Classifications

• • 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
  • A61K35/13—Tumour cells, irrespective of tissue of origin
• • 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
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/45—Bacterial antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 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 A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/57—Skin; melanoma
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells

Definitions

• the present invention relates to lymphocytes, preferably CD4 + T lymphocytes, which have been transinfected from dendritic cells with a bacterium, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor antigen.
• a bacterium preferably Listeria monocytogenes
• the present invention could be framed in the field of medicine. STATE OF THE TECHNIQUE
• CD8 + T cells in responses to cancer and infectious diseases is mainly carried out by dendritic cells (DCs), considered the most efficient and physiologically relevant antigen presenting cells (APCs) (Joffre et al., 2012).
• DCs dendritic cells
• APCs antigen presenting cells
• MCH-I major histocompatibility complex I
• This process is known as cross-presentation or cros-presentation (Joffre et al., 2012).
• cros-presentation was carried out exclusively by professional phagocytes of innate immunity, mostly by DCs, considered as the most efficient APCs.
• the antigen presentation occurs during the formation of immune synapses (IS), a structure formed by the intimate contact of a T cell (to be activated) with an APC loaded with the antigen that recognizes said T cell (Saito and Batista, 2010).
• IS immune synapses
• APC APC loaded with the antigen that recognizes said T cell
• CD4 + T cells are capable of capturing bacteria, during the formation of SI, that infect DCs in a process we call transfection (Cruz-Adalia et al., 2014). This transfection through IS is several orders of magnitude more efficient than direct infection.
• transinfected T cells tiT killed bacteria that engulfed more efficiently than professional phagocytes and also secreted large amounts of proinflammatory cytokines, such as IL-6, interferon- ⁇ and TNF- ⁇ ; these properties conferred protection in mice against infections by Listeria monocytogenes.
• DCs which are considered the best antigen presenting cells, must capture, process and cros-present the tumor antigens in their MHC-I to activate CD8 + T cells that are the ones that will eventually eliminate the malignant cells. Therefore, DCs have also been used as a cancer vaccine (by adoptive transfer) as they are capable of generating anti-tumor immune responses in vivo.
• chemotherapy-resistant leukemia who have presented lasting and complete clinical responses by injecting cells by adoptive transfer (Kalos and June, 2013), demonstrating that it is possible to achieve long-term sustainable immunotherapy against tumors.
• tumors express antigens, they are not eliminated by the host's immune system. It could be because the antigens do not present efficiently and therefore do not cause a sufficiently potent immune response or that there is a continuous selection in the cancer patient, so that the tumor cells can evade the immune recognition. In antitumor therapy, it is therefore necessary the appearance of a new tool that improves the response to tumors by activating specific CD8 + T cells against tumor antigens.
• the present invention demonstrates the use of lymphocytes transinfected with bacteria (used as proof of concept L monocytogenes) that express tumor antigens in antitumor therapy.
• lymphocytes transinfected with bacteria used as proof of concept L monocytogenes
• B-16 OVA an experimental model of aggressive melanoma
• transfected CD4 + T lymphocytes work by activating cytotoxic CD8 + T lymphocytes directly, such as APCs, that is by cros-presentation via MHC-I, and are surprisingly much better presenters than APCs known to date.
• the tiT cells are capable of presenting antigens.
• CD4 + T cells transinfected with Listeria-OVA Listeria monocytogenes expressing ovalbumin (Pope et al., 2001)
• CD8 + naf ' ve T cells from OT-I mice (transgenic mice where All T cell receptors (TCR) recognize an OVA peptide (257-264; SIINFEKL) in the context of H-2K b (Clarke et al., 2000)) (Figure 1), and therefore bona fide APCs.
• tiT cells are capable of internally processing the captured bacteria, and that the antigen presentation is independent of the DC, in other words, that the antigens do not come from the donor DCs but by cros-presentation by the tiT cells .
• CD4 + T lymphocytes a paradigm of adaptive immunity, are capable of carrying out roles thought to be exclusive to innate immunity cells.
• the present invention relates to a lymphocyte transinfected with a bacterium comprising a tumor antigen.
• a lymphocyte of the invention a lymphocyte transinfected with a bacterium comprising a tumor antigen.
• the lymphocyte is a CD4 + T cell or a B cell.
• CD4 + T cells are understood as those lymphocytes that express the T lymphocyte receptor (TCR), in addition to CD3 and the CD4 correceptor. They are also called T helper lymphocytes.
• TCR T lymphocyte receptor
• B lymphocyte is understood as those lymphocytes that express the B cell receptor; BCR Other molecules used as B cell markers are CD19 and CD20.
• the bacteria described in the present invention can be both Gram + and Gram -, and in addition the bacteria can be pathogenic or non-pathogenic.
• the bacterium is selected from the list consisting of: L. monocytogenes, enteric Salmonella, bacteria of the Mycobacterium tuberculosis complex, Staphylococcus aureus and Escherichia coli; preferably it is L monocytogenes.
• M. tuberculosis complex bacteria include M. tuberculosis, M. bovis (BCG), M. africanum, M. caprae, M. pinnipedii, M. canetti and M. microti. In a particular embodiment it is L. monocytogenes.
• cancer in the present invention encompasses solid tumors, as well as hematological tumors, such as leukemias, lymphomas and myelodysplastic syndromes.
• the lymphocyte of the first aspect of the invention is transinfected from a dendritic cell.
• the lymphocyte is from the same patient as the dendritic cell used for transfection of said lymphocyte, preferably both cells are originating from a patient with a tumor (wherein said tumor comprises the antigenic tumor peptide).
• Transfection is understood as the process by which a lymphocyte (T or B) captures bacteria from previously infected APCs (preferably DC).
• APC or antigen presenting cell
• APC is understood as the cells of the immune system whose function is to capture, process and, as the name implies, present antigens to the cells of the innate immune system in histocompatibility complexes.
• the APC is preferably a dendritic cell.
• Dendritic cell means an APC whose origin can be both myeloid and lymphoid specialized in presenting antigens. It expresses CD1 1c (in mice) and there are several subtypes, thymus DC, conventional or plasmacytoid, and each subtype with specific markers. In the experiments underpinning this invention, we worked with DC derived from bone marrow grown in the presence of GM-CSF (factor granulocyte and macrophage colony stimulant). The markers of these cells were: CD1 1c +, GR-, CD3-, CD19-, MHC-II +. In humans, preferred DCs will be derived from peripheral blood after treatment with GM-CSF and IL-4.
• the bacterium comprises a tumor antigen that can be any antigen that is specifically expressed in the malignant cells of any tumor, as it is being highlighted in recent works that propose how to identify tumor antigens (Schumacher and Schreiber, 2015; Yadav et al., 2014) for example antigens identified in melanoma peptides, lymphoma, chronic lymphocytic leukemia, myeloma, cancer of breast, ovary, uterus, cervix, testis, prostate, colon, colorectal, pancreatic, stomach and gastrointestinal tumors, gastric cancer, liver tumor, kidney (including clear cell renal carcinoma), bladder, oral cancer, pharynx, larynx , esophagus, lung (small and non-small cell), thyroid, glioblastoma, glioma, sarcoma, brain, brain, neuroblastoma and blastoma bone marrow, head and neck, bone and connective tissue.
• tumor antigens
• the tumor can be at any stage and can also be metastatic. Antigens derived from such tumors are known to the person skilled in the art (Schumacher and Schreiber, 2015; Yadav et al., 2014). Preferably the tumor has neo-antigens (antigens that result from a mutation in the tumor cells and therefore absent in the rest of the body's cells). It can also present fetal antigens, absent in the cells of the adult organism.
• melanoma derived antigen proteins include, but are not limited to: tyrosinase, gp75, gp100, Melan A / MART-1, TRP-2, MAGE (melanoma-associated antigen) GAGE and BAGE family proteins, NY-ESO-1, as well as mutations in CDk4, -catenin, MUM-1, p15.
• tumor antigen that specific for cancer, which can be of any size and that is absent in the healthy cells of the organism.
• a specific tumor specific antigen for example a “melanoma specific antigen,” it encompasses antigens of any size that are frequently associated with that tumor (eg melanoma).
• the lymphocyte is heterologous or autologous.
• the APC preferably the dendritic cell, is heterologous or autologous and can be from the same or different individual of the lymphocyte to which it transinfects.
• lymphocyte when we refer to a lymphocyte we also refer to fragments of said lymphocyte and derivatives thereof, such as any modification that favors contact between the tiT and the TIL (T lymphocytes infiltrated in the tumor, and therefore with tropism against tumor antigens).
• the lymphocyte of the invention is from a mammal, preferably a human of any race, age or sex.
• lymphocyte we also refer to a population of lymphocytes.
• a second aspect of the invention relates to a cell population comprising the lymphocyte of the first aspect of the invention.
• cell population is understood as a mixture of cells among which are the lymphocytes of the invention, preferably a mixture of blood cells.
• a third aspect of the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the lymphocyte of the first aspect of the invention or the cell population of the second aspect of the invention.
• the pharmaceutical composition is preferably a vaccine.
• pharmaceutical composition herein refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in humans or animals.
• the pharmaceutical composition of the invention can be used both alone and in combination with other pharmaceutical compositions.
• pharmaceutical composition and medicament are used interchangeably in this invention.
• the composition Pharmaceutical or medicament are characterized by understanding the lymphocyte of the invention in a therapeutically active amount, so that the lymphocyte exerts its function in the target tissue / cell.
• the expression "therapeutically effective amount” refers to the amount of lymphocyte calculated to produce the desired effect and, in general, will be determined, for the case of a therapeutic composition, by the characteristics of the compounds, the route , form and frequency of administration thereof, and other factors, including the age, condition of the patient, as well as the severity of the alteration or disorder.
• vehicle like the excipient, refers to a substance that is used in the pharmaceutical composition or medicament to dilute any of the components of the present invention comprised therein to a certain volume or weight.
• the function of the vehicle is to facilitate the incorporation of other elements, allow a better dosage and administration or give consistency and form to the composition.
• the pharmacologically acceptable carrier is the diluent.
• Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are vehicles known to those skilled in the art.
• said pharmaceutical composition is prepared in solid form or in aqueous suspension, in a pharmaceutically acceptable diluent.
• the therapeutic composition provided by this invention may be administered by any appropriate route of administration for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen.
• the administration of the therapeutic composition provided by this invention is carried out, for example, parenterally, intra-intravenously, intravenously, orally, intraperitoneally or subcutaneously, preferably intravenously.
• a fourth aspect of the invention relates to the use of the lymphocyte of the first aspect of the invention or of the cell population of the second aspect of the invention for the preparation of a medicament, preferably for the prevention or treatment of a tumor and / or stimulation. of the immune response against a tumor antigen.
• lymphocyte of the first aspect of the invention or of the cell population of the second aspect of the invention for use as a medicament, preferably a medicament for the prevention or treatment of a tumor and / or the stimulation of the immune response against a tumor antigen.
• prevention is to prevent or reduce the appearance of a tumor.
• treatment means to cure, mitigate, reduce or combat the damage caused by a tumor, reduce its size, reduce its aggressiveness, reduce or prevent its dispersion, or to stabilize the condition of individuals.
• the tumor to be treated or prevented is any tumor, for example those on this list: melanoma, lymphoma, chronic lymphocytic leukemia, myeloma, breast cancer, ovary, uterus, cervix, testis, prostate, colon, colorectal, pancreatic , stomach and gastrointestinal tumors, gastric cancer, liver tumor, kidney (including clear cell renal carcinoma), bladder, oral cancer, pharynx, larynx, esophagus, lung (small and non-small cell), thyroid glioblastoma, glioma, sarcoma, brain, brain, neuroblastoma and blastoma of the marrow, head and neck, bone and connective tissue.
• melanoma lymphoma
• chronic lymphocytic leukemia myeloma
• breast cancer ovary
• uterus cervix
• testis prostate
• colon colorectal
• the stimulation of the immune response against a tumor antigen comprises the stimulation of CD8 + lymphocytes.
• Said response can be measured by tests known to the person skilled in the art, for example ELISPOT (Enzyme-Lynked ImmunoSpof), by means of intracellular staining tests of cytokines, by tetramer tests, by antigen-specific detection, by follow-up tests of cell multiplication, and by the presence of cell activation markers (CD25, CD69) detected by flow cytometry.
• the cytotoxic capacity of activated CD8 + T cells with tiT lymphocytes can also be quantified.
• the lymphocyte of the present invention can be used in combination with other treatments known to the person skilled in the art, for example treatments with antibodies against CTLA-4, PD-1, PDL-1, as well as radiotherapy or chemotherapy treatments.
• a fifth aspect of the present invention relates to a kit or device comprising the lymphocyte of the first aspect of the invention or the cell population of the second aspect of the invention.
• a sixth aspect of the invention relates to the use of the kit or device of the fifth aspect of the invention for the prevention or treatment of a tumor and / or the stimulation of the immune response against a tumor antigen of any tumor, for example, but not exclusively, those on this list: melanoma, melanoma, lymphoma, chronic lymphocytic leukemia, myeloma, breast cancer, ovary, uterus, cervix, testis, prostate, colon, colorectal, pancreatic, stomach and gastrointestinal tumors, gastric cancer, tumor of liver, kidney (including clear cell renal carcinoma), bladder, oral cancer, pharynx, larynx, esophagus, lung (small and non-small cell), thyroid, glioblastoma, glioma, sarcoma, brain, brain, neuroblastoma and blastoma of marrow, head and neck, bone and connective tissue.
• the tumor has neo-antigens.
• the tumor
• the kit of the invention comprises the elements necessary to carry out the methods described in the present invention.
• the kit can also comprise the elements necessary to evaluate the immune response generated by the lymphocyte of the invention.
• This kit can comprise, without any limitation, buffer, enzymes, agents to prevent contamination, etc. It can also include the supports and containers necessary for its implementation and optimization. It may also contain positive or negative controls. You can also understand the instructions for carrying out its use.
• a seventh aspect of the invention relates to an in vitro method for transinfecting a lymphocyte comprising the following steps:
• Isolate antigen presenting cells preferably dendritic cells, preferably from peripheral blood, from the patient (by example by adding GM-CSF and IL-4) and isolating lymphocytes, preferably B lymphocytes and / or CD4 + T lymphocytes, from a biological sample; b. Differentiate the antigen presenting cell;
• step (b) infecting these antigen presenting cells from step (b) with a bacterium, where the bacterium comprises a tumor peptide;
• step (c) contacting the infected antigen presenting cells of step (c) with the lymphocytes of step (a) at 35-38 ° C, preferably at 37 ° C, for several hours, preferably 24-72 hours, more preferably 48h, to facilitate the transfection of lymphocytes.
• it further comprises a step (e) of isolation of the transfected lymphocytes.
• the bacteria can be both Gram + and Gram -, and in addition the bacteria can be pathogenic and non-pathogenic.
• the bacterium is selected from the list consisting of: L monocytogenes, enteric Salmonella, bacteria of the Mycobacterium tuberculosis complex, Staphylococcus aureus and Escherichia coli; preferably it is L monocytogenes.
• the bacteria of the M. tuberculosis complex they can be M. tuberculosis, M. bovis (BCG), M. africanum, M. caprae, M. pinnipedii, M. canetti and M. microti.
• it is L monocytogenes.
• the bacterium comprises a tumor antigen.
• the tumor antigen may be from a tumor that is selected from the list consisting of: melanoma, melanoma, lymphoma, chronic lymphocytic leukemia, myeloma, breast cancer, ovary, uterus, cervix, testis, prostate, colon, colorectal, pancreatic, Stomach and gastrointestinal tumors, gastric cancer, liver tumor, kidney (including clear cell renal carcinoma), bladder, oral cancer, pharynx, larynx, esophagus, lung (small and non-small cell), thyroid glioblastoma, glioma , sarcoma, brain, brain, neuroblastoma and blastoma of the marrow, head and neck, bone and connective tissue.
• the lymphocyte is heterologous or autologous.
• in vitro refers to the method of the invention being performed outside the subject's body. That is, it is performed on a biological sample of a subject.
• biological sample in the present invention refers to any sample comprising lymphocytes (CD4 + T lymphocytes and / or B lymphocytes) and includes, but is not limited to, biological fluids or tissues of an individual, obtained by any method known to an expert in the field that serves this purpose.
• the biological sample could be, for example, but not limited to, a sample of fluid, such as blood or serum.
• the biological sample in the present invention can be fresh or frozen.
• tumor antigen and "tumor peptide” is used interchangeably.
• Another embodiment of the present invention refers to a method for the prevention and / or treatment of a tumor and / or stimulation of the immune response against a tumor antigen characterized by comprising the administration of a therapeutically effective amount of the pharmaceutical composition.
• the tumor is selected from the list consisting of: melanoma, melanoma, lymphoma, chronic lymphocytic leukemia, myeloma, breast cancer, ovary, uterus, cervix, testis, prostate, colon, colorectal, pancreatic, stomach and gastrointestinal tumors, cancer gastric, liver tumor, kidney (including clear cell renal carcinoma), bladder, oral cancer, pharynx, larynx, esophagus, lung (small and non-small cell), thyroid, glioblastoma, glioma, sarcoma, brain, brain , neuroblastoma, bone marrow, head and neck blastoma, bone and connective tissue.
• the tumor has neo-antigens.
• antigens that stimulate the immune system are known, for example, but not exclusively tyrosinase, gp75, gp100, Melan A / MART-1, TRP-2, proteins of the MAGE family (melanoma-associated antigen) GAGE and BAGE , NY-ESO-1, as well as mutations in CDk4, -catenin, MUM-1, p15.
• FIG. 1 Transfected CD4 + T cells (tiCD4 + T) have bacterial antigens via MHC-I to "naive" CD8 + T cells in vitro.
• a CD4 + tiT transinfected with Liste ria-WT (wild strain; panels above) or with Listeria-OVA (strain expressing ovalbumin; panels below) were incubated with naive CD8 + T cells from OT-I mice (which recognize a ovalbumin peptide; OVAp-l 257-264; SIINFEKL (SEQ ID NO: 1) in the context of H2Kb).
• Histograms represent the fluorescence of CD8 + T cells marked with "CelITrace TM Violet".
• the cell divisions characteristic of activated cells, are observed as a shift to the left (in each division the cell dye is diluted) of the population.
• Gray-filled histograms show the fluorescence of naive CD8 + T cells, not activated.
• the proliferation of CD8 + T cells was analyzed at 24, 48 and 72 after contacts between CD4 + / CD8 + tiT cells.
• Histograms represent the expression of CD25 or CD69 of CD8 + T cells incubated with Listeria-WT (thin black line) or Listeria-OVA (thick black line).
• Histograms filled in gray show fluorescence of naive CD8 + T cells, not activated, c, Histograms show proliferation of activated CD8 + T cells with: anti-CD3 and CD28 antibodies, DCs loaded with soluble pOVA, infected DCs with Listeria-OVA, or tiCD4 + T cells transinfected with Listeria-OVA on day 2 or on day 3. Note that the activation of CD8 + T lymphocytes is much greater when activated with tiCD4 + T transinfected with Listeria-OVA than when activated with DCs infected with Listeria-OVA.
• Gray-filled histograms show the fluorescence of CD8 + T cells not activated, d, Histograms represent the Fluorescence ⁇ CellTrace TM Violet) of CD8 + T cells of C57 / BL6 WT mice or OT-I mice after conjugation with tiCD4 + transinfected with Listeria-OVA.
• TiCD4 + T cells process bacterial antigens, a, Proliferation of mouse CD8 + T-cells OT-I, 2 or 3 days after incubation with CD4 + tiT cells transinfected with Liste ria-WT or Listeria-OVA, which were captured from DCs loaded with the OVA peptide (OVAp-1).
• H-2K b / OVA detected using a specific antibody
• tiCD4 + T cells H-2K b + , black line; H-2K b " , gray
• H-2K b " gray
• e, f Western blots showing Tap1 expression in CD4 + naf ' ve T cells, activated (with DC loaded with soluble OVAp), or in tiCD4 + T cells transinfected with Listeria-OVA for 24 (E) or 48 h (F ) after activation.
• ERK2 and laminB were used as controls, g, Proliferation of CD8 + T cells from mouse OT-I, 3 or 4 days after conjugation with tiCD4 + T cells (from Tap1 KO mouse, or its wild isogenic, WT) transinfected with Listeria-OVA .
• FIG. 3 The tiCD4 + cells form immunological synapses with naive CD8 + T cells.
• a The histogram shows the expression of H-2Kb in CD4 + T cells before (black line) and after transfection (gray line).
• the gray-filled histogram shows the fluorescence of the negative control, b, CD86 expression in CD4 + T cells before (black line) and after transfection (gray line).
• the gray-filled histogram shows the fluorescence of the negative control, c, Expression of H-2Kb / OVAp-1 in TCD4 + T cells transfected with Listeria-OVA, 24 (line that does not move) or 48 h (line that moves on the right) post-transinfection and in non-transinfected T cells (gray-filled histogram), dye, confocal images of CD4 + tiT cells transinfected with Liste ria-WT (d) and Listeria-OVA (e) incubated with CD8 T cells + naf ' ve of OT-I mice for 1 h. The fluorescence of CD3 and actin is shown. The bars represent 10 ⁇ .
• CTLs effector CD8 + T cells activated by CD4 + tiT transinfected with Listeria-OVA (solid line) or for splenocytes loaded with pOVA (dotted line). Specific cytotoxicity was measured using different ratios of EL-4 (target cells): CTLs (0.5: 1, 1: 1, 2: 1, 5: 1).
• FIG. 4 The tiCD4 + cells activate the effector cytotoxic T cells in vivo, a, Histograms show the flowering (CelITrace TM Violef) of the CD8 + CD45.1 + T cells injected into recipient mice (CD45.2), which were inoculated with CD4 + tiT cells, transinfected with Liste ria-WT or Listeria-OVA.
• CD8 + CD45.1 + T cells from OT-I mice, stained with CelITrace Violet injected into wild C57BL / 6 recipient mice (whose bone marrow has been reconstituted after irradiation with cells of H-2K k mice).
• CD8 + CD45.1 + T cells from OT-I mice (4 x 10 6 cells / mouse) were transferred together with CD4 + T cells expressing H-2K b (right panels) or H-2K k ( left panels) of AND mice (4 x 10 6 cells / mouse) and MCC peptide (15 ⁇ g / mouse).
• mice were also infected with Listeria-OVA (10 3 bacteria / mouse). 5 days after infection the spleens were isolated and proliferation of the transferred CD8 + T cells was detected by flow cytometry (panels above). The panels below show the CD8 + T cells transferred from OT-I mice that are analyzed (CD45.1 + CD4 " ).
• OVA B16 melanoma cells (expressing ovalbumin) were injected subcutaneously on the right flank of recipient mice All groups (9 mice / group) were inoculated with CD8 + naf ' ve cells from OT-I mice (10 3 / mouse), together with phosphate buffered saline (PBS) in the group 1.5 x 10 5 tiCD4 + cells transinfected with Listeria WT in group 2 and 5x 10 5 tiT CD4 + cells transinfected with Listeria-OVA in group 3. The size of the tumors was monitored every two days (from day 5 to 13) and every 3 days from there.
• PBS phosphate buffered saline
• FIG. 5 B cells capture transinfection bacteria and cros-present bacterial antigens, a, mouse DC decorated + (or not -) with chicken egg lysozyme "Hen Egg Lysozyme (HEL)" were infected with Listeria monocytogenes, and incubated with B cells of MD4 transgenic mice, whose B cells have B cell receptors (BCR) that recognize HEL, under conditions that allow DC-cell B interactions (for 30 min), or in the presence of a polycarbonate barrier (transwell ) that prevents such interactions. After formation of the conjugates, the CD4 + T cells were re-isolated, and a classic gentamicin survival assay was carried out.
• BCR B cell receptors
• Colony forming units are shown (intracellular bacteria) per 50,000 CD4 + T cells.
• 0 marks the direct infection of the bacteria in the B. b, DC cells infected with L. monocytogenes and decorated with HEL were incubated with B cells of MD4 mice. After the formation of the conjugates, the B cells were re-isolated, and the survival (at different times) of the intracellular bacteria was quantified by gentamicin resistance assays. The data were normalized with respect to what was observed at time 0. As a control of bacterial "fitness", in parallel HeLa cells were infected.
• c Expression of CD86 in B cells before (left panel; na ' ⁇ ve B cells) and one day after transfection with Listeria-OVA (right panel). Both panels show the expression of IgM (channel FL-1) vs the expression of CD86 (channel FL-2).
• d Proliferation of CD8 + na ' ⁇ T cells from OT-I mice labeled with CelITrace Violet, after 3 days after contact with tiB cells that have captured by transinfection Listeria-WT (panels above) or Listeria-OVA (below) .
• the panels on the left show the fluorescence of CelITrace Violet vs. CD25 expression (a T cell activation marker).
• CTL CD8 + T cells activated by tiB cells transinfected with Listeria-OVA (top line) or with splenocytes decorated with the OVAp-1 peptide (bottom line).
• Different ratios of EL-4 (target cells): CTL were measured.
• T cells can capture bacteria from infected DCs through a process called transinfection (Cruz-Adalia., 2014), led us to wonder if it was possible that transinfected T cells (you) could act as true APCs . This was a risky question, because, if true, it would break a dogma of immunology, the strict separation of roles between innate and adaptive immunity, and would be a huge advance in the basic knowledge of the functioning of the immune system.
• CD4 + tiT cells transinfected CD4 + T cells
• Listeria-OVA L. monocytogenes expressing ovalbumin
• Listeria-WT isogenic wild strain, which does not express OVA
• BM-DCs bone marrow derived dendritic cells
• CD8 + T cells recognize an ovalbumin peptide (OVAp-l 257-264; SIINFEKL, SEQ ID NO: 1) in the H-2Kb context.
• Flow cytometric analysis of CD8 + T cells stained with "CelITrace TM Violet” shows a potent proliferation of CD8 + T cells that begins two days after contact with the CD4 + tiT cells (a movement towards the left of the fluorescence of "CelITrace TM Violet”), but only in those CD8 + T cells that were in contact with the CD4 + tiT cells transinfected with Listeria-OVA (Fig 1 a). This very proliferative population expresses high levels of CD8 (CD8 + hi9h ) (Fig. 1 a).
• CD8 + T cells that were incubated with Listeria-WT transinfected CD4 + tiT cells did not proliferate (Fig. 1a).
• the expression of the CD69 and CD25 activation markers was detected only in CD8 + T cells incubated with CD4 + tiT cells transfected with Listeria-OVA but not when incubated with CD4 + tiT cells transinfected with Listeria-WT (Fig. 1b).
• Ti4 CD4 + cells transinfected with Listeria-OVA were found to induce the proliferation of CD8 + T cells more potently than those produced by BM-DCs loaded with soluble OVA peptide, and still exceeded proliferation induced by polyclonal activation with CD3 / CD28 antibodies, and was much more potent than that produced by BM-DCs infected with Listeria-OVA (Fig. 1 c).
• Ti4 CD4 + cells transinfected with Listeria-OVA are not capable of inducing proliferation of CD8 + T cells from wild mice, which do not recognize any OVA antigen (Fig. 1 d), whereas if they induce very potent proliferation of CD8 + T cells from OT-I mice (Fig. 1a, c and d), indicating that the observed effects are antigen-specific.
• CD4 + tiT cells are capable of cros- presenting bacterial antigens to CD8 + naf ' ve T cells.
• tiCD4 + cells could recapitulate the different stages of cros-presentation that occur in professional APCs; endogenous processing of the antigens, antigen presentation via MHC-I, expression of co-stimulatory molecules, and interaction with target T cells through the formation of canonical immune synapses.
• the antigen presentation of the CD4 + tiT cells could be due to the capture of the MHC / antigen complexes from the surface of the DCs or to endogenous processing by the tiCD4 + cells themselves.
• CD4 + tiT cells from transgenic mice that express only the H-2Kk haplotype (MHC-I), and therefore are not able to stimulate OT-I CD8 + T cells) or their isogenic CD4 + tiT cells that they express H-2Kb, and by capturing both cell types the bacteria from DCs expressing H-2Kb, confirm that the activation of CD8 + T cells is mostly due to antigenic processing by the CD4 + tiT cells (Fig. 2b).
• the tiCD4 + T cells isolated from Tap1 KO mice had very reduced capacity as cros-presenting cells after bacterial capture (Fig. 2g), that is, the proliferation of CD8 + T cells was greatly reduced when the cells antigen presenting were tiCD4 + from Tap1 KO mice, compared to that observed when the presenting cells were tiCD4 + from WT mice.
• CD4 + tiT cells increased the expression of MHC-I (H-2Kb), H-2Kb coupled to OVA antigen (of bacterial origin) and co-stimulatory molecule ligands, such as CD86 (Fig. 3a-c), which is compatible with a canonical antigen presentation in MHC-I.
• the activation of CD8 + T cells involved the formation of CD4 + / CD8 + cell conjugates (Fig. 1c), indicative of the formation of immune synapses (IS), a seal of the activation of T cells by APCs.
• a mature SI is structured in multimolecular concentric rings called supramolecular activation groups (SMACs).
• cSMAC central zone of these SMACs
• pSMAC peripheral zone
• naive CD8 + T cells indeed form mature IS with CD4 + tiT cells transinfected with Listeria-OVA (Fig. 3e).
• CD4 + / CD8 + cell conjugates contain CD3 molecules, part of the TCR complex, recruited into the cSMAC, and show a massive accumulation of actin in the pSMAC; We found no evidence of the formation of these structures when CD4 + tiT cells transinfected with Liste ria-WT were used (in the few cell contacts detected) (Fig. 3d). Together, these data demonstrate that CD4 + tiT cells are professional APCs and activate CD8 + T cells by canonical cros-presentation.
• CD8 + T cells Activation of CD8 + T cells by tiCD4 + cells transinfected with Listeria-OVA gives them cytotoxic capacity, since they can eliminate El-4 target cells that express OVAp / OT-1 (Fig. 3f).
• CD4 + tiT cells it was investigated whether the CD4 + tiT cells are capable of activating naive CD8 + T cells in vivo.
• naive CD8 + T cells were isolated from OT-I / CD45.1 mice, stained ex vivo with "CelITrace TM Violet", transferred to a BL6 / CD45.2 mouse, and then the tiT cells were injected CD4 + to provoke a response.
• CD8 + CD45.1 T cells isolated from the spleen, proliferated in response to tiCD4 + cells transfected with Listeria-OVA but not if they were transinfected with Liste ria-WT (Fig. 4a).
• CD4 + cros-T cells have antigens in vivo, during a physiological activation of the immune system. To do this, the bone marrow of C57BL / 6 recipient mice were transplanted with H-2K k haplotype progenitor stem cells (the APCs of these mice will not be able to present antigens to the CD8 + T cells of OT-I mice).
• CD4 + T cells from AND mice are transferred into recipient mice together with CD8 + CD45.1 + T cells from OT-I mice, and MCC peptide (for favor transfection (Cruz-Adalia et al., 2014)).
• the recipient mice were also infected intravenously with Listeria-OVA. Only mice that received CD4 + H-2K b T cells were able to activate T cells CD8 + CD45.1 + of OT-I (Fig. 4b), confirming the presenting capacity of CD4 + T cells during the course of a bacterial infection.
• CD4 + tiT cells transinfected with Listeria-OVA.
• mice in the control groups treated with the vehicle or with CD4 + tiT cells transinfected with Listeria-WT, developed tumors in the first 1 1 days after injection of the B16-OVA cells (Fig. 4c).
• Treatment with CD4 + tiT cells transinfected with Listeria-OVA prevented the formation of tumors in 6 of the 9 mice and delayed tumor development in another mouse, that is, vaccination (a single inoculation) with the functioning tiCD4 + cells
• they conferred protection against melanoma in 7 of 9 cases Fig. 4c).
• B cells are also capable of capturing bacteria by transfection.
• the process of bacterial capture by B cells from infected DCs was quantified after re-isolating B cells after they formed conjugates with infected DCs, followed by classical tests of survival to gentamicin. This method allows us to analyze a large number of conjugates.
• the requirement of cell-cell contacts was tested using physical barriers (polycarbonate filters) that prevented DC-cell B contacts, and the role of antigen recognition by decorating DC with chicken egg lysozyme "Hen Egg lysozyme (HEL)" (Note that we use B cells isolated from MD4 transgenic mice, whose B cells have B cell receptors (BCR) that recognize HEL).
• CD25 activation marker was detected in CD8 + T cells incubated with tiB transinfected with Listeria-OVA, but not when tiB transinfected with Listeria-WT was used (Fig. 5d).
• TiB cells transinfected with Listeria-OVA induce the proliferation of CD8 + T cells more potently than even bone marrow-derived DC (BM-DC) loaded with soluble OVAp-l peptide, infected with Listeria-OVA, or than the polyclonal activation of the CD8 + T cells themselves with the anti CD3 / CD28 antibodies.
• BM-DC bone marrow-derived DC
• L. monocytogenes-OVA pPL2-LLO-OVA
• pPL2-LLO-OVA Listeria strain that expresses the OVA protein on its surface
• L. monocytogenes 10403S. Mice (1) The wild strain C57BL / 6.
• C57BL / 6-Tg (TcraTcrb) 425Cbn / J OTII which is a strain whose CD4 + T cells all expressed a specific TCR for OVA peptide 323-339 in the context of l-Ab.
• mice C57BL / 6- Tg mice (TcraTcrb) 1 100Mjb / J OTI whose CD8 + T lymphocytes all expressed a specific TCR for OVA peptide 257-264 in the context of H2Kb.
• AND-TCR transgenic mice that recognize peptide 88-103 (ANERADLIAYLKQATK; SEQ ID NO: 2) of moth cytochrome C (MCCp) in the context lE k , and expressing H-2K b , H-2K k or the two haplotypes.
• C57BL / 6-Tg (lghelMD4) 4Ccg / J, transgenic mice (MD4) with more than 99% of their B cells expressing a BCR specific for HEL (chicken egg lysozyme). Both males and females between 8 and 12 weeks were used. Mice were kept in pathogen-free conditions. in the CNB-CSIC animal farm. Animal experimentation was in accordance with Spanish and European laws and the guidelines indicated by FELASA were always followed.
• DCs were generated according to the method developed in (Inaba et al., 1992). Briefly, bone marrow cells (from tibiae and femurs of C57BL / 6 mice of 8-20 weeks) were grown in the presence of GM-CSF, at day 10 they were tested by flow cytometry for CD11 c, IA / IE and Gr1 to make sure they had differentiated correctly. They matured with LPS the day before use.
• the EL-4 lymphoma line was maintained in RPMI 1640 (Fisher Scientific), supplemented with 10% FCS, 0.1 U / ml penicillin, 0.1 mg / ml streptomycin (Lonza) and 0.05 mM 2-mercaptoethanol.
• the B16 OVA line of melanoma was maintained in RPMI 1640 with 0.4 mg / ml of geneticin. The antibiotics were washed 48 hours before inoculation.
• Antibodies :
• the antibodies we used were anti-CD69, -CD25, -CD4, -CD8, -CD1 1c, - IA / IE, -Gr1 (BD and Immunostep), biotinylated antibodies against CD45.1, CD3, CD4, CD8, CD28, IgM, B220, CD19, MHC class II (l-Ab), CD1 1 b, CD1 1c DX5, CD25 and CD16 / CD32 (BD and Immunostep), and anti-tubulin conjugated to FITC (Santa Cruz).
• the monoclonal antibody 25-D1.16 specific for SIINFEKL / H-2K b (SEQ ID NO: 1 / H-2K b ) and labeled with allophycocyanin (APC) was purchased from eBioscience.
• Anti-TAP-1 (M-18), -ERK-2 and -lam ⁇ n B were purchased from Santa Cruz.
• Secondary anti-mouse and goat anti-hamster antibodies conjugated to AlexaFluor488, 647, or 568 were purchased from Life Technologies; secondary antibodies conjugated with Goat anti-mouse IgG peroxidase and goat anti-rabbit IgG were purchased from Thermo Scientific.
• OVAp / OT-ll OVA 323-339; ISQAVHAAHAEINEAGR, SEQ ID NO: 3
• OVAp / OT-l OVA 257-264; SIINFEKL (SEQ ID NO: 1)
• CSIC Molecular Biology Center Severo Ochoa
• MCCp Peptide
• MCCp 88-103
• SEQ ID NO: 2 was purchased from GenScript.
• LPS Sigma-Aldrich
• streptavidin-coupled microparticles Miltenyi Biotec
• Streptavidin-PercP BD
• Alexa Fluor 568-Phalloidin Life Technologies
• Poly-L-Lysine Sigma-Aldrich
• CelITrace TM Violet (Life Technologies) and 7-AAD Viability staining solution from eBiosciences. Transfection of CD4 + T cells.
• OT-II mouse CD4 + T cells were transinfected with Listeria-OVA or Listeria-WT as described in (Cruz-Adalia et al., 2014). Briefly, BM-DC infected and loaded with OVAp (OT-ll) (to increase transfection) formed conjugates with CD4 + OT-II T cells.
• OVAp OVAp
• BM-DC and CD4 + T lymphocytes were isolated from AND mice expressing MHC-I, H-2K bk , or H-2K b negative haplotypes, H ⁇ K ⁇ . In these experiments, BM-DCs were loaded with MCCp to improve transfection.
• gentamicin 100 g / ml was added to the medium to remove extracellular bacteria. 24h later (in some 48h experiments) the tiCD4 + cells were purified by "cell sorting" (FACS Synergy; Cyt)
• CD8 + T cell proliferation assays The purified population of tiCD4 + cells was incubated with CD8 + naf ' ve cells isolated from OT-I mice. These CD8 + T cells were previously stained with CelITrace TM Violet in order to quantify their proliferation by flow cytometry (FACSAria; BD). In each cell division the population that proliferates loses fluorescence, and was observed as a left shift in the histogram. Only live cells, which do not capture the 7AAD dye, were analyzed.
• CD8 + T cells From CD45.1 + OT-I mice, stained with CelITrace TM Violet, were injected intravenously into recipient mice ( CD45.2 + C57BL / 6). After 24h, the tiCD4 + cells were transferred to the mice. Spleens were isolated after three days to analyze the proliferation of CD8 + T cells by flow cytometry.
• Fluorescence microscopy TiCD4 + cells and naive CD8 + T cells were allowed to form conjugates for 1 h, then fixed with 4% paraformaldehyde in PBS. CD8 + T cells were previously stained with CelITrace TM Violet. The samples were permeabilized with 0.1% Triton TM X-100 in PBS before staining with the indicated antibodies. F-actin was detected using a fluorophore coupled phalloidin. Samples were visualized by confocal microscopy using a Leica TCS-SP5 microscope equipped with 63x lenses and controlled by Leica LAS AF. The images were analyzed using ImageJ NIH; Bethesda, MD).
• CD8 + T cells were prepared from naive CD8 + T cells (from OT-I) activated with tiCD4 + cells transinfected with Listeria-OVA. As positive controls, CD8 + OT-I T cells activated with splenocytes loaded with OVAp were used.
• EL-4 cells were incubated with 0.5 ⁇ OVAp / OT-1 for 1 h (or not, as a control). After washing with PBS, the OVAp-loaded EL-4 cells were stained CelITrace TM Violet (5 ⁇ ). On the other hand, EL-4 cells without loading OVAp were stained with CelITrace TM Violet (0.5 ⁇ ). After washing with culture medium both populations were mixed and incubated with different ratios of CTLs (5: 1, 2: 1, 1: 1, 0.5: 1) (EL4 / CTLs). After 4h, the samples were analyzed by flow cytometry.
• CD4 + T cells transinfected with Liste ria-WT (as a negative control) or Listeria-OVA were prepared as described in previous sections. After 24h of transfection, the CD4 + tiT cells were re-isolated by cell sorter and resuspended in PBS. OT-I CD8 + naf ' ve T cells were purified by magnetic columns as previously described (Cruz-Adalia et al., 2014) and resuspended in PBS. 5 x 10 5 B16-OVA cells were injected subcutaneously in the right flank of recipient mice (C57BL / 6).
• mice were divided into three groups and transferred intravenously (iv) and in a single injection: group 1: PBS; group 2: 5 x 10 5 tiCD4 + cells transinfected with Liste ria-WT; and group 3: tiCD4 + cells transinfected with Listeria-OVA. All mice of all groups were also injected at the same time as the tiCD4 + or the PBS vehicle, 10 3 CD8 + T cells of OT-I (also via iv). Tumor growth was monitored every 2-3 days using a dial clamp, and the areas were determined by multiplying length and width. The experimental groups were randomly assigned and the experiment was performed in double blind form (people who measured the tumors did not know to which group each mouse belonged). The mice were sacrificed when the tumors reached 300 mm 2 according to the endpoint criteria according to the indications of the European Union, and FELASA for experimental animals, and in accordance with the legislation in force in Spain.
• BIBLIOGRAPHY Borroto, A., Arellano, I., Blanco, R., Fuentes, M., Orfao, A., Dopfer, EP, Prouza, M., Suchánek, M., Schamel, WW, and Alarcón, B. (2014). Relevance of Nck-CD3 epsilon interaction for T cell activation in vivo. J. Immunol. 792, 2042-2053.
• Pax3 functions at a nodal point in melanocyte stem cell differentiation. Nature 433, 884-887. Butler, J.I., Zorina, T., Storkus, W.J., Zitvogel, L, Celluzzi, C, Falo, L.D., Melief, C.J., lldstad, S.T., Kast, W.M., and DeLeo, A.B. (nineteen ninety five). Bone marrow-derived dendritic cells pulsed with synthetic tumor peptides elicit protective and therapeutic antitumor immunity. Nat. Med. 7, 1297-1302.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Epidemiology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Cell Biology (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Microbiology (AREA)
• Hematology (AREA)
• Virology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Developmental Biology & Embryology (AREA)
• Molecular Biology (AREA)
• Mycology (AREA)
• Oncology (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=56936436

Family Applications (1)

Country Status (6)

Families Citing this family (1)

• 2016
  • 2016-08-08 CA CA2995021A patent/CA2995021A1/en not_active Abandoned
  • 2016-08-08 JP JP2018525816A patent/JP2018522594A/ja active Pending
  • 2016-08-08 AU AU2016307370A patent/AU2016307370A1/en not_active Abandoned
  • 2016-08-08 US US15/750,787 patent/US20180228840A1/en not_active Abandoned
  • 2016-08-08 WO PCT/ES2016/070597 patent/WO2017025657A1/es not_active Ceased
  • 2016-08-08 EP EP16766331.9A patent/EP3333255A1/en not_active Withdrawn

Non-Patent Citations (24)

Also Published As

Similar Documents

Legal Events