EA040268B1 - Smallpox Vaccine for Cancer Treatment - Google Patents

Description

Cross-references to related applications

This application claims priority over U.S. Provisional Application No. 62/203835, filed August 2015, U.S. Provisional Application No. 62/216292, filed September 9, 2015, and U.S. Provisional Application No. 62/317226, filed April 1, 2016, the full contents of each of which are incorporated in this document by reference in its entirety.

Prior Art

Cancer is the second leading cause of death in the United States, behind only cardiovascular disease. In the United States, cancer causes one in four deaths. The relative 5-year survival rate for all patients in whom in 1996-2003. diagnosed with cancer was 66% compared with 50% in 1975-1977. (Cancer Facts & Figures American Cancer Society: Atlanta, GA (2008)). The development of highly effective cancer treatments is a major goal of cancer research.

The essence of the invention

Disclosed herein are methods and compositions for treating a solid tumor or hematologic malignancies in a subject.

In some embodiments, disclosed herein is a method of treating a proliferative disease (eg, a solid tumor or hematologic malignancy) in a subject, comprising administering a smallpox vaccine to the subject. In some embodiments, the smallpox vaccine is a replication-competent virus. In some embodiments, the smallpox vaccine does not contain a heterologous nucleic acid. In some embodiments, the smallpox vaccine is administered by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular injection. In some embodiments, the smallpox vaccine is a weakened strain of vaccinia virus from the New York City Board of Health (NYCBOH)). In some embodiments, the NYCBOH vaccinia strain may be ATCC VR-118 or CJ-MVB-SPX. In some embodiments, the smallpox vaccine is selected from Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, or a modified Ankara virus (MVA) vaccine. In some embodiments, the solid tumor is selected from glioblastoma, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, or melanoma. In some embodiments, the subject is a human. In some embodiments, the subject is a pediatric patient. In some embodiments, the proliferative disease is a childhood or pediatric tumor or cancer.

In some embodiments, disclosed herein is a method of treating a solid tumor or hematologic malignancy in a subject, comprising administering a pox vaccine to the subject simultaneously with a stem cell. In some embodiments, the stem cell is an autologous stem cell. In some embodiments, the stem cell is selected from the group consisting of adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells. cells, adipose stromal cells, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, small juvenile stem cells, skin fibroblast stem cells, and combinations thereof. In some embodiments, the stem cell is an adipose stromal cell.

In some embodiments, the stem cell is a modified stem cell. In some embodiments, the modified stem cell is an adult stem cell. In some embodiments, the modified stem cell is transformed with a lentivirus or a retrovirus. In some embodiments, the modified stem cell is transiently transfected with an artificial chromosome, viral DNA, or plasmid DNA. In some embodiments, the stem cell is autologous. In some embodiments, the smallpox vaccine does not contain a heterologous nucleic acid. In some embodiments, the smallpox vaccine is administered by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular injection. In some embodiments, the smallpox vaccine is a weakened strain of vaccinia virus from the New York City Board of Health (NYCBOH)). In some embodiments, the NYCBOH vaccinia strain may be ATCC VR-118 or CJ-MVB-SPX. In some embodiments, the smallpox vaccine is selected from Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, or a modified Ankara virus (MVA) vaccine. In some variants

The solid tumor is selected from glioblastoma, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor or melanoma. In some embodiments, the stem cell and the smallpox vaccine are administered to the subject simultaneously by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular injection. In some embodiments, the stem cell and the smallpox vaccine are placed in the same vial prior to injection into a subject. In some embodiments, the method further comprises simultaneously administering the genetically engineered virus to the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a pediatric patient. In some embodiments, the proliferative disease is a childhood or pediatric tumor or cancer.

In some embodiments, disclosed herein is a method of treating a proliferative disease in a subject, comprising administering the smallpox vaccine to the subject simultaneously with adipose-derived stromal vascular fraction (SVF). In some embodiments, the SVF of adipose origin is autologous. In some embodiments, the adipose-derived SVF is administered to the subject within 24 hours after the adipose tissue has been taken from the subject. In some embodiments, the smallpox vaccine is administered by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular injection. In some embodiments, adipose-derived SVFs are isolated from a subject using a Time Machine™ device. In some embodiments, the SVF of adipose origin is isolated from the subject using a 2.5 to 3 mm cannula. In some embodiments, the smallpox vaccine is a weakened strain of vaccinia virus from the New York City Board of Health (NYCBOH)). In some embodiments, the NYCBOH vaccinia strain may be ATCC VR-118 or CJ-MVB-SPX. In some embodiments, the smallpox vaccine is selected from Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, or a modified Ankara virus (MVA) vaccine. In some embodiments, the solid tumor is selected from glioblastoma, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, or melanoma. In some embodiments, the subject is a human. In some embodiments, the subject is a pediatric patient. In some embodiments, the proliferative disease is a childhood or pediatric tumor or cancer.

In some embodiments, disclosed herein is a method of treating a proliferative disease, such as a solid tumor or hematologic malignancy, in a subject, comprising administering a smallpox vaccine to the subject simultaneously with a carrier cell. In some embodiments, the carrier cell is an autologous cell from a subject. In some embodiments, the carrier cell is an immune cell selected from granulocyte, mast cell, monocyte, dendritic cell, natural killer cell, and lymphocyte. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the carrier cell contains a heterologous nucleic acid. In some embodiments, the smallpox vaccine and carrier cell are cultured together in vitro prior to administration to a subject. In some embodiments, the smallpox vaccine and carrier cell are placed in the same vial prior to administration to a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a pediatric patient. In some embodiments, the proliferative disease is a childhood or pediatric tumor or cancer.

In some embodiments, a composition is disclosed herein comprising: (a) a smallpox vaccine; and (b) a stem cell. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the stem cell is selected from the group consisting of adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells. cells, adipose stromal cells, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, cord blood stem cells, adult peripheral blood stem cells, myoblast stem cells, small juvenile stem cells, skin fibroblast stem cells, and combinations thereof. In some embodiments, the stem cell is an adipose stromal cell. In some embodiments, the stem cell is a modified stem cell. In some embodiments, the modified stem cell is an adult stem cell. In some embodiments, the modified stem cell is transformed with a lentivirus or a retrovirus. In some embodiments, the modified stem cell is transiently transfected with an artificial chromosome, viral DNA, or plasmid DNA. In some embodiments, the smallpox vaccine does not contain a heterologous nucleic acid. In some embodiments, the composition further comprises a genetically engineered virus.

In some embodiments, a composition is disclosed herein comprising: (a) a smallpox vaccine; and (b) an immune cell such as a granulocyte, mast cell, monocyte, dendritic cell, natural killer cell, or lymphocyte. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the implementation of the immune cell contains a heterologous nucleic acid.

In some embodiments, a composition is disclosed herein comprising: (a) a smallpox vaccine; and (b) an isolated stromal vascular fraction (SVF) of adipose tissue.

Additionally, in some embodiments, a host cell or tumor cell or mammalian organism is disclosed herein comprising any of the compositions comprising a smallpox vaccine and a stem cell; or comprising a smallpox vaccine and an immune cell selected from granulocyte, mast cell, monocyte, dendritic cell, natural killer, and lymphocyte; or comprising smallpox vaccine and isolated stromal vascular fraction (SVF) of adipose tissue.

Brief description of graphic materials

The set of materials of the patent or application contains at least one figure made in color. Copies of this patent or published patent application with color figure(s) will be made available by the Office upon request and upon payment of the fee.

In FIG. 1A and 1B in a panel of human tumor cell lines show an oncolytic effect after infection with the Lister and ACAM2000 virus strains, respectively.

Detailed description

Despite great advances in antitumor therapy over the past 30 years, cancer remains the leading cause of death in the world. Provided herein are methods and compositions for treating a tumor, cancer, or metastasis in a subject, such as a human or animal. In particular, disclosed herein are methods and compositions for activating the body's own immune system to fight cancer by administering a smallpox vaccine.

Definitions

As used herein, the term subject includes any animal for which diagnosis, screening, monitoring, or treatment is envisaged. Animals include mammals such as primates and domesticated animals. The primate is preferably a human. Patient refers to a subject, such as a mammal, primate, human, or farm animal, suffering from a disease or condition, or in which the disease or condition is to be determined or the risk of the disease or condition is to be determined.

As used herein, the term pediatric patient refers to individuals 21 years of age or younger at the time of diagnosis or treatment. In some embodiments, the pediatric patient may be selected from a neonate, infant, child, and adolescent. The term newborn refers to people from birth to 38 days of age. The term infant refers to people between the ages of 29 days and two years. The term child refers to people between the ages of two and 12. The term teenager refers to people between the ages of 12 and 21 (including but not including their twenty-second birthday). In some embodiments, the pediatric patient is greater than 12 months of age. In some embodiments, the pediatric patient is less than 18 years of age.

As used herein, the term simulta- neous administration of the smallpox vaccine and cell refers to administration at intervals of no more than 48 hours. no more than 12 hours, with an interval of no more than 10 hours, with an interval of no more than 8 hours, with an interval of no more than 6 hours, with an interval of no more than 4 hours, with an interval of no more than 2 hours, with an interval of no more than 1 hour

The terms autologous, autologous cell, or autologous transplantation, as used herein in relation to cell transplantation, indicate that the donor and recipient of the cells is the same individual. The term allogeneic, allogeneic cell, or allogeneic transplantation, as used herein in relation to cell transplantation, indicates that the donor and recipient of the cells are different individuals of the same species.

As used herein, the term adipose tissue refers to body adipose tissue, which is loose connective tissue composed primarily of adipocytes. In addition to adipocytes, adipose tissue contains a stromal vascular fraction (SVF) of cells, including preadipocytes, fibroblasts, vascular endothelial cells, a variety of immune cells, and regenerative stem cells.

As used herein, the term adipose stem cell or adult adipose-derived stem cell (ADASC) refers to stem cells isolated from adipose tissue, which are pluripotent stem cells capable of differentiating into various cell types. Adipose stem cells have proven to be equivalent, even superior, to bone marrow stem cells in terms of cell differentiation potential, angiogenesis, and anti-inflammatory effects.

As used herein, the term tumor or tumor cell refers to any type of tumor, including solid tumors or non-solid tumors, disseminated tumors, metastatic or disseminated tumors, or tumor cells from any tumor form.

As used herein, the term vaccine refers to any type of biological agent that promotes or induces an active immune response against a particular disease or pathogen. Such a biological preparation may include, without limitation, an antigen derived from a pathogenic agent, or a portion of an antigen derived from a pathogenic agent. Such a biological preparation may also be in the form of a live attenuated preparation, including live or attenuated or modified disease agents or pathogens; or in the form of inactivated or killed disease agents or pathogens. Alternative forms of such a biological preparation also include, without limitation, forms of subunits, toxoids, conjugates, DNA and recombinant vectors, or any suitable forms that may be developed or may be available in the future to activate an active immune response against them.

It should be noted that in some embodiments, when the term vaccine is used herein, the vaccine should not provide a strong immune response against smallpox (or any other pathogen), provided that it is effective against cancer or a proliferative disease as described herein. . For example, a vaccine may be an immunogenic or infectious composition that treats cancer. In some cases, the term is used to identify certain materials or compositions, and does not necessarily mean the ability of the material or composition to provide an immune response against smallpox, for example. In some embodiments, the composition or material administered may be a virus that elicits an immunogenic response against a tumor cell and/or that elicits an immunogenic response against smallpox. The virus may be any strain of virus, including, for example, one or more of those listed below and elsewhere herein, including viruses that are not part of approved or prospective vaccines. In some embodiments, for example, the virus may be vaccinia virus or any other type of virus described herein or having similar characteristics.

As used herein, the term virus refers to any of a large group of entities called viruses. Viruses, as a rule, contain a protein shell surrounding the core genetic material - RNA or DNA, but not a semipermeable membrane, and are capable of growth and reproduction only inside living cells. Viruses for use in the methods provided herein include poxvirus, adenovirus, herpes simplex virus, Newcastle disease virus, vesicular stomatitis virus, mumps virus, influenza virus, measles virus, reovirus, human immunodeficiency virus (HIV), hantavirus, virus myxomas, but not limited to cytomegalovirus (CMV), lentivirus, and any plant or insect virus.

As used herein, the term haematological malignancy refers to tumors of the blood and lymphatic system (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-associated lymphomas, immunoproliferative malignancies, multiple myeloma and plasma cell malignancies, lymphoid leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of a specific cell type, leukemias of an unspecified cell type, other and unspecified malignancies of lymphoid, hematopoietic and related tissues, for example, diffuse large cell lymphoma, T-cell lymphoma, or cutaneous T-cell lymphoma).

As used herein, the term heterologous nucleic acid refers to a nucleic acid, DNA or RNA that has been introduced into a virus or cell (or progenitor cell). Such a heterologous nucleic acid may include the sequence and functional regulatory elements for genes. For example, the heterologous nucleic acid may include a selectable marker gene, a suicide gene, or a gene expressing a desired protein product that is not endogenously expressed or is expressed endogenously at a low level.

Smallpox vaccines and anti-cancer compositions

In some embodiments, disclosed herein is a method of treating a solid tumor or hematologic malignancy in a subject, comprising administering to the subject a pox vaccine, another pox immunogenic composition, or another composition (immunogenic

- 4 040268 or other) capable of eliciting a similar response. While the terms smallpox vaccines may be used throughout the text, it should be understood that other compositions may also be used that may not be officially approved or labeled as vaccines. For example, other smallpox immunogenic compositions or other immunogenic compositions may be used. In some embodiments, the smallpox vaccine does not contain a heterologous nucleic acid. In some embodiments, the smallpox vaccine is administered by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular injection, or by intradermal injection, or any suitable means of delivery thereof.

The variola virus is the cause of smallpox. Unlike variola virus, vaccinia virus, which has been used for smallpox vaccination, usually does not cause systemic disease in immunocompetent individuals, and therefore it has been used as a live vaccine for smallpox immunization. Successful worldwide vaccination with the vaccinia virus led to the eradication of smallpox as a natural disease in the 1980s. Since then, vaccination has not been carried out for many years, except in cases of high risk of infection with poxvirus (for example, vaccination of laboratory workers). Although the United States stopped routine smallpox immunization of children in 1972, use of the smallpox vaccine is generally considered safe for treating children.

In some embodiments, an attenuated strain derived from a pathogenic virus is used to create a live vaccine. Non-limiting examples of viral strains that have been used as smallpox vaccine include Lister (also known as Elstree), New York City Board of Health (NYCBH strain), Dairen, Ikeda, LC16M8, Western Reserve (WR), Copenhagen, Tashkent, Tian Tan, Wyeth, IHD-J and IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16MO, LIVP, WR 65-16, EM63 and Connaught, but are not limited to. In some embodiments, the smallpox vaccine used in the methods disclosed herein is a weakened strain of vaccinia virus from the New York City Board of Health (NYCBOH)). In some embodiments, the NYCBOH vaccinia strain may be ATCC VR-118 or CJ-MVB-SPX.

In some embodiments, the smallpox vaccine is unattenuated.

In some embodiments, the smallpox vaccine is selected from Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, or a modified Ankara virus (MVA) vaccine. In some embodiments, the smallpox vaccine is not defective in any of the genes present in one or more of these strains.

In some embodiments, the smallpox vaccine is a replication-competent virus. In some embodiments, the smallpox vaccine is replication defective.

The methods disclosed herein can be used to treat any solid tumor or hematologic malignancy. Tumors that can be treated with the methods disclosed herein include bladder tumor, breast tumor, prostate tumor, carcinoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, CNS cancer, glioma, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, laryngeal cancer, leukemia, liver cancer , lung cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory system cancer, sarcoma, skin cancer, cancer stomach, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary system, such as lymphosarcoma, osteosarcoma, breast tumors, mastocytoma, tumor melanoma, adenosquamous carcinoma, lung carcinoid tumor, bronchial gland tumor, bronchiolar adenocarcinoma, small cell lung cancer, non-small cell lung cancer, fibroma, myxochondroma, lung sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilm's tumor, lymphoma Burkitt, microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma, transmissible venereal tumor, testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma, histiocytoma, chloroma, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma , hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal carcinoma, oral papillomatosis, hemangioendothelioma, cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma and squamous cell carcinoma of the lung, leukemia, hemangioperitsi toma, ocular neoplasia, preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma, connective tissue neoplasia, mastocytoma, hepatocellular carcinoma, lymphoma, lung adenomatosis, lung sarcoma, Rous sarcoma, reticuloendotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma, lymphoid leukemia, retinobla - 5 040268 stoma, hepatic neoplasia, lymphosarcoma, plasmacytoid leukemia, swim bladder sarcoma (in fish), caseous lymphadenitis, lung carcinoma, insulinoma, lymphoma, sarcoma, salivary gland tumors, neuroma, pancreatic islet of Langerhans tumor, gastric MALT lymphoma and adenocarcinoma stomach, but not limited to.

In some embodiments, the tumor is selected from metastatic melanoma; adenocarcinoma of the esophagus and stomach; cholangiocarcinoma (any stage); adenocarcinoma of the pancreas (any stage); gallbladder cancer (any stage); mucinous cancer of the appendix of high malignancy (any stage); gastrointestinal neuroendocrine cancer of high malignancy (any stage); mesothelioma (any stage); soft tissue sarcomas; prostate cancer; renal cell carcinoma; small cell carcinoma of the lung; non-small cell lung carcinoma; squamous cell carcinoma of the head and neck; colorectal cancer; ovarian carcinoma; hepatocellular carcinoma and glioblastoma.

In some embodiments, the tumor is selected from glioblastoma, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, and melanoma.

In some embodiments, the tumor or cancer that can be treated is a childhood or pediatric tumor or cancer. For example, a tumor or cancer may be leukemia, lymphoma, sarcoma, and the like. Non-limiting examples of leukemia include acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Non-limiting examples of types of lymphomas include Hodgkin's disease (or Hodgkin's lymphoma) and non-Hodgkin's lymphoma, B- and T-cell lymphomas. Non-limiting examples of solid tumors or cancers in pediatric patients include brain tumors, Ewing's sarcoma, eye cancer (retinoblastoma), embryonic cell tumors, kidney tumors (eg, Wilm's tumor), liver cancer, neuroblastoma, osteosarcoma, rhabdomyosarcoma, skin cancer ( melanoma), soft tissue sarcoma, and thyroid cancer. In some embodiments, the subject is a human. In some embodiments, the subject is a pediatric patient. In some embodiments, the subject is a neonate. In some embodiments, the subject is an infant. In some embodiments, the subject is a child. In some embodiments, the subject is a teenager. In some embodiments, the age of the subject is greater than 12 months. In some embodiments, the subject is less than 18 years of age.

Smallpox vaccine and composition carriers

Immunogenic inducers of cell death, such as viruses, undergo significant elimination and/or neutralization after systemic administration. Therefore, in some embodiments, suitable carriers are disclosed herein for protecting the disclosed smallpox vaccines from elements of humoral and cellular immunity in the bloodstream, as well as methods for their targeted delivery to tumor foci.

Thus, in some embodiments, disclosed herein is a method of treating a solid tumor or hematologic malignancy in a subject, comprising administering a smallpox vaccine to the subject simultaneously with a carrier cell. In some embodiments, the carrier cell is an autologous cell from a subject. In other embodiments, the cell may be non-autologous or allogeneic.

In some embodiments, the carrier cell is an insect cell, a reptile cell, an amphibian cell, a bird cell, or a mammalian cell. In some embodiments, the carrier cell is an immortalized cell. In some embodiments, the carrier cell is a neoplastic cell derived from a tumor. In some embodiments, the carrier cell is a human cell.

In some embodiments, the smallpox vaccine or other composition described herein and the carrier cell are administered simultaneously. In some embodiments, the smallpox vaccine and the carrier cell are administered at the same time using the same means of administration. In some embodiments, the smallpox vaccine and host cell are administered simultaneously using the same vessel, such as a syringe, by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular, or intradermal injection, or any suitable delivery method.

stem cells

Recent studies have shown that stem cells are characterized by pronounced homing to glioma, in addition, the possibility of placing genes into stem cells using viral vectors has been demonstrated. These studies indicate that stem cells are a promising candidate for the delivery of an anticancer drug carrier to tumor foci.

Accordingly, in some embodiments, the present document discloses the use of stem cells as carriers for the in vivo delivery of a smallpox vaccine to cancer cells or tumors. In some embodiments, the smallpox vaccine is mixed with stem

- 6 040268 carrier cells to avoid clearance of the virus by the immune system before the virus reaches the tumor. Thus, in some embodiments, disclosed herein is a method of treating a solid tumor or hematologic malignancy in a subject, comprising administering a smallpox vaccine to the subject simultaneously with a stem cell. In some embodiments, the smallpox vaccine does not contain a heterologous nucleic acid. In some embodiments, the method further comprises simultaneously administering the genetically engineered virus to the subject. Some embodiments relate to methods for formulating stem cell and smallpox vaccine compositions prior to administration.

In some embodiments, the carrier stem cell is an autologous stem cell. In other embodiments, the carrier stem cell is non-autologous or allogeneic.

In some embodiments, the carrier stem cells are selected from the group consisting of adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipose stromal cells, endothelial stem cells, induced pluripotent stem cells, bone marrow stem cells, cord blood stem cells, adult peripheral blood stem cells, myoblastic stem cells, small juvenile stem cells, skin fibroblast stem cells, and combinations thereof . In some embodiments, the modified stem cell is an umbilical cord-derived mesenchymal-like cell. In some embodiments, the umbilical cord-derived mesenchymal-like cell is an Immstem™ cell. In some embodiments, the stem cell is an adipose stromal cell. One or more of the above listed cells may be specifically excluded from the compositions and methods of some embodiments.

In some embodiments, the carrier stem cells are modified. In particular, in some embodiments, the modified stem cell is an adult stem cell (ASC). In some embodiments, the modified stem cell is transformed with a viral vector. In some embodiments, the modified stem cell is transformed with a lentivirus or a retrovirus. In some embodiments, the modified stem cell is transformed with a recombinant virus. In some embodiments, the modified stem cell is transiently transfected with an artificial chromosome, viral DNA, or plasmid DNA. In some embodiments, the virus is an oncolytic virus. In some embodiments, the implementation of the virus is a vaccinia virus. In some embodiments, the virus is a replicating oncolytic vaccinia virus (VACV). In some embodiments, the modified stem cell is capable of localizing to a tumor. In some embodiments, the modified stem cell is autologous. In some embodiments, the modified stem cell is allogeneic.

In some embodiments, the modified stem cell is an adult stem cell. In some embodiments, the modified stem cell is transformed with a lentivirus or a retrovirus. In some embodiments, the modified stem cell is transiently transfected with an artificial chromosome, viral DNA, or plasmid DNA. In some embodiments, the modified stem cell is capable of localizing to a tumor. In some embodiments, the modified stem cell is autologous. In some embodiments, the modified stem cell is allogeneic. In some embodiments, the modified stem cell is selected from the group consisting of adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipose stromal cells, endothelial stem cells, and combinations thereof. In some embodiments, the modified stem cell is an umbilical cord-derived mesenchymal-like cell. In some embodiments, the umbilical cord-derived mesenchymal-like cell is an Immstem™ cell.

ImmStem is an umbilical cord-derived mesenchymal-like cell that has a pluripotent differentiation capacity and is characterized by unique surface markers and production of growth factors. ImmStem has numerous advantages over other stem cell sources, including ease of preparation, faster proliferation rate, very low immunogenicity, and the ability to differentiate into tissues that are components of all three germ layers. Compared to other subtypes

- 7 040268 mesenchymal stem cells (MSC) ImmStem demonstrated increased anti-inflammatory and migratory capacity due to the cytokine priming step that precedes the introduction. ImmStem cells are isolated from the human umbilical cord, which is obtained from women who have given birth at term, immediately after giving birth. To stimulate the stress response, the cells are cultured for approximately 48 hours with interferon gamma. In some embodiments, the implementation of the cultivation with IFN-gamma can last from 1 to 72 h or for any time within this range.

In some embodiments, the smallpox vaccine and/or stem cell is administered to a subject by any suitable route of administration. In some embodiments, the smallpox vaccine and/or stem cell is administered by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular injection.

In some embodiments, the smallpox vaccine and the stem cell are administered simultaneously. In some embodiments, the smallpox vaccine and the stem cell are administered at the same time using the same means of administration. In some embodiments, the smallpox vaccine and the stem cell are administered simultaneously using the same vessel, such as a syringe, by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular, or intradermal injection, or any suitable delivery method.

In some embodiments, the tumor is selected from metastatic melanoma; adenocarcinoma of the esophagus and stomach; cholangiocarcinoma (any stage); adenocarcinoma of the pancreas (any stage); gallbladder cancer (any stage); mucinous cancer of the appendix of high malignancy (any stage); gastrointestinal neuroendocrine cancer of high malignancy (any stage); mesothelioma (any stage); soft tissue sarcomas; prostate cancer; renal cell carcinoma; small cell carcinoma of the lung; non-small cell lung carcinoma; squamous cell carcinoma of the head and neck; colorectal cancer; ovarian carcinoma; hepatocellular carcinoma and glioblastoma.

In some embodiments, the tumor is selected from glioblastoma, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, and melanoma. In some embodiments, one or more of the listed tumors, cancers, or malignancies may be specifically excluded from the compositions and/or methods described herein.

immune cells

Additionally, in some embodiments, the present document discloses the use of immune cells as a carrier for the in vivo delivery of a smallpox vaccine to cancer cells or tumors. In some embodiments, the anti-cancer agent is mixed with immune carrier cells to avoid clearance of the virus by the immune system before the virus reaches the tumor. Thus, in some embodiments, disclosed herein is a method of treating a solid tumor or hematologic malignancy in a subject, comprising administering the pox vaccine to the subject simultaneously with an immune cell selected from a granulocyte, a mast cell, a monocyte, a dendritic cell, a natural killer cell, and a lymphocyte. In some embodiments, the immune cell is an autologous cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the implementation of the immune cell contains a heterologous nucleic acid. In some embodiments, the smallpox vaccine and the immune cell are cultured together in vitro prior to administration to a subject.

In some embodiments, the smallpox vaccine and the immune cell are administered simultaneously. In some embodiments, the smallpox vaccine and the immune cell are administered at the same time using the same means of administration. In some embodiments, the smallpox vaccine and the immune cell are administered simultaneously using the same vessel, such as a syringe, by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular, or intradermal injection, or any suitable method of delivery thereof.

In some embodiments, the tumor is selected from metastatic melanoma; adenocarcinoma of the esophagus and stomach; cholangiocarcinoma (any stage); adenocarcinoma of the pancreas (any stage); gallbladder cancer (any stage); mucinous cancer of the appendix of high malignancy (any stage); gastrointestinal neuroendocrine cancer of high malignancy (any stage); mesothelioma (any stage); soft tissue sarcomas; prostate cancer; renal cell carcinoma; small cell carcinoma of the lung; non-small cell lung carcinoma; squamous cell carcinoma of the head and neck; colorectal cancer; ovarian carcinomas; hepatocellular carcinoma and glioblastoma. In some embodiments, one or more of the listed tumors, cancers, or malignancies may be specifically excluded from the compositions and/or methods described herein.

In some embodiments, the tumor is selected from glioblastoma, breast carcinoma

- 8 040268 glands, lung carcinomas, prostate carcinomas, colon carcinomas, ovarian carcinomas, neuroblastomas, tumors of the central nervous system and melanomas.

Adipose stromal vascular fraction

In some embodiments, disclosed herein is a method of treating a proliferative disease in a subject, comprising administering the pox vaccine to the subject simultaneously with an adipose-derived stromal vascular fraction (SVF), wherein the adipose-derived SVF is autologous. In some embodiments, the adipose-derived SVF is administered to the subject within about 24 hours after the adipose tissue has been taken. In some embodiments, the adipose-derived SVF may be administered at any time after receipt, up to about 48 hours after receipt, or at any time within this range. In some embodiments, the smallpox vaccine is administered by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular, or intradermal injection, or any suitable delivery method.

Adipose tissue is an alternative to bone marrow as a source of stem cells for the following reasons: a) cell isolation from adipose tissue is a simpler and less invasive procedure than cell extraction from bone marrow; b) the content of mesenchymal stem cells (MSC) in adipose tissue is higher than in the bone marrow; c) the number of adipose tissue MSCs does not decrease with age and therefore adipose tissue MSCs can serve as a source of autologous cells for all patients and d) adipose tissue is also a source of unique cell populations in addition to MSCs that have therapeutic potential, including endothelial cells and regulatory T cells.

MSCs are weakly immunogenic and have immunomodulatory activity, these properties are common to MSCs from different tissues. Due to their weak immunogenicity, allogeneic MSCs are believed to survive and remain active when administered therapeutically.

It is known that SVF of adipose origin, in addition to stem/progenitor cells, also contains monocytes/macrophages and endothelial cells.

Whole lipoaspirate-derived SVF cells do not require extensive processing, minimizing the number of steps where contamination can occur (Kurita et al., Plast Reconstr Surg 2008, 121:1033-1041; discussion 1042-1033; Yoshimura et al. al., Aesthetic Plast Surg 2008, 32: 48-55; discussion 56-47). The safety of administering cells derived from adipose tissue has been confirmed by autologous fat grafting, a common practice in cosmetic surgery (Hang-Fu et al., Aesthetic Plast Surg 1995, 19:427-437). Each of the above references is incorporated herein by reference in its entirety.

In some embodiments, the adipose-derived SVF is prepared by methods and in accordance with knowledge known to those skilled in the art. In some embodiments, adipose-derived SVFs are isolated from a subject using a Time Machine™ device. In some embodiments, the SVF of adipose origin is isolated from the subject using a 2.5 to 3 mm cannula. In some embodiments, one or more of the following devices may be used: PNC's Multi Station, CHA Biotech Cha-Station, Cytori Celution 800/CRS System, and Medi-Khan's Lipokit with MaxStem.

In some embodiments, smallpox vaccine and adipose-derived SVF are administered simultaneously. In some embodiments, smallpox vaccine and adipose-derived SVF are administered simultaneously. In some embodiments, the smallpox vaccine and the adipose-derived SVF are administered simultaneously by the same means of administration. In some embodiments, the smallpox vaccine and the adipose-derived SVF are administered simultaneously via the same vessel, e.g., a syringe, by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, or intraocular, or intradermal injection, or any suitable method of delivery thereof.

In some embodiments, the tumor is selected from metastatic melanoma; adenocarcinoma of the esophagus and stomach; cholangiocarcinoma (any stage); adenocarcinoma of the pancreas (any stage); gallbladder cancer (any stage); mucinous cancer of the appendix of high malignancy (any stage); gastrointestinal neuroendocrine cancer of high malignancy (any stage); mesothelioma (any stage); soft tissue sarcomas; prostate cancer; renal cell carcinoma; small cell carcinoma of the lung; non-small cell lung carcinoma; squamous cell carcinoma of the head and neck; colorectal cancer; ovarian carcinomas; hepatocellular carcinoma and glioblastoma. One or more of the above tumors may be specifically excluded from the compositions and methods of some embodiments.

In some embodiments, the tumor is selected from glioblastoma, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, or melanoma. One or more of the above components may be specifically excluded from the compositions and methods of some embodiments.

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Compositions

Disclosed herein are various compositions used in the methods described herein for the treatment of solid tumor or hematologic malignancy.

In some embodiments, a composition is disclosed herein comprising a smallpox vaccine and a carrier cell as disclosed herein.

In some embodiments, a composition is disclosed herein comprising (a) a smallpox vaccine and (b) a stem cell. In some embodiments, the stem cell is selected from the group consisting of adult stem cells, embryonic stem cells, fetal stem cells, mesenchymal stem cells, neural stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells. cells, adipose stromal cells, endothelial stem cells, and combinations thereof. In some embodiments, the stem cell is an adipose stromal cell. In some embodiments, the stem cell is a modified stem cell. In some embodiments, the modified stem cell is an adult stem cell. In some embodiments, the modified stem cell is transformed with a lentivirus or a retrovirus. In some embodiments, the modified stem cell is transiently transfected with an artificial chromosome, viral DNA, or plasmid DNA. In some embodiments, the smallpox vaccine does not contain a heterologous nucleic acid. In some embodiments, the composition further comprises a genetically engineered virus. One or more of the above components may be specifically excluded from the compositions and methods of some embodiments.

In some embodiments, a composition is disclosed herein comprising: (a) a smallpox vaccine; and (b) an immune cell selected from a granulocyte, a mast cell, a monocyte, a dendritic cell, a natural killer cell, and a lymphocyte. In some embodiments, the immune cell is a lymphocyte.

In some embodiments, the lymphocyte is a T cell. In some embodiments, the implementation of the immune cell contains a heterologous nucleic acid.

In some embodiments, a composition is disclosed herein comprising: (a) a smallpox vaccine; and (b) an isolated adipose stromal vascular fraction (SVF).

In some embodiments, the compositions disclosed herein include a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable carrier refers to solvents, diluents, preservatives, dispersing or suspending agents, isotonic agents, thickening or emulsifying agents, binders, and lubricants suitable for a particular dosage form. A person skilled in the art is aware of many different carriers that can be used in the preparation of pharmaceutical compositions and in known methods for their preparation (see Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995; which is incorporated herein by links in their entirety). Pharmaceutically acceptable carriers may include Ringer's solution, isotonic saline, starch, potato starch, sugars, glucose, powdered tragacanth, malt, gelatin, talc, cellulose and its derivatives, ethylcellulose, sodium carboxymethylcellulose, cellulose acetate excipients, cocoa butter, suppository waxes. , agar, alginic acid, oils, cottonseed oil, peanut oil, safflower oil, sesame oil, olive oil, soybean oil, corn oil, glycols, propylene glycol, esters, ethyl laurate, ethyl oleate, buffering agents, aluminum hydroxide, magnesium hydroxide, phosphate buffer solutions, pyrogen-free water, ethyl alcohol, other non-toxic compatible lubricants, sodium lauryl sulfate, magnesium stearate, colorants, releasing agents, coating agents, sweeteners, flavoring and flavoring agents, but not limited to. Pharmaceutically acceptable carriers may also include preservatives and antioxidants. One or more of the above materials may be specifically excluded from the compositions and methods of some embodiments.

A composition disclosed herein comprising a live smallpox vaccine may include an adjuvant. Optionally, one or more compounds having adjuvant activity may be included in the vaccine. Adjuvants are non-specific stimulants of the immune system. They enhance the host's immune response to the vaccine. Examples of adjuvants known in the art are complete and incomplete Freund's adjuvant, vitamin E, non-ionic block polymers, muramyl dipeptides, ISCOMs (immunostimulatory complexes), saponins, mineral oil, vegetable oil, and carbopol. Adjuvants particularly suitable for mucosal use are, for example, heat-labile E. coli toxin (LT) or cholera toxin (CT). Other suitable adjuvants are, for example, aluminum hydroxide, aluminum phosphate or alumina, oil emulsions (eg Bayol F® or Marcol 52®, saponins or vitamin E solubilisate). One or more of the above materials may be specifically excluded from

- 10 040268 compositions and methods of some embodiments.

The introduction of therapeutic agents

The effective dose of each of the therapeutic agents disclosed herein may vary depending on various factors, including, but not limited to, the particular treatment used, the compound or pharmaceutical composition used, the route of administration, the condition being treated, and/or the severity of the condition being treated. Thus, the dosage regimen of the combination of the invention is selected according to a variety of factors, including the route of administration and the renal and hepatic function of the patient. A physician, clinician or veterinarian of ordinary skill can easily determine and prescribe the effective amount of the individual active ingredients needed to prevent, counteract or arrest the progression of the disease. For optimum precision in achieving concentrations of active ingredients in a range that provides efficacy without toxicity, a regimen based on the kinetics of the availability of active ingredients to target areas is required.

Methods for preparing pharmaceutical compositions comprising appropriate therapeutic agents disclosed herein are known in the art and will be apparent in the art from well-known standard references such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th edition ( 1990), which are incorporated herein by reference in their entirety.

In some embodiments, the smallpox vaccine and/or cells disclosed herein are administered to a subject by any suitable method. In some embodiments, the smallpox vaccine and/or cells disclosed herein are administered by intratumoral, intravenous, intraperitoneal, intrathecal, intraventricular, intraarticular, intraocular, or intradermal injection, or by any suitable method of delivery thereof.

The amount of vaccine administered to an average-sized adult may be, for example, from 1x10 ² to 1x10 ¹⁰ plaque forming units, from 1x103 to 1x10 ⁸ plaque forming units, from 1x10 ⁴ to 1x10 ⁶ plaque forming units, or any other value or subrange within the values given. As a specific example, about 2.5x105 plaque forming units can be used.

It should be understood that the embodiments described herein are not limited to vaccination or vaccination per se, but are also associated with the activation of an immune response or response to cancer cells. Although the words vaccine, vaccination, or other similar terms are used for reasons of convenience, it should be understood that such embodiments also refer to immune compositions, immunogenic compositions, activation of the immune response, immunization, and the like, when complete prophylactic immunity not required or not enabled. For example, embodiments relating to vaccination may also refer to the activation or triggering of an immunogenic or immune response against a tumor cell or tumor, whether or not that response results in complete eradication or immunization against such tumor cell, tumor or cancer.

The disclosures described herein for purposes of illustration may be appropriately practiced without any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms including, including, containing, etc. should be understood in a broad sense and without limitation. In addition, the terms and expressions used herein have been used for the purpose of description and not limitation, and the use of such terms and expressions is not meant to exclude any equivalents of the features or parts shown and described, but it should be understood that various modifications are possible in the scope of the claimed essence of the invention.

Other embodiments are set forth in the claims.

Examples

Example 1 Preparation of ACAM2000 Vaccine

The vaccine vial should be removed from the refrigerator and warmed to room temperature before reconstitution. The flip caps of the vaccine and diluent vials are removed, each rubber stopper is swabbed with isopropyl alcohol and dried completely.

Using aseptic technique and a sterile 1 ml syringe fitted with a 25 G x 5/8 in. needle (supplied with the syringe), withdraw 0.3 ml of diluent and transfer the entire contents of the syringe to the vaccine vial. Shake gently to mix, being careful not to get the product on the rubber cap. The reconstituted vaccine should be a clear or slightly turbid, colorless or straw-yellow liquid that does not contain foreign matter. The color and presence of particulate matter in the reconstituted vaccine should be checked visually prior to administration. If particulate matter or discoloration is noted, the vaccine should not be used and the vaccine vial should be safely disposed of.

After reconstitution of the lyophilized preparation, each vial contains approximately 2.5-12.5x107 plaque-forming units (PFU) of vaccinia virus (live). After recovery

- 11 040268 ACAM2000 vaccine can be used within 6-8 hours if stored at room temperature (20-25°C, 68-77°F). Unused reconstituted ACAM2000 vaccine can be stored in a refrigerator (2-8°C, 36-46°F) for up to 30 days, after which it should be disposed of as a biohazard.

Those who receive and administer the vaccine should wear surgical or protective gloves and avoid contact of the vaccine with the skin, eyes, or mucous membranes.

The vaccine vial, rubber cap, diluent syringe, reconstitution needle, injection needle, and any material that has been in contact with the vaccine should be disposed of in sealed, puncture-proof containers for biological hazardous waste. These containers must then be properly disposed of.

Before use, the vaccine vial is gently shaken to mix, being careful not to get the product on the rubber cap.

Using aseptic technique and a sterile 1 ml syringe fitted with a 25 G x 5/8 inch needle, withdraw the entire contents of the vial and transfer it to a labeled 20cc syringe containing the SVF fraction.

Shake gently to mix well and incubate the syringe at 37°C for 2-4 hours.

Example 2 Isolation and Preparation of Adipose Stromal Vascular Fraction

The patient receives a local anesthetic comprising 0.5% lidocaine with epinephrine 1:400,000 with 8.4% HCO3 titrated to pH 7.4 (typically 5 cc ^HCO3 in a total volume of 60 ^cc ) and undergoes sterile preparation. Next, the patient undergoes a liposuction procedure using a Time-Machine™ device, a fat sampling device (syringe) and a 2.5-3 mm cannula. An ointment and a patch with bacitracin are fixed on the wound along with a pressure bandage.

SVF (ADSC) is obtained in a closed system according to the following protocol:

using TimeMachine® to select fat into a 60cc TR-101 syringe (a disposable sterile syringe for fat sampling);

centrifuge at 2800 rpm for 3 min;

remove free fatty acids and sediment (local/blood) using the closed system TP-109;

transfer 25 cm of condensed fat into a TP-102 syringe (syringe for SVF processing);

add pre-warmed (38°C) 25 ^cc Roche T-MAX® Time Machine Accelerator (GMP compliant collagenase) containing 12.5 Wunsch units;

incubate at 38°C for 30-45 min;

centrifuge at 200 g for 4 min;

remove the supernatant except for the lower 3-10 cm ³ ;

add 50 cc ^of D5LR as a wash solution to remove collagenase residues and centrifuge at 200 g for 4 min;

repeat washing two more times (3 washings in total);

remove all the supernatant, leaving 3-10 cm ³ of sediment, this is the stromal-vascular fraction;

transfer the SVF into a labeled 20cc syringe through a 100 micron filter;

collect a sample of SVF and determine the number of cells in it, their viability, check for the absence of lumps and sediment;

remove aliquots from each cell suspension for testing for endotoxins and sterility; SVF will be available for injection only after endotoxin test results confirm that the EU level is less than or equal to 5 EU/kg/h and Gram stain results are negative;

resuspend cells in 20 ml Isolyte;

select cell suspensions with a 18G injection syringe;

up to 100 million viable cells can be used for injections;

the syringe is then placed in a sealed sample bag labeled with the patient's name and medical record number for transport to the treatment room for injection.

Example 3 Administration of Adipose SVF with Smallpox Vaccine

Methods of administration

Intravenous: Unfoamed autologous stromal vascular fraction (SVF) extracted from 500 ml or less of lipoaspirate, purified by collagenase digestion followed by a series of washes, and containing up to 100 million cells incubated with vaccinia virus, is delivered by intravenous injection in a volume of 20 ml.

Intratumoral: Non-foamed autologous stromal vascular fraction (SVF) extracted from 500 ml or less of lipoaspirate purified by collagenase digestion followed by a series of washes and containing cells incubated with vaccinia virus is delivered by intratumoral injection. The volume of injection and the number of cells injected will vary and depend on the type of tumor, its size and location.

Example 4

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Oncolysis of human tumor cell lines after infection with Lister and

ACAM2000 in vitro and comparison of solid tumor regression in tumor-bearing A549 and PC3 nude mice colonized with ACAM2000 or Lister vaccinia virus strains in vivo and in clinical studies.

In vitro study

For a comparative study of the antitumor activity of the ACAM2000 smallpox vaccine, a panel of human cancer cell lines were infected with vaccinia virus strains ACAM2000 and Lister with an MOI of 0.01.

MDA-MB-231 breast cancer cells, A549 and NCI-H1299 lung adenocarcinoma cells, PC3 prostate cancer cells, and SiHa cervical cancer cells were seeded in 96-well plates 24 hours before infection. The next day the cells formed an almost continuous layer and were infected with 50 µl of DMEM supplemented with 2% fetal bovine serum (FBS) containing the appropriate amount of vaccinia virus (MOI is 0.01). Control cells were left untreated. After 1 h of incubation, the medium was changed to DMEM supplemented with 10% FBS. After 96 h, the death of tumor cells was analyzed by the MTT method.

Cell death was measured 96 hours after infection with vaccinia viruses ACAM2000 or Lister (MOI is 0.01). Viability was measured using a colorimetric MTT test. Optical density was measured at 570 nm. n=3. (A) Results are presented as percentage (%) of dead cells normalized to untreated cells. (B) The table compares the cell killing efficiency of ACAM2000 and Lister viruses (+=1-25%, ++=26-50%, +++=51-75%, ++++=76100%).

As shown in FIG. 1A and 1B, the ACAM2000 virus strain effectively killed all human cancer cell lines except NCI-H1299, which was comparable to the Lister virus strain (FIGS. 1A and B). Similar data were obtained for Copenhagen, MVA and other selected vaccinia virus strains.

In vivo study

To further compare the antitumor activity of the ACAM2000 and Lister smallpox vaccines, A549 and PC-3 tumor cells were injected subcutaneously into nude (athymic) mice. After the tumor volume reached 350 mm ³ , the animals were injected with a viral inoculum (5x106 PFU) by retroorbital injection (n=5). Tumor-bearing control mice were injected with saline buffer. Comparable tumor growth inhibition was observed for both vaccinia virus samples at 20 days post-injection in A549 tumor-bearing mice and 14 days after virus injection in PC-3 tumor-bearing mice (data not shown).

Clinical researches

Twenty-five patients with advanced metastatic solid tumors and hematologic malignancies received the autologous stem cell/vaccinia virus combination in the recent StemImmune study. None of the 25 patients in this study experienced any side effects resulting from stem cell/vaccinia administration. In particular, no fever, chills or other side effects associated with the virus were reported, whereas in previous clinical studies such side effects were observed 6-12 hours after administration of naked virus without stem cells. Importantly, all patients in the current study experienced a burning sensation localized to the tumor site approximately 1-3 weeks after stem cell/oncolytic virus injection, demonstrating efficient delivery of the virus to tumor foci and successful infection of target tumors. In some patients, a decrease in tumor size and complete disappearance of the tumor were observed.

A series of blood samples were taken from treated patients: before treatment (baseline sample), as well as 1 day, 1 week, 1 month, 3 months and 6 months after the start of treatment. Analysis of these samples showed effective induction of cytokine release approximately 1 week after the start of treatment. Importantly, flow cytometry analysis confirmed the induction of activated T cells and memory T cells one month after the start of treatment.

Thus, these results confirmed i) the high safety of this treatment without any treatment-related side effects; ii) documented efficacy in reducing the size of the tumor and even the complete disappearance of the tumor in some patients; and iii) effective induction of cytokine release and a pronounced cellular response after this treatment.

Example 5 Clinical Studies of SVFs of Adipose Origin with Vaccinia Virus

Study design

The study evaluates the safety of administering adipose-derived SVF in combination with ACAM2000 (SVF/ACAM2000) to patients with advanced solid tumors based on subjective and objective patient outcomes. Also, when possible, the antitumor effects of adipose-derived SVF/ACAM200 administration and the presence of vaccinia virus in malignant tumors following SVF/ACAM200 injection are evaluated by examining a resected/biopsied tumor sample. In addition, the immune response against vaccinia virus and antitumor immune response after administration of SVF/ACAM2000 were evaluated.

All methods of administration (intravenous and intratumoral) will be classified regardless of the condition in order to obtain information on specific methods of administration. The study will continue to monitor each patient to determine if any side effects occur after treatment with SVF/ACAM2000.

Inclusion Criteria

Patients (>18 years) with histologically confirmed primary or recurrent solid organ cancer in stage III or IV (staged according to the American Joint Committee on Cancer (AJCC; 7th edition)), and/or aggressive solid organ cancer (aggressive cancer is defined if published survival rates, directly related to the underlying disease, are less than 20% at 5 years despite the best currently available therapy).

Patients provide written consent to the collection of needle biopsy samples of tumor tissue (primary or metastatic) before the start of the study for the analysis of mutations associated with the patient's malignant tumor, for the analysis of the sensitivity of tumor cells to the vaccinia virus and for correlation studies. Instead, if available, the patient may provide a block of formalin-fixed, paraffin-embedded tissue (from his/her primary or metastatic tumor) obtained 45 days or less prior to written consent to participate in a clinical trial involving paraffin blocks .

Criteria include:

1) Ability to understand and willingness to sign written informed consent.

2) Histologically confirmed diagnosis of advanced solid organ cancer (AJCC 7th edition: stage III or IV) or aggressive solid organ cancer (aggressive cancer is defined if published survival rates directly related to the underlying disease are less than 20% in 5 years despite the best therapy currently available).

Such cancers include metastatic melanoma; adenocarcinoma of the esophagus and stomach (stage III/IV); cholangiocarcinoma (any stage); adenocarcinoma of the pancreas (any stage); gallbladder cancer (any stage); mucinous cancer of the appendix of high malignancy (any stage); gastrointestinal neuroendocrine cancer of high malignancy (any stage); mesothelioma (any stage); soft tissue sarcoma of high malignancy (any stage); prostate cancer; renal cell carcinoma; small cell carcinoma of the lung; non-small cell lung carcinoma; squamous cell carcinoma of the head and neck; colorectal cancer; ovarian carcinoma; hepatocellular carcinoma; glioblastoma, but is not limited to them.

3) Absence of persistent acute toxic effects due to any prior therapy, including but not limited to biological therapy, radiotherapy, chemotherapy, or surgery, i.e. all such effects must be defined according to the Common Terminology Criteria for Adverse Events (CTCAE, version 4.0) as grade < 1. Any other surgery (excluding biopsy) must be performed at least 28 days before the start research.

4) General condition on the ECOG scale is from 0 to 2.

5) Life expectancy is at least 3 months.

6) Proper functioning of organs and bone marrow in accordance with the indicators below:

absolute neutrophil count (ANC) > 1.5x109;

platelets > 100x109 (no platelet transfusion);

hemoglobin > 9.0 g/dl (with or without red blood cell (RBC) transfusion);

serum creatinine < 1.5 x upper limit of normal (ULN);

bilirubin <1.5xULN;

ALT and AST<2.5xULN (in case of liver metastases AST/ALT<5.0xULN);

LDH<1.5xULN.

7) Women of childbearing age and men with partners of childbearing potential must agree to use adequate contraception (hormonal or barrier method of preventing pregnancy, abstinence) prior to study entry, during study participation, and for 90 days after completion of therapy. If a woman becomes pregnant or suspects she is pregnant while participating in this study, she should tell her doctor immediately.

A woman of childbearing age is any woman (regardless of sexual orientation, tubal ligation, or unmarried) who meets the following criteria:

has not undergone a hysterectomy or bilateral oophorectomy, or has not gone through natural menopause for at least 12 consecutive months (i.e., has had a period at any time in the previous 12 months).

- 14 040268

8) Women of childbearing age have a negative pregnancy test before dosing study drug

9) Consent and ability to perform scheduled visits, treatment plan, imaging and laboratory tests.

Exclusion Criteria

1) Current use or intended use of other investigational drugs or marketed anticancer drugs during the study.

2) Patients who received chemotherapy or radiotherapy within 4 weeks prior to study entry or who did not recover from adverse events due to agents administered more than 4 weeks prior to study entry.

3) Patients who had undergone surgery (excluding biopsy) less than 4 weeks prior to the start of the study or had not recovered sufficiently from surgical trauma or from wound healing.

4) Having known disorders of the immune system (including acquired immunodeficiency syndrome (AIDS), HIV infection, or hepatitis B or C). Eligible patients must have tested negative for HIV within 4 weeks prior to study entry.

5) Patients receiving additional immunosuppressive therapy or any steroids (with the exception of the simultaneous use of corticosteroids, provided that the use of no more than 20 mg per day of the equivalent of prednisolone).

6) Previously subjected to gene therapy or therapy with any type of cytolytic virus.

7) Have clinically significant cardiovascular disease (New York Heart Association class III or IV), including pre-existing arrhythmias, uncontrolled angina, and myocardial infarction in the year prior to study entry, or a reduced left ventricular ejection fraction of grade 2 or greater.

8) Pulse oximetry oxygen saturation < 90% at rest.

9) Having dementia or an altered mental state that does not allow informed consent.

10) Severe or uncontrolled medical disorder that, in the investigator's opinion, would impair the ability to receive study treatment (i.e., uncontrolled diabetes, chronic kidney disease, chronic or active lung disease, fever, systemic and/or uncontrolled infections, mental illness/social situations that limit adherence to treatment requirements).

11) Concomitantly receiving a vaccinia antiviral (eg, cidofovir, vaccinia immunoglobulin, imatinib, ST-246) during the treatment period.

12) Those who are known to be allergic to ovalbumin or other egg products.

13) Having clinically significant dermatological disorders (eg, eczema, psoriasis, or any non-healing skin sores or ulcers) as judged by the clinical trial director at the time of screening and during the study.

14) Having a history of allergy to iodinated contrast medium.

15) Patients with known brain metastases should be excluded from this clinical trial because of their poor prognosis and because they often develop progressive neurological dysfunction that may interfere with evaluation of neurological and other adverse events.

16) Pregnant or lactating. This treatment can cause birth defects and harm babies.

17) Patients who are not accepted into this study because their condition cannot be improved with SVF/ACAM2000 in the opinion of one or more of the medical specialists in the study team.

Procedures, administration and duration of treatment

The following procedures are provided

The patient receives a local anesthetic comprising 0.5% lidocaine with epinephrine in a ratio of 1:400,000 with 8.4% HCO3 titrated to pH 7.4 (usually 5 cc ^HCO3 in a total volume of 60 ^cc ).

The patient undergoes sterile preparation.

The patient undergoes a liposuction procedure using a Time-Machine™ device, a fat harvester (syringe) and a 2.5-3 mm cannula.

An ointment and a patch with bacitracin are fixed on the wound along with a pressure bandage.

SVF (ADSC) receive in a closed system in accordance with the established protocol (see pages 26-27).

Methods of administration

IV: unfoamed autologous stromal vascular fraction (SVF) extracted from 500 ml or less of lipoaspirate, purified by collagenase digestion followed by

- 15 040268 series of washes and containing up to 100 million cells incubated with vaccinia virus, delivered by intravenous injection in a volume of 20 ml.

Intratumoral: SVF incubated with vaccinia virus is delivered by intratumoral injection to Desert Medical Imaging in Palm Desert, CA. The volume of injection and the number of cells injected will vary and depend on the type of tumor, its size and location. Dosage will depend on individual outputs. If we find specific correlations with a certain number of cells, then we can try to optimize such indicators for future patients. This, however, is not critical to the nature of this study and should not affect its outcome.

Approved concomitant therapy

Medicines needed to treat adverse events and control symptoms of cancer, stable comorbid disease (eg, controlled hypertension), and supportive care such as erythropoietin or blood transfusions, and pain medications are allowed. The patient must notify the study site of any new drugs that the patient is taking after the start of the study. All medicinal products (other than the investigational drug) and significant non-drug therapy (including physical therapy and blood transfusion) used after the patient begins treatment with the investigational drug must be noted in the patient's medical record.

The following are permitted concomitant therapies/procedures:

antiemetics;

antidiarrheals;

analgesics (eg, non-steroidal anti-inflammatory drugs (NSAIDs) or oral or transdermal narcotic analgesics);

antibiotics;

food and liquid additives;

myeloid growth factors (excluding the usual use of G-CSF and GM-CSF);

bisphosphonates;

blood transfusion and use of erythropoietin is allowed at the discretion of the attending physician;

prophylactic anticoagulant therapy (low dose) and complete inhibition of blood coagulation are allowed.

Prohibited concomitant therapy

Other experimental treatments should not be used while the patient is participating in this study. Patients taking study drug should not be subjected to other anticancer therapy (eg, chemotherapy, biological or radiation therapy, surgery) other than the current treatment. If such agents are necessary for the patient, then the patient should be excluded from the treatment part of the study.

The following are prohibited concomitant therapies:

surgical intervention (except for the permitted surgical resection of the primary tumor);

other experimental treatment;

other anti-cancer treatment;

systemic immunosuppressants (with the exception of the simultaneous use of corticosteroids, provided that no more than 20 mg per day of the equivalent of prednisolone is used);

immunotherapy;

regular prophylactic use of growth factor (G-CSF or GM-CSF).

Toxicity and Dosing Delay/Dose Change

Any patient treated under this protocol will be subjected to toxicity testing. Each patient will be assessed for the development of toxicity according to the NCI Common Toxicity Criteria for Adverse Events (CTCAE), version 4.03. Treatment delay due to toxicity greater than 2 weeks is considered DLT requiring treatment withdrawal.

Safety/Tolerability I

Tests will be performed on all patients receiving at least one dose of study drug. The study will use CTCAE version 4.03 (http://ctep.cancer.gov/repordng/ctc.html) for reporting adverse events (Appendix B).

Antitumor effect - solid tumors

Patients' disease status will be monitored by transverse imaging.

Assessment of tumor response (from the Stanford I.T. study)

Tumor response rate will be calculated based on immune-mediated response criteria (irRC) (Wolchok et al., Guidelines for the evaluation of immune therapy activity in solid tumors: immunerelated response criteria, Clin. Cancer Res. 2009, 15:7412-7420) of solid tumors , and secondarily by post-hoc response assessment based on the Choi test for gastrointestinal stromal tumors (GIST). See Choi et al., Correlation of computed tomography and positron emission tomography in patients with metastatic gastrointestinal stromal tumor treated at a single institution with imatinib mesylate: pro-

Claims (17)

posal of new computed tomography response criteria, J. Clin. oncol. 2007, 25:1753-1759. Necessary imaging studies of tumors will be performed before the start of the study, during treatment and after 1 month, 3 months and then every 3 months after treatment for 12 months and annually thereafter. Since immunotherapy can lead to an increase in tumor mass (1) due to either continued tumor growth until an immune response occurs, or due to a transient immunocellular infiltrate with or without edema, or (2) due to the appearance of new lesions (there are difficulties with differentiation of new lesions from original undetectable lesions due to T cell infiltration into existing radiographically undetectable tumor metastases), treatment should be continued, as tumors may begin to shrink after medical observation at a later time point to confirm PD. Patients with a stable general condition and not much deteriorating laboratory parameters, or patients who have moderate tumor growth on physical examination or radiographic imaging, should repeat the scan to determine the true progression of the disease and stop treatment. For long-term treatment, the potential risks of toxicity from long-term treatment should be assessed. The primary activity assay is based on immune-mediated response criteria. The best overall response (BORR) is calculated as the number of patients with BOR on CR or PR divided by the number of patients treated. The disease control rate (DCR) is calculated as the number of patients with CR, PR, or SD divided by the number of patients treated. Overall survival (OS) is defined as the time from randomization to death from any cause. Disease-free survival (DFS) is defined as the time period from randomization to tumor recurrence or death from any cause. Time to relapse is defined as the time period from the start of treatment to the first occurrence of a relapse. Relapse is defined as clinical, radiographic, or pathological evidence of new or persistent local, local, or distant disease. CLAIM 1. A method of treating a solid tumor or hematologic malignancy in a subject, comprising administering to the subject an oncolytic virus, which is a vaccinia virus, and an adipose-derived stromal vascular fraction (SVF), where the vaccinia virus and the adipose-derived SVF are incubated together prior to administration. 2. A method of treating a solid tumor or hematologic malignancy in a subject, comprising administering to the subject vaccinia virus, which is a smallpox vaccine, and adipose stromal cells, where the virus and cells have been co-incubated. 3. The method of claim 2, wherein the adipose stromal cells are derived from adipose-derived stromal vascular fraction (SVF). 4. The method of claim 2 or 3, wherein the adipose stromal cells are autologous to the subject. 5. The method of claim 2 or 3, wherein the adipose stromal cells are allogeneic to the subject. 6. The method according to any one of claims 2 to 5, wherein the adipose stromal cells contain an artificial chromosome, a virus, or a plasmid. 7. The method according to any one of claims 1 to 6, wherein the vaccinia oncolytic virus does not contain a heterologous nucleic acid that encodes a heterologous product. 8. The method according to any one of claims 1 to 7, wherein the vaccinia virus is a weakened strain of vaccinia virus from the New York City Board of Health (NYCBOH)). 9. The method of claim 8, wherein the NYCBOH vaccinia virus strain has all of the distinguishing characteristics of the strain known as ATCC VR-118 or CJ-MVB-SPX. 10. The method according to any one of claims 1 to 9, wherein the vaccinia virus is selected from Dryvax, ACAM1000, ACAM2000, Lister, EM63, LIVP, Tian Tan, Copenhagen, Western Reserve, Modified Vaccinia Ankara (MVA), New York City Board of Health, Dairen, Ikeda, LC16M8, Tashkent, Wyeth, IHD-J, IHD-W, Brighton, Dairen I and Connaught. 11. The method of any one of claims 1 to 10, wherein the solid tumor is a glioblastoma, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, or melanoma. 12. The method according to any one of claims 1-6 and 8-11, wherein the oncolytic vaccinia virus encodes a heterologous gene product. 13. The method according to any one of claims 1-12, wherein the subject is a human. 14. The method of claim 13 wherein the subject is a pediatric patient. 15. The method of claim 1 wherein the SVF of adipose origin is autologous to the subject. 16. The method of any one of claims 2 to 14, wherein the vaccinia oncolytic virus and the adipose stromal cells are cultured together in vitro prior to administration to a subject. 17. The method according to any one of claims 1 to 16, wherein the oncolytic vaccinia virus is os- -