EP1226235A2 - Constructions cellulaires adaptees a l'immunotherapie, leur production et leur utilisation - Google Patents

Constructions cellulaires adaptees a l'immunotherapie, leur production et leur utilisation

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Publication number
EP1226235A2
EP1226235A2 EP00972821A EP00972821A EP1226235A2 EP 1226235 A2 EP1226235 A2 EP 1226235A2 EP 00972821 A EP00972821 A EP 00972821A EP 00972821 A EP00972821 A EP 00972821A EP 1226235 A2 EP1226235 A2 EP 1226235A2
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Prior art keywords
cells
cell
tumor
mhc
donor
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EP00972821A
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German (de)
English (en)
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Peter Leskovar
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Individual
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Individual
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Priority claimed from DE19951072A external-priority patent/DE19951072A1/de
Priority claimed from DE10016846A external-priority patent/DE10016846A1/de
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Publication of EP1226235A2 publication Critical patent/EP1226235A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • 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
    • 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
    • A61K39/4611—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
    • A61K39/4612—B-cells
    • 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
    • A61K39/4614—Monocytes; Macrophages
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • 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
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y117/00—Oxidoreductases acting on CH or CH2 groups (1.17)
    • C12Y117/03—Oxidoreductases acting on CH or CH2 groups (1.17) with oxygen as acceptor (1.17.3)
    • C12Y117/03002—Xanthine oxidase (1.17.3.2)
    • 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
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells

Definitions

  • the invention relates to cell constructs which are suitable for immunotherapy, and to processes for their preparation and their use.
  • the principle of the present invention is based on the knowledge that the cause of many diseases is based on the multiplication of undesired activated T cells and, in some cases, additionally on the presence of cells which protect unwanted cells, namely protective suppressor cells.
  • a method was therefore developed to effectively remove the unwanted T cells and, if necessary, the suppressor cells and thus to combat the underlying disease.
  • Many different embodiments are possible for this general principle, which depend, inter alia, on the type of disease, the type of cells to be controlled and the severity of the disease. These various embodiments are described below.
  • a preparation is provided with which malignant tumors can be combated and a recurrence or the occurrence of metastases can be prevented.
  • a basic principle of the method according to the invention is to remove both undesired activated T cells and protective suppressor cells.
  • Undesired activated T cells are understood to mean those cells which have a pathological effect, for example cells infected by viruses, autoaggressive cells, cells which induce the occurrence of atherosclerosis, cells which trigger allergic reactions and other cells whose reproduction disturbs the organism.
  • diseases are combated according to the invention in which protective cells are present in addition to undesired T cells.
  • protective cells include neoplastic and virus-related diseases.
  • T cells In a first stage, the majority of the cells protecting the tumor or virus are removed. Since these protective cells belong to the class of T cells, which in turn belong to the hematological cells, one could destroy the hematological cells by radiation or by administration of cyclophosphamide by methods known per se, which destroys the entire immune system and thus also the T Cells are depleted. However, in order not to overly weaken the patient, it is preferable to be more selective and only remove the T cells. For the selective removal of the T cells, the person skilled in the art is aware of methods and means which are suitable here. A preferred means according to the invention is treatment with monoclonal antibodies which are directed against T cells, if appropriate conjugated with immunotoxins. This leads to a humoral control of the T cells.
  • anti-CD3 antibodies which are commercially available as OKT3 (Orthoclone), ATG or ATGAM. These are administered according to the manufacturer's instructions, e.g. up to 4 weeks daily or at regular intervals. It is often sufficient to administer the monoclonal antibodies 3 to 15 times, in particular 3 to 10 times, on consecutive days once a day in order to deplete the T cells.
  • the amounts to be administered here are those recommended by the manufacturer.
  • the activated T cells are therefore attacked in a second stage using lymphocytes with predetermined cell death.
  • PBLs ie peripheral blood lymphocytes
  • mitomycin C ie peripheral blood lymphocytes
  • the lymphocytes in the patient attack the activated T cells before they can trigger a GvH reaction. This removes a further significant proportion of the T cells.
  • cell constructs are used. These are intended to direct the patient's immune system to these cells and are therefore used as a type of vaccine.
  • the cell constructs are fused cells that result from the fusion of tumor cells or virus-infected cells and MHC II-positive cells and from the fusion of suppressor cells and MHC II-positive cells. The former prevent the tumor from forming again and the latter prevent new suppressor cells from forming.
  • both fusions with autologous MHC II-positive cells and with allogeneic are possible.
  • both autologous and allogeneic MHC II-positive cells are used for the fusion.
  • Triomas in which a foreign MHC II-positive cell, a patient's own MHC II-positive cell and a tumor cell or a suppressor cell have been fused are particularly preferred. This can be achieved by collecting MHC II cells from a donor and the patient.
  • Methods for the selective extraction of the individual cell types mentioned are known to the person skilled in the art and require no further explanation. A variety of methods are explained in the literature. Miltenyi's technique is suitable, for example. The latter is used, for example, to obtain tumor cells in a process which is described in US Pat. No.
  • the cell constructs obtained according to the invention e.g. Hybridomas, triomas or quadromas are usually administered to the patient by infusion, preferably subcutaneously.
  • he is preferably treated in a further stage with cells which have been clonally postexpanised in a manner known per se.
  • a blood sample is taken from the patient, washed, mixed with fetal calf serum, IL2 and mitogenic antibodies, e.g. Anti CD3 or Anti CD2 or Anti CD28, incubated with tumor cells, selects the most efficient cytotoxic T cells (Tc), which are directed against the tumor cells, and then infuses these selected T cells into the patient after clonal post-expansion.
  • Tc cytotoxic T cells
  • a further embodiment of the invention relates to a receptor-independent activation of cells, which is used both in addition to the stages described above and can also be used in general to strengthen the immune system in general. It was surprisingly found that the ratio of ROI, ie reactive oxygen intermediates, to RNI, ie reactive nitrogen intermediates (reactive oxygen intermediates / reactive nitrogen intermediates) has an influence on cell activation and, moreover, an influence on whether T cells in the form of Ts Cells or Tc cells are present. It has been found that cells can be activated or Ts cells can be converted into Tc cells by contacting them with xanthine oxidase and its substrate substrate.
  • xanthine or hypoxanthine can be used as the substrate for xanthine oxidase.
  • membrane-permeable substances which increase the intracellular pHi have an immunostimulating effect and promote the Ts-Tc conversion. Examples are NH 3 and triethanolamine in a sub-toxic dose.
  • Activation can take place in vitro and in vivo.
  • the cells are incubated with the enzyme and substrate in a suitable manner.
  • xanthine oxidase and substrate must be brought to the right place. Means for this are known to the person skilled in the art, for example enzyme and substrate in a protective matrix or enzyme bound to PEG are suitable.
  • the suppressor cells required for the fusion are obtained from the patient's T cells. To do this, one simply has to isolate the peripheral blood lymphocytes from the patient's blood, which is known per se. These PBLs also contain the suppressor cells, which are elevated in tumor patients. The PBL can then be incubated with tumor cells, which activates all cells which react with tumor cells, in particular tumor-specific Ts and Th2 cells. These cells can then be used for the fusion.
  • MHC II-positive cells and tumor cells or T cells are used for the fusion. The following happens here: If an allogeneic MHC II-positive cell is fused with the tumor, this cell increasingly presents foreign MHC II and tumor antigen on its surface. This activates the patient's T cell response. MHC I is also presented by the tumor. This combination of MHC II, MHC I and tumor antigen may be particularly helpful.
  • the MHC II does not activate the T cell response, but the fact that own MHC II positive cells are used means that the binding with the T cells is stronger and in turn the contact with the tumor antigen. A combination of the two is considered particularly advantageous.
  • the fusion cells used which are used like a vaccine, are primarily intended to help against recurrences.
  • the invention also relates to a principle for removing unwanted cells, namely autogressive, allergic cells and GvH cells. In this case no suppressor cells need to be removed.
  • Triomas which in turn consist of autologous and allogeneic MHC II-positive cells and the unwanted cell, can be used here. These triomas in turn stimulate the T cell response against the unwanted cell presented, which then removes these cells.
  • a means and a method for the treatment of cancer and viruses are provided.
  • One principle is to remove the tumor or virus-bearing cells as well as these protective cells. If only the tumor is removed without removing the protective cells at the same time, a new tumor can form after the treatment, since it can never be possible to destroy all tumor cells.
  • the use of fused cells is proposed which increase the immune response against both cell types. Surprisingly, it was found that a combination of autologous and allogeneic fused MHC II-positive cells with the unwanted tumor or virus-carrying cells increases the immune response in such a way that the latter cells can no longer grow, which prevents later recurrences.
  • the fused cells proposed according to the invention lead to a very effective presentation of the unwanted cells, which can greatly enhance the immune response.
  • a minimum limit concentration of peroxide anion (0 2 2 ) or hydrogen peroxide (H 2 0 2 ) in the cytosol or in the mitochondrial area is critical for the maintenance and function of Th1 vs. Th2 and Tc / CTL vs. Ts-subsets.
  • the antigen-presenting cells primarily macrophages
  • APC antigen-presenting cells
  • H 2 0 2 peroxide anion
  • xanthine oxidase XOD
  • XOD xanthine oxidase
  • H02 horseradish peroxidase
  • SOD superoxide dismutase
  • Peroxide formation can also be achieved with the enzyme glucose oxidase (GOD) and the corresponding substrate (glucose).
  • Suitable scavengers are described, for example, in "The Oxygen", Biochemistry, Biology, Medicine (author: EF Elstner),ticiansverlag Mannheim / Vienna / Zurich, 1 990.
  • "enzymatic" scavengers are also used to intercept H0 2 ' and / or H 2 0 2 signals and thus for precise immunomodulation.
  • the H0 2 signals can be intercepted by Mn, Cu / Zn and / or Fe SOD, the H 2 0 2 signals by catalase and / or peroxidase.
  • NO neutralization which is also secreted by the activated and specially hyperactivated or chronically activated APC (macrophages) by NO savengers, which are offered commercially by several manufacturers.
  • NO savengers which are offered commercially by several manufacturers.
  • subtoxic doses of ammonia (NH3) and ammonium salts on the one hand or NH3 scavengers for example a combination of glutamine synthase plus glutamate and / or asparagine synthase plus aspartate
  • NH3 scavengers for example a combination of glutamine synthase plus glutamate and / or asparagine synthase plus aspartate
  • hyper- or chronically activated macrophages by secretion of H0 2 and / or H 2 0 2 degrading or H0 2 in H 2 0 2 hyperdismuting SOD exoenzymes at the inappropriate time may give rise to unwanted immunosuppression.
  • these exoenzymes are therefore neutralized as undesired suppressor factors with appropriate antibodies.
  • Tc interconversion can (a) be carried out by "preprogramming" the APCs (macrophages) for simultaneous superoxide and peroxide formation or advantageously for dominant peroxide formation and (b) by adding sub-toxic amounts of H 2 0 2 or H 2 0 2 ' and H0 2 -generating enzymes. The addition of is also preferred
  • a Th1: Th2 or Tc: Ts interconversion is aimed at when targeting an antigen-specific or non-specific tolerance induction, for example in bone marrow or organ transplantation, then the use of agonists Gs-coupled receptors and / or antagonists of Gi-coupled receptors as well iNOS / cNOS stimulators with / without NO-releasing preparations preferred.
  • T cell subset interconversion in vitro can furthermore be achieved by receptor-independent action of Ca influx-promoting preparations of the Ca ionomycin and Ca ionophore A23187 type and by various phorbol esters (TPA / PMA) become.
  • TPA / PMA various phorbol esters
  • cAMP-specific PDEs which are divided into 3 subclasses (type IV, rolipramine-sensitive type VII and IBMX-insensitive type VIII), but also with inhibitors of non-specific PDEs (types I, II and III), the Th1: Th2- or Tc: Ts conversion.
  • the increase in cAMPi induced by agonists of Gs-coupled receptors and / or antagonists of Gi-coupled receptors and / or iNOS / cNOS stimulators in the target cells can be increased and maintained over a longer period of time.
  • stimulators of the cAMP-specific and non-specific PDEs can further reduce a cAMPi level in the target cells which is kept low by antagonists of Gs-coupled receptors and / or Ca antagonists and / or iNOS / cNOS inhibitors.
  • these PDE stimulators can strongly support the Th2: Th1 or Ts: Tc conversion described above.
  • the inhibitors of the cAMP-specific and non-specific PDEs discussed above could be of particular importance in the prevention of the HvGR in organ transplantations and the GvHD in the bone marrow transplantation (BMT, SC-T, PBPC-T).
  • test cells can be achieved not only by the combination of H02 and H202, but also by NH3 in sub-toxic doses, which even in PBS (contains only Na / K-phosphate), ie do not come to a standstill in the absence of serum (BSA / FCS) and other medium components. It is therefore proposed to generate e.g. suppressive layer (a) with H02 and / or H202, e.g. to stimulate the immune cells with the system XOD / xanthine (hypoxanthine) and / or NH3 in sub-toxic doses.
  • suppressive layer a
  • the present invention relates to preparations and methods which are based on a method known as "microimmune surgery" (MIS).
  • MIS microimmune surgery
  • This MIS method is described in more detail in the application EP 0 705 1 1 1 by the present applicant. For details of this method, reference is therefore made to this application.
  • An object of the invention is an embodiment based on the MIS method, namely the marking, more precisely
  • MIT microimmuno targeting
  • the new MIT technique is based on the transfer ("upoculation") of the cytokine profile of donor PBLs, more precisely donor T cells, on the "wrongly programmed" patient T cells.
  • the in vivo depletion or inactivation of the pathological patient T cells in the MIS technique is at least partly. based on a residual cytotoxic or cytolytic effect of the pre-programmed alloreactive donor cells.
  • Th1 cytokine profile In contrast, in the newer MIT technique according to the invention, donor T cells seem to transfer the Th1 cytokine profile to the Th2 suppressor T cells (Ts) of the tumor patient during intimate cell cell contact.
  • Ts Th2 suppressor T cells
  • the therapeutic effect of MIT effectors is likely to be based on the inactivation of pre-activated (autoaggressive) T cells by the local excess of type 1 (pro-inflammatory) cytokines; further activation of cytokine, e.g. IL-2 pre-incubated T cells at their death (by apoptosis).
  • cytokine e.g. IL-2 pre-incubated T cells at their death (by apoptosis).
  • the pathologically hyperactivated (incorrectly programmed) patient T cells are recognized by their obligatory post-expression of class II MHC (HLA-DR / DQ) antigens.
  • an overactivated T cell acts as a non-professional antigen-presenting cell (APC), which "takes over" the cytokine pattern of the latter cell in intimate cell contact with the preprogrammed donor T cell, in analogy to APC influence mature, immune-competent T cells during the activation process by the processed foreign antigen.
  • APC antigen-presenting cell
  • the donor effector cells are programmed to MIT effectors; MIS preprogramming of the donor effectors results at mitomycin C concentrations below 10 // g / ml.
  • a further embodiment of the present invention consists in a method for preventing the tolerance (re) induction (a) towards residual tumor cells and / or (b) against all types of infections, especially (retro) viral infections (eg EBV or CMV infections).
  • retro viral infections
  • T- (and B-) cells which can also be called pre-T- (or pre-B-) cells or T- or B-precursor cells may refer to mature T cells of the Th2 and Ts subtypes.
  • pre-T- (or pre-B-) cells which can also be called pre-T- (or pre-B-) cells or T- or B-precursor cells may refer to mature T cells of the Th2 and Ts subtypes.
  • T- (and B-) cells which can also be called pre-T- (or pre-B-) cells or T- or B-precursor cells may refer to mature T cells of the Th2 and Ts subtypes.
  • pre-T- (or pre-B-) cells which can also be called pre-
  • This undesired contact or misdirection of the maturing precursor cells in the direction of Th2 and Ts cells can be prevented by consequent in vivo depletion (a) of Th2 and / or Ts cells, (b) of immature T- (and B -) - Cells, or (c) can be achieved by simultaneously depleting both subsets, whereby in the latter case (point (c)) a partial, less consistent code plating of both subsets is sufficient.
  • a temporary in vivo depletion of the Th (T4) subset as such must be considered.
  • the immature precursor T cells can be selectively eliminated in vivo by Mabs such as OKT6, OKT9 and / or OKT10.
  • anti-CD2 Mabs which recognize both mature T cells and thymocytes, as well as CD5, CD6, CD7.
  • the anti-DC4 Mabs are of particular interest because they can eliminate both the Th2 and 75% of the pre-T cells (thymocytes) in vivo (example: the two complement-binding OKT4 and OKT4A Mab), with Anti -CD4-Mabs can generally be combined with the Anti-CD8-, with the Anti-CD3- and / or the Anti-CD2-Mab.
  • the above-mentioned anti-CD6 Mab (OKT1 7) is particularly interesting because it recognizes activated helper T cells and can therefore selectively deplete the Th2 fraction.
  • Another Mab, directed against CD2R, should be emphasized because of its selectivity for the activated T (Th2 and TS) cells.
  • the sole depletion, more precisely code-plating of Th2 cells or the combined elimination of T4 (Th2) - plus pre-T cells e.g. with OKT6, OKT9 or OKT10) promises an efficient suppression of tolerance (re) induction both towards the residual ones Tumor cells (after treatment of the primary tumor) as well as against reactivated (retro) viral infections, if the observations in the animal model are also confirmed in the clinic.
  • Another important point is the improvement of the allogeneic bone marrow transplantation through the successful transfusion of the bone marrow cells or the CD34 + stem cell (SC / PBPC) subpopulation (a) first from the MHC-incompatible bone marrow donor and then (optimally: 7 to 10 days later) from the MHC-identical donor.
  • the former donor cells are to be used for "microimmune surgery” and the latter for better cooperation with autologists, ie patient APCs (macrophages).
  • the latter HLA-identical PBLs can be partially or completely replaced or combined with Ts or Th2- (T4) -predpleted patient PBLs in patients with solid tumors.
  • the particular "strength" of autologous PBLs is their MHC identity with patient APCs and its tumor cells, while HLA-identical donor lymphocytes have the advantage of a lack of a common history with tumor cells and the associated absence of any pre-generated tumor-specific Th2 and / or Ts Bring subpopulation with it.
  • Another object of the invention is also a new principle for the treatment of tumors, infections with the help of vaccines.
  • the new principle according to the invention is based on the knowledge that the development of cytotoxic T cells (CTLs / Tc), which are directed, for example, against tumor cells or (retro) virally infected cells, is closely accompanied by the cytokine-producing helper T cells and that the latter only in the 1st Phase after their activation, ie as so-called "activated naive Th cells” as the only cytokine to produce the type 1 lymphokine IL-2 required by the precursor Tc cells (pCTLs).
  • CTLs / Tc cytotoxic T cells
  • pCTLs cytotoxic T cells
  • ThO cells the T4 cells secrete IL-4 and other Th2-type cytokines in addition to IL-2;
  • these activated, antigen-specific T4 cells are differentiated into either the Th1 or Th2 subtype.
  • Ts cells suppressor T cells
  • anti-T4 (CD4) -Mabs and corresponding immunotoxins can create a situation in which all the antigen (eg TSTA TATA) is activated by decoupled, ie 2 to 5 days later, activation of helper T4 cells.
  • treatment with cyclophosphamide 2 to 7 days before priming with the relevant antigen can also result in a decoupling of the parallel activation of T4 and T8 cells, which consists in the fact that activated pCTLs are completely decoupled hit the only activated IL-2 secreting "activated naive Th cells”.
  • the targeted generation of a specific antigen of the corresponding T cells (CTLs) with simultaneous suppression of the corresponding Ts cells succeeds in particular if the macrophage or the APC cells at the time of presentation of this antigen (eg the tumor or virus antigen) has an increased level of ROS / ROI, which directs the precursor T cells in the direction of the Th1, not Th2 subtype.
  • CTLs corresponding T cells
  • the ROl / ROS level automatically increases as a result of the "respiratory burst"
  • particulate antigens low-dose erythrocytes of foreign blood group, inactivated bacteria or their lysates, complete Freund's adjuvant / CFA, lyposes, denatured foreign proteins
  • T4 cells type 1 (IL-2) lymphokine-producing helper (T4) cells is critical for the generation of antigen (eg tumor or virus antigen) -specific CTLs while at the same time suppressing the corresponding Ts cells the surface or in the micromilieu of APCs presenting this antigen at the time of antigen presentation.
  • antigen eg tumor or virus antigen
  • T4 cells type 1 lymphokine-producing helper (T4) cells
  • antigen eg tumor or virus antigen
  • This goal can be achieved according to the invention, similarly as mentioned under point (6), by foreign proteins (eg bacterial lysate) or by the use of histo-incompatible (preferably MHC-I plus MHC-II-incompatible) donor T cells (lymphocytes, PBLs) can be achieved with preprogrammed cell death (ie, donor T cells or lymphocytes pretreated for microimmunosurgery).
  • preprogrammed cell death ie, donor T cells or lymphocytes pretreated for microimm
  • the T4 subpopulation of the donor recognizes the patient's own MHC-I and MHC-II structures on the patient macrophage (APCs) and reacts with the IL-2 secretion in both cases, which among other things. the tumor or virus-specific CTLs or their precursors (pCTLs) benefit.
  • APCs patient macrophage
  • pCTLs tumor or virus-specific CTLs or their precursors
  • the target cells for gene therapy are the macrophages, the dendritic cells and the like.
  • APCs because they have the great advantage of simultaneous MHC-II expression (in addition to MHC-I expression) on their cell surface compared to the previously used tumor cells and / or TIL cells.
  • patent protection should apply to all genes used to date (e.g. for cytokines, LAPs / adhesins / integrins etc.) as they are derived from the transfection of tumor cells, TIL cells and others.
  • Target cells are known, extend.
  • a modification provides for hybrid cells between patient and donor APCs (or B cells); others recommend MHC-II expression (along with other conventionally used genes e.g. for cytokines and their receptors) on tumor and TIL cells, achievable by cDNA or fusion.
  • tumor-specific Tc Tc
  • Another object of the invention is the combination of an MIS / MIT treatment with the HTT technology, whereby in addition to the basic therapy, all modifications and refinements of both strategies (MIS / MIT and HTT strategy) are to be regarded as components of this combined therapy.
  • Both basic therapies were explained in detail in EP 0 705 1 1 1, to which reference is made.
  • MIS / MIT which was introduced later, stands for “cell death preprogrammed allogeneic effector cells”
  • HTT technology which was also introduced subsequently, stands for "hybridoma-CTrioma, quadroma) cells".
  • the present application also relates to combinations of the described MIS / MIT and / or HTT technology with other immunotherapeutic methods for the treatment of cancer, bacterial and (retro) viral infections, autoimmune diseases, allergies, arteriosclerosis, and of GvH and HvG complications for organ and bone marrow or stem cell / progenitor cell transplants.
  • the aim of the hybridoma or trioma formation from patient tumor cells and autologous and / or allogeneic MHC-II-positive cells mentioned at the beginning is to present tumor antigens on the surface of the common tumor: APC hybrid cell, which leads to the breaking of the pre-existing tolerance leads to tumor cells.
  • autoimmune diseases for example, the fusion of autoantigen-specific, ie autoaggressive T cells with autologous and / or allogeneic MHC-II-positive cells is intended to use the special effect of the resulting cell construct (hybridoma or trioma cells), which consists of that after subcutaneous or intradermal inoculation or iv injection of the hybridoma or trioma cells mentioned, a strong anti-idiotypic defense reaction builds up, which in a highly selective clonotypical manner depletes or inactivates the patient's auto-aggressive T cell subpopulations.
  • the fusion of autologous and / or allogeneic MHC-II positive cells (APC) with allergen-specific T cells (mostly of the Th2 subtype) and / or with allergen-specific plasma (B) cells can be too high efficient and highly selective (clonotypical) in vivo elimination or inactivation of the allergy-inducing and maintaining immune cells.
  • APC autologous and / or allogeneic MHC-II positive cells
  • T and / or B cells with specificity e.g. a cell construct is formed for OxLDL, which can lead to the selective in vivo depletion of the immunocyte subclones actively involved in atherogenesis.
  • the new principle, the HTT-AIT technique can also be used in organ and bone marrow transplantation by subcutaneous vaccination or i.v. Injection of the hybridoma or trioma cells, formed by the fusion of MHC-II positive cells (macrophages, dendritic cells) of autologous and / or allogeneic origin with alloaggressive T cell subpopulations, in the selective inactivation of the HvGR or GvHR-inducing T Cell subclones result.
  • hybridoma or trioma cells with Fc-epsilon-R positive B, T and / or mast cells or basophils can be provided, after the FACS or MACS pre-separation (in T and B Cells) can be enriched with IgE-loaded cell affinity chromatography columns.
  • Another variant of the allergy treatment with the HTT-AIT technique provides that the MHC-II positive cells are fused with IgE-loaded, Fc-epsilon-R positive cells (B cells, mast cells, basophils).
  • Fc-epsilon-R B cells, mast cells, basophils
  • cells expressing Fc-epsilon-R B cells, mast cells, basophils
  • vaccination against allergy is generally carried out using the HTT-AIT technique.
  • the patient-specific allergen spectrum must first be determined in a conventional manner in order to then carry out the allergen-specific T and / or B cell enrichment by incubating the patient PBLs in the presence of the relevant allergens and IL-2.
  • the further steps are the same as above.
  • the completed HTT-AIT technique also brings additional improvements in the treatment of solid tumors and leukaemias / lymphomas.
  • bcr-abl fusion protein eg CML
  • immunization sc, id or iv
  • hybridoma / trioma cells consisting of bcr-abl-expressing malignant cells plus autologous and / or allogeneic MHC-II positive cells (macrophages, dendritic cells) induce a highly efficient and selective in vivo depletion of (rest) tumor cells.
  • tumor-specific Ts (and Th2) cells of the patient with the MHC-II-positive autologous and / or allogeneic APCs (macrophages, dendritic cells) enables the in vivo elimination of tumor protectors Ts - (and Th2) cells are made even more efficient and selective at the cellular level;
  • APC hybridomas combine two important concepts in the Tumor control, the in vivo generation of new tumoricidal CTLs (“HTT technique”) with the consequent elimination of tumor protective Ts (“HTTAIT technique”).
  • MIS / MIT cell death preprogrammed alloreactive donor cells
  • donor T cells or donor PBLs are processed in a kind of one-way MLC ( Donor: responder, recipient: stimulator) co-incubated for 3 to 5 days, then preprogrammed with mitomycin C or incorporation of 3-H-thymidine (suicide dose) cell death and used as MIS / MIT in vivo.
  • MLC Donor: responder, recipient: stimulator
  • MIS / MIT effectors are used to eliminate tumor-protective Ts (and Th2) cells subset- but not tumor-specific
  • HTT -AIT technology enables one to a certain extent tumor-specific depletion of protective T cells.
  • APCs macrophages
  • Type2- (Th2) - reprogrammed to type1- (Th1) function which also makes it naive.
  • Tumor-specific T cells are preprogrammed in the Th1 direction, (d)
  • the HTT technology enables the in vivo formation of the tumor-specific Tc (Th1) cells in two ways, (a) by recruiting new, naive Tc and / or (b) by reprogramming pre-existing Ts into Tc cells.
  • the new combined technique which is characterized by minimal side effects, is also recommended according to the invention in the treatment of LAS / ARC / AIDS.
  • a further increase in efficiency is achieved by the in vivo medication influencing the patient's immune competent cells e.g. by preparations which actively modulate the cAMP / cGMP ratio in the immune cells and / or the calnflux in the Ts (Th2) cells. Therefore, according to the invention, the combination with these preparations, which is recommended in all details in EP 705 1 1 1. Combinations of this pharmacological / medicinal therapy concept with the MIS / MIT and / or HTT or HTT-AIT technology are recommended according to the invention since an additive or synergistic therapeutic effect is to be expected. This pharmacological / drug therapy concept showed 80 to 85% tumor regression in the clinic and a complete disappearance of metastases.
  • the vaccines can be administered either subcutaneously / intradermally or iv.
  • Ts, TH2 pathogen or tumor-specific suppressor cells
  • the invention also relates to two preparations which are used in bone marrow or Stem cell / progenitor cell transplantation can be used.
  • a first embodiment unlike conventional transplantations, only mature, cell death preprogrammed donor T cells (or PBLs / PBMs) or their combination with mature recipient T cells (or PBLs) are used, which in the case of malignant diseases of the recipient similar to how the donor cells are to be pretreated;
  • the immature bone marrow or stem / progenitor cells should only be post-infused after 7 to 14 days according to this protocol.
  • the patient's partial immune competence is maintained even in the critical phase after conditioning, which significantly reduces the susceptibility to infections and the risk of re-induction of the tumor-specific tolerance.
  • the partial damage to immature immunocytes, especially HLA-DR / DQ-positive ones can be prevented by mature alloreactive T cells.
  • a second form of performance concerns the introduction of the allogeneic, i.e. Donor LAK cells both in tumor therapy and in bone marrow or stem cell / progenitor cell transplantation.
  • these allogeneic LAK cells must first be freed from the approximately 10% non-MHC-strained T cells with anti-panT (CD3) antibodies and / or anti-CD8 antibodies.
  • the allogeneic LAK cells can be preprogrammed for cell death, similar to MIS / MIT effector cells described in detail in EP 705 1 1 1.
  • Another object of the invention relates to the regulation of Th1, Th2, Tc / CTL and Ts subsets of the T lymphocytes.
  • Th1 - but also Tc subsets compared to Th2 and Ts cells an increased intracellular or mitochondrial ROI / RNI ratio.
  • Pharmacological / medicinal interventions and manipulations are therefore also considered that can directly or indirectly change the Th1 / Th2 or Tc / Ts or T4 / T8 ratio in vitro and / or in vivo.
  • Examples are various inhibitors of RNI-increasing NO synthetases (iNOS, cNOS), which lead, for example, to an increase in Th1 cytokine and a reduction in Th2 cytokine production, as well as all ROI and RNI-generating or secreting systems and all ROI and RNI scavengers, enzymatic or chemical in nature.
  • An increased intracellular NO level in the Ts cell prevents the Cai-dependent translocalization of the preformed perforin and DC95L (Apo-IL / FasL) molecules to the cell surface, which prevents the cytolytic effect of Tc.
  • the intimate contact between the Tc or Ts cell and the MHC-1 positive target cell also enables the membrane-bound excess NO 'radical to be penetrated from the Ts into the target cell, resulting in the cytosolic H02 radical molecule is chemically "neutralized", which is associated with an immunosuppressive effect on the target cell.
  • All pharmacological / medicinal interventions for the in vitro or in vivo conversion of Ts to Tc cells are also considered and claimed here.
  • the APCs (macrophages / monocytes) are also switched from the so-called "suppressor-monocyte" function to the tumoricidal / virucidal / bactericidal effector function by changing the intracellular quotient and also have a major influence on the Th1 - vs.
  • Th2 preprogramming of the antigen-specific naive (nonprimed) T cells the pharmacological / medicinal APC influence by changing the ROI / RNI quotient is also considered and claimed.
  • Another object of the invention are further technical improvements to the HTT and HTT-AIT, as set out below.
  • This includes the modification of both technologies based on ZelhZell fusion, in which the cell nucleus: cell fusion and the cell nucleus: cell nucleus fusion take the place of the ZelhZell fusion. This applies to two (hybridoma), three (trioma) and four (quadro) combinations. All fusion techniques, the "conventional" cell and the modified cell nucleus: cell or cell nucleus: cell nucleus fusion techniques result in the fusion of the cell nuclei and the chromosomes as well as the surface co-expression of the genes of both fusion partners.
  • cell fusion processes are the introduction of foreign cell genes (for example the missing genes of professional or wild-type foreign cells into deficient autologous cells) without the risk of rejection of foreign cells due to MHC-I and / or MHC -II- Mismatch.
  • a particularly current special case is the fusion of the donor cell nucleus with recipient thymus cell, which, after homing of the autologous thymus cell in the recipient thymus, leads to simultaneous recipient and donor-specific thymal training of the maturing T cells.
  • the resulting immunocompetent T cells are, similar to (AxB) F1 hybrids, double, i.e. Receiver and donor MHC restricted.
  • the fusion according to the invention can either be chemical (e.g. PEG or PEG / DMSO or PVP or PEG / PVP), high-frequency current (electrofusion / electroporation) or viral (e.g. SV40).
  • a further substantial improvement of all technologies based on fusion can be achieved according to the invention by targeted, highly selective approximation of the partner cells to be fused by means of bifunctional antibodies which bind to typical surface structures of the cells to be fused.
  • bifunctional antibodies instead of the bifunctional antibodies, other, bi-, tri- or tetravalent antibody constructs can also be used, which can result in hybridoma, trioma or quadroma cells of the desired composition.
  • the concentration of the chemical fusogens must be kept relatively low (for PEG, for example, 35 to 40%) or the conditions for the electrofusion selected so that the fusion of the pre-approached partner cells takes place selectively, before the fusion of other, pre-approached partner cells can.
  • bivalent antibodies In the fusion of patient tumor cells with the autologous and / or allogeneic MHC-II positive cells (macrophages, dendritic cells), bivalent antibodies must be used, which are TSTA / TSA / TAA-specific on the one hand and structures on macrophages on the other hand (e.g. CD1 6 / Detect CD32 / CD64, ie Fc-gamma-RI / RII / RIII) or on dendritic cells (eg CD83 or T6).
  • hybridomas consisting of dysregulating immunocytes plus autologous or allogeneic APCs (MHC-II-expressing cells), bispecific Mabs or.
  • Antibody constructs with double specificity are used, on the one hand recognize typical surface structures (eg differentiation antigens) on the pathological (dysregulated) immune cells and on the other hand the membrane structures mentioned on the APCs.
  • Th2 or Th1 cells If one wants to immunize against dysregulated Th2 or Th1 cells, then in vitro - as in the Th1 and Th2 FACS analysis - the sapon perforation of the Th2 cell membrane must first take place and then a bifunctional MAB or antibody construct which uses the Th2 - or Th1 cells approximates or binds to the autologous or allogeneic APCs. The selective fusion is then carried out with low-dose PEG, thus providing the basis for a highly selective in vivo immunization against the Th2 or Th1 subset. In the special case of allergies, the use of bifunctional Mabs or antibody constructs with specificity for IgE or Fc-epsilon-R on the one hand and for CD1 6.
  • T6 on dendritic cells
  • IgE-bearing B and T cells or mast cells / basophils can enable the selective formation of hybridoma - Or trioma constructs can be achieved, which enables highly selective immunization against IgE-bearing B and T cells or mast cells / basophils.
  • the efficiency of the allergy treatment described can be further increased according to the invention if (a) with Fc-epsilon subunits of IgE and / or (b) with soluble IgE receptors (sFc-epsilon-R) and / or (c) with IgEs without allergen specificity and / or (d) with IgEs whose Ag-binding domain, for example is disturbed by the use of one or more amino acid residues, the allergen-specific IgEs are displaced from the surface of mast cells and basophils.
  • sFc-epsilon-R soluble IgE receptors
  • IgEs without allergen specificity
  • IgEs whose Ag-binding domain for example is disturbed by the use of one or more amino acid residues
  • the circulating and (mast cell) bound can be shifted according to the invention in such a way that the density of the (mast cell) bound IgE molecules decreases, which likewise leads to a weakening of the allergy symptoms.
  • bispecific and / or hybrid antibodies which on the one hand recognize the IgE or the Fc-epsilon-R receptor and on the other hand bind MHC-II positive cells (APCs), by approaching the partner cells To strive for high selectivity in hybridoma / trioma formation.
  • APCs MHC-II positive cells
  • Detailed laboratory tests by the applicant have shown yet another phenomenon, namely the strong immune stimulation by low, subfusogenic concentrations of chemical fusogens (PEG, PVP and their combinations).
  • the principle is likely to be the intensified interaction of involved immunocompetent cells in the presence of fusogenic polymers in subfusogenic concentrations.
  • HTT or HTT-AIT technique relate to the use of bifunctional and / or hybrid antibodies with specificity on the one hand for cytokeratin, on the other hand for differentiation antigens and other surface structures, typically for MHC-II positive cells (APCs such as dendritic cells, macrophages / Monocytes and / or B cells). Since cytokeratin is expressed on all solid tumors, but not on leukocytes (eg APCs), these antibodies enable an intimate approximation of the desired fusion partner cells and thus a highly selective one
  • APCs MHC-II positive cells
  • Hybridoma / trioma / quadroma formation at suboptimal concentrations of fusogens e.g. PEG, PVP, PEG / PVP combinations.
  • tumor antigen-specific antibodies can be used with the APC-recognizing antibodies in the form of bifunctional and / or hybrid antibodies. Another important point is the simplification according to the invention of the classic Mab extraction according to Köhler and Milstein, which makes the time-consuming and costly cloning in selective media such as HAT, HT etc. unnecessary.
  • the new principle consists of a targeted intimate approximation of both fusion partners, the antigen-specific B cells with immortalized transformed standard cell lines.
  • the B cells of the animal immunized with the special antigen can be pre-selected using FACS or MACS technology, for example, and pre-treated with a bifunctional and / or hybrid antibody that recognizes B cell differentiation antigens and specific structures on the surface of the immortalizing cell lines mentioned ,
  • the partner cells approximated in this way can now be fused selectively. Greater selectivity will be achieved by replacing the B cell-specific antibodies with those that only recognize the activated B cells.
  • the splenocytes of the immunized animal must first be preactivated in vitro in the presence of the specific antigen.
  • the microtitration plates In this clonotypic fusion, the microtitration plates must first be coated directly or indirectly (via poly-L-lysine treatment) with the specific antigen.
  • all other selection methods used in conventional Mab extraction can be used.
  • the antigen-specific B cells are now roughly or finely enriched, the use of bifunctional and / or hybrid antibodies, which on the one hand bind B cell-typical differentiation antigens or activated B cells and on the other hand interact with APC surface structures, the requirement for a selective merger of the fusion partners is fulfilled.
  • (b) the use of (b1) cell death preprogrammed allogeneic, MHC-II positive cells and / or (b2) of cell death preprogrammed hybridoma / trioma cells, consisting of autologous plus allogeneic, MHC-II, is a novel class of immunomodulators (BRMs) -positive cells (macrophages, dendritic cells, B cells) recommended.
  • BRMs immunomodulators
  • the Th1 / Tc- vs. Th2 / Ts pre-programming can be done by pre- or post-treatment of the hybridoma / trioma cells, consisting of tumor cells, autologous APC and / or allogeneic APC, in the sense of type 1 - vs. Achieve type 2 profiles (e.g. via modulation of the intracellular cAMP level or via cytokine modulation).
  • a modification of the fusion technique provides for the patient's tumor cell to be fused with autologous and - separately from it - with allogeneic MHC II-positive partner cell and the resulting 2 types of hybridoma cells to be fused again to quadroma cells, whereby electrofusion or chemofusion (with PEG ) can be used.
  • the MHC II + cells (APCs such as DCs or macrophages) should be activated beforehand, e.g. with IFNgamma or MDP (Muramyl dipeptide). This preactivation of the fusion partner cells is generally recommended according to the invention.
  • MIT effectors which differ from the aforementioned MIS effectors only in the concentration of the mitomycin C used for their preprogramming (20ug / ml / 1,106 cells, instead of 10ug / ml / 1,106 cells), one can do a lot here desired effect can be achieved, namely the one-sided Th1 cells), preprogramming of the APCs and indirectly (via these APCs) of the recruiting antigen-specific (eg TSA) T cells.
  • TSA recruiting antigen-specific
  • Th1 / Tc recruitment succeeds because the MIT effectors to be successfully injected die as a result of the cell death preprogramming before they reach the transition from pTh to Tho; thus they only secrete IL-2 (and possibly some IL-12 and IFNgamma), but not yet Th2 cytokines (IL-4, IL-13, IL-10). If, on the other hand, type 2 (Th2) programming of the recruiting T cells is sought, the APCs must be preprogrammed directly or indirectly (via mature Th2 / Ts cells) in the Th2 sense.
  • Tumor vaccines which are based on DC transfection with tumor-specific antigens (for example MART-1, MAGE-1, gp1 5, gp95, gp100), viruses serving as a gene ferry, such as vaccinia virus, can be improved according to the invention if instead of autologous ones DCs allogeneic DCs or both DC types can be used. In addition to DCs, other MHC II + cells can also be used. After transfection of autologous and allogeneic DCs (and other MHC II + cells), these can be combined (fused) into hybrid cells by electrofusion or chemofusion.
  • tumor-specific antigens for example MART-1, MAGE-1, gp1 5, gp95, gp100
  • viruses serving as a gene ferry such as vaccinia virus
  • the transfected DCs can result from the fusion of patient tumor cells with autologous and / or allogeneic APCs (DCs, macrophages), as well as with MIS or MIT effectors be combined.
  • the tumor vaccine is further improved when co-vaccinating with TSA-encoding cDNA.
  • a further improvement in the tumor vaccine relates to the coincubation of the TSA-encoding cDNA with MHC II- «cells (APCs such as DCs and macrophages). This leads to type 1 (ThD tumor defense, which is characterized by a cellular rather than humoral response.
  • the conversion of the Th2 / Tc into the Th2 / Ts subset / e.g. for the purpose of tolerance induction or stabilization) can be achieved according to the invention by incubating the cells with Mn or Cu / Zn SOD, further with catalase and / or (GSH) peroxidase.
  • the effect can be enhanced by inhibitors of NO synthase (iNOS, cNOS).
  • iNOS, cNOS inhibitors of NO synthase
  • a novel possibility of donor-specific immune tolerance arises from the combination (a) of pre-irradiated donor thymus cells with (b) immature bone marrow or stem cells or T-depleted PBLs, the latter preferably being injected into the recipient only after a 2-5 day delay become.
  • flavonoids / polyphenols which selectively inhibit the enzyme COX-2 and thus prostaglandin synthesis, a drift from Th2 to Th1 or Ts to Tc can be achieved.
  • Other flavonoids which inhibit both the synthesis of prostaglandins and that of leukotrienes, an anti-inflammatory effect can be achieved.
  • the infusion of F1 hybrid cells in the P1 or P2 recipients proves to be very cheap. It is therefore recommended according to the invention to replace the transfusion of donor cells previously used in bone marrow transplantation (KMT) with the transfusion of a 1: 1 mixture of donor plus recipient bone marrow or stem cells.
  • KMT bone marrow transplantation
  • This technology which is to be introduced routinely, would often take years Prevent immunocompromization of the KMT recipient due to the impaired T-cell APC cooperation.
  • a special embodiment of this method provides for the use of hybrid cells from donor and recipient bone marrow or stem cells, PBLs or their T-cell and APC fraction; this technique can further increase donor tolerance and precede injection of the 1: 1 mixture of immature donor plus recipient cells.
  • preprogrammed / pre-immunized mature T cells show the greatest effect when they come into contact with immature precursor cells (e.g. T-depleted PBLs) in vivo.
  • immature precursor cells e.g. T-depleted PBLs
  • the tailor-made cells can be pre-programmed with cell death treatment (e.g. mitomycin C, s.MIS and MIT effector cells) or used intact. Examples of such tailored cells are (antigen-specific) Th1, Th2, Ts and Tc (Tc1 / Tc2 / Tc3) cells.
  • a new method for in vivo reprogramming of suppressor monocytes into tumoricidal (virucidal / bactericidal) effector macrophages consists in the successive injection of MIT effector cells, originating from donors A, B, C, D etc., with the cell death preprogramming the change the cytokine production from type 1 (IL-2, IFNgamma, IL-1 2) to type 2 (IL-4, IL-13, IL-10) was prevented (cell death before reaching the tho-maturation level).
  • HTT cell fusion or cell hybrid technology
  • anti-idiotype vaccine in which not, as in the classic HTT technique against tumor cells per se, but against the dysregulated T (and / or B) - Cells are vaccinated, for example against the tumor-protective Ts and / or Th2 cells, which offer the malignant cells protection from access by the immune system from the time they are formed.
  • the combination of these "anti-idiotypic vaccines” is used in the special aspect of tumor treatment the “antitumor vaccine", consisting of tumor cell plus autologous and / or allogeneic APC (DC, macrophage), is recommended.
  • this "anti-idiotype vaccine” is suitable for the treatment of autoimmune diseases (HTT-AID), allergies (HTT-ALL), atherosclerosis (HTT-ATH), viral and bacterial infections (HTT-INF), including AIDS, and to prevent or combat GvHD
  • the dysregulated, ie pathologically (hyper) activated can be enriched by cell affinity chromatography, FACS or MACS, based on (hyper) activation marker-recognizing Mabs, before they are autologous and / or allogeneic MHC II + cells (DCs , Macrophages).
  • This “atiidiotypic vaccine” is therefore based on “T-subset” -specific fusion.
  • the so-called “clonotypic” anti-idiotype vaccine goes even further in the selectivity and thus freedom from side effects of the vaccination.
  • the antigen-specific T and / or B cell clones with autologous and / or allogeneic MHC 11 + cells.
  • This "clonotypical" vaccine was made possible by the introduction of new techniques in which the Ag-activated (and only these) T (and B) cell clones were labeled and FACS using special bifunctional Mabs - or MACS- can be separated. These bifunctional Mabs show specificity for CD45 (pan-Leu) and for cytokines, e.g. IL-2 or IL-4.
  • two cell constructs are particularly suitable for exerting a type of catalytic effect on the immune defense against tumor cells even in very low concentrations.
  • the first cell construct concerns triomas or Quadromas, created by fusion of tumor cells, tumor-specific Ts or Th2 cells and autologous and / or allogeneic MHC II + cells (DC, macrophages).
  • DC autologous and / or allogeneic MHC II + cells
  • the second cell construct is put together somewhat more simply; it consists of a tumor cell, autologous and / or allogeneic APC (DC, macrophage).
  • a modification of the HTT technique which can be used in vaccines against (retro) viral and bacterial infections, provides for the fusion of pathogens (e.g. bacteria) and autologous or allogeneic APCs.
  • Bone marrow and stem cell transplantation can be improved with 2 new strategies.
  • a controlled GvHD is first induced by KM or stem cells of the first, MHC-incompatible donor, which is then brought to a standstill in the next step by in vitro prealloimmunized recipient PBLs or T cells.
  • the recipient first receives cells from a histocompatible donor and then (1-3 weeks later) cells from a histocompatible second donor.
  • the recipient first receives histocompatible bone marrow and then a more incompatible graft from a second donor;
  • the strategy principle is the induction of the Ts (Th2) cells under sub-GvHD conditions, which then enable the tolerance to the 2nd, histo-incompatible donor.
  • All these 3 strategies can be used under myeoloablative (KMT, SCT, PCT) and non-ablative (MIS / MIT, DLI / DLT) preparative conditions.
  • T cells By pre-immunization of the donor and recipient T cells (or PBLs) and by varying the MHC incompatibility and the mixing ratio These cells (eg from 0.1: 1 to 10: 1) can have interesting effects in vivo, but the recipients (patients) must be preconditioned beforehand in a myeloablative or non-ablative manner; in the latter, the in vivo depletion of mature T cells by Mabs or Mab combinations is sufficient.
  • the above-mentioned intermediate phase of the donor-type chimerism for the purpose of eradicating the residual tumor cells, followed by the stabilizing phase of the real "mixed allogeneic chimerism" can be elegantly realized by means of this new fine-metering technique.
  • T-depleted donor PBLs into T-depleted recipients, this T-depletion being carried out by specific Mabs; u.U. a more radical T depletion through additional treatment with MIS effectors (in vitro and / or in vivo) may be necessary.
  • This tolerance induction can be further stabilized by adding allospecific Ts and / or Th2.
  • Another approach is the simultaneous transfer of mature allospecific Ts or more Th2 cells plus immature precursor cells, the tolerance induction for the tolerance of donor organ transplants in vitro, the injection of pre-generated, donor-specific Th21 (Ts) cells of the recipient into the T - Requires depleted recipients, while in KMT and HSCT allospecific donor and recipient Th2 (Ts) cells together with the 1: 1 mixture of immature donor plus recipient precursors have to be transfused into the myeloablative or non-ablatively pretreated recipient.
  • Ts Th21
  • Ts Th2
  • tumor cells with costimulatory structures (B 7.1 / B 7.2; CD 40) are transfected. According to our ideas, this is only sufficient for post-stimulation of existing, TSA-specific. CTLs and, in the best case, for the partial reprogramming of existing Ts into Tc cells, but not for a novo re-recruitment of tumorous CTLs; not even if cytokines such as IL-2 or IFN gamma are additionally post-transduced.
  • the gene therapy mentioned (a) by cotransfection with MHC2 antigen and / or (b) by simple fusion of the tumor cell with MHC II + cells (DCs, macrophages) could be significantly improved.
  • a modification of this technique provides for either fusing the transfected tumor cells with APCs (DCs, macrophages) of the autologous and / or allogeneic origin, or for subsequently combining the hybridomas, triomas or quadroma cells formed in the HTT technique with costimulatory and / or cytokine cells. to transfect coding genes.
  • a further improvement in the KMT and HSCT provides for the MHC2 + cells of the donor and recipient to be transfected with Th2 cytokines (IL-4, IL-13, IL-10) or with Th2 subset, which secretes these cytokines to merge. In the case of organ transplants, the corresponding transfection of donor APCs is sufficient.
  • immunosuppression glucocorticoids, CSA etc.
  • T-cell subclones under control
  • ex vivo clonal post-expansion eg in vitro stimulation of TSA-specific Ts or Th2 cells with Con A or in vitro post-expansion
  • autoantigen-specific CTLs in autoimmune diseases and aggressive CTLs in GVHD
  • GVHD autologous or allogeneic APCs
  • MIS effector cells that would possibly escape the pre-programmed cell death, which cannot be ruled out during MIS pretreatment with extremely low doses of mitomycin C (-5ug / ml / 1,106 cells) in vivo depletion by allospecific., ie Donor MHC specif. Mabs or with anti-DNP or anti-TNP Mabs; in the latter case, the MIS effector cells used would have to be labeled with DNP or. TNP reagents are pretreated. An example is the treatment of established GvHD by 3rd party MIS effector cells.
  • a significant improvement of the MIS / MIT and HTT technology is the subsequent stimulation with (a) BRMs (b) interleukins / interferons (c) antagonists of the Gs-coupled receptors (d) immunostimulating ROI (H202, H02) - + NO ) Inhibitors and others Immune stimulants.
  • cAMPi-reducing e.g. propranolol
  • Th2 / Ts inhibiting substances e.g. ca antagonists
  • anti-CD3 and anti-CD8 Mabs used in the MIS / MIT technique can be extended to all those Mabs, Mab combinations and immunotoxins derived therefrom which have been described in detail in.
  • a special variant of the anti-idiotypic in vivo elimination of pathologically dysregulated T cells provides for the donor to first use PBLs, T cells, and possibly -i- B cells of the patients with autoimmune and allergic diseases against the patient's clonally pre-expanded idiotype sensitize and after the previous tolerance induction towards this donor to infuse the anti-idiotype-containing T (and B) cells (PBLs) of the presensitized donor in this way into the patient.
  • This technique can be used with that in EP 374 207 or EP 705 1 1 1 treated "clonotypical" HTT technique can be combined.
  • idiotype-expressing T and B cells Generate allogeneic anti-idiotypes in a standardized form, for example allogeneically generated anti-idiotypes against ß (B) cells of the pancreas, alone or in combination with "clonotypic" hybridomas of the same specificity in patients with type 1 diabetes mellitus.
  • DLI donor lymphocyte infusion
  • CLL patients can be explained by poor to missing in vivo alloreactivation of the donor lymphocytes in the first group. Therefore, according to the invention, it is recommended that the DLI and the MIS effector cells be allopreimmunized in vitro before use against the recipient (patient).
  • the DLs must be replaced urgently by MIS effectors, since the allo-preimmunization significantly increases the GvHD induction by DLs.
  • An important improvement in the technique, in which the CTL clones with the highest tumoricidal activity are preselected in vitro and clonally post-expanded relates to the introduction of an upstream patient vaccination (id) by hybridoma or trioma cells, consisting of tumor cells plus autologous and / or allogeneic MHC II + Cells (e.g. DCs).
  • an upstream patient vaccination id
  • hybridoma or trioma cells consisting of tumor cells plus autologous and / or allogeneic MHC II + Cells (e.g. DCs).
  • id upstream patient vaccination
  • DCs allogeneic MHC II + Cells
  • lethalen GvHD free if (a) the cell death preprogrammed MIS effector cells are used instead of untreated donor lymphocytes (DLs), and (b) if the DLI / DLT procedure is carried out in several stages by first using a 1: 1 - Mixture of donor and recipient MIS effector cells established a kind of temporary "mixed allogeneic chimerism", which then enables the use of non-existent DLs.
  • a similar mechanism is based on the finely graduated, repeated transfusion of donor lymphocytes, which (a) pretreated either by decreasing mitomycin C concentration (from 20 to 15 to 10 to 5 or even 2 ⁇ g / ml / 1,106 cells), or but (b) have been prepared by donors with increasing MHC mismatch.
  • the last step can always be the use of untreated DLs, which has the advantage that the GvM (GvL / GvT) reaction increases gradually.
  • GVHD under control An interesting way to get the GVHD under control is to use (a) Mabs, directed against activated T cells, plus (b) autologous and / or allogeneic Nk and LAK cells.
  • CD3-Mab which can pre-eliminate about 10% of non-MHC-restricted T cells in the LAK association.
  • the MIS effector cells mentioned can be all-pre-immunized in a three-day MLC by inactivated recipient PBLs (optimally 1 / 10-1 / 2 of the donor PBLs) and by increasing the donor: recipient histoin incompatibility and by reducing the concentration of the MIS treatment Mitomycin C used can be increased in their efficiency.
  • An additional effect is the formation of allotolerant, mature Th2 or Ts cells, which later induce them to mutual tolerance when transfusing immature donor or donor plus recipient precursor cells.
  • MIS effector cells to eliminate tumor cells and the Ts (Th2) cells protecting them in a radical manner
  • MIT effectors to reprogram the APCs from the suppressor monocytes into the to accomplish tumoricidal effector function.
  • a further possibility of inducing tolerance is based on (a) a 48-hour incubation of the PBLs in the presence of Con A, Gs agonists and / or strong immunosuppressants such as dexamethasone, cortisol, methylprednisolone, prednisone, c ⁇ closporin A, FK 506 or rapamycin (b) on the coincubation of the Ts effector cells thus formed with immature cells (eg T-depleted or non-depleted PBLs of the same person) in the presence of the PBLs of the person against which tolerance is sought, or in the presence of lectins (PHA or ConA), and (c) on the infusion of these allotolerant (mature) T cells into the recipient, immediately before or with immature cells (T cell precursors) of the donor or a mixture (1: 1) of donor and recipient T- Cell precursors (eg T-depleted PBLs or KM or stem cells).
  • the basic principle here is
  • double vaccination directed (a) against tumor cells and (b) against tumor-protective Ts / th2 cells, can be carried out in one step in patients with hematological tumors.
  • the patient's PBLs are mixed with antologic and / or allogeneic MHC II positive cells (APCs such as DCs or macrophages) and fused by adding PEG or by electrofusion.
  • APCs antologic and / or allogeneic MHC II positive cells
  • APCs antologic and / or allogeneic MHC II positive cells
  • hybrid cells from APCs with malignant plus those with tumor-specific Ts or Th2 cells are also hybrid cells from APCs with malignant plus those with tumor-specific Ts or Th2 cells.
  • An ideal cell construct contains, for example, malignant (leukemia) cells, antologous plus allogeneic MHC II-positive cell or e.g. Th2 / Tc plus autologous and / or allogeneic MHC II-positive cell or e.g. malignant cell plus tumor-specific Th2 / Ts cell plus autologous and / or allogeneic MHC II expressing (APC) cell. Since the effect is rather a catalytic one, a small number of such random constructs is sufficient for a positive reaction. Moreover, the resulting catalytic cell constructs do not need to be cloned or enriched.
  • All somatic hybrid cells based valezin can be further improved according to the invention if the partner cells (tumor cells, APCs) or hybrid cells with strong surface haptens (DNCB, DNBrB, TNCB, TNBrB, DNTS, TNTS) and / or viral structures (eg NCD viruses) are xenogenized become.
  • a further increase in vaccine efficiency can be achieved by injecting allogeneic, pre-inactivated PBLs into the vaccine.
  • This "allostimulating" effect can be more targeted by using MIT effector cells.
  • MIS effector cells injected 5-10 days after vaccination, can be used to eliminate TSA or pathogen-specific Th2 or TS cells at the cellular level; these MIS effectors can be combined with the Mabs directed against Th2 or Ts.
  • xenogenization with strong haptens eg DNCB
  • viruses eg NCD virus
  • All therapeutic methods based on the in vitro selection and post-expansion of the cytolytic CTLs can be substantially improved according to the invention if the patients have previously been treated by s.d./ i.d. Vaccination with hybrid cells, consisting of tumor cells plus autologous and / or allogeneic APC (e.g. DC), would be stimulated to generate "qualitatively better", i.e. more efficient tumor-specific CTLs in vivo.
  • APC allogeneic APC
  • BMT bone marrow and stem cell transplantation
  • spleen cells could be achieved (a) by adding the above-mentioned 1: 1 mixture of D and R cells with (b) mature D cells complete; however, the latter must be replaced by D-derived MIS effector cells because of the GvHD risk.
  • a further optimized method envisages making the allogeneic BMT and HSCT (ie PBSCT, PBPCT and BMSCT) and dependent on unpredictable fluctuations between entrained donor and non-depleted recipient T cells, by a priori after myeloablative or not - Ablative pretreatment of the recipient (patient) first a 1: 1 mixture of mature D and R cells (PBLs or T cells) or, preferably, is transfused from corresponding MIS effector cells and only after that, 3-10 days later , the immature cells (optimally the 1: 1 mixture of D plus R bone marrow or stem cells) are post-injected. This prevents both GVHD and HVG (rejection of transplanted cells) and a common allogeneic chimerism is established.
  • T-cell-depleted D and R-PBLs can also be used as immature cells.
  • the 1: 1 (D: R) -MIS effector can also be used in the presence of various immunosuppressants such as dexamethasone, cortisol, cyclosporin A, agonists of G-coupled receptor subtypes and Th2 cytokines (IL-4.IL- 10, TGFß, IL-13) to accelerate mixed allogeneic chimerism.
  • immunosuppressants such as dexamethasone, cortisol, cyclosporin A, agonists of G-coupled receptor subtypes and Th2 cytokines (IL-4.IL- 10, TGFß, IL-13) to accelerate mixed allogeneic chimerism.
  • leukocytes separated after treatment e.g. B.
  • mitomycin C in the patient, so as to bridge the critical labile intermediate phase, in which tolerance to the remaining tumor cells can be re-established.
  • the ex vivo treatment of the patient's leukocytes which can contain large amounts of malignant cells in leukemia / lymphoma and tumor cells contaminating solid tumors, can solve the problem of the lack of tumor-specific Mabs when “purging” the autologous bone marrow and thus the autologous bone marrow - Transplantation for hematological and solid tumors allow wider clinical use.
  • Immature bone marrow or stem cells could partially can be replaced by T cell-depleted donor PBLs (PBMCs).
  • PBMCs T cell-depleted donor PBLs
  • the efficiency of the HTT effectors can be further increased according to the invention if, instead of untreated patient tumor cells, the tumor cells transduced with cDNA coding for IL-2, IFNgamma or IL-12 are provided as fusion partners of autologous and / or allogeneic APCs (eg DCs) , This could combine the advantages of two important techniques.
  • the tumor cells transduced with cDNA coding for IL-2, IFNgamma or IL-12 are provided as fusion partners of autologous and / or allogeneic APCs (eg DCs) , This could combine the advantages of two important techniques.
  • a variant of this method provides for the cDNA for TSA antigens to be introduced into the tumor cell first by means of viral vectors (for example vaccinia or adeno-associated virus) in order to increase the TSA membrane density and in the second step the fusion with autologous and / or allogeneic APCs (e.g. DCs).
  • viral vectors for example vaccinia or adeno-associated virus
  • APCs autologous and / or allogeneic APCs
  • ROS / ROI namely both the H 2 0 2 (0 2 2 ⁇ anion / peroxide anion) and the H0 2 (0 2 " anion / superoxide anion) for strong cell stimulation even in the absence of otherwise essential Culture medium components such as Ca 2+ , Mg 2+ , NEA, FCS / BSA etc.
  • ROS withdrawal in the medium through ROS-degrading enzymes such as catalase, peroxidase and / or Mn-Cu / ZnSOD
  • ROS-degrading enzymes such as catalase, peroxidase and / or Mn-Cu / ZnSOD
  • ROS scavengers enzyme and chemical
  • ROS / ROI can be combined with other, receptor-independent activation mechanisms, eg via canonophore or Ca ionomycin and / or phorbol esters.
  • Th1 vs.Th2 and Tc vs.Ts interconversions and functional polarizations can also be achieved.
  • the particular advantage is a rapid immune intervention even in the absence of appropriate receptors, such as those required for Th1 or Th2 type cytokines.
  • Nh3 leads to cell stimulation due to its membrane permeability and binding of the intracellular H + ion
  • NH3 and NH3-releasing systems eg glutaminase / glutamine, asparginase / aspargin
  • ROS / ROI receptor-independent receptor-independent (ROS / ROI, phorbol esters, Ca lonophore) and - dependent cell stimulators (a) for cell activation and (b) for Th1 or Use Tc subset polarization.
  • the HTT technique with all its modifications can be significantly improved if pre-existing tumor-specific Th2 and Ts cells are pre-eliminated in vivo. Therefore, the HTT combination with Th2 or Ts-specific Mabs (and polyclonal Ab) with / without MIS / MIT effector cells is recommended.
  • T-cell disease-promoting or disease-promoting immunocyte
  • the hybridization (fusion) of deficient cells with proficient cells or wild-type cells is alternatively recommended according to the invention.
  • all forms of hybridization i.e. Zeil: cell, core: Zeil and core: core mergers within the scope of the invention.
  • the defect in the cell can be remedied, at least in the heterozygous sense .
  • allogeneic partner cells must be used.
  • tolerance towards the donor must be established beforehand or allogeneic partner cells that do not express MHC must be provided.
  • MHC-negative partner cells are reticulocytes and K562 cells (human erathroleukaemic cell line).
  • a "dilution" of the membrane MHC must be sought by increasing the mixing ratio of the autologous (deficient) with the allogeneic (professional) fusion partner cells (2: 1, 3: 1, 4: 1 etc.).
  • Another HTT variant provides that, instead of patient tumor cells, those of another patient with the same type of tumor (e.g. melanoma or renal cell carcinoma) be fused with autologous and / or allogeneic APCs (DCs) of this patient or a third person.
  • DCs autologous and / or allogeneic APCs
  • a mixture of the tumor cells from patient I and II can also be assumed, or hybrid cells with tumor cells from patient I can be mixed with those from patient II.
  • Inactivation e.g. with 10,000-20.
  • T cells T cells
  • B cells mature T cells
  • T cells with the desired properties e.g. Th1, Th2, Tc, or Ts subset with or without antigen specificity
  • “Cocktails” from precisely defined starting sub-populations can also be used as a kind of "primer” for the targeted generation ("multiplication") of the desired T cell suspensions.
  • the bone marrow and Replace stem cell transplantation by transfusing T cells and transfer the pre-separated APC (macrophage) and B cell fraction to the recipient later (3-14 days after T cell transfusion).
  • this can prevent or delay the generation of GvHD; on the other hand, a higher yield of Ag-specific Th1 and Tc memory cells is to be expected due to the aforementioned shifting out of the counter-regulating Th2 and / or ts cells.
  • the lowest common denominator of all tumor cells at least in their active, not in the "dormant" state, regardless of their lineage, type and individuality (a) is a constitutively increased H0 2 / H 2 0 2 ' or 0 2 70 2_ quotient and / or increased H0 2 / NO or H0 2 (NO + H 2 0 2 ) - vehicle (b) an increased Go / Gp - or PI-PLC quotient, more precisely a constitutively increased degree of activation Gi PLA2
  • PLA PKG where DAG stands for diacylglycerol, PKC for protein kinaseC and PKG for protein kinase G.
  • DAG stands for diacylglycerol
  • PKC protein kinaseC
  • PKG protein kinase G.
  • the desired patent protection concerns all measures derived from this knowledge (pharmacological interventions) which lead to the controlled proliferation of degenerated cells by reducing the quotients mentioned. Examples include antagonists of the alpha-1 AR type and agonists of the alpha2-AR type.
  • Embodiments for allergy treatment according to the invention are also given below: Since the allergy is associated with a T-subset shift in favor of the Th2 subset, it is recommended (a) with T-depleting Mabs plus MIT / MIS effectors that Th2 / Th1 - Correct (normalize) the ratio (b) with Ca antagonists (e.g. nifedipine), cAMPi and NOi-lowering pharmaceuticals (e.g.
  • Ca antagonists e.g. nifedipine
  • cAMPi and NOi-lowering pharmaceuticals e.g.
  • these fusion proteins with IL-1 2, IFNgamma, IFNalpha and TNFalpha are also recommended for other methods in which type 1 (pro-inflammatory) immune response is sought, e.g. in the treatment of tumor in infections, whereby the antigen must be known in each case.
  • Ag-specific T cells for example allergen or TSA / TSTA-specific T cells
  • TSA / TSTA-specific T cells have recently been able to be activated by an elegant method in which they are activated in the presence of the specific antigen or allergen or alloantigen T cells can be recognized based on the secreted cytokines and sorted by FACS or MACS.
  • Secreted cytokines are retained by bispecific Mabs, which recognize the T cell on the one hand and the cytokine on the other hand, in order to subsequently label them with a second Mab (with specificity for the same cytokine) and to make the cell separation accessible.
  • IgE and the receptor on mast cells and eosinophils can be weakened by proteolytically pre-cleaved IgE subunits that cannot bridge 2 Fc epsilon receptors.
  • allergy-promoting cytokines eg IL-4, IL-5, IL-1 3, sometimes IL-10) can be obtained by appropriate Mabs be neutralized.
  • a stable R: D tolerance can be established by preceding in vivo T-depletion (with Mab + MIS / MIT effectors) and / or by post-injection of immature donor cells (BMCs, HSCs), but also T-dependent donor PBLs , Instead of D cells, a 1: 1 mixture of (immature) R + D cells can be post-injected.
  • MIS / MIT effector cells prepared from D and R * PBLs, which can also be prealloimmunized to the respective partner (D or R), to use both GvHD and HvGR (Bone marrow or organ rejection) are to be brought under clinical control.
  • D or R the respective partner
  • GvHD the respective partner
  • HvGR Hemarrow or organ rejection
  • pan-T-specific Mab eg Anti-Thy1 .2-Mab
  • this method could be used enable both the outcome of a cell transplantation and subsequent corrections in vivo, for example, the temporary “D-type allogeneic chimerism” aimed at fighting residual tumor cells us “later peu-a-peu to be replaced by the D: R tolerance-stabilizing” mixed allogeneic chimerism ".
  • the excess of D cells can be "compensated” by R cells. This method is of great clinical importance because it can be used, for example, to combat established GvHD and acute organ rejection.
  • autologous NK cells or the LAK cells derived therefrom as well as allogeneic LAK cells which either have pan-T-specific Mabs depleted from their T cell portion or according to the type of MIS - Effectors cell preprogrammed must be used.
  • Donor-type allogeneic chimerism can be intervened.
  • Another variant provides for the infusion of the MIT-MACH effectors (i.e. 1: 1 mixture of D + R-MIS effectors) immediately (3-10 days) before DLI / DLT.
  • MIS-BM / SC effectors The MIS effectors used to eliminate dysregulated T cell subclones (eg Ts / Th2 cells in tumors or Tc cells in autoimmune diseases) are called MIS-BM / SC effectors and those used for R: D tolerance stabilization referred to as MIS-MACH ("mixed allogeneic chimerism") effector cells.
  • MIS-MACH mixed allogeneic chimerism
  • H0 2 promotes proliferation and H 2 0 2 promotes differentiation
  • H 2 0 2 promotes differentiation
  • all systems that H0 2 vs. Generate H 2 0 2 , of particular interest.
  • Two systems are protected in this context: (a) H0 2 formation by XOD / xanthine or XOD / hypoxanthine, combined with catalase and / or (GSH) peroxidase, and (b) Molsidomin / Sydnonimin / SIN-1, which forms H0 2 plus NO at decay, whereby NO is bound by various scavengers (eg hemoglobin).
  • various scavengers eg hemoglobin
  • MIS effectors allow a gradual introduction of "sharper”, ie MIS effectors pretreated with a lower MitomycinC concentration and finally the use of untreated D -Lymphocytes, however, unlike the DLI technique, without GvHD complications.
  • MIS-MACH_effectors can be replaced peu-a-peu by "sharper" D + R effectors (1: 1 mixture) and for Efficiency increase can be provided by allotolerant Ts and Th2 cells pre-enriched in vitro (in the MLC in the presence of Gs agonists, ConA and / or CsA).
  • the MIS / MIT technology follows two basic principles: (a) With the 1st In principle, the MIS effectors are used exclusively for in vivo depletion or inactivation of the dysregulated T cell subsets (Ts / Th2 for tumor or Tc for AID, GvHD and BM and organ rejection reaction). The BMT or HSCT can be omitted here; nevertheless an optimal T-cell: APC cooperation (after MIS treatment) is guaranteed, (b) the second principle eliminates not only the mature, dysregulated T-cell subsets, but also the immature precursors in vivo.
  • the efficiency of the MIT, more precisely MIT-BM / SC effectors can be increased further if the in vitro preall immunization in one-way MLC with a D: R mixing ratio of 10: 1 (or> 1: 1 in general) instead of 1: 1.
  • allogeneic BMT and HSCT (a) to use a 1: 1 mixture of donor plus recipient cells instead of the "pure" donor cells, and (b) a 1: 1 before each (allogeneic) BMT or HSCT -A mixture of MIS effector cells of the donor plus recipient to establish a kind of preparatory "mixed allogeneic chimerism" in the recipient for 3-10 days. Both measures can both prevent GvHD or HvGR induction and guarantee optimal post-transplant immune defense.
  • HTT autologous or allogeneic APCs
  • idiotype-expressing T cell e.g. Ts / Th2 in tumor patients
  • MHC II and costimulator structures e.g. B7.1 /B7.2, CD40 etc.
  • a 4-6 hour incubation with cytokines (IFN gamma), H 2 0 2 + iNOS / cNOS inhibitors can be used for this.
  • An important goal in gene therapy is standardization, ie independence from the patient's material. This goal can be achieved by pre-establishing the donor-specific tolerance according to one of the present application described method can be achieved. After induction of tolerance, bone marrow and stem cells as well as fibroblasts of the donor or pooled donor cells can be used as secretor cells, for example for the coagulation factor VIII or IX or for insulin (in IDDM patients), but also for various enzymes, the replacement of which "normalizes” a number of genetic diseases ", into which patients are injected, the laws of" cell homing "often ensuring that these allogeneic secretory cells settle spontaneously in organs of origin.
  • autologous gene-deficient cells can also be fused with similar allogeneic (proficient or Wildt ⁇ p-) cells or the missing gene can be substituted by transduction (eg by means of viral gene transports).
  • the HTT and HTT-AIT technique can also be increased in efficiency by transfection of either the fused cell constructs or the fusion partners used (e.g. tumor cell) with cytokines such as IL-2, IFNgamma, TNFalpha or IL-12.
  • cytokines such as IL-2, IFNgamma, TNFalpha or IL-12.
  • the "freezing" of tailored cell properties (eg secretion of cytokines, growth factors, hormones etc.) by fusion with immortalized partner cells is also the subject of the present application.
  • Specialized cell subtypes eg Th1, Th2, Tc, Ts etc.
  • cell death can be preprogrammed, for example, with mitomycin C pretreatment.
  • Th1 cells using a 1-3 day MLC approach can be combined with a pre-programming of cell death within 1-3 days.
  • the patient can successively receive these short-lived T4 cells (or PBLs) from different donors, with the early death preventing the conversion to Tho cells that secrete Th2 cytokines.
  • Ts in Th2 arise.
  • These cells too, with or without mitomycin C pretreatment, can be used as "primer cells" for in-vivo recruitment of new Ts (Th2) cells from precursors or naive T cells.
  • the problem of transduction of target cells with more than 3 cytokines can be solved by fusing tumor or TIL cells, each transferred with 1-2 cytokines.
  • tumor-specific CTLs can be significantly improved if the patient previously with HTT and / or HTT-AIT effectors, i.e. Hybrids from tumor cells plus APCs and / or hybrids from tumor-specific Ts and / or Th2 cells plus APCs are vaccinated and if the pre-existing tumor-specific Ts (Th2) cells are predeplicated in vivo by Mabs plus MIS / MIT effectors.
  • the number of tumoricidal CTLs can be increased further if the reinjection of the in vitro pre-cloned CTLs is combined with the infusion of larger numbers of immature or naive T cells.
  • NK cells of the recipient can induce HvGR and those of the donor GvHD (with), it is recommended to use antibodies against NK cells and some Th1 cytokines (e.g. IFNgamma, TNFalpha, IL-1 2) for BMT and HSCT.
  • Th1 cytokines e.g. IFNgamma, TNFalpha, IL-1 2
  • D-specific R and R-specific D-Ts or Th2 cells Rapid addition of D-specific R and R-specific D-Ts or Th2 cells is possible by adding immature cells (BMG, HSCs, PBSCs / PBPCs) to pre-generated Ts and, even better, Th2 cells and subsequent mitogens (Lectin) or alloantigen stimulation of the cell mixture.
  • the Ts or Th2 cells can be pretreated with mitomycin C or radiation.
  • the MIS effectors can be freed of these by Mabs, directed against HLA-DR surface structures on accompanying suppressor subpopulations; the cytotoxic MIS effectors are (in the activated state) HLA-DQ +.
  • T cells or PBLs
  • APCs hybrid cells from allopa-preactivated recipient T cells (or PBLs) plus APCs (DCs) are to be pre-vaccinated before the actual transplantation
  • This highly selective strategy can be expected from the combination with other HTT and MIS / MIT methods.
  • a special HTT-AIT modification enables vaccination against autoaggressive T cells, which are responsible for the demise of the insulin-producing ß or B cells (Langerhaus islet cells) of the pancreas.
  • IDDM T cells from one or more type 1 (juvenile) diabetics as fusion partners; in this case, the hybrid cells must be preprogrammed according to the type of MIS / MIT effectors and injected into the T-cell-depelted patients. Regardless of the provenance of the autoantigenic-recognizing fusion partner cells (T cells), pretreatment of the IDDM patient with Mabs plus MIS effectors is recommended.
  • Tumor cells or TIL cells mostly serve as target cells for gene transduction. It is recommended to use this e.g. to combine with IL-2, IL-4, IL-7, IL-12, IFNgamma, TNFalpha etc. target cell transfected with HTT and / or HTT-AIT effectors.
  • target cell transfected with HTT and / or HTT-AIT effectors e.g. to combine with IL-2, IL-4, IL-7, IL-12, IFNgamma, TNFalpha etc.
  • target cell transfected with HTT and / or HTT-AIT effectors e.g. to combine with IL-2, IL-4, IL-7, IL-12, IFNgamma, TNFalpha etc.
  • target cell transfected with HTT and / or HTT-AIT effectors e.g. to combine with IL-2, IL-4, IL-7, IL-12, IFNgamma, TNFalpha etc.
  • a method according to the invention provides for either transfecting the tumor-specific CTLs which have been pre-selected and clonally post-expanded ex vivo in a “conventional” manner with Th1 cytokines such as IL-2, IFNgamma and / or IL-1 2 and with autologous and / or allogeneic APCs ( DCs, macrophages) to inject the resulting hybrid cells sc or id into the patient.
  • Th1 cytokines such as IL-2, IFNgamma and / or IL-1 2
  • autologous and / or allogeneic APCs DCs, macrophages
  • Another (better) variant involves vaccinating the patient with HTT and / or HTT-AIT affectors first in order to “CTLs” higher quality "in vivo, to transduce these tumoricidal CTLs with Th1 cytokines and to treat the patients again with these genetically modified CTLs or CTL secretor cells
  • the same multi-step process can also significantly increase the efficiency of "classic" TIL cells, these TIL cells being partially identical to the tumoricidal CTLs.
  • the CTLs can be secreted after vaccination with Th1 - cytokine-secreting agents Constructs can be post-expanded ex vivo clonally and reinjected into the patient.
  • the stabilization of cells with tailored properties, eg antigen-specific or non-specific Th1, Th2, Tel, Tc2, Tc3, Ts etc., without malignancy can be achieved either by fusion of cell mixtures in which the number of untransformed versus that of the transformed partner cells is gradually increased, or it can be done by several successive fusion steps. With this, the malignancy of the cell constructs can be "thinned out" bit by bit, while the stability or dependency on growth factors can nevertheless be influenced favorably.
  • Another point concerns the simplified transmission of information e.g. via cytokine, growth factor or hormone secretion, by replacing the cDNA information, which is usually introduced individually by means of viral vectors, by simple fusion with specialized secretor cells.
  • both modern cellular techniques, the MIS / MIT and the HTT technique, can also be used according to the invention in addition or partially to replace recently described IDDM therapies based on the Mab daclizumab approved for kidney transplants (since 1999) and the two immunosuppressants tacrolimus and sirolimus become.
  • An interesting new approach in the induction of D-specific tolerance is the (a) transfection of autologous thymus cells with D-MHC II and MHC l-cDNA (of the donor D1, D2, D3 etc.) or (b) fusion of autologous thymus cells with Partner cells expressing MHC I and MHC II (of donor D1, D2, D3 etc.).
  • Examples of such cells are BMCs, HSCs / PBSCs / PBPCs, embryonic / fetal SCs, T cells and APCs.
  • the thymus cells modified in this way settle in the thymus and at the same time enable the R- and D (D1, D2, D3, etc.) - specific "thymic education", which can be found in R- and D (D1, etc.) -specific MHC- Restriction of the maturing T cells is expressed:
  • BMT or HSCT of 1 1 mixture of donor and recipient cells, the ideal constellation now arises that mature D and R cells have mutual tolerance and also T cells and APCs from D and R cooperate optimally with each other.
  • D-specific tolerance induction results from immunization or vaccination with MHC 11 + and MHC 1 + cells of the donor under strongly immunosuppressive conditions, for example after injection of cyclophosphamide, glueocorticoids and / or cyclosporin A, rapamycin or FK506 in the recipients.
  • HTT vaccines could be increased by xenogenization, i.e. Pretreatment with strong haptens such as DNCB, DNBB, TNCB, TNTS etc., but also by pre- or cofusion with xenogenic cells (a) either the tumor or (b) the hybrid cell can be further increased.
  • xenogenization i.e. Pretreatment with strong haptens such as DNCB, DNBB, TNCB, TNTS etc.
  • hat carriers such as trinitrotoluenesulfonic acid (TNTS) or e.g. BCG post-treatment of the bladder, if BCG germs (inactivated) have been used for the xenogenization, additional tumoricidal effects of the HTT basic treatment are sought.
  • the invention therefore also relates to their combination with the immunostimulants listed in EP 374 207 and EP 705 1 1 1 or the cAMP or cAMP / cGMP-reducing pharmaceuticals described therein,
  • Another object of the present invention relates to the combination (a) of all variants for Ts and / or Th2 elimination on a humoral basis (with Mabs, Mab conjugates, Mab fusion proteins, immunotoxins etc.), which are the same in the above-mentioned patent applications Applicants (EP 374 207 and 705 1 1 1) have been described in detail, with (b) MIS / MIT technology and its modifications and / or (c) HTT technology and its modifications.
  • MIS / MIT technology i.a.
  • HTT technology HTT technology and its modifications.
  • Another object of the invention is also the intended substitution of chemotherapy and / or radiotherapy for hematological tumors by specific Mabs and possibly leukapheresis as a preparative step in all of the methods discussed here, which are based on MIS / MIT and HTT technology
  • Combination of Mab treatment and leukapheresis is an essential preparative step not only for leukemia and lymphoma, but also for solid tumors; in the latter case, it should not directly serve to eradicate malignant cells, but rather to co-eliminate tumor-protective Ts and Th2 cells.
  • V. Schirrmacher with a low-dose combination of IL-2 plus IFNgamma and the increase in tumor therapy by infection of the tumor cells with ND (Newcastle Disease) virus can be significantly improved by humoral and cellular Ts / Th2 depletion.
  • ND Newcastle Disease
  • Rapid Th2 and / or Ts generation can be achieved by receptor-independent stimulation with PMA TPA + Calonophore / Ionomycin + ROS (H02 + H202) - releasing substances or Enzymes can be achieved when additional Gs agonists + glucocorticoids + some cytostatics (cyclophosphamide) + CsA (rapamycin, FK506) are present.
  • Th1 and / or Tc cells can be generated quickly if, instead of GS agonists, GS antagonists, iNOS / cNOS inhibitors and / or Gi agonists are present during the incubation. Further modulation can be achieved with various agonists of the 7 cAMP or cGMP-PDE enzymes.
  • NH3 and NH3 generators e.g. glutaminase / glutamine
  • RNS NO and NO-releasing systems
  • the pre-formed Th1 / Tc or Th2 / Ts cells can lead to further clonal expansion of tailor-made T cell subsets after T depletion or injection of immature T cells.
  • Th2 / Ts cells A simplified procedure provides for Th2 / Ts cells to be generated by cAMPi-increasing plus cAMP-PDE-inhibiting additives and to be stimulated in a 1: 1 mixture with fresh PBLs from the same person.
  • Th1 / Tc cells can be generated "in a rapid process" by coincubation with cAMPi-inhibiting and cAMPi-degrading PDE agonists.
  • a special "punctual" / "clonotypic” tolerance induction can be achieved by transfection of tailor-made secretor cells with IL-4 / IL-13, IL-10 and / or ß-TGF. (Alternatively, these secretor cells with Th2 cells hybridized / fused). The recipient (patient) must be temporarily T-depleted in order not to reject these cell constructs.
  • the constitutive secretion of Th2-type cytokines helps to induce and stabilize tolerance to these genetically engineered cells of allogeneic origin.
  • the secretor cells used can secrete important hormones and factors (e.g. dopa, dopamine, factor VIII or IX).
  • A2 / P1 (purinergic) agonists plays a special role in maintaining the immunosuppressive state. For this reason, appropriate A2 / P1 antagonists are recommended for the primary suppression of suppression.
  • the immunization against all (soluble) antigens except for the alloantigens in vitro only succeeds after previous in vivo immunization.
  • the in vivo pre-immunization can be omitted if in vitro autologous and / or allogeneic APCs are first pre-incubated with the (soluble) antigen and then fused.
  • the fusion of the APCs can additionally be co-fused with the cells as the source of these membrane-associated antigens. It is advantageous to stimulate the APCs before or during the coincubation with the antigen by Th1 cytokines (IFNgamma, IL-1 2, IL-2, etc.). With the help of these hybrid cells, which still need not be cloned, a new batch with autologous and / or allogeneic PBLs can now be used for in vitro immunization against (soluble) antigens. The next point concerns a supplement to the gene therapeutic techniques.
  • Th1 cytokines IFNgamma, IL-1 2, IL-2, etc.
  • the fusion of cells is to be supplemented by fusion of isolated cell nuclei, of individual chromosomes and of isolated genes and gene fragments with whole cells, with cell nuclei and / or individual genes and gene fragments. All types of fusion with cytoplasts should also be protected; these are cytoplasmic residual structures without a core, which can be obtained technically by cell treatment (coring) using cytokalasin B, whereby in addition to the cytoplasts, cores with a partially remaining core membrane (karyoplasts) are formed.
  • Chromosome Chromosome and (g) all of the above combinations with purified gene (incl. corresponding cDNA) and gene fragments.
  • a variant provides for the introduction of foreign DANN via erythrocyte membranes, which are opened under hypotonic conditions for the foreign nucleus or for the foreign chromosome and sealed in the hypertonic medium. This technique could be of interest in inducing tolerance to donor MHC.
  • the fusion of foreign (donor) cell nuclei or karyoplasts with autologous cells or cytoplasts pursues a similar goal, two special cases to be mentioned, the thymic epithelial cell and the HSC.
  • Chromosome fragment can be provided by appropriate material from efficient or wild-type cells.
  • MIS and. MIT technology recommended mitomycin C can be supplemented by two other products, BMY 25282 and BMY 25067, and some of them. be replaced.
  • the quality of the MIS and MIT effector cells can be increased (a) by prematurely (2-5d.) Terminated in vitro MLC approach for R-specific allopreimmunization of the MIS / MIT effectors, (b) by high-level pre-activation of the stimulator ( R) cells in the above-mentioned MLC approach and (c) by a high responder (D): stimulator (R) ratio (2: 1 to 10: 1). All three Measures prefer the MIS / MIT effectors with the highest affinity for R target cells and thus enable a selection of the most efficient effectors.
  • Another technique for in vivo generation of the "mixed" allogeneic chimerism is the in vivo use of anti-CD4 Mabs followed by the infusion of mature PBLs plus immature bone marrow and stem cells (T-depleted PBMCs).
  • T-depleted PBMCs immature bone marrow and stem cells
  • the anti CD3 (or the anti-CD4 plus anti-CD8) Mabs are used instead of anti-CD4.
  • T-cell-containing cell suspensions are incubated with bispecific antibodies (“catch matrix”), which are attached to the T cells or leukocytes (CD45R) on the one hand, and to a cytokine (mostly IFNgamma or IL-4) on the other ), the antigen-specific T cells are labeled and sorted using a second antibody (against the cytokine), the "detection antibody", FACS or MACS.
  • a second antibody asgainst the cytokine
  • FACS fluor MACS
  • the specific antigens are pathogens, TSTA / TSA, autoantigens and allergens.
  • This new "clonotypical" HTT modification thus gains topicality, not least because of the particularly low complication rate in the treatment of residual tumor and metastatic cells, bacterial and (retro) viral infections (including HIV), and also of GvHD, by HvGR (rejection reaction), organ transplantation and BMT / HSCT, autoimmune diseases (AID) and allergies, where the clonotypic vaccine in tumor patients is directed against dysregulated, tumor-protective Ts and / or Th2 cells, whereas in AID it is directed against autoaggressive ones Tc cells: In the fight against GvHD vaccination against the alloreactive Tc cells of the donor and in the HvGR treatment against those of the recipient. In the case of allergies, the HTT-CL vaccine is directed against allergenic T cells, primarily the TH2 subset All of these HTT-based modifications are increased in their efficiency by upstream MIS / MIT treatment.
  • the clonotypic HTT technique in combination with MIS / MIT simultaneously combines a GvHD-free GvM (GvL or GvT) effect with a recurrence-preventing vaccination against dysregulated T cell subclones.
  • GvL or GvT GvHD-free GvM
  • a combination of hybrid cells resulted from the fusion (a) of patient tumor cells and (b) of his Ts / Th2 cells (pre-selected in a subset or clonotype-specific manner), both with MHC 11 + cells (autologous and / or of allogeneic origin) can be optimally dealt with against tumor cells and T cells protecting them.
  • the fusion with MHC 11+ cells can simultaneously generate both types of hybrid cells in one step; what is important here is a kind of "catalytic" effect of the smallest number of hybrid cells, which is why they are neither enriched nor clonally post-expanded have to; moreover, they can even be pretreated with MitomycinC without losing their effectiveness.
  • a special case of clonotypic HTT vaccination concerns vaccination against allospecific T cells, which enables an elegant, low-side-effects route to D: R tolerance.
  • D D tolerance
  • the tolerance of the recipient T cells towards the donor in contrast with BMT and HSCT, a mutual D: R or R: D tolerance is sought.
  • the new technique of tolerance induction is based on the principle of clonotypic HTT vaccination described above.
  • the effector cells of the type responsible for organ rejection are first used in a "one way" MLC with R as responder and D as inactivated stimulator cells.
  • BMT and HSCT have to use two separate "one way" MLCs, once , as just described for organ transplantation and once in the MLC with D as responder and R as inactivated stimulator cells, the fusion partner cells for autologous and / or allogeneic APCs are embossed.
  • BMT and HSCT are pre-immunized against both GvHD and GvGR.
  • the vaccines in general can be improved with MIT effectors. This is based on the observation that the pre-existing tolerance to a model antigen can be broken simply by adding allogeneic cells. The same principle (addition of MIT effectors) can also support the in vitro immunization against (soluble) antigens discussed above.
  • Mabs against T differentiation antigens which are only expressed on certain precursor subsets, are particularly suitable for this purpose, e.g. Anti-T6, Anti-T9, Anti-T10 and / or Anti T1 1 -Mabs.
  • the inefficient removal of contaminating malignant cells can be solved by immature bone marrow or stem cells susceptible to tolerance to residual tumor cells by immune-competent, tolerance-resistant (re) MIS effectors, followed by BMC or HSC reinjection, and the contaminating tumor cells are rendered harmless during MIS preparation (with mitomycin C etc.).
  • the treatment described can be supplemented by ex vivo clonally post-expanded tumor-specific CTLs, this sub-step being carried out immediately after MIS treatment.
  • the “quality” of these tumoricidal CTLs is increased if they are removed from the patient, post-expanded and reinjected after the HTT vaccination (against tumor cells alone or against tumor plus Ts / Th2 cells).
  • the therapy scheme described above is special because it is directed against both tumor cells and their protector cells and because it could later effectively prevent relapses by simultaneous vaccination against both "actors".
  • a simplified version provides that the clonotypic HTT vaccination against tumor (TSTA / TSA) and / or tumor-protective Ts / Th2 cells must be accompanied by simultaneous APC costimulation with allospecific signals, which is due to the combination of the vaccine with MIT effectors is achieved.
  • ApoE could become a scarce commodity in dietary or otherwise caused hyperlipidemia.
  • H0 2 -reducing, H 2 0 2 -increasing, iNOS / cNOS-stimulating and cGMP-PDE-inhibiting substances alone or in combination with cytostatics, intervention can be tumoristatic or tumoridic.
  • modulators of various cAMPs - and cGMP-specific phosphodiesterases are used, and the combination of the cAMP or cGMP-generating system with inhibitors of the cAMP or cGMP-specific PDEs is often of interest, since in this way the concentration of the cyclic nucleotide and its duration of action The degradation of the pre-existing intracellular cAMP or cGMP levels by stimulators of corresponding PDEs is also of interest.
  • Another suggestion for more efficient control of the immunocyte function is the switching off of the immunocyte (hyper) activity by combined treatment with NO-releasing drugs plus inhibitors of the cGMP-specific PDE (dhPDE V).
  • dhPDE V cGMP-specific PDE
  • auto-aggressive T cells in AID or all-aggressive CTLs in GvHD can be influenced favorably.
  • the hyperpolarization effects that increase cAMPi and are based on cGMPi exaggeration can be bundled for the rapid reduction of cell overactivation.
  • okadaic acid okadaic acid
  • a significant improvement in conventional therapies for hematological tumors implies the following steps according to the invention:
  • panT panT
  • anti-CD4 + anti-CD8-Mabs or corresponding polyclonal Ab ATG, ATGAM, ALG etc.
  • anti-T6, anti-T9, anti -T10- and / or Anti T1 1 -Mab or polyclonal Ab which also include T precursors, are recommended.
  • the dysregulated T cell subsets which may the main culprit for the development and / or maintenance of the hematological tumors, also of the B or myeloid type, must be radical, i.e. be removed on a humoral (with Abs) and cellular (with MIS / MIT effectors) level, in all, not only T-cell-based tumor types.
  • the HTT vaccine directed against tumor- and tumor-specific Ts / Th2 cells can be used, the cell fusion by adding autologous and / or allogeneic MHC II + cells (eg DCs) to the patient PBLs can be done in one step.
  • autologous and / or allogeneic MHC II + cells eg DCs
  • an approx. 1 1 mixture of mature and immature donor cells with finely dosed Anti-T or Anti-T2 + Anti-T8-Mab can be allogeneic after infusion Create chimerism of the donor type, but also of the mixed type. If the exact Mab concentrations were found clinically, they would have a general validity, ie they would be the same or similar in different people, so that the method could be standardized.
  • an R: Dx and a D: Rx one-way MLC is recommended enables the correction factor to be determined.
  • the 1: 1 mixture of D and R MIS effectors can be used without or with a 5-7 day preincubation, the MIS preparation from the D and R PBMCs should only take place after this preincubation.
  • ornithine, arginine and putrescine inhibit Tc generation, they are of interest for (a) tolerance induction and (b) BMT / HSCT / PBPCT as well as other immunosuppressive agents as Th2 or Ts stimulating additives.
  • the cDNA or the so-called “naked DNA” encoding the antigen of interest can be brought into autologous and / or allogeneic MHC II + cells (as plasmid DNA , free or bound to apatite or enclosed in (cationic) liposomes. Now it can be used as a vaccine sc, id or im or with different HTT-, HTT / AIT- and / or HTT-CL (HTT / AIT-CL ) Hybrid cells can be combined.
  • This vaccine to be used for tumor, AID, allergies, infections and atherosclerosis can, in the absence of the specific cDNA, be fused by fusion of the cDNA-containing cell nucleus or the corresponding chromosome, or else the intact cells containing cDNA be fused with the autologous and / or allogeneic MHC II -4- cell, for example not only choose the 1: 1 mixing ratio on the other hand and can combine them.
  • the same technique can also be used for double vaccination against tumor cells and against tumor-specific Ts or Th2 cells, alone or in combination with "conventional" vaccine (based on hybrid cells from APC plus tumor or APC plus Ts / Th2 cell) , use.
  • cDNA can also simply be added before or after fusion of the tumor cell with the APC in order to further increase the antigenicity of the construct.
  • a special technique provides for presensitizing the recipient PBLs or T cells against donor A, B, C, D etc. in separate MLC approaches, the activated receptor antidonor T cells with different cytokines, growth factors, hormones etc. to transduce and reinject into the recipient. Later, the desired cytokine secretion can be induced in vivo by (inactivated) PBLs of the donor A, B, C etc.
  • MIS / MIT effectors of different origins should always be followed by repeated pretreatment with Mabs, i.e. MIS / MIT effectors should always be combined with Mab pretreatment should.
  • a further improvement relates to the repeated injection of the cell hybrids from patient tumor cells and APVs (eg DCs) from different, most histo-incompatible donors A, B, C etc., whereby this step can be combined with ex vivo pre-expanded tumor-specific CTLs.
  • the respective donor can also have been pretreated beforehand with IL-12 or IL-2 and / or IFNalpha in order to increase the MHC and integrin expression on the allogeneic MHC II + fusion partner cell.
  • Patent protection is also sought for all improvements (a) in tumor therapy with tumor-specific Mabs and (b) in cellular tumor therapy using LAK, TIL and / or CTL effector cells in which the inhibitory effect of tumor-specific Ts and / or Th2 cells are overcome according to the invention with the combination of (a) anti-CD3 or anti-CD4 plus anti-CD8 Mabs plus (b) MIS / MIT effectors.
  • Another variant of the HTT technique provides for hybridizing the patient's tumor cells not only with DCs, but also with macrophages and / or B cells of autologous and / or allogeneic origin.
  • HTT and / or MIS effectors can also be combined with cell constructs based on immortalized specialized subclasses (e.g. Th1, Th2, etc.).
  • immortalized specialized subclasses e.g. Th1, Th2, etc.
  • the H 2 0 2 increase can be induced via H0 2 synthesis, various stimulants of NADPH oxidase being of interest: (a) soluble stimulants, such as PMA (1 -2ug / ml), N-formyl met Leu Ph ( 0.5-5uM), Ca-ionophore A23187 (10uM), LPS (10ug / ml), TNFalpha (250mg / ml), IFNgamma (3.103 U / ml), AA (arachidonic acid) (50uM); (b) Particulate stimulants, such as heat-aggregated IgG (1.5 mg / ml), zymosan (1.5 mg / ml).
  • soluble stimulants such as PMA (1 -2ug / ml), N-formyl met Leu Ph ( 0.5-5uM), Ca-ionophore A23187 (10uM), LPS (10ug / ml), TNFalpha (250mg /
  • the fusion of mature, specialized cells e.g. Thl, Th2, macrophages, NK cells, B cells etc.
  • a) embryonic a hematopoietic stem cells results in long-lived but not transformed cells.
  • fibroblasts cell lines.
  • tissue-specific cells for example heart muscle cells, liver cells etc. with embryonic or hematological stem cells, makes it possible, according to the invention, to grow long-lived hybrid cells with tissue-specific and multipotent / tutipotent properties.
  • cytokine-secreting Th1 partner cells should be activated beforehand, e.g. with inactivated allogeneic PBLs.
  • the problem of ex vivo post-expansion (a) of the embryonic (b) the hematological and (c) the adult stem cells without the undesired (partial) cell differentiation can be solved by supplying H02, e.g. by XOD / xanthine (hypoxanthine) (with simultaneous H202 elimination using catalase or GSH peroxidase).
  • H02 e.g. by XOD / xanthine (hypoxanthine) (with simultaneous H202 elimination using catalase or GSH peroxidase).
  • the fusion with (a) embryonic (b) adult (c) hematological stem cells gives the hybrid cells longevity or unhindered reproduction without malignancy.
  • the fusion of deficient cells e.g. cells with gene deletion or point mutation
  • genetic diseases the number of which is around 4000 (!)
  • A) with nucleated precursor cells of erythrocytes (b) with (pre-inactivated) K562 cells (MHC I and ll-negative) and (c) with MHC I and MHC II not expressing efficient or wild-type cells of allogeneic origin, in which the corresponding MHC-coding genes have been inactivated either by "gene targeting" or "gene knock-out” leads to hybrid cells that now have the missing gene in a heterozygous manner.
  • These cell hybrids can replace the time-consuming and, with many additional problems, transfection of the missing gene by means of retroviral vectors.
  • APCs are programmed or reprogrammed in vivo by repeated injection of anti-CD8-pretreated MIS or MIT effectors from donor A, B, C etc. in the sense of Th1-type cytokines. If the cell death of the MIS or MIT effectors is preprogrammed for 3-4 days, the APCs of IL-2 (and IFN gamma), but not IL-4, IL-13 or IL-10, are continuously "washed around".
  • Previous experience in the animal model and in the clinic generally shows that modern methods of immune control without simultaneous or preceding radical in vivo elimination of the tumor-protective Ts and / or Th2 cells only lead to a temporary improvement (tumor regression, decrease in metastases).
  • Every form of immune stimulation e.g. with BRMs, cytokines etc.
  • each of the new immunotherapeutic methods e.g. LAK / IL-2, TIL / IL-2-, all variants of cellular / adaptive immunotherapy with in vitro pre-selected and post-expanded tumor-specific CTLs, etc.
  • Ts / Th2 pre-depletion whereby all combinations with these Ts / Th2-depleting methods such as MIS / MIT and Ts-, Th2-, T4-, T8-, T3 etc. specific Mabs are to be considered as the subject of the present application.
  • the particular advantage of combining existing anti-tumor therapies with Ts / Th2 co-elimination is that (a) temporary effects are replaced by long-term
  • the subject of the present application are also all combinations of existing immunotherapeutic methods with techniques that prevent later relapses, to which e.g. vaccination against the disease-inducing or - maintaining T-subsets, i.e. the Ts or Th2 cells in the case of the tumor, the auto-aggressive and allergen-specific T subpopulations in the case of AID or allergies, etc. (HTT-AIT technique).
  • a greatly simplified therapy variant provides for the PBLs of patients to be mixed with those of one or more donors in vitro in various ratios (from 0.1: 1 to 1: 1 to 1: 0.1) with cell death preprogramming Substances, e.g. Treat mitomycin C and reinject it in the PBMCs or T cell depelted patients.
  • cell death preprogramming Substances e.g. Treat mitomycin C and reinject it in the PBMCs or T cell depelted patients.
  • the therapeutic effect can be further enhanced by (prealloimmunized MIS / MIT effectors and various HTT vaccines).
  • naive allospecific T cells in the first phase i.e. Securing only IL-2 and some IFNgamma 1-3 days after activation is used for APC reprogramming in the Th1 sense e.g. after tumor excision, chemotherapy and radiotherapy, practically used, with preprogrammed T cells or PBLs from donors A, B, C, D ... with or without T8 predeplication used in vivo for 2-4 days life expectancy.
  • the immune defense is generally suppressed in AIDS patients and in patients with advanced tumors, so that the immune response against neoantigens (for example new infections) is also impaired, according to the invention, in all Diseases associated with chronic progression suggested, from time to time, to eliminate the dysregulated or unwanted T cells in vivo by Mabs plus MIS effectors, the APCs with MIT treatment and with Gs blockers, Ca antagonists and NOS -Reprogramming inhibitors in subclinical doses and stimulating the now unblocked immune system of the patient with low-dose BRMs.
  • preparations that correct the ROS / RNS ratio such as XOD / xanthine (hypoxanthine), GOD / glucose or various sub-dosed ROS-releasing substances, can also be used.
  • osteoclast activity leads to increased osteoclast activity.
  • Theoretical considerations suggest that an APC reprogramming from the suppressor monocyte to the tumoricidal / bactericidal effector function, i.e. a type 2 (Th2): type 1 (ThD interconversion of the osteoclast: osteoblast conversion corresponds.
  • Th2 Th1 - or Ts: Tc- reprogramming systems, such as XOD / xanthine (hypoxanthine) or GOD / glucose (c) of Gs blockers + Gi agonists, Ca antagonists and NOS inhibitors and of all others Representatives of this Th2: Th1 - or Ts: Tc-modulating substance class are recommended.
  • Example is the simultaneous partial inhibition of the alphal, alpha2, ß1 - and ß2-adrenoceptors or the H1 - H2-histamine receptors, which prevents the hypoergy or anergy of the immunocompetent cells by overactivation. There are signs that the cell is blocked by reducing excess "background signals" to Th2: Th1 - or Ts: Tc reprogramming leads.
  • the hybrid cells discussed in detail in the earlier application EP 705 1 1 1 are to be expanded by two further cell constructs: (a) the first novel cell construct consists of Th1 - plus non-MHC-restricted Tc / CTL cells (b) the second cell construct is made up of Th1 and APC.
  • Another way to induce tolerance is to generate allotolerant donor-specific receptor T cells (for organ transplants) or mutually tolerant T cells (for KMT) by using in vitro mature T cells or PBLs from one partner Pre-incubated with immature B cells from the other partner (veto effect). The coincubation of APC-depleted donor and recipient PBLs also leads to the generation of allotolerant T cells.
  • T-depleted PBLs of donor and recipient to be provided with a 1: 1 mixture of donor and recipient-derived MIS effectors and to be injected into the recipient, the immunocompetent PBMCs thereof having to be pre-replicated in vivo.
  • the ways of tolerance induction discussed here can be combined with one another, but also with other methods for tolerance induction discussed in the present application, which could further increase the efficiency.
  • Th1 and Ts: Tc reprogramming is based on the combination (a) of ROS / RNS modulators such as XOD / xanthine (hypoxanthine) + SOD and / or calcium ion, with (b) sub-dose decouplers of respiratory chain phosphorylation (e.g. DNP, fatty acids, ADP) to be expected.
  • ROS / RNS modulators such as XOD / xanthine (hypoxanthine) + SOD and / or calcium ion
  • sub-dose decouplers of respiratory chain phosphorylation e.g. DNP, fatty acids, ADP
  • the efficiency of the HTT effectors can be further increased by xenogenizing the individual partner cells, primarily the autologous and / or allogeneic APCs or else the hybridoma or trioma cells. All of the literature, e.g. in the “tumor immunology” of Warnatz. Alternatively, heterologous cells can be used as additional fusion partner cells for the purpose of xenogenizing the cell constructs.
  • the fusion technique could also partially or completely transfect the plant germ or sperm cells with genes, for example, for the breeding of new plant species replace higher resistance to drought, frost etc.
  • the advantage of this technique is that the resistance-coding cDNA need not be known.
  • Tolerance can also be achieved by in vitro or in vivo co-incubation of donor and recipient PBLs in the presence of (a) IL-10 and other type 2 / Th2 cytokines (b) Gs agonists (c) inhibitors of cAMP-specific PDE ( Phosphodiesterase) and (d) NOS stimulators or NO-releasing substances can be achieved.
  • Th2 cytokines e.g. IL-10
  • tolerance to individual transmitted cells can be achieved.
  • the tolerance e.g. the tumor-specific tolerance by (a) IL-1 2, IFN, TNF and other Th1 cytokines (b) by Gs antagonists (c) by stimulating the cAMP-specific PDEs and (d) by NOS inhibitors.
  • a combination with MIS and anti-T-Mab pretreatment (for the purpose of Ts and Th2 pre-elimination) enables a significant improvement in all historical (Coley) and modern immunotherapeutic methods which are based on immunostimulation by means of inactivated bacteria (for example, bladder tumor treatment with BcG and chemotherapy ) or bacterial lysates and extracts (as BRMs).
  • a special technique enables the maturing, antigen-specific and / or non-specific T cells to be controlled in the direction of the Th1 or Tc subset by removing the T4 cells from the in vitro and / or in vivo with anti-CD4 Mabs Surface of the Ag-presenting accessory cells "keeps away” to them 3-5 days later (as a pure T4 population) in the presence of IL-2 (and IFNgamma) - secreting Th1, more specifically activated pTH1 cells, unilaterally in the direction to "program” the CTL / Tc subset.
  • MIS / MIT effectors whose life expectancy is "pre-programmed” for 3-5 days. Both techniques can be combined.
  • An alternative method to the MIS / MIT technique which does not require the cell death preprogramming of the MIS / MIT effectors or could be used to eliminate the GvHR effectors in a GvHR that is hardly to be expected after MIS / MIT treatment, is the treatment of Donor -PBLs or T cells with strong haptens, followed by their transfusion into the recipient and immunization of the same or a different donor with its own haptenized PBLs or donor MIS / MIT effectors can now use the donor MIS / MIT- Effectors (after a mission or to prevent an unexpected GvHD side effect) are removed from the recipient.
  • the donor is first immunized with PBLs, B cells, T cells or blood of the recipient after organ transplantation has taken place. If organ rejection is imminent, the plasma or the IgG isolated from it can now. Fraction (with or without the donor-derived MIS / MIT effectors) injected into the recipient, whereby the alloreactive T cells of the recipient responsible for organ rejection are eliminated.
  • the same principle can also be used to combat GvHD or HvGR in bone marrow recipients.
  • Th1 is the T cell or. PBL activation by mitogenic lectins (e.g. PHA) and / or mitogenic Mabs (anti-CD3, anti-CD2, anti-CD28 Mabs) in the presence of Gs blockers (e.g. propranolol) and / or stimulators of cAMP-PDE or Inhibitors of cGMP-PDE.
  • mitogenic lectins e.g. PHA
  • mitogenic Mabs anti-CD3, anti-CD2, anti-CD28 Mabs
  • Gs blockers e.g. propranolol
  • Th2 generation is achieved through mitogenic stimulation in the presence of Gs agonists and / or cAMP-PDE blockers.
  • Th1 and Ts cells An elegant way to quickly generate Th1 and Ts cells in vivo is the combination of (a) radical T cell depletion (using anti-T cell Mabs plus MIS effector cells) (b) the subsequent treatment with MIT effectors and (c) post-injection from immature precursor T cells or T-depleted PBLs of the recipient (patient).
  • Th1 and Tc generation treatments (a) with ROS (H02, H202) -releasing systems (for example XOD / xanthine; GOD / glucose; low-dose, ROS-generating cytostatics such as mytomycin C, bleomycin, Cyclophosphamide and their combinations plus (b) with various cell stimulators such as BRMs, cytokines or lectins.
  • ROS H02, H202
  • Th1 or Ts Tc reprogramming can also be achieved by combining ROS-releasing systems plus cAMP-PDE stimulators or cGMP-PDE inhibitors.
  • FIG. 1 shows the strong activation of lymphocytes by subtoxic doses of
  • Figure 3 shows the activation of PBLs by adding XOD / xanthine to the medium
  • FIG. 4 shows the dependence of the activity of lymphocytes on the superoxide content
  • Figure 5 shows the strong activation of PBMCs with a combination of ammonia
  • Figure 6 shows the influence of XOD / xanthine on the activity of PBLs
  • Figure 7 shows the improvements in activity of PBLs with a combination of
  • Figure 8 shows the influence of the GOD / glucose system on immune-competent cells
  • FIG. 9 shows the receptor-independent activation of PBLs with XOD / substrate or
  • Figure 1 the stimulation of lymphocytes with a subtoxic dose of
  • Figure 12 shows the influence of superoxide concentration on cell activity
  • Figure 13 shows the strong immunosuppressive effect of catalase on PBMCs
  • Figure 14 shows the immunosuppressive effect of SNAP on lymphocytes
  • Figure 1 5 shows the immunosuppressive effect of sodium prusside on PBLs
  • Figure 16 shows the influence of different SNOG concentrations on PBL activity
  • Figure 17 shows the suppressive effect of promethazine on lymphocytes
  • Figure 18 shows the effect of diethylamide NONOat on PBLs
  • Figure 19 shows the effect of hydroxyguanidine on PBMC activity
  • Figure 20 shows the effect of the phosphodiesterase inhibitor MDL-1 2, 330 A on the
  • Figure 21 shows the effect of XOD / xanthine on PBMC growth
  • Figure 22 shows the influence of a H0 2 scavenger on the cell activity of lymphocytes
  • Figure 23 shows the influence of the GOD / glucose system on the activity of PBLs.

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Abstract

La présente invention concerne des constructions cellulaires, leur procédé de production et leur utilisation dans le traitement de maladies induites par des cellules T activées indésirables.
EP00972821A 1999-10-22 2000-10-20 Constructions cellulaires adaptees a l'immunotherapie, leur production et leur utilisation Withdrawn EP1226235A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19951072A DE19951072A1 (de) 1999-10-22 1999-10-22 Neue Wege der Immunmodulation und deren therapeutische Anwendung
DE19951072 1999-10-22
DE10016846 2000-04-05
DE10016846A DE10016846A1 (de) 2000-04-05 2000-04-05 Neue Strategien und Technologieverbesserungen in der Immuntherapie
PCT/EP2000/010340 WO2001029193A2 (fr) 1999-10-22 2000-10-20 Constructions cellulaires adaptees a l'immunotherapie, leur production et leur utilisation

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Publication number Priority date Publication date Assignee Title
WO1993023080A1 (fr) * 1992-05-13 1993-11-25 The Beth Israel Hospital Association Cytotoxicite d'especes activees ciblees
WO1996030030A1 (fr) * 1995-03-31 1996-10-03 Universite Libre De Bruxelles Hybrides et hybridomes de cellules dendritiformes/cellules tumorales servant a provoquer une reponse antitumorale
WO2000057705A1 (fr) * 1999-03-31 2000-10-05 University Of Pittsburgh Of The Commonwealth System Of Higher Education Induction in vitro de cellules t specifiques a un antigene a l'aide d'immunogenes derives de cellules dendritiques-cellules tumorales ou de cellules dendritiques-cellules virales

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WO1990003185A1 (fr) * 1988-09-28 1990-04-05 Ideon Corporation Immunotherapeutiques enzymatiques de combinaison
US5310541A (en) * 1992-08-27 1994-05-10 Montgomery Robert E Antimicrobial rawhide animal chew containing an oxidoreductase and oxidoreductase substrate
AU744042B2 (en) * 1997-04-15 2002-02-14 Dana-Farber Cancer Institute, Inc. Dendritic cell hybrids
EP1107781A2 (fr) * 1998-08-28 2001-06-20 University Of Bath Ameliorations du traitement des lesions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023080A1 (fr) * 1992-05-13 1993-11-25 The Beth Israel Hospital Association Cytotoxicite d'especes activees ciblees
WO1996030030A1 (fr) * 1995-03-31 1996-10-03 Universite Libre De Bruxelles Hybrides et hybridomes de cellules dendritiformes/cellules tumorales servant a provoquer une reponse antitumorale
WO2000057705A1 (fr) * 1999-03-31 2000-10-05 University Of Pittsburgh Of The Commonwealth System Of Higher Education Induction in vitro de cellules t specifiques a un antigene a l'aide d'immunogenes derives de cellules dendritiques-cellules tumorales ou de cellules dendritiques-cellules virales

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