen hematopoietic and immune cells will be transported to the outpatient Stanford Cancer Center BMT Infusion Treatment Area (ITA), thawed in a warm water bath, and infused through a central venous catheter as rapidly as possible. Patients will be pre-medicated with hydrocortisone 100 mg IV and diphenhydramine 50 mg IV 30 minutes prior to cell infusion.
  • ITA Stanford Cancer Center BMT Infusion Treatment Area
  • a CD3+/CD4+/CD8+ absolute numbers/percentages including naive
  • CD62L + CCR7 + CD45RA + central memory
  • CD62L + CCR7 + CD45RO + effector memory
  • c Serum IL-7 and IL-15 assessment of cytokine-associated homeostatic expansion of T cells d % Proliferating T cells: Ki-67, TREC
  • Anti-tumor immune responses will assessed in vitro: purified total T cells or purified CD4 + and purified CD8 + T cells (and their memory subsets) will be used as responder cells (50 x 10 3 /well). Stimulator cells will be irradiated (5,000 cGy in vitro) single cell suspensions of tumor cells (50 x 10 3 ) or purified dendritic cells pulsed with the tumor cell lysate obtained by sonication and freeze/thaw.
  • Measurements of responses include proliferation ( 3 H-thymidine incorporation) and production of the cytokines IL-2, IFN- ⁇ , TNF-a, and IL-10 assayed by intracellular staining or in triplicate culture well supernatants by ELISA.
  • g Autoimmunity panel ANA, anti-ds-DNA antibodies, anti-thyroid antibodies, anti- microsomal antibodies, anti-thyroglobulin antibodies, anti-cardiolipin antibodies, rheumatoid factor, thyroid stimulating hormone, thyroxine, and triiodothyronine levels
  • Nausea, vomiting, or both may be controlled with antiemetic therapy.
  • Mild to moderate allergic or hypersensitivity reactions can be treated with antihistamines such as diphenhydramine.
  • Pentastatin will be avoided during fludarabine administration given the high incidence of pulmonary toxicity when given concomitantly
  • Apheresis is not a routine part of colon cancer management and is thus considered an investigational component. This procedure will enable us to collect the unstimulated immunized cytotoxic T lymphocytes (CTLs) (apheresis 2), and the G-CSF stimulated hematopoietic progenitor cells (apheresis 3) as well as perform immune monitoring (apheresis 1, 2, &3). Cells from aphereses 2 and 3 will be transplanted back into the lymphodepleted patient with the autologous hematopoietic and immune cell rescue. The re-infused immunized CTLs will undergo homeostatic expansion which will ideally induce regression in residual tumor. The re-infused hematopoietic progenitor cells will aide in cell recovery after the fractionated TBI, reducing the duration of cytopenia.
  • CTLs cytotoxic T lymphocytes
  • apheresis 3 the G-CSF stimulated hematopo
  • Potential risks and/or discomforts of apheresis include:
  • Adverse effects will be treated symptomatically as clinically indicated.
  • Vaccine preparation requires extensive handling of resected tumor to generate single cell suspensions.
  • the cells are combined with recombinant enzymes and CpG during processing. Release testing of the product to ensure its safety for injection will be according to the following schedule.
  • Sterility testing will use the BioMeneux BactAlert system with both Fluid Thioglycollate Medium and Tryptic Soy Broth. This system has been validated within the Clinical Microbiology Laboratory at Stanford Hospital and Clinics and has been shown to be at least as sensitive to microbial contaminants as standard USP compliant assays. Vaccines showing evidence of microbial contamination either by detectable growth in sterility cultures or endotoxin levels above the stated limit will not be released for injection.
  • Vaccines showing evidence of microbial contamination either by detectable growth in sterility cultures or endotoxin levels above the stated limit will not be released for injection.
  • the CpG will be kept in a secured location within the Blood and Marrow Transplantation Lab where only the investigators will have access to it.
  • Injections will be administered by a person designated by the investigator (e.g., study nurses, physicians) in response to written orders from the investigator.
  • the investigator e.g., study nurses, physicians
  • CpG agents The most frequent adverse events seen with CpG agents have been those related to the expected immunomodulatory pharmacologic effects. These include local injection reactions, systemic flu-like symptoms, and hematologic changes.
  • acetaminophen for pain or inflammation, antihistamines for pruritis.
  • Options for future injections splitting injection into more than one site, rotating the site of injection, avoiding injection into a site with an existing local reaction, using small gauge needles.
  • Fludarabine monophosphate is a purine analogue used extensively in the treatment of lymphoid malignancies, particularly CLL and low grade NHL. Fludarabine is phosphorylated intracellularly in several steps to its active form 2-fluoroadenosine arabinoside triphosphate (93). Although fludarabine functions primarily as an antimetabolite it does affect both dividing and non- dividing cells. Fludarabine may function as a radiosensitizer (94), which may enhance the immunosuppressive properties of our regimen but also the toxicity.
  • Gastrointestinal mild nausea/vomiting, anorexia, diarrhea, gastrointestinal bleeding (3-13%)
  • Hematologic myelosuppression (nadir 10-14 days; recovery 5-7 weeks), anemia, neutropenia (grade 4: 59%; nadir: ⁇ 13 days), thrombocytopenia (50-55%;
  • fTBI is not a normal part of clinical management for colon cancer. However, its use as part of a hematopoietic cell preparatory regimen is longstanding and we will follow our institution's well-established guidelines.
  • TBI will be given in 200cGy fractions daily using up to a 15mV linear accelerator at a rate of ⁇ 15cGy/minute. Dosimetry calculations will be performed by the radiation physicist. Patient positioning and treatment planning procedures will be performed according to institutional standard practice.
  • fTBI will be initiated the day after the last dose of fludarabine which depending on the dose cohort will either be day -1 if in the 400cGy cohort, day -2 if in the 600cGy cohort or day -3 if in the 800cGy cohort.
  • Toxicities associated with FTBI include:
  • DMSO DMSO will be infused along with the cells. It is excreted through the
  • grade 1 skin reaction occurs at the injection site, subsequent injections should be given in a different site or adjacent to the first injection site.
  • Patients who experience toxicity according to that described in Table 6-1 may have their doses held or discontinued. If any grade 3 injection site reaction or systemic reaction occurs as characterized by hypotension, anaphylaxis, laryngeal edema, or hospitalization, no further vaccinations will be given.
  • An Adverse Event is the development of an undesirable medical condition or the deterioration of a pre-existing medical condition following or during exposure to a pharmaceutical product, whether or not considered causally related to the product.
  • An undesirable medical condition can be symptoms (e.g., nausea, chest pain), signs (e.g., tachycardia, enlarged liver) or the abnormal results of an investigation (e.g., laboratory findings, ECG).
  • an AE can include an undesirable medical condition occurring at any time, including run-in or washout periods, even if no study treatment has been administered.
  • New cancers are those that are not the primary reason for administration of study treatment and have been identified after inclusion of the patient into the clinical study.
  • a Serious Adverse Event is an AE occurring during any study phase (i.e., run-in, treatment, washout, follow-up), and at any dose of the investigational product, comparator or placebo, that fulfills one or more of the following criteria:
  • Adverse events will be recorded at each visit, which is the first day of each cycle. If an adverse event occurs mid-cycle requiring medical attention, this will be recorded as well.
  • the variables to be recorded for each adverse event include, but are not limited to, onset, resolution, intensity, action taken, outcome, causality rating, and whether it constitutes an SAE or not.
  • the intensity of the adverse event should be captured using CTCAE criteria, version 3.0, when possible.
  • Pregnancy should be excluded before enrollment. Should a pregnancy occur, it must be reported in accordance with the procedures described in Section 8.2. Pregnancy in itself is not regarded as an AE unless there is a suspicion that an investigational product may have interfered with the effectiveness of a contraceptive medication.
  • a cover page should accompany the MedWatch form indicating the following:
  • the SAE report will designate the causality of events in relation to all study medications and if the SAE is related to disease progression, as determined by the principal investigator.
  • CT computed tomography
  • C/A/P chest/abdomen/pelvis
  • CMP complete metabolic panel
  • Cond conditioning
  • D day
  • Flud. fludarabine
  • fTBI fractionated total body irradiation
  • HICR hematopoietic and immune cell rescue
  • Mo. month
  • MRI magnetic resonance imaging
  • Blood pressure and heart rate will be assessed before and 30 minutes after vaccinations.
  • d Weight will be assessed at screening, day of resection/vaccinations/autologous HSCT, and at monthly clinic visits.
  • hepatitis panel HepBsAgA, HepB total core Ab, Hep total Ab
  • Hep C Ab qualitative Hep C PCR
  • HIV-1 Ag HIV 1 &2 antibody
  • HIV PCR HIV PCR
  • HSV 1 & 2 HSV 1 & 2
  • h Autoimmunity panel ANA, anti-ds-DNA antibodies, anti-thyroid antibodies, anti- microsomal antibodies, anti-thyroglobulin antibodies, anti-cardiolipin antibodies, rheumatoid factor, thyroid stimulating hormone, thyroxine, and triiodothyronine levels i
  • ANA anti-ds-DNA antibodies, anti-thyroid antibodies, anti- microsomal antibodies, anti-thyroglobulin antibodies, anti-cardiolipin antibodies, rheumatoid factor, thyroid stimulating hormone, thyroxine, and triiodothyronine levels
  • Wk 7 apheresis for unstimulated cytotoxic killer lymphocytes (CTLs)
  • HPCs hematopoietic progenitor cells
  • Tumor measurements clinically for palpable lesions and radiographically using RECIST criteria; Also to be done at week 18 if still on chemotherapy. 1 Tumor assessment for all lesions will be evaluated according to RECIST criteria. m Within 4 weeks of the baseline apheresis, pulmonary function testing with spirometry and diffusing capacity, and transthoracic echo or MUGA scan will be performed to ensure .
  • Vaccinations will be performed subcutaneously at a minimum of seven days after tumor resection and after hematopoietic and immune cell rescue.
  • the second vaccination will be given a minimum of 7 days after the first vaccination.
  • Standard chemotherapy will be given only if needed for progressive disease.
  • the regimen will be at the discretion of the investigator.
  • the dose of the fTBI will be determined according to the cohort the patient enters into and will be given once daily for 2-4 days
  • HICR Autologous hematopoietic and immune cell rescue
  • Acyclovir prophylaxis if HSV-1 or-2 positive, Acyclovir 400 mg p.o. bid starting on the first day of fTBI
  • Bacterial prophylaxis When ANC ⁇ 500, start Ciprofloxacin 500mg orally daily. Days +30- 60: Bactrim 160mg/800mg p.o. bid, Saturday and Sunday only for Pneumocystis carinii prophylaxis.
  • Immune monitoring See separate schedule in section 4.1.16.3. Immune monitoring will be done at month +1 , +3, +6, +12, +18, and at termination of the study.
  • Patients will undergo re-staging CT scans of the chest/abdomen/pelvis at week 4 and week 8 to decide if standard chemotherapy is needed. If chemotherapy is initiated or continued at week 8, then the patient will then have a repeat CT scan of the chest/abdomen/pelvis after completion of at least 3 cycles of standard chemotherapy. After transplant, patients will undergo monthly CT scans of the chest/abdomen/pelvis for 2 months then every 2 months and as needed per the Investigators' discretion to assess for progressive disease or response. The post- transplant month 2 CT scan will be considered the confirmatory response scan.
  • the post-rescue CT scans will be compared to both the pre-conditioning and screening CT scans.
  • Responses from the pre-conditioning scans (week 8 or 18) as compared to the post-rescue scans will be the primary assessment of response to our investigational therapy as the comparison to the study entry (screening) scan may be biased by any chemotherapy intervention.
  • All lesions not considered measurable by the definition above including small lesions (longest diameter ⁇ 20 mm with conventional techniques or ⁇ 10 mm with spiral CT) and truly non- measurable lesions.
  • Truly non-measurable lesions include: bone lesions, leptomeningeal disease, malignant ascites, malignant pleural or pericardial effusion, inflammatory breast disease, lymphangitic spread of tumor, and abdominal masses that are not pathologically confirmed metastases and followed solely by imaging techniques.
  • CT and MRI scans are currently the best available and most reproducible methods for measuring target lesions.
  • Conventional CT and MRI should be performed with contiguous cuts of 10mm or less in slice thickness.
  • Spiral CT should be performed using a 5mm contiguous reconstruction algorithm.
  • Chest X-ray lesions on chest X-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung.
  • Clinical examination clinically detected lesions will only be considered measurable if they are superficial and readily palpable on repeated clinical examination.
  • Target lesions will be defined as all measurable lesions up to a maximum of five lesions per organ and 10 lesions in total, representative of all involved organs. Target lesions should be selected based on the largest size and best suitability for accurate repeated measurements. [00261] The sum of the longest diameters of all target lesions will be calculated at baseline and reported as the baseline sum longest diameter. This baseline sum longestdiameter will be used as the reference to characterize objective tumor response. For lesions measurable in 2 or 3 dimensions, the longest diameter at the time of assessment will be reported.
  • CR Complete response
  • Partial response At least a 30% decrease in the sum of the longest diameters of target lesions, taking as reference the baseline sum longest diameter.
  • PD Progressive disease
  • Stable disease Neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease. To be assigned a status of stable disease, measurements must have met the stable disease criteria at least once after study entry at a minimum interval of six weeks.
  • Symptomatic deterioration Patients with global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having symptomatic deterioration.
  • the best overall response is the best response recorded from registration until disease progression/recurrence.
  • CR complete response
  • PR partial response
  • SD stable disease
  • Duration of overall response is the period measured from the time that measurement criteria are met for complete or partial response (whichever status is recorded first) until the first date that recurrent or progressive disease is objectively documented, taking as reference the smallest measurements recorded since treatment started.
  • First documentation of Response The time between initiation of therapy and first documentation of PR or CR.
  • Duration of Stable Disease is the measurement from registration until the criteria for disease progression is met, taking as reference the smallest measurements recorded since registration. To be assigned a status of stable disease, measurements must have met the stable disease criteria at least once after study entry at a minimum interval of six weeks.
  • TTP Time to Progression
  • Tumor markers alone cannot be used to assess response. However, if a tumor marker is initially above the upper limit of normal, it must normalize for a patient to be considered in complete CR when all tumor lesions have disappeared. The tumor markers that will be assessed in this study are CEA and CA 19-9.
  • Baseline demographic characteristics will be recorded on each patient including age, gender, race, ECOG performance status, location of primary cancer, location and number of metastases, presence or absence of primary tumor, dates of initial diagnosis and recurrence if applicable, and presence of other co-morbidities.
  • T cells targeted against a single minor histocompatibility antigen can cure solid tumors. Nature Medicine. 1 1 : 1222-1229.
  • Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling. J Clin Invest 117:2197-2204.
  • O oxaliplatin
  • FOLFOX-6 metastatic colorectal cancer
  • Ciardiello F Tortora G. EGFR antagonists in cancer treatment. N Engl J Med 2008;358: 1 160-74.
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  • Bolotin E Smogorzewska M, Smith S, Widmer M, Weinberg K. Enhancement of thymopoiesis after bone marrow transplant by in vivo interleukin-7. Blood 1996;88: 1887-94.
  • Bolotin E Annett G, Parkman R, Weinberg K. Serum levels of IL-7 in bone marrow transplant recipients: relationship to clinical characteristics and lymphocyte count. Bone Marrow Transplant 1999;23:783-8.

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Abstract

Selon un aspect, la présente invention concerne un procédé de traitement du cancer consistant en une vaccination de cellules tumorales associée à une transplantation de cellules tumorales et immunitaires. Dans certains modes de réalisation, le procédé passe par une vaccination de cellules tumorales avant transplantation de cellules hématopoïétiques et immunitaires autologues. Selon un autre aspect, l'invention concerne un procédé consiste à purifier des cellules tumorales chez un sujet en vue de leur transplantation.
PCT/US2009/006342 2009-12-01 2009-12-01 Vaccination tumorale combinée à une transplantation de cellules hématopoïétiques (hct) pour cancérothérapie WO2011068491A1 (fr)

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WO2014172532A3 (fr) * 2013-04-17 2014-12-31 The Board Of Trustees Of The Leland Stanford Junior University Procédés permettant un traitement du cancer

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