WO2015077532A1 - Thérapie et méthodes d'introduction de cellules dendritiques immatures et/ou de lymphocytes t cytotoxiques et anticorps anti-tnf pour traiter les tumeurs - Google Patents

Thérapie et méthodes d'introduction de cellules dendritiques immatures et/ou de lymphocytes t cytotoxiques et anticorps anti-tnf pour traiter les tumeurs Download PDF

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WO2015077532A1
WO2015077532A1 PCT/US2014/066762 US2014066762W WO2015077532A1 WO 2015077532 A1 WO2015077532 A1 WO 2015077532A1 US 2014066762 W US2014066762 W US 2014066762W WO 2015077532 A1 WO2015077532 A1 WO 2015077532A1
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patient
ctls
cells
immature dendritic
dendritic cells
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Kenichiro Hasumi
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Hasumi International Research Foundation
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Priority claimed from US14/549,706 external-priority patent/US9907819B2/en
Publication of WO2015077532A1 publication Critical patent/WO2015077532A1/fr

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/47—Brain; Nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/49—Breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/58—Prostate
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages
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    • C12N2510/00—Genetically modified cells

Definitions

  • Nonprovisional Application 13/928,844 which claims priority from Provisional Application 61/664,998, both entitled "Therapy and Method for Intratumorally Introducing Cytotoxic T Lymphocyte and/or N T Cell With Anti-TNF and or Anti-IL-10" filed on June 27, 2013 and June 27, 2012, respectively.
  • the invention relates to tumor cell and tumor tissue therapy, and methods for applying the therapy to treat a cancer patient.
  • the invention includes introducing intratumorally and/or through vessel immature dendritic cells and/or cytotoxic T lymphocytes to the patient, and introducing intratumorally and or through vessel and/or subcutaneously anti-tumor necrosis factor (anti-TNF) antibody to the patient.
  • anti-TNF subcutaneously anti-tumor necrosis factor
  • the tumor therapy and methods of the invention are effective to treat the patient in the presence or in the absence of conventional therapy, such as chemotherapy and/or radiotherapy.
  • Cytotoxic T lymphocytes are an important component of cellular immunity. They play a critical role in the control of many infections and cancers. These T cells are responsible for "hunting down" other cells of the body that are infected by viruses or are cancer-containing, and destroying them. For example, when a virus or cancer is using a cell to reproduce, the cell displays some of the viral proteins or cancer components on its surface. The cytotoxic T cells can recognize these proteins or components and hone-in to destroy the infected or cancer-containing cells before they can release the new infection or cancer into the bloodstream. Many vaccines are effective, at least in part, by stimulating this type of T cell activation or response. CTLs can also create chemicals known as cytokines which assist in coordinating how the immune system fights against disease.
  • TNF Tumor necrosis factor
  • Anti-TNF antibody also known as TNF blockers or inhibitors, interfere with the body's production of TNF.
  • the invention solves the above need by providing effective therapy and methods for regression, reduction or elimination of tumor cells in tumor tissue of a patient, as well as tumor cells in metastasized tumors.
  • the invention provides a method of introducing intratumorally and/or through vessel a
  • the method can further include collecting monocyte cells and/or the CTLs from a patient, culturing the monocyte cells and/or the CTLs, forming immature dendritic cells from the monocyte cells, and re-introducing the cultured immature dendritic cells and/or the cultured CTLs back into the patient.
  • the re-introducing of the cultured CTLs can be conducted prior to or following or coincident with the introducing of the cultured immature dendritic cells.
  • the monocyte cells can be cultured in a medium including IL-4, GM-CFS, and mixtures thereof to form immature dendritic cells.
  • the CTLs can be cultured in a medium including IL-2, CD3, and mixtures thereof.
  • the introducing of the anti-TNF antibody can be coincident with the introducing of the immature dendritic cells and/or CTLs. In other embodiments, the introducing of the anti-TNF antibody can be immediately following or following a short time thereafter the introducing of the immature dendritic cells and/or CTLs. A short period of time can include seconds or minutes or hours or days.
  • the anti-TNF antibody is introduced when there is a sufficient amount of CTLs present in the auto-immune system of the patient, such that the immune response of the CTLs is supported by suppressing the activity of the TNF.
  • the introducing of the immature dendritic cells and/or CTLs, and/or the introducing of the anti-TNF antibody can be in conjunction with an anti-inflammatory agent.
  • the introducing of the immature dendritic cells and/or CTLs can be in conjunction with an adjuvant.
  • the immature dendritic cells and/or CTLs and adjuvant can be combined to form a composition and the composition can be introduced intratumorally and/or through vessel into the patient.
  • the adjuvant can be selected from the group consisting of lipid-based, protein-based and polysaccharides- based adjuvants, such as lymphocyte cultured medium, Marignase, Agaricus,OK432, BCG, Lentinan (shiitake),Reishi,Sarunokoshikake, TNF Meshimakobu, Froint's complete or incomplete adjuvant, LPS, fatty acids, TW80, phospholipids, cytokines or a virus, and mixtures thereof.
  • the adjuvant can be a leukocyte cultured medium (LCM) adjuvant.
  • the LCM adjuvant can include at least three cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNy, IP10, ILlp, ILlra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, ⁇ ⁇ , ⁇ ⁇ , PDGFbb, RANTES, TNFa and VEGF.
  • cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNy, IP10, ILlp, ILlra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, ⁇ ⁇ , ⁇ ⁇ , PDGFbb, RANTES, TNFa
  • the invention provides a method of regressing, reducing or eliminating tumor cells in a patient, which includes obtaining monocyte cells from the patient by isolating the monocyte cells from peripheral blood mononuclear cells, differentiating the monocyte cells to produce immature dendritic cells, combining a first sample of the immature dendritic cells with adjuvant and keyhole limpet to form a first mixture of the immature dendritic cells, introducing intratumorally and/or through vessel the first mixture of the immature dendritic cells into the patient, preparing CTLs from the monocyte-depleted peripheral blood mononuclear cells, introducing intratumorally and/or through vessel a first sample of the CTLs into the patient subsequent to introducing the first mixture of immature dendritic cells, combining a second sample of the immature dendritic cells with adjuvant to form a second mixture of the immature dendritic cells, introducing intratumorally and
  • the invention includes introducing into a patient immature dendritic cells and/or cytotoxic T lymphocytes (CTLs) and anti-tumor necrosis factor (anti-TNF) antibody.
  • CTLs cytotoxic T lymphocytes
  • anti-TNF anti-tumor necrosis factor
  • the immature dendritic cells and/or CTLs are introduced to the patient intratumorally and/or through vessel, and the anti-TNF antibody is introduced to the patient intratumorally and/or through vessel and/or subcutaneously.
  • the time between introducing the immature dendritic cells and or CTLs and introducing the anti-TNF antibody can vary. The time period may range from zero (they are introduced simultaneously) to several seconds to several minutes to several hours to several days.
  • the anti-TNF antibody is introduced coincident with introduction of the immature dendritic cells and/or CTLs. In other embodiments, the anti-TNF antibody is introduced immediately following or a short time thereafter the introduction of the immature dendritic cells and/or CTLs. In general, the anti- TNF antibody is introduced when there is a sufficient amount of CTLs present in the auto-immune system of the patient, such that the immune response of the CTLs is supported by suppressing the activity of the TNF.
  • the time between introducing the immature dendritic cells and the CTLs can vary.
  • the time period may range from zero (they are introduced
  • the CTLs are introduced coincident with introduction of the immature dendritic cells. In other embodiments, the CTLs are introduced
  • the therapy and methods of the invention are effective to induce regression, reduction or elimination of tumor cells, tumor tissue, and one or more tumors including tumors formed by metastasis. Further, this regression, reduction or elimination can be achieved in the presence or in the absence of conventional therapy, such as, but not limited to, radiotherapy and chemotherapy. In certain embodiments, therapy and methods of the invention is administered to a patient without additionally subjecting the patient to radiotherapy and/or chemotherapy.
  • patient(s) include mammal(s), which include human(s).
  • the term "therapeutically effective amount” refers to that amount of CTLs, anti-TNF antibody, immature dendritic cells, anti-inflammatory agent, adjuvant, or combinations thereof, required to bring about a desired effect in a human or other mammal. In all instances, at its most basic level, the desired effect is a regression, reduction or elimination of tumor cells in tumor tissue of the patient when compared to the tumor cells in the tumor tissue of the patient prior to employing the therapy and methods of the invention.
  • the term "subcutaneous” and related terms employing "subcutaneous” or “subcutaneously” refer to therapy that includes the introduction of anti-TNF antibody beneath the skin of a patient, e.g., hypodermic.
  • through vessel and related terms employing "through vessel” refer to therapy that includes the introduction of immature dendritic cells and/or CTLs and/or anti-TNF antibody into a channel, such as, a vein or artery, in a patient's body for carrying fluid.
  • intravascular therapy and related terms employing "intratumoral” or “intratumorally” refer to therapy that includes the introduction (e.g., injection) of immature dendritic cells and/or CTLs and/or anti-TNF antibody directly into the tumor tissue of a patient.
  • the anti-TNF antibody can be in various forms known in the art.
  • the anti- TNF antibody can be incorporated into a delivery mechanism, such as a carrier or medium, to facilitate introduction into the patient.
  • the anti- TNF antibody can be included or contained in a liquid for ease of introduction, e.g., injection.
  • TNF circulates throughout the body of a patient, contributes to effective immune surveillance, and is required for proper proliferation and function of natural killer cells, T cells, e.g., CTLs, B cells, macrophages, and dendritic cells, as well as, the regulation of immune cells. Further, it is known that the presence of TNF can cause systemic inflammation.
  • the introduction of immature dendritic cells in a patient can positively promote an immunoresponse to induce CTLs.
  • introduction of the immature dendritic cells can induce the TNF level in the patient's bloodstream and increase inflammation at the tumor site(s), which can interfere with or hinder the immunoresponse of the CTLs and, in turn, induce inflammation at the tumor site, and progress the growth of the tumor.
  • anti-TNF antibody can be combined (e.g., co-introduced or co-injected) with the CTLs to inhibit the induced inflammation.
  • introduction of anti-TNF antibody intratumorally and/or through vessel and/or subcutaneously is effective to suppress the systemic inflammation caused by TNF.
  • the CTLs e.g., induced by the introduction of the immature dendritic cells and/or CTLs, are not limited in their immunological function and therefore, are effective to destroy tumor cells both locally and systemically in the patient.
  • the result of this immunological function can vary and may depend on the amount of tumor cells, CTLs and TNF in the patient's body.
  • these factors are used to determine a therapeutically effective amount of anti-TNF antibody to be introduced or administered to the patient in order to achieve a favorable outcome, e.g., to maximize the regression, reduction or elimination of tumor cells (local and metastasized) in the patient. It is contemplated that an appropriate balance of the amount of tumor cells and TNF present in the patient's body, the amount of CTLs induced by the introduction intratumorally and/or through vessel of immature dendritic cells and/or CTLs, and the amount of anti-TNF antibody intratumorally and/or through vessel and/or subcutaneously introduced into the patient's body results in maximizing the effect of the therapy and the regression of tumors.
  • the regulatory T-cell is a suppressing factor against local and/or systemic immunological response
  • the therapy and methods of the invention which control, e.g., suppress, the TNF-based inflammation (instead of the regulatory T-cell) are effective to treat tumors.
  • the therapy and methods of the invention are particularly effective to treat multiple metastasized tumors.
  • CTLs which are naturally induced as part of the autoimmune response above- described, may not be potent against tumors especially when the tumors are at an advanced stage or aggressively growing. The reasons may be that these CTLs are not induced in an adequate quality and/or in an adequate quantity and or in a timely manner to defend the patient's body from the tumor invasion.
  • CTLs are produced by the patient, in certain embodiments, CTLs can be collected from the patient, cultured, and then introduced, e.g., returned, to the same patient's body.
  • the culture medium can vary and may be selected from those known in the art. Non-limiting examples include, but are not limited to, IL-2, CD3, and mixtures thereof.
  • the invention includes collecting monocyte cells from the patient. The monocyte cells are cultured to form immature dendritic cells which are then introduced, e.g., returned, to the same patient's body.
  • the culture medium can vary and may be selected from those known in the art. Non-limiting examples include, but are not limited to, IL-4, GM-CFS, and mixtures thereof.
  • the CTLs are collected from the patient following collection, culture and return of the immature dendritic cells into the patient.
  • the CTLs are cultured in a suitable medium and then introduced, e.g., returned, to the same patient's body.
  • the immature dendritic cells and/or CTLs are introduced intratumorally and/or through vessel into the patient.
  • only immature dendritic cells are introduced or only CTLs are introduced or a combination of immature dendritic cells and CTLs are introduced into the patient.
  • the CTLs can be introduced prior to, coincident with or following the intratumoral and/or through vessel introduction of the immature dendritic cells.
  • the anti-TNF can be introduced intratumorally and or thorough vessel and/or subcutaneously into the patient in a therapeutically effective amount prior to, coincident with, or following introducing the cultured CTLs and or immature dendritic cells.
  • an adequate quantity and quality of CTLs are provided in the patient's body and, in particular, at the tumor site(s), to regress, reduce or eliminate tumor cells. Further, the CTLs are combined with anti-TNF to inhibit potential inflammation at the tumor site(s). It is contemplated that the quantity and quality of the natural CTLs (i.e., induced by the introduction of immature dendtritic cells into the patient) may be sufficient (e.g., a therapeutically effective amount) to accomplish this objective.
  • the invention provides for collecting the natural CTLs from the patient, culturing or activating these collected cells, and re-introducing them, e.g., in combination with anti-TNF, into the same patient in a quantity and quality which is sufficient (e.g., a therapeutically effective amount) to regress, reduce or eliminate tumor cells and to inhibit inflammation at the tumor site(s).
  • the anti-TNF antibody is introduced when there is a sufficient amount of CTLs presenting in the autoimmune system of the patient, such that the immune response of the CTLs is supported by suppressing the activity of the TNF.
  • the CTLs include CD8+N T cell population.
  • the invention is a human-initiated therapeutic vaccine with immature dendritic cells and/or CTLs in combination with anti-TNF antibody.
  • the immature dendritic cells and/or CTLs are introduced in conjunction with an adjuvant.
  • the adjuvant can be introduced intratumorally and/or through vessel into the patient prior to, coincident with or following intratumoral and/or through vessel introduction of the immature dendritic cells and/or CTLs.
  • the immature dendritic cells and/or CTLs and the adjuvant can be combined to form a composition and the composition can be introduced intratumorally and/or through vessel into the patient.
  • the immature dendritic cells and/or CTLs, and optionally adjuvant are introduced intratumorally and/or through vessel prior to, coincident with or following introducing intratumorally and/or through vessel and or subcutaneously anti-TNF antibody into the patient.
  • Suitable adjuvants for use in the invention can include, without limitation, lipid-based, protein-based and polysaccharides-based adjuvants, such as lymphocyte cultured medium, Marignase, Agaricus,O 432, BCG, Lentinan (shiitake),
  • the adjuvant can be a leukocyte cultured medium (LCM) adjuvant.
  • LCM leukocyte cultured medium
  • the LCM adjuvant can include at least three cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNy, IP 10, ILlp, ILlra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, 1L15, IL17, MCP1, ⁇ , ⁇ ⁇ , PDGFbb, RANTES, TNFa and VEGF.
  • cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNy, IP 10, ILlp, ILlra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, 1L15, IL17, MCP1, ⁇ , ⁇ ⁇ , PDGFbb, RANTES, TNFa and VEGF.
  • the immature dendritic cells and/or CTLs can be introduced to the patient in conjunction with an anti-inflammatory agent.
  • Suitable anti-inflammatory agents can include those that are known in the art.
  • the antiinflammatory agent can be introduced prior to, coincident with or following introduction of the immature dendritic cells and/or CTLs. It is typical for introduction of the immature dendritic cells, CTLs and anti-inflammatory agent to be simultaneous or substantially simultaneous, or for the elapsed time between introducing the immature dendritic cells and/or CTL and the anti-inflammatory agent to be relatively short in duration.
  • the invention can optionally include a precursor treatment. That is, prior to introducing the dendritic cells and/or CTLs, the patient may be administered a treatment selected from chemotherapy, radiotherapy, antibody therapy, and combinations thereof. These regimens are well known in the art. Further, optionally, it is contemplated that the use of these regimens may be employed at various other times throughout the method of the invention. However, it is further understood, that these regimens are not necessary.
  • the therapy and methods of the invention are effective to regress, reduce or eliminate tumor tissue in a patient in the absence of chemotherapy, radiotherapy, antibody therapy, and combinations thereof.
  • the method of administering the therapy includes the following steps.
  • Step 1 Collecting monocyte cells and/or CTLs from a patient.
  • Step 2 Culturing the monocyte cells and/or culturing/activating the
  • the monocyte cells form immature dendritic cells.
  • Various conventional techniques known in the art can be employed to culture the monocyte cells and/or to culture/activate the CTLs, and various culture mediums known in the art can be used, such as those disclosed herein.
  • Step 3 Introducing intratumorally and/or through vessel, such as but not limited to injecting, a therapeutically effective amount of the immature dendritic cells and/or CTLs into the patient.
  • the immature dendritic cells and/or CTLs can be mixed or combined with an adjuvant and the mixture or combination can be introduced intratumorally and/or through vessel into the patient.
  • the adjuvant can be selected from those known in the art and disclosed herein.
  • an anti-inflammatory agent may be present with the immature dendritic cells and/or CTLs and adjuvant as disclosed herein.
  • Step 4 Introducing intratumorally and/or through vessel and/or subcutaneously, such as, but not limited to, injecting, a therapeutically effective amount of anti-TNF antibody into the patient.
  • the anti-TNF antibody can be mixed or combined with anti-IL- 10 and this mixture or combination can be introduced intratumorally and/or through vessel and/or subcutaneously into the patient.
  • the anti-TNF antibody can be introduced coincident with or immediately following or a short time after the introduction of the immature dendritic cells and/or CTLs into the patient.
  • Step 3 can be performed coincident with Step 4 and in other alternate embodiments, Step 4 can be performed following Step 3, such as a couple of seconds, hours, days, or weeks after Step 3.
  • the time allowed to lapse within Step 3, i.e., between the introduction of the immature dendritic cells and CTLs, can also vary. In certain embodiments, their introduction is simultaneous or substantially simultaneous. In other embodiments, there is a pre-determined or desired time period which is allowed to lapse between the introduction of each. It is typical for this period of time to be relatively short in duration.
  • the CTLs can be collected from the patient coincident with collection of the monocyte cells or at a time thereafter.
  • the monocyte cells are collected and cultured to form immature dendritic cells and the dendritic cells are introduced into the patient prior to collection of CTLs from the patient.
  • conventional therapy such as, radiation or chemotherapy, may be conducted at any time during Steps 1 through 4.
  • the invention can include the treatment steps of: introducing intratumorally and/or through vessel immature dendritic cells in a therapeutically effective amount into the tumor tissue of the patient; collecting from the patient CTLs induced by the introduction of the immature dendritic cells; reintroducing intratumorally and/or through vessel the collected cytotoxic T lymphocyte into the tumor tissue of the same patient; and introducing intratumorally and/or through vessel and/or subcutaneously anti-TNF and/or anti-IL-10 into the patient.
  • the introduction of the immature dendritic cells is a prerequisite to the collection and introduction of the CTLs cells with the anti-TNF and/or anti-IL-10 for inducement of the natural CTLs.
  • the inducement of CTLs by the immature dendritic cells is sufficient (e.g., a therapeutically effective amount) such that the natural CTLs are not removed from the patient and not re-introduced into the same patient.
  • the anti-TNF and/or anti-IL-10 can be introduced to inhibit inflammation in the absence of the introduction of CTLs. That is, the introduction of immature dendritic cells can be in combination with the introduction of the anti-TNF and/or anti-IL-10.
  • immature dendritic cells which are formed by culturing monocyte cells collected from a patient and CTLs which are produced by a patient and collected from the patient, provide for an enhanced desired effect when injected into the same patient as compared to immature dendritic cells and CTLs produced and obtained by other means. It appears that immature dendritic cells which are formed from the patient's own monocyte cells which have been collected and CTLs which have been collected, cultured and re-introduced intratumorally and/or through vessel, provide improved coupling or interaction with other cells in the body of the patient.
  • the invention provides regression, reduction or elimination of tumor cells in tumor tissue which can be visually detected by MRI and/or CT and/or Echo scan.
  • a combination of immunotherapy and local radiation is administered to a cancer patient.
  • this combination of treatments provides for a therapeutic protocol to stimulate a systemic adaptive immune response against malignant cells.
  • monocyte cells are obtained from the patient for differentiation into immature dendritic cells.
  • the monocyte cells are isolated from peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the monocyte cell-depleted, T-cell enriched fraction of the PBMCs is then used to prepare activated T-cells, e.g., CTLs.
  • the immature dendritic cells are combined with LCM, a multi-cytokine-based adjuvant, and keyhole limpet hemocycanin ( LH). This mixture is injected into the patient. Subsequently, such as but not limited to, on the following day, activated T-cells, e.g., CTLs, are infused. After a period of time, such as, but not limited to, about seven days, local radiation is administered to the patient. After a period of time, such as, but not limited to, about another seven days, a second sample of immature dendtric cells and activated T-cells, e.g., CTLs, is administered to the patient.
  • LCM keyhole limpet hemocycanin
  • immature dendritic cells suspended in LCM are injected and then, e.g., on the following day, activated T-cells, e.g., CTLs, are infused. It has been found that this protocol results in an increase in CD8 + CD56 + cells. Without intending to be bound by any particular theory, it is believed that these cells are capable of killing cancer cells.
  • a MRI was performed on a 71 year-old male patient and the patient was diagnosed with Stage IV prostatic cancer and multiple bone metastasis. The patient had advanced cancer and progressive disease that had not responded to conventional standard therapies. Apheresis was performed on the patient to collect moncyte cells from the patient. The monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of immature dendritic cells.
  • a cocktail was prepared containing between about 10 7 to 10 s immature dendritic cells and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline.
  • PR is defined as a 30% reduction in the size of the injected tumor, a decline in serum markers, no increase in tumor size at other metastatic sites or appearance of new metastasis.
  • SD is defined as showing less than a 20% increase in tumor size and less than a 30% reduction in tumor size, with no increase in serum tumor markers.
  • a 74 year-old male patient was diagnosed with Stage III prostatic cancer and multiple bone metastasis.
  • the patient had advanced cancer and progressive disease that had not responded to conventional standard therapies.
  • Apheresis was performed on the patient to collect moncyte cells from the patient.
  • the monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of immature dendritic cells.
  • a cocktail was prepared containing between about 10 7 to 10 8 immature dendritic cells and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline. Depending on the size of the tumor, between 2.0 and 5.0 cc of normal saline was injected into multiple tumor sites of the patient. The patient was also administered radiotherapy and a subsequent intratumoral injection of the cocktail containing the immature dendritic cells and LCMadj.
  • a 51 year-old female patient was diagnosed with Stage IV breast cancer and Lt. axilla LN metastasis.
  • the patient had advanced cancer and progressive disease that had not responded to conventional standard therapies.
  • Apheresis was performed on the patient to collect moncyte cells from the patient.
  • the monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of immature dendritic cells.
  • a cocktail was prepared containing between about 10 7 to 10 8 immature dendritic cells and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline.
  • LCMadj a cocktail was prepared containing between about 10 7 to 10 8 immature dendritic cells and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline.
  • between 2.0 and 5.0 cc of normal saline was injected into multiple tumor sites of the patient.
  • the patient was also administered radiotherapy and,
  • the patient was evaluated by RI image analysis.
  • the treated tumors of the patient showed PR and CR.
  • a 79 year-old female patient was diagnosed with Stage II lung cancer and brain metastasis.
  • the patient had advanced cancer and progressive disease that had not responded to conventional standard therapies.
  • Apheresis was performed on the patient to collect moncyte cells from the patient.
  • the monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of immature dendritic cells.
  • a cocktail was prepared containing between about 10 7 to 10 8 immature dendritic cells and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline. Depending on the size of the tumor, between 2.0 and 5.0 cc of normal saline was injected into multiple tumor sites of the patient.
  • the patient was also administered radiotherapy and a subsequent intratumoral injection of the cocktail containing the immature dendritic cells and LCMadj.
  • Apheresis was performed on the patient to collect CTLs from the patient.
  • the CTLs were cultured and a cocktail was prepared containing from lOxlO 8 to 40xl0 8 of the cultured CTLs and 12.5 mg to 25.0 mg of anti-TNF.
  • the cocktail was injected into multiple tumor sites of the patient.
  • the detailed protocol is shown in Table 4.
  • the patient was evaluated by RI image analysis.
  • the treated tumors of the patient showed CR.
  • Protocol HITV & CTL-II Intra- tumoral
  • Prostatic Cancer Stage IV
  • Protocol HITV & CTL-II Intra- tumoral Lung Cancer (Stage II), brain metastasis

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Abstract

L'invention concerne une thérapie et des méthodes d'application de thérapie à un patient. L'invention concerne l'introduction de cellules dendritiques immatures et/ou de lymphocytes T cytotoxiques (CTL) dans le patient, et l'introduction d'un anticorps anti-TNF dans le patient. Les cellules dendritiques immatures et/ou les CTL sont introduits de manière intratumorale et/ou par l'intermédiaire d'un vaisseau, et l'anticorps anti-TNF est introduit de manière intratumorale et/ou par l'intermédiaire d'un vaisseau et/ou de manière sous-cutanée. Les cellules dendritiques immatures et/ou les CTL peuvent être formés par collecte de cellules de monocyte provenant du patient et culture dans un milieu de culture. L'invention peut être efficace pour faire régresser, réduire ou éliminer les cellules tumorales d'un tissu tumoral de patients, notamment les tumeurs métastasées. Le traitement est également efficace en l'absence de thérapie classique, telle qu'une radiothérapie et une chimiothérapie.
PCT/US2014/066762 2013-11-21 2014-11-21 Thérapie et méthodes d'introduction de cellules dendritiques immatures et/ou de lymphocytes t cytotoxiques et anticorps anti-tnf pour traiter les tumeurs WO2015077532A1 (fr)

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