Invented by Samuel Strober, Alexander Filatenkov, Leland Stanford Junior University
The Leland Stanford Junior University invention works as followsThe present invention offers a method of treating cancer that includes tumor cell vaccination combined with hematopoietic or immune cell transplantation. The method may include autologous tumor cells vaccination before autologous hematopoietic or immune cell transplantation. The present invention also provides a method for purifying tumor cells from subjects in preparation to vaccination.
Background for Tumor vaccination combined with hematopoietic cells transplantation for cancer treatment
Cancer, or malignant neoplasm as it is also called, is an abnormal growth in cells that exhibits uncontrolled cell division. It can also cause invasion of tissues and destroy them, and even spread to other parts of the body. Breast cancer, skin-cancer, lung-cancer, colon-cancer, prostate cancer and lymphoma are just a few of the more than 100 different types of cancer. About 13% of deaths in America are caused by cancer. It is the second most common cause of death. Cancer can affect anyone at any age, including fetuses. However, the risk of most cancers increases with age. All animals can be affected by cancer.
Chemotherapy is now the standard treatment for many cancers.” The term chemotherapy refers to the use of antineoplastic drugs to treat cancer, or the combination with other drugs to create a standard cytotoxic treatment regimen. The most common way chemotherapy works is by killing cancerous 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). Targeted therapy is a newer form of anticancer drug that targets abnormal proteins within cancer cells.
The current treatment for cancer is not effective in many cases, even with the new agents or improved combinations. “Cancer therapy is in dire need of improved regimens and treatment.
In one aspect of the invention, a method is provided for treating cancer. This comprises: (a), obtaining purified tumour cells; (b), vaccinating with their purified cancer cells that have been combined with an adjuvant and irradiated; (c), collecting immune and hemopoietic cell from the vaccinated subject; and (d), injecting the collected immune cells and hematopoietic from the subject intravenously following total body irradiation. In some embodiments the tumor cells can be purified by removing a tissue from a subject with a cancer. In certain embodiments, tumor cells are separated from stromal cell. In certain embodiments, tumor cells are separated from immune-suppressive cells. In certain embodiments, purified tumor cells may be irradiated or stimulated before vaccination. In certain embodiments, purified tumor cell are combined with adjuvant. Adjuvants can include CpG, GM-CSF, or other immunostimulants. In certain embodiments, the subject of the donor is a tumor-bearing. In certain embodiments, immune cells include T cells. In certain embodiments, immune cells can be added to progenitor hematopoietic cells, such as CD34+. In certain embodiments, hematopoietic progenitor cells are mobilized and enriched in the subject after vaccination. In some embodiments the recipient is irradiated with a single dose or multiple doses prior to the transplant, either locally or total body. In some embodiments the subject is a cancer patient. In a preferred form, the autologous T cells and hematopoietic stem cells are transplanted. In some embodiments the method includes vaccinating a subject with irradiated cancer cells and adjuvant prior to the transplantation. In some embodiments the cancer is solid. Solid tumors can be treated using the methods of the invention. Examples include colorectal, lung, breast, pancreatic, liver, prostate, and ovarian carcinomas. In some embodiments the tumor cells come from a metastatic or primary tumor. Cancer can be primary or metastatic in some embodiments.
In another aspect of the invention, the method of purifying tumor cell from vaccination comprises: (a), obtaining a tissue of a tumor from a patient; (b), making a cell suspension from the tissue of the patient; (c), separating the tumor cells from this cell suspension; (d), obtaining purified cells of a purity at least 30%. In certain embodiments, the cancer is either a primary or metastatic tumor. In some embodiments the tumor can be a solid tumour. The solid tumor may be a colorectal cancer, breast tumors, lung tumors, liver tumors, pancreatic tumours, prostate tumors, or ovarian carcinomas. In some embodiments the subject is a cancer patient. In certain embodiments, tumor cells are separated by other components of the cell suspension. In certain embodiments, tumor cells are separated from immunosuppressive factors and cells present in the suspension. In certain embodiments, tumor cell purity can be greater than 30%. 40%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%. In some embodiments the purity of tumor cells is higher than 90%. In certain embodiments, purified tumor cells can be used to vaccinate the original subject. In some embodiments the purified cancer cells are irradiated with an adjuvant and then stimulated. Adjuvants can include CpG, GM-CSF, or other immunostimulants.
Another aspect of the invention is a composition containing purified and irradiated cancer cells from a patient. In certain embodiments, tumor cells can be purified from stromal cell. In certain embodiments, tumor cells can be purified from immune-suppressive cells. In certain embodiments, the irradiated and purified tumor cells are stimulated before vaccination. In certain embodiments, purified tumor cells can be combined with an adjuvant. Adjuvants can include CpG, GM-CSF, or any other immunostimulant. In some embodiments the subject is a cancer patient. In some embodiments the purified and radio-irradiated cancer cells are used for vaccinating the subject. In some embodiments the purified and Irradiated Tumor Cells are taken from a solid tumour. Solid tumors include but are not limited to colorectal cancer, lung cancer, breast tumour, pancreatic carcinoma, liver tumors, prostate tumors, and ovarian carcinomas. In some embodiments the irradiated and purified tumor cells come from a metastatic or primary tumor. Purified tumor cells in some embodiments have a purity of greater than 30%. 40%. 50%. 60%. 70%. 80%. 90%. or 95%.
INCORPORATION BY RESEARCH
All publications, patents and patent applications mentioned herein are herein incorporated as if each publication, patent, and/or patent application were specifically and individually indicated that they would be incorporated by refer.
The present invention also provides a method for purifying tumor cells from vaccination, which comprises: a) obtaining a tumor tissue from a subject; b) making cell suspension of the tumor tissue; and c) separating tumor cells from this cell suspension; and d), transplanting collected hematopoietic and immune cells of the donor to a recipient with a tumor after total body irradiation. The present invention also provides a method of purifying cancer cells from vaccination, which comprises: a), obtaining a tissue of a tumor from a patient; b), making a cell suspension from the tissue; and, c) separating the tumor cells from this cell suspension.
In some embodiments, tumor cells can be purified by centrifugation from a tissue tumor in a subject with a cancer. In certain embodiments, tumor cells can be purified using Ficoll and Percoll density gradients followed by centrifugation. In certain embodiments, tumor cells can be purified using cell surface markers to identify tumor cells and then separating the positive stained cells. After purification, the tumor cells are then irradiated with a radioactive substance and stimulated by an adjuvant. Examples of adjuvant that can be used in the subject methods of the present invention 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).
In some embodiments of the invention, a method for treating cancer is provided. This includes obtaining purified cancer cells from a patient who has cancer; vaccinating that patient with the cancer cells mixed with adjuvants; collecting hematopoietic and immune cells from that patient; and autologous transplantation back into the patient of those collected hematopoietic and immune cell after total body irradiation. In some embodiments the immune cells are called T cells. In some embodiments the hematopoietic cell is CD34+ progenitor cell. The subject methods can treat cancers including but not limited to colorectal, lung, pancreatic, breast, prostate, liver, ovarian, and other solid tumors. The tumor may be primary or secondary.
The present invention discloses, in one aspect of the invention, a method to treat cancer by tumor vaccination followed autologous hematopoietic cell and immune transplantation. The method includes a) obtaining pure tumor cells, b) vaccinating with purified cancer cells a recipient subject; c), collecting immune cells of the vaccinated recipient; and d), transplanting the immune cells collected from the donor to a recipient bearing a tumor after total body irradiation.
Cancer immunotherapy involves using the immune system to fight cancer. The principle is to stimulate the patient’s own immune system so that it attacks the malignant tumour cells responsible for the illness. The patient can be immunized, which will train the immune system to identify tumor cells and destroy them, or therapeutic antibodies administered as drugs. In the latter case, the patient’s immune is recruited to kill tumor cells.
The immune system is able to differentiate between self and nonself and therefore, tumor cells that develop as a result from cancer can be tolerated more or less by the patient?s immune system. This is because the tumor cells are the patient?s own cells, which are growing, multiplying and spreading without any regulatory control. Despite this, however, many types of tumor cells exhibit unusual antigens, which are either inappropriate or only present in the environment of the organism (e.g. fetal antigens). These antigens can include, but are not restricted to, the glycosphingolipid GD2, a diialoganglioside which is expressed in significant amounts on the outer membranes of neurons. Its exposure to the immune systems is then limited by the blood brain barrier. GD2 can be found on the surface of a variety of tumor cells, including neuroblastomas and sarcomas of soft tissues, such as osteosarcomas, small-cell lung carcinomas, medulloblastomas and astrocytomas. Some tumor cells have cell surface receptors which are absent or rare on healthy cells. These receptors are responsible for activating signal transduction pathways, which cause unregulated growth and cell division. ErbB2, for example, is a constitutively-active cell surface receptor produced in abnormally high amounts on the surface breast cancer tumor cells.
Vaccines were tested for their ability to induce an integrated immune response composed of CD4+ and CD8+T cells, which is considered essential for the effective control of cancer over a long period of time. These studies have provided a framework that can be used to select the components of an ideal therapeutic cancer vaccination. This includes multiple cancer-antigens, delivered in different forms with powerful adjuvants. All of these are administered in a prime boost setting with a modulator for cancer immunosuppression. In one case, skin cancer patients were treated with immune cells that had been cloned directly from their own immune systems. Autologous cells were then reinjected into the patient. The term “autologous” is used. In the context of transplantation, the term “autologous” is used to describe the situation where the recipient and donor are the same individual. Autologous grafts are grafts, such as skin, that are given to the person who obtained the graft. After being injected autologous cells, the patient with advanced skin cancer was free of tumors in eight weeks. This suggests that autologous cell transplantation is an effective cancer treatment in general.
Another approach to therapeutic anticancer vaccines is to generate an immune response in situ within the patient. OncoVEX GM-CSF is one example. OncoVEX GM CSF is a herpes simplex version that has been engineered so it can replicate only in tumor tissue, and expresses the immune stimulating protein GM CSF. It enhances the immune response against tumors by releasing antigens after lytic virus replication. This results in an anti-tumor patient-specific vaccine. Cancer vaccines that are effective target antigens specific to tumors and different from self-proteins. Adjuvants, which are molecules that activate antigen presenting cells and stimulate immune responses, must be selected. At the moment, only BCG salts, aluminum-based compounds, and squalene oil-water emulsions have been approved for use in clinical trials. A vaccine that is effective should also provide a long-term memory to prevent tumors from recurring. Both the adaptive and innate immune systems are best activated. (Pejawar Gaddy S, Finn O. Critical Reviews in Oncology Hematology (2008). 67: 93-102).
Tumor Antigens are divided into two categories: shared tumor and unique tumor antigens. Shared antigens can be found in many tumors. Unique tumor antigens are the result of mutations caused by physical or chemical carcinogens. They are expressed only by specific tumors.
In one approach vaccines are made up of whole tumor cells. However, these vaccines were less effective at eliciting an immune response in spontaneous cancer models. Defined antigens in tumors reduce the risk of autoimmunity, but because the immune system is focused on a single epitope tumors may be able to avoid destruction due to antigen loss variation. This process is called “epitope spread”. A process called?epitope spreading? This weakness may be mitigated by the fact that an immune response against a single antigen can lead to immunity to other antigens found on the same tumour. The majority of cancer vaccines under development target specific cancer types, and are therapeutic vaccines. Several cancer vaccines, including those developed by Antigenics Inc., Geron Corporation, Dendreon Corp, BN ImmunoTherapeutics, GlobeImmune, Advaxis, Inc., Lovaxin C, Accentia Biopharmaceuticals, majority-owned subsidiary Biovest (GMXX), Apthera, Inc., and GeneMax Corp, are in development. (NeuVax).
Despite the potency of the immune system and its specificity, vaccination with tumor-specific antigens fails to eradicate most cancers in humans and mice (1,2). Adoptive cell therapy is the most effective form of immunotherapy at present. This involves activating tumor-infiltrating cells (TILs), re-infusing these cells with high doses cytokines, and then re-infusing them. This approach is restricted by cytokine toxicities and the limited number of tumors (melanomas) from which TILs are available (3).
Bone marrow transplantation is now a well-established treatment for malignant disorders. For most hematological cancers, and for certain solid tumors, high-dose chemotherapy combined with hematopoietic cells is used. Recent developments in blood progenitor cells harvest, namely the availability of large amounts of blood stem cell, mobilized by Granulocyte Colony-Stimulating Factor and collected by leukapheresis can overcome histocompatibility in HLA mismatched patients. “Other recent developments, including but not restricted to new methods of blood progenitor cell mobilization, ex vivo proliferation of progenitor and immune cells, as well as other molecular technologies, support the effective treatment of cancer by autologous transplantation hematopoietic cells and immune.
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