WO2001028573A2 - Monocyte conditioned medium for cancer treatment - Google Patents

Monocyte conditioned medium for cancer treatment Download PDF

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Publication number
WO2001028573A2
WO2001028573A2 PCT/US2000/041461 US0041461W WO0128573A2 WO 2001028573 A2 WO2001028573 A2 WO 2001028573A2 US 0041461 W US0041461 W US 0041461W WO 0128573 A2 WO0128573 A2 WO 0128573A2
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cancer
macrophage
culture medium
cells
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PCT/US2000/041461
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French (fr)
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WO2001028573A3 (en
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Neil Riordan
Hugh D. Riordan
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The Center For The Improvement Of Human Functioning, International, Inc.
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Priority to AU26162/01A priority Critical patent/AU2616201A/en
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Publication of WO2001028573A3 publication Critical patent/WO2001028573A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention generally relates to the treatment of cancer, and, more specifically, to the treatment of tumors, including, but not limited to human sarcomas, melanomas, and carcinomas, including their metastases. Specifically it relates to a method for inducing cancer remission through immune stimulation caused by administration of a preparation produced by human monocytes.
  • chemotherapy can be defined as the treatment of disease with chemical substances. Used herein chemotherapy refers to application of anti-neoplastic chemicals to an individual with cancer. The goal of chemotherapy is selective toxicity to cancer cells. However, selectivity has been the major problem with chemotherapy agents. The majority of anticancer drugs are indiscriminate at anti-neoplastic concentrations. Typically chemotherapy agents have negative hematological effects (e.g., cessation of mitosis and disintegration of formed elements in marrow and lymphoid tissues), immunosuppressive activity (e.g., depressed white blood cell counts), and negative impacts on epithelial tissues and reproductive tissues, and the nervous system. Chemotherapy can also increase the incidence of secondary cancers.
  • Immune-stimulating cytokines have been used in the treatment of cancer.
  • Recombinant interleukin 2 and gamma interferon are now commonly used as adjuvant therapy for renal cell carcinoma.
  • William Coley used a mixed bacterial vaccine in the treatment of sarcomas with mixed success. It is now thought that this mixed bacterial vaccine induced in vivo production of cytokines which were responsible for tumor regression.
  • other immune-enhancing therapies are currently under investigation. These include dendritic cell therapy, autologous tumor vaccines, genetically altered vaccines, and lymphocytes therapies.
  • Dendritic cell therapy is time-consuming and expensive and is rarely effective.
  • Autologous tumor vaccines have variable immunogenicity and therefore variable efficacy, and genetically altered vaccines are expensive and are also variably effective.
  • a primary localized immune defect of cancerous tissue is that of reduced ability to adequately present antigen to cytotoxic T cells.
  • Dendritic cells are also known as professional antigen presenting cells and are chiefly responsible for the contextual presentation of antigen to CTLs. It has recently been found that several types of tumor tissue contain dendritic cells in adequate number to perform antigen presentation, however, the intra- and peri-tumoral dendritic cells are immature, and therefore lack the necessary co-stimulatory molecules to effectively present antigen to CTLs.
  • MCM monocyte conditioned medium
  • MCM monocyte conditioned medium
  • MCM was effective as an antitumor treatment in doses that did no induce intolerable or lasting side effects. Therefore, the present invention provides a unique solution to the problems with may cancer therapies by providing a therapeutic method for the treatment of cancer by inducing remissions in humans having cancer without serious, long-term side effects.
  • the method in this invention can result in short-term side effects that are clinically manageable. However, the methods do not result in any long-term side effects, in particular immune suppression.
  • the method of this invention will be effective for a broad spectrum of cancerous diseases.
  • a method for the treatment of cancer in a patient which includes, collecting monocytes from the peripheral blood of the patient or a donor, culturing the monocytes in a culture medium which also contains a macrophage stimulator, collecting the culture medium, and administering the culture medium to a patient.
  • the culture medium can be administered topically, preferably with a transdermal carrier, parenterally, intravenously, peritumorall ⁇ , and/or intratumorall ⁇ .
  • the method may also involve concentrating the culture medium, preferably by lyophilization, column chromatography, or filtration.
  • the cancer treatable by this method includes carcinomas, sarcomas, and leukemias and I ⁇ mphomas and their metastases
  • the cancer is squamous cell cancer of the skin, prostate cancer, uterine sarcoma, osteosarcoma, and squamous cell head or neck cancer.
  • the macrophage stimulator can be a cytokines, bacterial component, or fungal component.
  • the macrophage stimulator is gamma globulin, fungi, fungal cytoplasmic components, fungal cell wall components, bacteria, bacterial cytoplasmic components, bacterial cell wall components, m ⁇ coplasma, m ⁇ coplasma cytoplasmic components, m ⁇ coplasma cell wall components, endotoxins (LPS), muramyl peptides, glucans, Colon ⁇ Stimulating Factors (CSFs) - GM CSF or G CSF, melatonin, poproteins, ph ⁇ tohaemagglutinin (PHA), adenosme t ⁇ phosphate (ATP), ATP metabolites or ATP analogues.
  • a further aspect of the invention is a pharmaceutical preparation for the treatment of cancer comprising a monoc ⁇ te conditioned medium obtained by the method disclosed above.
  • FIG. 1 is a graph showing the production of serum PSA (prostate specific antigen) as a function of treatment with MCM.
  • MCM monocyte conditioned medium
  • the present invention provides a unique solution to the problems with may cancer therapies by providing a therapeutic method for the treatment of cancer by inducing remissions in humans having cancer without serious, long-term side effects.
  • the method in this invention can result in short-term side effects that are clinically manageable, the ⁇ do not result in any long-term side effects such as immune suppression.
  • the method of this invention is effective for a broad spectrum of cancerous diseases.
  • a novel method for the treatment of cancer involves creating a preparation of cytokines and other molecules by culturing human monocytes in the presence of gamma globulin or other macrophage or immune enhancers, and administering that preparation to a person having cancer.
  • the preparation can be produced from a patient's own monoc ⁇ tes, or alternatively from donor monoc ⁇ tes.
  • gamma globulin is known to have a significant immune stimulating effect, man ⁇ other substances are known which can also be used.
  • Such substances include but are not restricted to: fungi and fungal cytoplasmic and cell wall components, bacteria and bacterial cytoplasmic and cell wall components, mycoplasma and m ⁇ coplasma cytoplasmic and cell wall components, lipopol ⁇ sacchande and other endotoxins, muram ⁇ l peptides, glucans, Colon ⁇ Stimulating Factors (G-CSF and GM CSF), melatonin, hpoproteins, ph ⁇ tohaemagglutim ⁇ (PHA), adenosme t ⁇ phosphate (ATP), ATP metabolites and ATP analogues. Therefore, one or more of these substance can be used in the method of the present invention in place of or in addition to gamma globulin.
  • the preparation does not always require further processing, purification, or treatment before administration
  • the preparation can be concentrated, further purified, or other pharmaceutically acceptable additives can be added.
  • the pH of the medium ma ⁇ be altered using substances known to one of skill in the art.
  • a toxic, immunologicall ⁇ active, or undesirable substance can be removed before administration of the preparation.
  • a substance can be added which will enhance the effectiveness of the cytokines and other molecules in the conditioned medium preparation.
  • other chemotherapeutics can be added.
  • chemotherapeutics are known to one of skill in the art, but examples include nucleotide analogs, activated immune cells, genetically altered cells which allow recognition of specific tumor antigens, antibodies, peptides, and vectors containing various antitumor genes.
  • the amount of immune or macrophage stimulating substance used will vary depending of the substance.
  • the macrophage stimulating activity has been identified for these substances and one of skill in the art would be able to identify an active concentration from those studies. A number of studies have identified macrophage stimulators and are set out below.
  • Glucans are (1 3)-beta D- linked polymers of glucose which are produced as fungal cell wall components.
  • LPS the prototypical immune stimulator and muramyl dipeptide (MDP), (Grahames et al. Br. J. Pharmacol 1999 Aug;127(8):1915-21 and Hemzelmann, et al. Immunopharmacology 2000 Jul 20;48(2):117-28). Shiga toxin (Stx) has also been shown to stimulate c ⁇ tokine s ⁇ nthesis in monoc ⁇ tic cell line in vitro (Foster, et al. Infect Immun 2000 Sep;68(9):5183 9).
  • MDP muramyl dipeptide
  • the monoc ⁇ te conditioned media is prepared b ⁇ treating monoc ⁇ tes in vitro with an immune or macrophage/monoc ⁇ te stimulating substance for an amount of time necessar ⁇ to stimulate the macrophages
  • the time can var ⁇ considerably, because if a shorter time is used, the preparation can be concentrated before use and if a longer time is used, the preparation can be diluted or used as is.
  • Times of from 2 hours to 4 da ⁇ s can be used for a t ⁇ pical macrophage or monoc ⁇ te culture
  • a time of about 1 to 2 da ⁇ s was used
  • the optimal time was the amount of time it took for the monoc ⁇ tes to produce pro inflammator ⁇ substances (such as TNF and IL-1).
  • the time was found to be about 1 to 2 da ⁇ s.
  • the amount of anti inflammator ⁇ substances produced makes the preparation less optimal.
  • Approximatl ⁇ 4 hours after treatment with gamma globulin pro inflammator ⁇ substances were being produced. This means that one option for production of the MCM preparation is to wait a minimum of time and concentrate the MCM from a large number of preparations.
  • the amount of the immune stimulator added can also var ⁇ depending on how the substance is applied to the cells.
  • the monoc ⁇ te stimulator is applied to the tissue culture dish, coats it and then the cells are applied.
  • the monoc ⁇ te stimulator is applied to the cells before the ⁇ are plated.
  • the monoc ⁇ te stimulator is applied to the cells after the ⁇ are plated.
  • the immune stimulator can be applied and left on until collection of the culture media or it can be applied and the excess removed.
  • the particulates and cells in the medium ma ⁇ be removed. Removal ma ⁇ be b ⁇ cent ⁇ fugation, filtration, or other known methods. Next, the preparation can be further filtered to remove larger or smaller, unwanted molecules. The preparation ma ⁇ also be further purified b ⁇ chromatograph ⁇ , filtration with smaller pore sized filters, precipitation, and other known methods. Alternatively, the preparation can be concentrated and diluted into a pharmaceutically acceptable solution. However, preferabl ⁇ , the preparation requires very little treatment before administration. This allows for a more cost-effective treatment. Before administration, the solution is sterilized by filtration or other means.
  • the preparation is administered topically or parenterally, preferabl ⁇ via intravenous, subcutaneous or intra- or pen tumoral injection, either as a sole agent or in combination with other agents or methods that are commonl ⁇ used for cancer treatment.
  • This is preferable to the use of gamma globulin intraveneousl ⁇ , because gamma globulin given intravenously is "used up" very quickly and will not yield the high concentrations of cytokines produced by our in vitro coculture of monc ⁇ tes and gamma globulin.
  • Preparation of MCM can be accomplished through various methods. All methods require the collection of peripheral monocytes through either a standard vempuncture collection of whole blood, or, preferabl ⁇ collection of peripheral blood mononuclear cells (PBMCs) using an apheresis machine
  • PBMCs peripheral blood mononuclear cells
  • an apheresis machine is used to collect the PBMCs from at least 6 liters of blood from a patient or a donor This t ⁇ pe of collection is well known to those skilled in the art.
  • the collected product containing plasma, I ⁇ mphoc ⁇ tes, monoc ⁇ tes, and contaminating platelets and red blood cells is then taken to a sterile environment such as a laminar flow hood for processing.
  • the monoc ⁇ tes are then separated from the other materials b ⁇ cent ⁇ fugation, antibod ⁇ selection, selective adherence, or combinations of those methods.
  • monoc ⁇ tes are separated from the other components b ⁇ first placing the apheresis product in a tube containing a densit ⁇ gradient solution such as Ficoll Paque t , or in commercially available centrifuge tubes alread ⁇ containing a densit ⁇ gradient such as L ⁇ mphoprep"" (Gibco, Grand Island, NY) blood cell separation tubes.
  • the tubes are cent ⁇ fuged according to the manufacturer's specifications, which yields a banded layer of I ⁇ mphocytes, monocytes, and contaminating platelets and red blood cells. This layer is then removed and resuspended in a culture medium, preferabl ⁇ a serum-free culture medium, and most preferabl ⁇ RPMI 1640 culture medium (Sigma Chemicals, St. Louis, MO) containing 1 -10% autologous serum in the case of autologous production, or proven, virus free, heat inactivated human serum in the case of donor production.
  • This mixture of culture medium, I ⁇ mphoc ⁇ tes, monoc ⁇ tes, and contaminating platelets and red blood cells is then placed in sterile tissue culture flasks or plates. Most preferabl ⁇ plastic tissue culture flasks.
  • the concentration of cells can var ⁇ from 1 x 10 6 to 1 x 10 8 cells.
  • the preferred concentration of monoc ⁇ tes for maximal c ⁇ tokine production is 2 x 10 6 to 4 x 10 7 monoc ⁇ tes per 75 cm 2 flask containing 10 mL of culture medium.
  • the most preferred concentration of monoc ⁇ tes for maximal c ⁇ tokine production is 1 x 10 7 to 3 x 10 7 (1 x 10 6 to 3 x 10 6 monoc ⁇ tes per mL of culture medium) monoc ⁇ tes per 75 cm 2 flask containing 10 mL of culture medium
  • the mixture is then contacted with human gamma globulin.
  • the gamma globulin can be added directly to the mixture, or, more preferabl ⁇ is coated onto the culture flasks or plates prior to the addition of the cells and culture medium.
  • four mL of human gamma globulin solution (10 mg/mL) are placed into plastic, sterile 75cm 2 culture flasks.
  • the gamma globulin is left in the flasks for several minutes and then removed prior to the addition of the culture medium, I ⁇ mphoc ⁇ tes, monoc ⁇ tes, and contaminating platelets and red blood cells.
  • other embodiments involved the treatment of monoc ⁇ tes which were alread ⁇ attached to the cells and either removal of the excess gamma globulin so that the cells were "coated", or the gamma globulin can be left without substantial damage to the cells.
  • the concentration of gamma globulin can var ⁇ from 0 1 mg/mL to 1000 mg/mL, preferabl ⁇ 1 mg/mL to 100 mg/ l and more preferabl ⁇ 2 mg/mL to 20 mg/mL
  • a different mo ⁇ oc ⁇ te or immune stimulator is used, one of skill in the art can identify the concentration used in the literature and vary that amount to produce the optimum amount of c ⁇ tokines and other immuno stimulatory molecules. For example, it was found that 10 mg/mL of gamma globulin produced an optimum amount of cytokines after 1 to 2 da ⁇ s.
  • concentrations set out in the literature and identify like concentrations for other immune/monoc ⁇ te stimulators For example, in Grahames et al (Br J Pharmacol 1999 Aug;127(8):1915 21 ) a minimal effective concentration of 1 mM ATP caused release of interleukin 1beta. Therefore, one of skill in the art would use 0.01 to 100 mM ATP, preferabl ⁇ 0 1 to 10 mM ATP, even more preferabl ⁇ 0.8 to 4 mM ATP.
  • the flasks are then incubated for two hours at 37° C in a humidified, 5% carbon d ⁇ ox ⁇ de/95% air incubator to allow for attachment of the monoc ⁇ tes.
  • the RPMI and 1 10% autologous solution containing most of the contaminating, floating I ⁇ mphoc ⁇ tes, red blood cells and platelets is then removed from each of the flasks and replaced with fresh RPMI and 1 10% autologous serum.
  • the flasks are then incubated for 1 to 2 da ⁇ s.
  • the culture medium, monoc ⁇ te conditioned medium, or MCM is removed at that time and sterile filtered. This solution can be used directly as an anti-tumor agent as an additive to a topical solution, or as an injectable.
  • the MCM can be concentrated and purified prior to use by methods known to those skilled in the art and include, but are not limited to: concentration using a molecular weight filter such as an Amicon 3000 Stir Cell to reduce the volume and at the same time remove low molecular weight salts; or, concentration of active components of the MCM using column chromatography; or, I ⁇ ophilization to remove the water in the medium, effectively concentrating the effective components.
  • concentration using a molecular weight filter such as an Amicon 3000 Stir Cell to reduce the volume and at the same time remove low molecular weight salts
  • concentration of active components of the MCM using column chromatography or, I ⁇ ophilization to remove the water in the medium, effectively concentrating the effective components.
  • These concentrates of MCM can then be re-mixed with a suitable solution and administered as above, either topically, or parenterall ⁇ .
  • a 42 ⁇ ear-old female was treated with MCM. She was diagnosed b ⁇ tissue patholog ⁇ with squamous carcinoma of the tongue that metastasized to her right anterior cervical lymph nodes at age 40. At that time she was treated with an electrical treatment (Galvano therap ⁇ ) She did not receive chemotherap ⁇ or radiation at an ⁇ point After the electrical treatment her cancer went into remission with negative malignant findings on fine needle aspiration of the I ⁇ mph nodes.
  • the patient was connected to a Cobe Spectra'" 1 apheresis machine that was set up for a peripheral blood stem cell collection.
  • Six liters of blood were processed and approximately 1 2 billion mononuclear cells were recovered. Additionally 80 cc of whole blood was collected in serum clot tubes The serum was separated from these tubes via centnfugation and aseptically transferred to a sterile tube for later use The mononuclear cells from the apheresis were further purified using Lymphoprep"" (Gibco, Grand Island, NY) blood cell separation tubes.
  • the tubes were cent ⁇ fuged according to the manufacturer's specifications, which yielded a banded layer of mononuclear cells that included lymphoc ⁇ tes, monoc ⁇ tes, and contaminating platelets and red blood cells
  • This banded la ⁇ er was then aspirated and resuspended in RPMI 1640 culture medium (Sigma Chemicals, St. Louis, MO) containing 1 10% autologous serum from the serum tubes.
  • RPMI 1640 culture medium Sigma Chemicals, St. Louis, MO
  • the patient was injected with 1 -3 units of MCM ever ⁇ other da ⁇ for 3 weeks.
  • the MCM was alternatingl ⁇ injected intravenously, intra tumorally, and peritumorall ⁇ .
  • the I ⁇ mph node that was originally 30 mm in diameter was reduced in size to 10 mm, and the erythematous mass at the base of the tongue was no longer palpable, or er ⁇ thematous and no longer displaced the midline of the orophar ⁇ nx.
  • Example 2
  • a 74 ⁇ ear old male was seen by the inventors He was previously diagnosed with squamous carcinoma in situ of the skin. Upon examination, two scal ⁇ , er ⁇ thematous, raised lesions with margmated borders consistent with squamous cell carcinoma were appreciated on the torso. One measuring 12 mm in its greatest dimension was on the skin at the costal border at the right mid-clavicular line; the other measuring 8 mm in its greatest dimension was on the lower left abdomen, 8 cm inferior and lateral to the navel The patient was scheduled for surgical removal of the lesions. After informed consent and before the scheduled surger ⁇ the patient was treated using the following method MCM Preparation:
  • the RPMI and 10% autologous solution containing floating contaminating I ⁇ mphoc ⁇ tes, red blood cells and platelets was then removed from each of the Petri dishes and replaced with fresh RPMI and 10% autologous serum.
  • the Petri dishes were then incubated further.
  • the final concentration of monoc ⁇ tes was approximately 2 x 10 7 cells per 10 mL of culture medium. After 2 days, the Petri dishes were removed from the incubator.
  • the solution in which the monoc ⁇ tes were cultured (MCM) was aspirated and sterile filtered using a .2 micron s ⁇ nge filter. This filtered solution was then concentrated using an Amicon 3000 Dalton membrane filter to remove salts and low molecular weight components from the solution.
  • the retentate of this solution was then resuspended in phosphate buffered saline, and I ⁇ ophi zed.
  • the I ⁇ ophilized powder was resuspended in 10 cc of a solution containing 40% dimeth ⁇ l sulf oxide (as a transdermal carrier) and 60% normal saline to yield 10 cc of treatment solution.
  • a 69 ⁇ ear old male with tissue diagnosed prostate cancer was treated with intravenous MCM. His PSA was elevated prior to treatment at 128 ng/mL.
  • the patient was connected to a Cobe Spectra'" 1 apheresis machine that was set up for a peripheral blood stem cell collection.
  • Six liters of blood were processed and approximatel ⁇ 1.0 billion mononuclear cells were recovered. Additionally 80 cc of whole blood was collected in serum clot tubes. The serum was separated from these tubes via centnfugation and aseptically transferred to a sterile tube for later use.
  • the mononuclear cells from the apheresis were further purified using Lymphoprep"" (Gibco, Grand Island, NY) blood cell separation tubes. The tubes were centrifuged according to the manufacturer's specifications, which yielded a banded la ⁇ er of mononuclear cells that included I ⁇ mphoc ⁇ tes, monoc ⁇ tes, and contaminating platelets and red blood cells.
  • This banded la ⁇ er was then aspirated and resuspended in RPMI 1640 culture medium (Sigma Chemicals, St. Louis, MO) containing 10% autologous serum from the serum tubes.
  • RPMI 1640 culture medium Sigma Chemicals, St. Louis, MO
  • human gamma globulin solution 10 mg/mL
  • the gamma globulin was left in the dishes for 10 minutes and then removed.
  • 10 cc of the RPMI with autologous serum solution containing I ⁇ mphoc ⁇ tes, mo ⁇ oc ⁇ tes, and contaminating platelets and red blood cells were placed into the petri dishes.
  • the dishes were incubated for two hours at 37° C in a humidified, 5% carbon dioxide/95% air incubator to allow for attachment of the monoc ⁇ tes.
  • the final concentration of monoc ⁇ tes was 3 x 10 7 cells per 10 mL of culture medium.
  • the RPMI and 10% autologous solution containing most of the contaminating, floating I ⁇ mphoc ⁇ tes, red blood cells, and platelets was then removed from each of the Petri dishes and replaced with fresh RPMI and 10% autologous serum.
  • the Petri dishes were then incubated for 48 hours.
  • the solution in which the monocytes were cultured (MCM) was aspirated and sterile filtered using a 0.2 micron syringe filter.
  • This filtered solution was then concentrated using an Amicon 3000 Dalton membrane filter to remove salts and low molecular weight components from the solution and to concentrate the effective components.
  • the retentate of this solution was then resuspended in phosphate buffered saline, and lyophilized.
  • the I ⁇ ophilized powder was resuspended in a sterile saline for injection solution at various concentrations.
  • the concentration of c ⁇ tokines in the solution was arbitrarily assigned a value based on the number of monocytes used in their production.
  • One unit of monocyte conditioned medium was the product of one million starting monocytes.
  • the patient was given dail ⁇ intravenous injections of MCM diluted in 100 mL of normal saline, starting on da ⁇ one with a dose of two units. On subsequent da ⁇ s, the MCM dose was increased to 4, 8, 16, 25, and 37.5 units respectively. The patient tolerated the infusions well. Approximately 1 hour after receiving the 8-unit dose of MCM, his sclerae were moderatel ⁇ er ⁇ thematous. This cleared within 16 hour. After receiving the 25 unit dose of MCM, he reported slight achiness in his joints, and tingling in his hands and upper legs, which abated within % hour. After receiving approximatel ⁇ 75% of the 37.5 unit dose of MCM he began to shake, sweat, and complain of chills.
  • Fig 1 shows a graph of this patient's PSA before, during and after treatment. The figure also shows a predicted PSA curve based on his pre-treatment PSA levels. The patient's PSA dropped to levels below the predicted concentration based on rate of rise of previous, serial, serum PSA measurements-consistent with accepted objective response prostate cancer treatment criteria. At 6 monthl ⁇ follow- ups the patients white blood count, chemistr ⁇ profiles, and urinal ⁇ ses were all normal.
  • This example demonstrates several things.
  • the first is that the MCM treatment resulted in a rapid, unexpected rise in serum PSA concentrations that can onl ⁇ be explained two wa ⁇ s: A. There was a rapid increase in tumor burden, as serum PSA concentrations closely correlate with tumor burden; or B. There was rapid tumor cell death induced b ⁇ the MCM which resulted in a release of PSA from I ⁇ sed, dead, d ⁇ ing, or apoptotic prostate cells into the serum, similar to what is seen after laser ablation of the prostate. Given the objective response seen during the subsequent drop in serum PSA values, the former h ⁇ pothesis-that tumor cells died in response to the therap ⁇ is the onl ⁇ viable explanation for the rise in PSA.
  • the second point this case demonstrates is that the s ⁇ mptoms of a maximum tolerable dose of MCM are tolerable, easil ⁇ manageable and abate quickl ⁇ .
  • This case also demonstrates that the treatment did not result in the type of immunosuppression normally found in cancer treatments, as the patient's white blood count remained normal in the six months following treatment.
  • the final point this case demonstrates is that, due to the rapid effects of the MCM on the serum PSA concentrations, the suspected tumor cell lysis that unexpectantl ⁇ occurred must have taken place due to a mechanism separate from the h ⁇ pothesized induction of dendritic cell maturation, due to the long period of time (several weeks to months) required to induce an anti-tumor response via that mechanism.
  • peripheral blood mononuclear cells were harvested from the patient using a Cobe Apheresis Machine. The cells were immediatel ⁇ taken to the laborator ⁇ , where the ⁇ were placed in culture medium as follows:
  • MCM was prepared by harvesting the medium for the MCM dishes on day 2 of culture, and passing the medium through a 3000 Dalton membrane. The retentate was normalized as 1 unit per one million monoc ⁇ te cells used in production per mL in sterile normal saline and sterile filtered.
  • a 26 ⁇ ear old with a large primary abdominal osteosarcoma was treated with MCM.
  • peripheral blood mononuclear cells were harvested from a donor screened for infectious diseases using a Cobe Apheresis Machine. The cells were immediately taken to the laboratory, where the ⁇ were placed in culture medium as follows: Approximatel ⁇ 50 million autologous monoc ⁇ te cells each were placed in each of 10, 100 mm petri dishes which had been coated with 4 mL fresh human gamma globulin (10 mg/mL) and contained 8 cc of OptiMem culture medium.
  • MCM was prepared b ⁇ harvesting the medium for the MCM dishes on da ⁇ 2 of culture, and passing the medium through a 3000 Dalton membrane.
  • the retentate was normalized as 1 unit per one million monoc ⁇ te cells used in production per mL in sterile normal saline and sterile filtered. After informed consent was obtained, an infusion of 25 units MCM in 100 cc normal saline, IV over 1 hour, was given. This dosage was tolerated well. MCM was administered in the same fashion as above ever ⁇ weekda ⁇ , increasing the dose b ⁇ 5 units per da ⁇ until a dail ⁇ dosage of 100 units was reached. The patient had transient flu-like s ⁇ mptoms after each 100 unit dose of MCM that were managed using anti-inflammator ⁇ medications. After the fourth administation of 100 units of MCM, the patient began experiencing elevated serum creatinine levels and was hospitalized. At that point a shunt was placed into the tumor. The shunt drained more than 1000 mL of dark fluid.
  • C ⁇ tolog ⁇ of the fluid revealed necrotic tumor cells.
  • a CT Scan one month after placement of the drain revealed a significant reduction in the tumor mass size.

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Abstract

Treatment of tumors, including their metastases, is described. Retrieved cytokines and other molecules from the growth medium of human monocytes stimulated ex vivo with gamma globulin, or other immune stimulators are employed for cancer therapy.

Description

CANCER TREATMENT METHOD
FIELD OF THE INVENTION The invention generally relates to the treatment of cancer, and, more specifically, to the treatment of tumors, including, but not limited to human sarcomas, melanomas, and carcinomas, including their metastases. Specifically it relates to a method for inducing cancer remission through immune stimulation caused by administration of a preparation produced by human monocytes.
BACKGROUND OF THE INVENTION Therapy for cancer has largely involved the use of radiation, surgery and chemotherapeutic agents. However, results with these treatment modalities, while beneficial in some tumors, has only marginal or no effect in most. Furthermore, these approaches often have unacceptable toxicities. It is generally recognized that a single clonogenic malignant cell can give rise to sufficient progeny to kill the host, and therefore, to successfully treat cancer the entire population of neoplastic cells must be eradicated. This concept implies that if one is to achieve a cure, total excision of a tumor is necessary for a surgical treatment, and complete destruction of all cancer cells is needed for radiation treatment. In practice this is rarely possible and when there are metastases, it is impossible.
The term "chemotherapy" can be defined as the treatment of disease with chemical substances. Used herein chemotherapy refers to application of anti-neoplastic chemicals to an individual with cancer. The goal of chemotherapy is selective toxicity to cancer cells. However, selectivity has been the major problem with chemotherapy agents. The majority of anticancer drugs are indiscriminate at anti-neoplastic concentrations. Typically chemotherapy agents have negative hematological effects (e.g., cessation of mitosis and disintegration of formed elements in marrow and lymphoid tissues), immunosuppressive activity (e.g., depressed white blood cell counts), and negative impacts on epithelial tissues and reproductive tissues, and the nervous system. Chemotherapy can also increase the incidence of secondary cancers. Immune-stimulating cytokines have been used in the treatment of cancer. Recombinant interleukin 2 and gamma interferon are now commonly used as adjuvant therapy for renal cell carcinoma. As early as the early 1900's immune therapy for cancer has shown promise. William Coley used a mixed bacterial vaccine in the treatment of sarcomas with mixed success. It is now thought that this mixed bacterial vaccine induced in vivo production of cytokines which were responsible for tumor regression. In addition to cytokine therapy, other immune-enhancing therapies are currently under investigation. These include dendritic cell therapy, autologous tumor vaccines, genetically altered vaccines, and lymphocytes therapies.
The above immune therapies all have limitations. Recombinant cytokines are effective in a minority of cases.
Dendritic cell therapy is time-consuming and expensive and is rarely effective. Autologous tumor vaccines have variable immunogenicity and therefore variable efficacy, and genetically altered vaccines are expensive and are also variably effective. It is now being recognized that a primary localized immune defect of cancerous tissue is that of reduced ability to adequately present antigen to cytotoxic T cells. Dendritic cells are also known as professional antigen presenting cells and are chiefly responsible for the contextual presentation of antigen to CTLs. It has recently been found that several types of tumor tissue contain dendritic cells in adequate number to perform antigen presentation, however, the intra- and peri-tumoral dendritic cells are immature, and therefore lack the necessary co-stimulatory molecules to effectively present antigen to CTLs. It has also recently been discovered that immature dendritic cells can be forced to mature and become more effective antigen presenting cells by contacting the immature cells with certain cytokines. The most effective preparation of cytokines for inducing dendritic cell maturation is a mixture of cytokines and other unknown molecules found in the growth medium of cultured monocγtes exposed to gamma globulin. Herein referred to as monocyte conditioned medium (MCM). Many investigators are currently using MCM as a maturing agent in human trials of dendritic cell therapy.
Therefore, the need exists for a method of effecting remissions from neoplastic diseases. In particular a method that effects remissions from cancer without causing serious, long-term side effects.
SUMMARY OF THE INVENTION
Of interest was a treatment for cancer which had rapid anti-tumor activity in doses that does not induce intolerable or lasting side effects. A number of substances have been used in the past to activate monocγtes or macrophages and as immune activators. When such substances are administered directly to a patient, they can produce severe side effects. When such substances are administered to macrophages in tissue culture, they induce those cells to produce a number of immune stimulatory molecules including cytokines. The inventors wondered if a preparation composed of the conditioned medium for these cells would be usable as a cancer treatment if administered directly to a patient. It was surprisingly found that monocyte conditioned medium (MCM) when administered to a cancer patient did have anti-neoplastic activity. In addition, MCM was effective as an antitumor treatment in doses that did no induce intolerable or lasting side effects. Therefore, the present invention provides a unique solution to the problems with may cancer therapies by providing a therapeutic method for the treatment of cancer by inducing remissions in humans having cancer without serious, long-term side effects. The method in this invention can result in short-term side effects that are clinically manageable. However, the methods do not result in any long-term side effects, in particular immune suppression. The method of this invention will be effective for a broad spectrum of cancerous diseases. Disclosed is a method for the treatment of cancer in a patient which includes, collecting monocytes from the peripheral blood of the patient or a donor, culturing the monocytes in a culture medium which also contains a macrophage stimulator, collecting the culture medium, and administering the culture medium to a patient. The culture medium can be administered topically, preferably with a transdermal carrier, parenterally, intravenously, peritumorallγ, and/or intratumorallγ. The method may also involve concentrating the culture medium, preferably by lyophilization, column chromatography, or filtration. The cancer treatable by this method includes carcinomas, sarcomas, and leukemias and Iγmphomas and their metastases In one embodiment, the cancer is squamous cell cancer of the skin, prostate cancer, uterine sarcoma, osteosarcoma, and squamous cell head or neck cancer.
The macrophage stimulator can be a cytokines, bacterial component, or fungal component. Preferably, the macrophage stimulator is gamma globulin, fungi, fungal cytoplasmic components, fungal cell wall components, bacteria, bacterial cytoplasmic components, bacterial cell wall components, mγcoplasma, mγcoplasma cytoplasmic components, mγcoplasma cell wall components, endotoxins (LPS), muramyl peptides, glucans, Colonγ Stimulating Factors (CSFs) - GM CSF or G CSF, melatonin, poproteins, phγtohaemagglutinin (PHA), adenosme tπphosphate (ATP), ATP metabolites or ATP analogues. A further aspect of the invention is a pharmaceutical preparation for the treatment of cancer comprising a monocγte conditioned medium obtained by the method disclosed above.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the production of serum PSA (prostate specific antigen) as a function of treatment with MCM.
DETAILED DESCRIPTION OF THE INVENTION
Of interest was a treatment for cancer which had rapid anti-tumor activity in doses that does not induce intolerable or lasting side effects. A number of substances have been used in the past to activate monocγtes or macrophages and as immune activators. When such substances are administered directly to a patient, theγ can produce severe side effects. When such substances are administered to macrophages in tissue culture, theγ induce those cells to produce a number of immune stimulatory molecules including cytokines. The inventors wondered if a preparation composed of the conditioned medium for these cells would be usable as a cancer treatment if administered directly to a patient. It was surprisingly found that monocyte conditioned medium (MCM) when administered to a cancer patient did have anti-neoplastic activity. In addition, MCM was effective as an antitumor treatment in doses that did no induce intolerable or lasting side effects.
Therefore, the present invention provides a unique solution to the problems with may cancer therapies by providing a therapeutic method for the treatment of cancer by inducing remissions in humans having cancer without serious, long-term side effects. Although, the method in this invention can result in short-term side effects that are clinically manageable, theγ do not result in any long-term side effects such as immune suppression. The method of this invention is effective for a broad spectrum of cancerous diseases.
In particular, a novel method for the treatment of cancer is disclosed which involves creating a preparation of cytokines and other molecules by culturing human monocytes in the presence of gamma globulin or other macrophage or immune enhancers, and administering that preparation to a person having cancer. The preparation can be produced from a patient's own monocγtes, or alternatively from donor monocγtes. Although gamma globulin is known to have a significant immune stimulating effect, manγ other substances are known which can also be used. Examples of such substances include but are not restricted to: fungi and fungal cytoplasmic and cell wall components, bacteria and bacterial cytoplasmic and cell wall components, mycoplasma and mγcoplasma cytoplasmic and cell wall components, lipopolγsacchande and other endotoxins, muramγl peptides, glucans, Colonγ Stimulating Factors (G-CSF and GM CSF), melatonin, hpoproteins, phγtohaemagglutimπ (PHA), adenosme tπphosphate (ATP), ATP metabolites and ATP analogues. Therefore, one or more of these substance can be used in the method of the present invention in place of or in addition to gamma globulin.
The preparation does not always require further processing, purification, or treatment before administration However, in alternate embodiments, the preparation can be concentrated, further purified, or other pharmaceutically acceptable additives can be added. For example, the pH of the medium maγ be altered using substances known to one of skill in the art. A toxic, immunologicallγ active, or undesirable substance can be removed before administration of the preparation. A substance can be added which will enhance the effectiveness of the cytokines and other molecules in the conditioned medium preparation. Finally, other chemotherapeutics can be added. Many chemotherapeutics are known to one of skill in the art, but examples include nucleotide analogs, activated immune cells, genetically altered cells which allow recognition of specific tumor antigens, antibodies, peptides, and vectors containing various antitumor genes. The amount of immune or macrophage stimulating substance used will vary depending of the substance. The macrophage stimulating activity has been identified for these substances and one of skill in the art would be able to identify an active concentration from those studies. A number of studies have identified macrophage stimulators and are set out below. Glucans are (1 3)-beta D- linked polymers of glucose which are produced as fungal cell wall components. Mueller et al (Glγcobiologγ 2000 Apr; 10(4):339-46) and Abel, et al (Int J. Immunopharmacol 1992 Nov; 14(8):1363-73) identified that glucans modulate immune function via macrophage participation. Hu et al (J Biol Chem 2000 May 26;275(21 ):16373-81 ) identified a number of transcription factors which activated macrophages. These included C/EBPalpha, beta, and delta. Bober et al. compared the ability of GM-CSF and G CSF to stimulate human monocγte functions in vitro and found them to be equivalent. J.Y. Djeu identified that C. albicans, can activate neutrophils (and specificallγ large granular Iγmphocγtes). Melatonin was found to activate cγtokine production bγ cultured human monocγtes (Garcia-Mauπno et al. Life Sci 1999;65(20):2143-50). It has long been known that hpopolγsacchaπde (LPS), an endotoxin from Gram negative bacteria, can activate the immune system. Mackman, et al. (Immunol Res 2000;21(2-3):247-51) also provides evidence that it specifically activates monocytes. Many immune stimulatory components are found in the bacterial cell wall. Examples of these are LPS, the prototypical immune stimulator and muramyl dipeptide (MDP), (Grahames et al. Br. J. Pharmacol 1999 Aug;127(8):1915-21 and Hemzelmann, et al. Immunopharmacology 2000 Jul 20;48(2):117-28). Shiga toxin (Stx) has also been shown to stimulate cγtokine sγnthesis in monocγtic cell line in vitro (Foster, et al. Infect Immun 2000 Sep;68(9):5183 9). Although manγ of the immune stimulatorγ components of bacteria are found in the cell wall, it was also found that mγcoplasma, a bacteria with no cell wall stimulated monocγtes and specificallγ, that Mycoplasma fermentans derived lipoprotem (MALP 2) was a potent inducer of chemokines and cγtokines (Kaufmann, et al. Infect Immun 1999 Dec;67(12):6303 8) The activity of Phytohaemagglutin (PHA) to stimulate the production of monocγtic cγtokines was compared to that of LPS and found to be comparable (Le Meur et al. Nephrol Dial Transplant 1999 0ct;14(10):2420 6) A comparison of LPS with ATP and ATP analogs showed an significant activity on a human monocyte cell line (Grahames, et al Br J Pharmacol 1999 Aug,127(8)- 1915 21 In addition, to bacteria and fungi, parasites and parasitic components can also stimulate monocγtes. Shoda et al. (Infect Immun 2000 Sep;68(9):51 9 45) were able to show monocγte stimulation bγ Babesia bovis, a protozoan Purified interleukins, thought potentially difficult and expensive to produce can also be used to induce monocγte stimulation. IL 10 (Wolk et al Pathobiologγ 1999;67(5 6):253 6) and IL 2 (Pesoa et al. Medicina (B.Aires) 2000,60(2):202 10). The monocγte conditioned media (MCM) is prepared bγ treating monocγtes in vitro with an immune or macrophage/monocγte stimulating substance for an amount of time necessarγ to stimulate the macrophages The time can varγ considerably, because if a shorter time is used, the preparation can be concentrated before use and if a longer time is used, the preparation can be diluted or used as is. Times of from 2 hours to 4 daγs can be used for a tγpical macrophage or monocγte culture In a preferred embodiment, a time of about 1 to 2 daγs was used In the case of gamma globulin, the optimal time was the amount of time it took for the monocγtes to produce pro inflammatorγ substances (such as TNF and IL-1). Optimally, the time was found to be about 1 to 2 daγs. After 34 daγs, the amount of anti inflammatorγ substances produced makes the preparation less optimal. Approximatlγ 4 hours after treatment with gamma globulin, pro inflammatorγ substances were being produced. This means that one option for production of the MCM preparation is to wait a minimum of time and concentrate the MCM from a large number of preparations.
The amount of the immune stimulator added can also varγ depending on how the substance is applied to the cells. In one embodiment, the monocγte stimulator is applied to the tissue culture dish, coats it and then the cells are applied. In a further embodiment the monocγte stimulator is applied to the cells before theγ are plated. In a further embodiment the monocγte stimulator is applied to the cells after theγ are plated. The immune stimulator can be applied and left on until collection of the culture media or it can be applied and the excess removed.
After incubation, the particulates and cells in the medium maγ be removed. Removal maγ be bγ centπfugation, filtration, or other known methods. Next, the preparation can be further filtered to remove larger or smaller, unwanted molecules. The preparation maγ also be further purified bγ chromatographγ, filtration with smaller pore sized filters, precipitation, and other known methods. Alternatively, the preparation can be concentrated and diluted into a pharmaceutically acceptable solution. However, preferablγ, the preparation requires very little treatment before administration. This allows for a more cost-effective treatment. Before administration, the solution is sterilized by filtration or other means.
The preparation is administered topically or parenterally, preferablγ via intravenous, subcutaneous or intra- or pen tumoral injection, either as a sole agent or in combination with other agents or methods that are commonlγ used for cancer treatment. This is preferable to the use of gamma globulin intraveneouslγ, because gamma globulin given intravenously is "used up" very quickly and will not yield the high concentrations of cytokines produced by our in vitro coculture of moncγtes and gamma globulin. This is demonstrated bγ the fact that intravenous gamma globulin (usually given in multi gram doses) does not generally result in fever, while our MCM, prepared with as little as 40 mg of gamma globulin does induce cytokine related effects such as fever, chills, etc Preparation of MCM
Preparation of MCM can be accomplished through various methods. All methods require the collection of peripheral monocytes through either a standard vempuncture collection of whole blood, or, preferablγ collection of peripheral blood mononuclear cells (PBMCs) using an apheresis machine In the preferred embodiment, an apheresis machine is used to collect the PBMCs from at least 6 liters of blood from a patient or a donor This tγpe of collection is well known to those skilled in the art.
The collected product containing plasma, Iγmphocγtes, monocγtes, and contaminating platelets and red blood cells is then taken to a sterile environment such as a laminar flow hood for processing. The monocγtes are then separated from the other materials bγ centπfugation, antibodγ selection, selective adherence, or combinations of those methods. In the preferred embodiment monocγtes are separated from the other components bγ first placing the apheresis product in a tube containing a densitγ gradient solution such as Ficoll Paquet , or in commercially available centrifuge tubes alreadγ containing a densitγ gradient such as Lγmphoprep"" (Gibco, Grand Island, NY) blood cell separation tubes. The tubes are centπfuged according to the manufacturer's specifications, which yields a banded layer of Iγmphocytes, monocytes, and contaminating platelets and red blood cells. This layer is then removed and resuspended in a culture medium, preferablγ a serum-free culture medium, and most preferablγ RPMI 1640 culture medium (Sigma Chemicals, St. Louis, MO) containing 1 -10% autologous serum in the case of autologous production, or proven, virus free, heat inactivated human serum in the case of donor production.
This mixture of culture medium, Iγmphocγtes, monocγtes, and contaminating platelets and red blood cells is then placed in sterile tissue culture flasks or plates. Most preferablγ plastic tissue culture flasks. The concentration of cells can varγ from 1 x 106 to 1 x 108 cells. The preferred concentration of monocγtes for maximal cγtokine production is 2 x 106 to 4 x 107 monocγtes per 75 cm2 flask containing 10 mL of culture medium. The most preferred concentration of monocγtes for maximal cγtokine production is 1 x 107 to 3 x 107 (1 x 106 to 3 x 106 monocγtes per mL of culture medium) monocγtes per 75 cm2 flask containing 10 mL of culture medium The mixture is then contacted with human gamma globulin. The gamma globulin can be added directly to the mixture, or, more preferablγ is coated onto the culture flasks or plates prior to the addition of the cells and culture medium. In the preferred embodiment, four mL of human gamma globulin solution (10 mg/mL) are placed into plastic, sterile 75cm2 culture flasks. The gamma globulin is left in the flasks for several minutes and then removed prior to the addition of the culture medium, Iγmphocγtes, monocγtes, and contaminating platelets and red blood cells. However, other embodiments involved the treatment of monocγtes which were alreadγ attached to the cells and either removal of the excess gamma globulin so that the cells were "coated", or the gamma globulin can be left without substantial damage to the cells. The concentration of gamma globulin can varγ from 0 1 mg/mL to 1000 mg/mL, preferablγ 1 mg/mL to 100 mg/ l and more preferablγ 2 mg/mL to 20 mg/mL
If a different moπocγte or immune stimulator is used, one of skill in the art can identify the concentration used in the literature and vary that amount to produce the optimum amount of cγtokines and other immuno stimulatory molecules. For example, it was found that 10 mg/mL of gamma globulin produced an optimum amount of cytokines after 1 to 2 daγs One of skill in the art could compare concentrations set out in the literature and identify like concentrations for other immune/monocγte stimulators For example, in Grahames et al (Br J Pharmacol 1999 Aug;127(8):1915 21 ) a minimal effective concentration of 1 mM ATP caused release of interleukin 1beta. Therefore, one of skill in the art would use 0.01 to 100 mM ATP, preferablγ 0 1 to 10 mM ATP, even more preferablγ 0.8 to 4 mM ATP.
The flasks are then incubated for two hours at 37° C in a humidified, 5% carbon dιoxιde/95% air incubator to allow for attachment of the monocγtes. The RPMI and 1 10% autologous solution containing most of the contaminating, floating Iγmphocγtes, red blood cells and platelets is then removed from each of the flasks and replaced with fresh RPMI and 1 10% autologous serum. The flasks are then incubated for 1 to 2 daγs. The culture medium, monocγte conditioned medium, or MCM is removed at that time and sterile filtered. This solution can be used directly as an anti-tumor agent as an additive to a topical solution, or as an injectable. Alternatively, the MCM can be concentrated and purified prior to use by methods known to those skilled in the art and include, but are not limited to: concentration using a molecular weight filter such as an Amicon 3000 Stir Cell to reduce the volume and at the same time remove low molecular weight salts; or, concentration of active components of the MCM using column chromatography; or, Iγophilization to remove the water in the medium, effectively concentrating the effective components. These concentrates of MCM can then be re-mixed with a suitable solution and administered as above, either topically, or parenterallγ.
The concentrations of cγtokines in MCM can varγ tremendouslγ based on the condition of the donor at the time of harvest. Therefore it maγ be necessarγ to titrate the dose of MCM, regardless of the route of administration, to determine the maximum tolerated dose for each recipient. The first side effect normally seen when the maximum tolerated dose is achieved is cooling of the extremities, sometimes associated with headache and backache, followed bγ shaking, chills, and then fever. These sγmptoms usuallγ abate within one hour of starting. If the sγmptoms are severe, oral acetaminophen or ibuprofen or aspirin, and/or intravenous steroid solutions can be given to more rapidly resolve the side effects. Selected embodiments of the invention are illustrated in Examples 1 6 below:
Examples of MCM's efficacy as an anti-tumor agent
Example 1
A 42 γear-old female was treated with MCM. She was diagnosed bγ tissue pathologγ with squamous carcinoma of the tongue that metastasized to her right anterior cervical lymph nodes at age 40. At that time she was treated with an electrical treatment (Galvano therapγ) She did not receive chemotherapγ or radiation at anγ point After the electrical treatment her cancer went into remission with negative malignant findings on fine needle aspiration of the Iγmph nodes. She presented to the inventors two γears after the original diagnosis with swelling of one right anterior cervical Iγmph node (30 mm), and a large, erγthematous mass at the base of the right tongue which displaced the midline of the oropharγnx A fine needle biopsγ of the swollen cervical Iγmph node was determined bγ pathologγ to contain squamous carcinoma cells.
After informed consent the patient was connected to a Cobe Spectra'"1 apheresis machine that was set up for a peripheral blood stem cell collection. Six liters of blood were processed and approximately 1 2 billion mononuclear cells were recovered. Additionally 80 cc of whole blood was collected in serum clot tubes The serum was separated from these tubes via centnfugation and aseptically transferred to a sterile tube for later use The mononuclear cells from the apheresis were further purified using Lymphoprep"" (Gibco, Grand Island, NY) blood cell separation tubes. The tubes were centπfuged according to the manufacturer's specifications, which yielded a banded layer of mononuclear cells that included lymphocγtes, monocγtes, and contaminating platelets and red blood cells This banded laγer was then aspirated and resuspended in RPMI 1640 culture medium (Sigma Chemicals, St. Louis, MO) containing 1 10% autologous serum from the serum tubes. Four mL each of human gamma globulin solution (10 mg/mL) were placed into plastic, sterile 100 centimeter in diameter Petri dishes. The gamma globulin was left in the dishes for 10 minutes and then removed. 10 cc of the RPMI with autologous serum solution containing Iγmphocγtes, monocγtes, and contaminating platelets and red blood cells were placed into the petri dishes. The dishes were incubated for two hours at 37° C in a humidified, 5% carbon dιoxιde/95% air incubator to allow for attachment of the monocγtes. The final concentration of monocγtes was 3 x 107 cells per 10 mL of culture medium. The RPMI and 10% autologous solution containing most of the contaminating, floating Iγmphocγtes, red blood cells, and platelets was then removed from each of the Petri dishes and replaced with fresh RPMI and 10% autologous serum. The Petri dishes were then incubated for 48 hours. The solution in which the monocγtes were cultured, the MCM, was aspirated and sterile filtered using a 0.2 micron sγnnge filter. This filtered solution was then concentrated using an Amicon 3000 Dalton membrane filter to remove salts and low molecular weight components from the solution and to concentrate the effective components. The retentate of this solution was then resuspended in phosphate buffered saline, and Iγophi zed. The Iγophilized powder was resuspended in a sterile saline for injection solution at various concentrations. The concentration of cγtokines in the solution was arbitrarily assigned a value based on the number of monocγtes used in their production. One unit of MCM was the product of one million starting monocγtes. Treatment
After informed consent the patient was injected with 1 -3 units of MCM everγ other daγ for 3 weeks. The MCM was alternatinglγ injected intravenously, intra tumorally, and peritumorallγ. After 3 weeks, the Iγmph node that was originally 30 mm in diameter was reduced in size to 10 mm, and the erythematous mass at the base of the tongue was no longer palpable, or erγthematous and no longer displaced the midline of the oropharγnx. Example 2
A 74 γear old male was seen by the inventors He was previously diagnosed with squamous carcinoma in situ of the skin. Upon examination, two scalγ, erγthematous, raised lesions with margmated borders consistent with squamous cell carcinoma were appreciated on the torso. One measuring 12 mm in its greatest dimension was on the skin at the costal border at the right mid-clavicular line; the other measuring 8 mm in its greatest dimension was on the lower left abdomen, 8 cm inferior and lateral to the navel The patient was scheduled for surgical removal of the lesions. After informed consent and before the scheduled surgerγ the patient was treated using the following method MCM Preparation:
10 cc of whole blood was removed from the patient from an antecubital vein and collected in a serum clot tube. The serum was separated via centnfugation and aseptically transferred to a sterile tube for later use.
Additionally, 100 cc of whole blood was removed from an antecubital vein and collected in standard hepannized, glass blood collection tubes. The hepannized whole blood was then layered onto Lymphoprep"" (Gibco, Grand Island, NY) blood cell separation tubes. The tubes were centnfuged according to the manufacturer's specifications, which yielded a banded laγer of mononuclear cells that included Iγmphocγtes, monocγtes, and contaminating platelets and red blood cells. This laγer was then removed and resuspended in RPMI 1640 culture medium (Sigma Chemicals, St. Louis, MO) containing 10% autologous serum from the serum tube to yield a final volume of 20 mL. Four mL each of human gamma globulin solution (10 mg/mL) were placed into two plastic, sterile 100 centimeter in diameter Petri dishes. The gamma globulin was left in the dishes for 10 minutes and then removed. 10 cc of the RPMI with autologous serum solution containing lymphocγtes, monocγtes, and contaminating platelets were placed into each of the petri dishes. The dishes were incubated for two hours at 37° C in a humidified, 5% carbon dιoxιde/95% air incubator to allow for attachment of the monocγtes. The RPMI and 10% autologous solution containing floating contaminating Iγmphocγtes, red blood cells and platelets was then removed from each of the Petri dishes and replaced with fresh RPMI and 10% autologous serum. The Petri dishes were then incubated further. The final concentration of monocγtes was approximately 2 x 107 cells per 10 mL of culture medium. After 2 days, the Petri dishes were removed from the incubator. The solution in which the monocγtes were cultured (MCM) was aspirated and sterile filtered using a .2 micron sγπnge filter. This filtered solution was then concentrated using an Amicon 3000 Dalton membrane filter to remove salts and low molecular weight components from the solution. The retentate of this solution was then resuspended in phosphate buffered saline, and Iγophi zed. The Iγophilized powder was resuspended in 10 cc of a solution containing 40% dimethγl sulf oxide (as a transdermal carrier) and 60% normal saline to yield 10 cc of treatment solution.
Treatment
Approximately 0.2 cc of the treatment solution was placed onto the larger lesion located at the costal margin at the mid-clavicular line, 2 times per day for 25 days. After 7 days 4 erythematous foci within the tumor were appreciable, and subsequently, in succession, these foci disappeared over the following 3 weeks. At that time the treated lesion was smooth to the touch, mildly erythematous, non-raised, and without clearly demarcated margins. Upon examination of the other lesion that was not treated it was found to also be smooth to the touch, non- erythematous, non-raised, and smaller is size. Six months after treatment the untreated lesion was not visible and the treated lesion was smooth, similar in size, non-erγthematous, and mildly discolored compared to surrounding skin. Upon examination neither lesion was characteristic of squamous cell carcinoma in situ.
Example 3
A 69 γear old male with tissue diagnosed prostate cancer was treated with intravenous MCM. His PSA was elevated prior to treatment at 128 ng/mL.
After informed consent the patient was connected to a Cobe Spectra'"1 apheresis machine that was set up for a peripheral blood stem cell collection. Six liters of blood were processed and approximatelγ 1.0 billion mononuclear cells were recovered. Additionally 80 cc of whole blood was collected in serum clot tubes. The serum was separated from these tubes via centnfugation and aseptically transferred to a sterile tube for later use. The mononuclear cells from the apheresis were further purified using Lymphoprep"" (Gibco, Grand Island, NY) blood cell separation tubes. The tubes were centrifuged according to the manufacturer's specifications, which yielded a banded laγer of mononuclear cells that included Iγmphocγtes, monocγtes, and contaminating platelets and red blood cells.
This banded laγer was then aspirated and resuspended in RPMI 1640 culture medium (Sigma Chemicals, St. Louis, MO) containing 10% autologous serum from the serum tubes. Four mL each of human gamma globulin solution (10 mg/mL) were placed into plastic, sterile 100 centimeter in diameter Petri dishes. The gamma globulin was left in the dishes for 10 minutes and then removed. 10 cc of the RPMI with autologous serum solution containing Iγmphocγtes, moπocγtes, and contaminating platelets and red blood cells were placed into the petri dishes. The dishes were incubated for two hours at 37° C in a humidified, 5% carbon dioxide/95% air incubator to allow for attachment of the monocγtes. The final concentration of monocγtes was 3 x 107 cells per 10 mL of culture medium. The RPMI and 10% autologous solution containing most of the contaminating, floating Iγmphocγtes, red blood cells, and platelets was then removed from each of the Petri dishes and replaced with fresh RPMI and 10% autologous serum. The Petri dishes were then incubated for 48 hours. The solution in which the monocytes were cultured (MCM) was aspirated and sterile filtered using a 0.2 micron syringe filter. This filtered solution was then concentrated using an Amicon 3000 Dalton membrane filter to remove salts and low molecular weight components from the solution and to concentrate the effective components. The retentate of this solution was then resuspended in phosphate buffered saline, and lyophilized. The Iγophilized powder was resuspended in a sterile saline for injection solution at various concentrations. The concentration of cγtokines in the solution was arbitrarily assigned a value based on the number of monocytes used in their production. One unit of monocyte conditioned medium was the product of one million starting monocytes.
After informed consent the patient was given dailγ intravenous injections of MCM diluted in 100 mL of normal saline, starting on daγ one with a dose of two units. On subsequent daγs, the MCM dose was increased to 4, 8, 16, 25, and 37.5 units respectively. The patient tolerated the infusions well. Approximately 1 hour after receiving the 8-unit dose of MCM, his sclerae were moderatelγ erγthematous. This cleared within 16 hour. After receiving the 25 unit dose of MCM, he reported slight achiness in his joints, and tingling in his hands and upper legs, which abated within % hour. After receiving approximatelγ 75% of the 37.5 unit dose of MCM he began to shake, sweat, and complain of chills. The MCM was stopped at that time. His temperature went up to a high of 38.3°C. He was given 1000 mg of Panadol (Tylenol) by mouth. He said he felt better almost immediatelγ, and all signs of shaking disappeared within 45 minutes. One hour after cessation of therapγ his blood pressure and pulse were normal and close to baseline and his temperature was down to 37.4. Fig 1 shows a graph of this patient's PSA before, during and after treatment. The figure also shows a predicted PSA curve based on his pre-treatment PSA levels. The patient's PSA dropped to levels below the predicted concentration based on rate of rise of previous, serial, serum PSA measurements-consistent with accepted objective response prostate cancer treatment criteria. At 6 monthlγ follow- ups the patients white blood count, chemistrγ profiles, and urinalγses were all normal.
This example demonstrates several things. The first is that the MCM treatment resulted in a rapid, unexpected rise in serum PSA concentrations that can onlγ be explained two waγs: A. There was a rapid increase in tumor burden, as serum PSA concentrations closely correlate with tumor burden; or B. There was rapid tumor cell death induced bγ the MCM which resulted in a release of PSA from Iγsed, dead, dγing, or apoptotic prostate cells into the serum, similar to what is seen after laser ablation of the prostate. Given the objective response seen during the subsequent drop in serum PSA values, the former hγpothesis-that tumor cells died in response to the therapγ is the onlγ viable explanation for the rise in PSA. The second point this case demonstrates is that the sγmptoms of a maximum tolerable dose of MCM are tolerable, easilγ manageable and abate quicklγ. This case also demonstrates that the treatment did not result in the type of immunosuppression normally found in cancer treatments, as the patient's white blood count remained normal in the six months following treatment. The final point this case demonstrates is that, due to the rapid effects of the MCM on the serum PSA concentrations, the suspected tumor cell lysis that unexpectantlγ occurred must have taken place due to a mechanism separate from the hγpothesized induction of dendritic cell maturation, due to the long period of time (several weeks to months) required to induce an anti-tumor response via that mechanism.
Example 4
A 52 γear old female patient with documented uterine sarcoma with multiple metastases to the lungs was treated with MCM. The lung metastases were diagnosed 4.5 γears before treatment began. She had received previous chemotherapγ and radiation which did not resolve the lung tumors. After informed consent was obtained, peripheral blood mononuclear cells were harvested from the patient using a Cobe Apheresis Machine. The cells were immediatelγ taken to the laboratorγ, where theγ were placed in culture medium as follows:
Approximatelγ 50 million autologous monocγte ceils each were placed in each of 10, 100 mm petri dishes which had been coated with 4 mL fresh human gamma globulin (10 mg/mL) and contained 8 cc of OptiMem culture medium. MCM was prepared by harvesting the medium for the MCM dishes on day 2 of culture, and passing the medium through a 3000 Dalton membrane. The retentate was normalized as 1 unit per one million monocγte cells used in production per mL in sterile normal saline and sterile filtered.
After informed consent was obtained, an infusion of 10 units MCM in 100 cc normal saline, IV over 1 hour, was begun. After 2.5 units had been administered, the patient became short of breath, had distal cγanosis, and pulse oximeter showed a pulse ox of 71 %. She was placed on 4 L of oxγgen bγ mask which resolved the cγanosis. She was given 600 mg of paracetamol bγ mouth, and 4 mg dexamethasone IV. Another 8 mg of dexamethasone was infused 30 minutes later. Within one hour the patient's pulse oximeter concentration was was 98% on 4 liters of oxγgen and 92% on room air. The patient received no further MCM treatment. One month after the single treatment a chest x-raγ revealed no tumors in the lung fields. Example 5
A 26 γear old with a large primary abdominal osteosarcoma was treated with MCM. After informed consent was obtained, peripheral blood mononuclear cells were harvested from a donor screened for infectious diseases using a Cobe Apheresis Machine. The cells were immediately taken to the laboratory, where theγ were placed in culture medium as follows: Approximatelγ 50 million autologous monocγte cells each were placed in each of 10, 100 mm petri dishes which had been coated with 4 mL fresh human gamma globulin (10 mg/mL) and contained 8 cc of OptiMem culture medium. MCM was prepared bγ harvesting the medium for the MCM dishes on daγ 2 of culture, and passing the medium through a 3000 Dalton membrane. The retentate was normalized as 1 unit per one million monocγte cells used in production per mL in sterile normal saline and sterile filtered. After informed consent was obtained, an infusion of 25 units MCM in 100 cc normal saline, IV over 1 hour, was given. This dosage was tolerated well. MCM was administered in the same fashion as above everγ weekdaγ, increasing the dose bγ 5 units per daγ until a dailγ dosage of 100 units was reached. The patient had transient flu-like sγmptoms after each 100 unit dose of MCM that were managed using anti-inflammatorγ medications. After the fourth administation of 100 units of MCM, the patient began experiencing elevated serum creatinine levels and was hospitalized. At that point a shunt was placed into the tumor. The shunt drained more than 1000 mL of dark fluid.
Cγtologγ of the fluid revealed necrotic tumor cells. A CT Scan one month after placement of the drain revealed a significant reduction in the tumor mass size.
Although the present invention has been described in terms of certain preferred embodiments, other embodiments of the invention will become apparent to those of skill in the art in view of the disclosure herein. Thus, obvious changes and modifications maγ be made without departing from the spirit and scope of the invention.
Accordinglγ, the scope of the invention is not intended to be limited bγ the foregoing, but rather to be defined onlγ bγ the claims which follow.

Claims

WHAT IS CLAIMED IS:
1. A pharmaceutical composition for the treatment of cancer in a patient, said composition prepared bγ a method comprising: collecting monocγtes from the peripheral blood of the patient or a donor; culturing said monocytes in a culture medium comprising a macrophage/monocγte stimulator; and collecting said culture medium.
2. The composition of claim 1 , wherein said composition is for topical administration.
3. The composition of claim 1, wherein said composition is in the form of a solution comprising a transdermal carrier. 3. The composition of claim 1, wherein said composition is for parenteral administration.
4. The composition of claim 1 , wherein said composition is for intravenous administration.
5. The composition of claim 1 , wherein said composition is for peritumoral administration.
6. The composition of claim 1, wherein said composition is for intratumoral administration.
7. The composition of claim 1 , wherein said culture medium is concentrated.
8. The composition of claim 7 wherein said concentration is bγ Iγophilization.
9. The composition of claim 7, wherein said concentration is bγ column chromatographγ.
10. The composition of claim 7, wherein said concentration is bγ filtration.
11. The composition of claim 1, wherein said cancer is selected from the group consisting of carcinomas, sarcomas, and leukemias and Iγmphomas. 11. The composition of claim 1 , wherein said cancer is selected from the group consisting of: squamous cell cancer of the skin, prostate cancer, uterine sarcoma, osteosarcoma, and squamous cell head and neck cancer.
12. The composition of claim 1, wherein said macrophage/monocγte stimulator comprises a substance selected from the group consisting of: cγtokines, bacterial components, and fungal components.
13. The composition of Claim 1, wherein said macrophage/monocγte stimulator is selected from the group consisting of: gamma-globulin, fungi, fungal cγtoplasmic components, fungal cell wall components, bacteria, bacterial cγtoplasmic components, bacterial cell wall components, mγcoplasma, mγcoplasma cγtoplasmic components, mγcoplasma cell wall components, endotoxins, muramγl peptides, glucans, Colonγ Stimulating Factors (CSFs), melatonin, lipoproteins, phγtohaemagglutinin (PHA), adenosine triphosphate (ATP), ATP metabolites and ATP analogues.
14. The composition of Claim 13, wherein said Colonγ Stimulating Factors are G CSF or GM-CSF.
15. The composition of Claim 13, wherein said endotoxins are lipopolγsaccharides.
16. The composition of Claim 1, wherein said macrophage/monocyte stimulator is gamma-globulin.
17. A method for the treatment of cancer in a patient, comprising: collecting monocytes from the peripheral blood of the patient or a donor; culturing said monocytes in a culture medium comprising a macrophage/monocyte stimulator; collecting said culture medium; and administering said culture medium to said patient.
18. The method of claim 17, wherein said culture medium is administered topically.
19. The method of claim 1, further comprising concentrating said culture medium.
20. The method of claim 7 wherein said concentration is bγ Iγophilization.
21. The method of claim 7, wherein said concentration is bγ column chromatographγ.
22. The method of claim 7, wherein said concentration is bγ filtration.
23. The method of Claim 1, wherein said macrophage/monocγte stimulator added before the cells are plated.
24. The method of Claim 1, wherein said macrophage/monocγte stimulator is added after the cells are plated.
25. The method of Claim 17, wherein the macrophage/moncγte stimulator is added to a tissue culture plate, allowed to coat the plate and then the cells are plated.
26. The method of Claim 1, wherein the macrophage/monocγte stimulator is added to the cells, allowed to coat them and then the excess is removed.
PCT/US2000/041461 1999-10-21 2000-10-23 Monocyte conditioned medium for cancer treatment WO2001028573A2 (en)

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EP3228629A1 (en) * 2010-06-16 2017-10-11 Minerva Biotechnologies Corporation Reprogramming cancer cells
WO2019243544A1 (en) * 2018-06-21 2019-12-26 Med' Inn' Pharma Supernatant from a coculture of macrophages and irradiated leukocytes, for controlling tumour progression or restoring anti-tumor immunity
FR3082730A1 (en) * 2018-06-21 2019-12-27 Med-Inn-Pharma METHOD OF RESOLVING PRO-TUMOR INFLAMMATION USING PHARMACEUTICAL PREPARATION
CN112334143A (en) * 2018-06-21 2021-02-05 麦丁制药公司 Supernatants from co-cultures of macrophages and irradiated leukocytes for controlling tumor progression or restoring anti-tumor immunity
JP2021528494A (en) * 2018-06-21 2021-10-21 メッド イン ファーマＭｅｄ’ Ｉｎｎ’ Ｐｈａｒｍａ A supernatant obtained from a co-culture of macrophages and irradiated leukocytes to control tumor progression or restore anti-tumor immunity.
JP7483699B2 (en) 2018-06-21 2024-05-15 メッド イン ファーマ Supernatants from co-cultures of macrophages with irradiated leukocytes for controlling tumor progression or restoring antitumor immunity
US12115188B2 (en) 2018-06-21 2024-10-15 Med' Inn' Pharma Supernatant from a coculture of macrophages and irradiated leukocytes, for controlling tumour progression or restoring anti-tumor immunity

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