WO2001029193A2

WO2001029193A2 - Cell constructs that are suitable for immunotherapy, the production and the use thereof

Info

Publication number: WO2001029193A2
Application number: PCT/EP2000/010340
Authority: WO
Inventors: Peter Leskovar
Original assignee: Peter Leskovar
Priority date: 1999-10-22
Filing date: 2000-10-20
Publication date: 2001-04-26
Also published as: EP1226235A2; AU1141901A; WO2001029193A3

Abstract

The invention relates to cell constructs and to methods for producing the same and to their use for the treatment of diseases that are caused by undesirable activated T cells.

Description

Cell constructs suitable for immunotherapy, their production and

use

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. Among other things, a preparation is provided with which malignant tumors can be combated and a recurrence or the occurrence of metastases can be prevented.

Many tumor therapies fail because the surgical and / or chemotherapeutic removal of the tumor never destroys all tumor cells and, in addition, the tumor-protecting "tumor-protective" suppressor cells are not detected. A similar problem also occurs with chronic viral diseases that are difficult to combat. According to the invention, therefore, a method and a suitable means are provided to combat these diseases. 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.

In one embodiment, diseases are combated according to the invention in which protective cells are present in addition to undesired T cells. Examples include neoplastic and virus-related diseases.

According to the invention, the following method is carried out for this:

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. Examples of suitable antibodies are 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.

Normally, not all T cells and especially not all suppressor cells are detected in this treatment. According to the invention, the activated T cells are therefore attacked in a second stage using lymphocytes with predetermined cell death. For this purpose, PBLs, ie peripheral blood lymphocytes, of a donor are preferably pretreated with mitomycin C in such a way that they can only divide about 3 to 15 times, preferably 3 to 5 times, and then die. In this way, 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. In order to prevent the disease from occurring again, ie the formation of new tumors and metastases or the development of viruses, the formation of new suppressor cells which would protect the tumor or the undesired cells again is prevented according to the invention. For this purpose, preparations called 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. In a preferred embodiment, 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. 6,110,244 and is also suitable for the present invention. A method as described in US-A 6008002 is suitable for obtaining MHC-II positive cells. Various methods are known for obtaining T cells, including one that is described in US Pat. No. 5,965,535.

The cell constructs obtained according to the invention, e.g. Hybridomas, triomas or quadromas are usually administered to the patient by infusion, preferably subcutaneously.

In order to further strengthen the patient's natural immune defense, he is preferably treated in a further stage with cells which have been clonally postexpanised in a manner known per se. To do this, 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.

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. For example, xanthine or hypoxanthine can be used as the substrate for xanthine oxidase. This creates ROI and increases the RIO / RNI quotient. This activates the cells. In addition, it has been found that 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. For in vitro activation, the cells are incubated with the enzyme and substrate in a suitable manner. For in vivo activation, 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.

It has now been found that if RNIs predominate in the cell, the number of suppressor cells increases, while if ROIs predominate, the number of Tc cells increases. The ratio of RNS to ROS thus influences the ratio of Th to Th ₂ or Tc to Ts.

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.

If autologous cells of the patient are used, 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.

According to one embodiment of the invention, 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. In order to destroy both tumor cells and the cells protecting the tumor, 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.

In order to further improve the tumor treatment, removal of the tumor is proposed before the treatment according to the invention, in particular by excision.

The methods proposed in EP 705 1 1 1 are particularly suitable for removing the suppressor cells. Therefore, these need no further explanation here.

It was also found that a high superoxide / peroxide (0 ₂ 70 ₂ ^{2 "} or H0 ₂ / H ₂ O ^{2 '} ) quotient in the cytosol or in the mitochondrial and / or microsomal endoplasmic compartment promotes cell differentiation. In other words: cell proliferation (cell division) is associated with high or elevated superoxide (0 ₂ ^' , H0 ₂ ) levels, while cell differentiation is associated with increased peroxide (0 ₂ ^2' , H ₂ 0 ₂ ) levels all tumor cells, regardless of the type and type of tumor, are characterized by a constitutively increased H0 ₂ / H ₂ 0 ₂ (superoxide / peroxide) quotient.

According to the invention, it is important to differentiate between the Th1 versus Th2 subpopulations of the DC4-positive helper / inducer T cells on the one hand and between the Tc / CTL versus Ts subclasses of the CD8 positive suppressor / cytotoxic T cells on the other hand. According to this, 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. If this critical peroxide concentration is reduced by a reduced 0 ₂ ^{2 "} replica (eg by increasing cAMP1) or by chemical neutralization of the immediate precursor (H0 ₂ ) If NO is exceeded, the formation of Th2 or Ts instead of Th1 or Tc / CTL subpopulations is promoted.

The applicant also found that the antigen-presenting cells (APC), primarily macrophages, at the critical moment of antigen presentation to naive, antigen-specific T cells by secretion of peroxide anion (H ₂ 0 ₂ ) the Th1 - or Tc- Induce subset, while the secretion of NO instead of H ₂ 0 ₂ leads to Th2 or Ts predetermination in this critical early phase. The following therapeutic consequences result from the new findings presented above:

(1) By manipulating the absolute or relative proportion of superoxide or peroxide anion in the target cells, these can be controlled either in the direction of proliferation or differentiation. In other words, either an increase in the number of target cells or an increase in efficiency, ie refinement of the function of existing target cells, can be achieved from the outside. For this purpose, one can use, for example, the enzyme xanthine oxidase (XOD) with the corresponding substrate xanthine and / or hypoxanthine, whereby the superoxide is primarily used in combination with other enzymes, catalase or peroxidase (glutathione peroxidase or horseradish peroxidase) - (H02) formation and with another enzyme, the superoxide dismutase (SOD), be it Mn, Cu / Zn or Fe SOD, conversely the peroxide (H ₂ 0 ₂ ) formation can be achieved. Peroxide formation can also be achieved with the enzyme glucose oxidase (GOD) and the corresponding substrate (glucose).

Another consequence of the latest findings is the in vitro and in vivo control of Th1 - vs. Th2 and / or Tc vs. Ts formation via interception of the H0 ₂ ^' and / or H ₂ 0 ₂ signals emitted by APC (macrophages) using specific scavengers. These scavengers are chemically well defined, not least with regard to the mechanism of action (binding of 0 ₂ - vs. 0 ₂ ² anion; some have been used as radical scavengers in vivo). Suitable scavengers are known to the person skilled in the art and therefore require no further explanation. Examples of suitable scavengers are described, for example, in "The Oxygen", Biochemistry, Biology, Medicine (author: EF Elstner), Wissenschaftsverlag Mannheim / Vienna / Zurich, 1 990. Experiments have shown that, in addition to the "chemical" scavengers mentioned above, "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. Another possibility is the 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. In a further embodiment, 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) are added, as a result of which the T cell modulation is controlled can. This has been confirmed by laboratory tests carried out by the applicant.

Since on the one hand 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, for example during the antigen presentation to neoantigen-specific naive T cells, may give rise to unwanted immunosuppression. In a preferred embodiment, these exoenzymes are therefore neutralized as undesired suppressor factors with appropriate antibodies.

(2) According to the invention, for the first time a possibility is provided to influence the direction of the immune defense (Th1 / Tc vs. Th2 / Ts) during the first contact with the foreign antigen ("priming") in vitro and in vivo, and on the other hand a subpopulation of the T cells to convert to the other at least functionally. It should be emphasized that this conversion does not depend on pre-existent receptors because, unlike in the case of the cytokines, the modulation is mediated by membrane-bound mediators.

The selective promotion of the Th1 and Tc subsets as well as the in vitro and in vivo Th2, Th1 and Ts: 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) Ca antagonists, in particular nicardipine and niphedipine),

(b) antagonists of the Gs (Gsalpha) -conjugated membrane receptors, such as 2-AR and A2-purinergic (adenosine) receptors, which can still be combined with agonists of the Gi (Gial-pha) -conjugated receptor subtypes, and

(c) the addition of inhibitors of NO synthesis (for example inhibitors of constitutive and / or inducible NO synthetases), which are also commercially available. If, on the other hand, 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. The fine regulation of the T cell subset interconversion in vitro (not in vivo) 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. (3) In a further embodiment of the present invention, representatives of an important class of substances, namely the various cAMP- or cGMP-specific phosphodiesterases (PDEs), are used as T-cell modulators.

With inhibitors of 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. In addition, 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.

Analogously, stimulators of the cAMP-specific and non-specific PDEs (see above) 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. In addition, 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). Since a temporary increase in cAMPi is also observed during cell activation by interferon (IFNgamma), the inhibition of cAMPi degradation by cAMP.PDE inhibitors in the critical phase of GvHD and HvGR induction could prevent these fatal side effects in the transplantation.

(4) As the detailed investigations in the applicant's laboratory showed, high proliferation rates of test cells (PBLs / PBMs) 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.

(1) The present invention relates to preparations and methods which are based on a method known as "microimmune surgery" (MIS). 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

Transfection / transduction of the donor effector cells (lymphocytes, PBLs / PBMCs, agranulocytes, "buffy coat" / - "lymphocyte concentrate") with the viral (HSV) Thymidine kinase (TK / tk), followed by later selective in vivo elimination of the labeled donor cells using ganciclovir. The use of this so-called "suicide" technique, which is based on HSV-TK / ganciclovir, can be used according to the invention for all areas of application which are specified for the MIS method in EP 0 705 1 1 1.

(2) Another object of the present application relates to the so-called "microimmuno targeting" (MIT) technology. It has many similarities to the (MΙS) technique described in detail in EP 0 705 1 1 1. The main difference lies in the in vitro pretreatment of the donor lymphocytes (PBSs / PBMCs), more precisely in the concentration of the bivalent alkylating antibiotic mitomycin C (and others, DNA) used to bridge the two DNA strands of the donor PBSs harmful compounds). While the MIS technique on the prevented proliferation or based on the pre-programmed cell death for the purpose of an increased GvL / GvT reaction while preventing GvH disease at the same time, 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. As detailed in-house experiments have shown, 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. 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. In autoimmune diseases, 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). The pathologically hyperactivated (incorrectly programmed) patient T cells are recognized by their obligatory post-expression of class II MHC (HLA-DR / DQ) antigens.

Interestingly, such 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.

The direct inactivation of incorrectly programmed T cells by the so-called "veto effect" is also likely to play a certain role, with the additional interaction of B7.1 - (CD80) -, B7.2- (CD86) - and B7. 3- (BB1) antigens on the hyperactivated patient T cells with the corresponding CD28 and CTLA-4- Structures on the donor effector cells lead to the selective elimination of the former. As detailed tests have shown, the critical concentration of mitomycin C - after an 18-hour incubation - is 10 / g / ml or 10 μg / 1,106 donor PBLs / PBMCs. If the mitomycin concentration is at or above 10 / g / ml, the donor effector cells are programmed to MIT effectors; MIS preprogramming of the donor effectors results at mitomycin C concentrations below 10 // g / ml.

(3) 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). It must be prevented that in the immunosuppressed organism, e.g. after tumor excision, after chemotherapy and radiotherapy as well as bone marrow and organ transplantation immature 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. One thinks primarily of the tumor- or (retro) virus-specific T- (and B -) - subpopulations. The consequence of such an in vivo meeting of "incorrectly programmed" immunocompetent immunocytes with their precursors is a tolerance re-induction towards the remaining tumor cells or viruses, which enables their unimpeded multiplication.

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. As there are no Th1 and Th2 specific Mabs to date, 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.

According to the invention, the interruption of the misguided precursor T cell differentiation (“commitment”) to Th2 or Ts cells by the selective in vivo depletion (“cutting out”) of these pre-T cells with appropriate Mabs (OKT6, OKT9, OKT10 ) to bridge the unstable phase until immune competence is regained. The fact that when polyclonal antibodies against T cells, such as ATG or ALG, are used, the complications with secondary infections and / or lymphoproliferative diseases (based mostly on the reactivated EBV infections) are much less common than when using monoclonal Mabs (eg OKT3) can occur due to the simultaneous, although temporary depletion of mature T (Th2, Ts) cells plus their precursors in the former case (polyclonal AK) or the sole elimination of mature T cells in the latter case (monoclonal AK / Mab) can be explained.

For the same reason, in the conditioning of tumor patients, both in the treatment of solid tumors with "microimmune surgery" and in bone marrow transplantation (KMT) of the tumor patients (allogeneic KMT in leukemia and lymphoma patients and autologous KMT in certain types of solid tumors) according to the invention Use of polyclonal antibodies (ATG, ALG, ALS) preferable to that of the panT or CD3-specific Mabs.

Among the Mabs, however, there are those that, in addition to mature T cells, at least partially also detect immature precursor T cells. Examples are 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.

(4) 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. (5) 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). Already in the second phase of their development, ie as so-called ThO cells, the T4 cells secrete IL-4 and other Th2-type cytokines in addition to IL-2; In the third phase, these activated, antigen-specific T4 cells are differentiated into either the Th1 or Th2 subtype. In order to exclude the Th2-type lymphokines which direct young T4 cells in the direction of suppressor T cells (Ts cells), it is recommended according to the invention to treat the precursor or naive T4 cells in vivo in such a way that only until the 1st Stage ("activated naive Th cells") can develop and die before reaching the Th2 cytokine-type-secreting ThO stage. To achieve this goal, the same procedures and substances are used as for the pre-programming of cell death / suicide in the "microimmune surgery" described in EP 705 1 1 1. As an alternative to this, the addition of 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. specific CTLs on the exclusively IL-2 (not IL-4) secreting Th ("activated naive Th cells") on or in the vicinity of macrophages (and other APCs) presenting the relevant antigen, which leads to an increased to exclusive Ts -free CTL generation leads. According to the invention, treatment with cyclophosphamide 2 to 7 days before priming with the relevant antigen (eg tumor and virus 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".

(6) According to the invention, it was found that 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. Since in the phagocytosis of corpuscular / particulate, pre-aggregated, pre-naturalized and hydrophobicized antigens (eg proteins) the ROl / ROS level automatically increases as a result of the "respiratory burst", this is achieved according to the invention by simultaneous administration of particulate antigens (low-dose erythrocytes of foreign blood group, inactivated bacteria or their lysates, complete Freund's adjuvant / CFA, lyposes, denatured foreign proteins) the desired effect, ie a temporary increase in ROI in APCs during the presentation of the tumor - or virus antigen.

(7) The presence of 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. 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). In the former case, 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.

(8) In the gene therapy approach, it is recommended according to the invention that the target cells for gene therapy are the macrophages, the dendritic cells and the like. To provide 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. In this case, 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.

(9) The HTT technology described in the patent application PCT / EP 94/01992 (EP 705 1 1 1 », based on (a) on the fusion of tumor cells of the patient with autologous and / or allogeneic, MHC-II positive cells ( Macrophages, monocytes, dendritic cells, B cells), and (b) on the fusion of specialized immune cells (Th1, Th2, Tc, Ts etc.) with malignantly transformed cells / cell lines (for the purpose of immortalizing the specialized immune cells), should now be carried out by others new findings and important improvements are to be added, and this new technology, which has now proven to be very successful in the clinic, is also to be included all of their variants on other indication areas are protected by patent.

Because of the common principle of action, i.e. The novel in-vivo elimination of the pathologically dysregulated immunocyte subsets, combined with the re-establishment of disease-fighting effector cells, is intended to increase the range of indications for HTT technology and its refinements, e.g. the HTT-AIT technique, in addition to the treatment of tumors (solid tumors and all forms of blood cell cancer), now also the treatment of autoimmune diseases, allergies, infections (bacterial and (retro) viral infections including HIV infection), arteriosclerosis, rejection reaction in organ transplants as well as GvHD and HvGR (rejection) in bone marrow, stem cell and progenitor cell transplantation. Since only hybridomas consisting of e.g. from tumor cells plus allogeneic APC (macrophage, dendritic cell) the recruitment of new tumor-specific Tc (Th1) does not work, it is recommended to load autologous APCs with tumor cells, their lysates or, if available, tumor-specific peptides and with the hybridoma cells in the patients i.v. or s.c. to inject.

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. It should also be pointed out here that the term MIS / MIT, which was introduced later, stands for "cell death preprogrammed allogeneic effector cells" and the term 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. After clinical studies clearly confirmed the high efficiency of the principle protected in EP 705 1 1 1, this principle in a completed form as the so-called HTT-AIT technology is now also to be used for other indicators such as autoimmune diseases, allergy, atherosclerosis, and for treatment of bacterial and (retro) viral infections, including HIV, as well as in the fight against GvHD and HvG complications in organ and bone marrow transplants, but taking into account some peculiarities of the individual clinical pictures. In 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.

Also in the treatment of allergies, 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. Similarly, in atherosclerosis, the fusion of autologous and / or allogeneic MHC-II positive cells with 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.

In all those clinical pictures in which autoaggressive or alloaggressive T cells are actively involved (autoimmune diseases, graft rejection and GvHD), it is recommended according to the invention that the in vivo depletion of this disease or complication-causing T cell subsets with a further step, namely to supplement the stabilization of the non-aggressive state. Antigen-specific Ts (and Th2) cells are suitable for this purpose. PGE2, etc.) and in the case of transplant complications using the one-way MLC approach (in the presence or absence of mentioned immunosuppressive additives) subsequent inactivation of the alloreactive T cell subpopulations, be it with radiation (50 to 100 gy) or mitomycin C (20 to 50 // g / 106 cells), in vitro. In the above-mentioned in vitro enrichment of the antigen-specific T cell clones, which are required, for example, in the treatment of autoimmune diseases, but also of allergies with the HTT-AIT technique as a fusion partner, the co-incubation of the patient's PBLs with the Autoantigen or allergen as well as with IL-2 recommended, whereby the low-dose IL-2 can still be combined with IFNgamma and / or IL-1 2. In the special case of allergy treatment, 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). For this purpose, cells expressing Fc-epsilon-R (B cells, mast cells, basophils) can be loaded with IgR or the Fc subunit in vitro and then fused with autologous cells. In this way, vaccination against allergy is generally carried out using the HTT-AIT technique. If you want to treat with allergen selectivity, 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 (fusion and SC vaccination or IV injection) are the same as above.

The completed HTT-AIT technique also brings additional improvements in the treatment of solid tumors and leukaemias / lymphomas. In the special case of all those blood cancer forms which are characterized by the presence of bcr-abl fusion protein (eg CML), immunization (sc, id or iv) with 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.

The fusion of 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; In addition, the first-time use of two types of fused cells, namely the TumorAPC hybridomas ("HTT technique") and the Ts: APC hybridomas ("HTT AIT technique") 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"). The additional use of the MIS / MIT strategy suggests a further increase in efficiency. An important improvement of the MIS / MIT effectors, which is referred to in EP 705 1 1 1 as "cell death preprogrammed alloreactive donor cells", consists according to the invention in their prealloimmunization. For this purpose, 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. The advantages of this combination of MIS / MIT, HTT and HTT-AIT treatment are briefly described again: (a) MIS / MIT effectors are used to eliminate tumor-protective Ts (and Th2) cells subset- but not tumor-specific, (b) HTT -AIT technology enables one to a certain extent tumor-specific depletion of protective T cells. (c) With MIT effector cells that are prevented from cytolytic activity by higher-dose (20 to 50 //g/1.106 cells) mitomycin-C pretreatment, but are not impaired in their metabolic performance, APCs (macrophages) are affected by the 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. With the MIS and the HTT-AIT technique, HIV-specific, but also opportunistic infections protecting Ts (Th2) are eliminated; With the MIT and HTT technology, APCs (macrophages) are reprogrammed in the Th1 direction and Th1 - and Tc- instead of Th2 and Ts subsets are induced.

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.

Another important point needs to be addressed. Since the fusion with autologous MHC-II positive cells (APC like macrophages and dendritic cells) An exceptionally efficient presentation of the surface antigens of the fusion partner cell (e.g. tumor cell), which has also been clinically confirmed in the meantime, enables one in co-fusion (trioma formation) with allogeneic APCs or when combining hybridoma cells based on autologous APCs with hybridoma cells based on allogeneic APCs Another positive effect, namely introducing the Th1 programming of the new cell construct, is recommended according to the invention to strengthen all conventional immunizations with this type of fusion construct. Here one thinks primarily of all forms of vaccines, ie those that are directed against infections (bacterial or (retro) viral), but also of all types of tumor vaccines. The vaccines can be administered either subcutaneously / intradermally or iv. In HTT-AIT refinement, the premature weakening of the immunization process, initiated by counter-regulating pathogen or tumor-specific suppressor cells (Ts, TH2), can be achieved by using hybridomas / triomas consisting of autologous and / or allogeneic MHC-II positive cells, plus pathogen or tumor-specific Ts (Th2), are prevented or at least stopped.

(10) The invention also relates to two preparations which are used in bone marrow or Stem cell / progenitor cell transplantation can be used. In 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. As a result, 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. In addition, 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. However, 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. Alternatively, 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.

(1 1) Another object of the invention relates to the regulation of Th1, Th2, Tc / CTL and Ts subsets of the T lymphocytes. According to the applicant's knowledge, 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.

In addition, the inventor found that the Tc / CTL and the Ts cells represent the same CD8 + cell, which is in two "frozen" functional states, the Tc and Ts states, respectively. 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. Even more, 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 (e.g. in the case of tolerance induction or autoimmune diseases) are also considered and claimed here. Since 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.

(1 2) 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. As in the case of viral cell infestation and (malignant) cell transformation, gene products (peptides / polypeptides / proteins) of both fusion partners are expressed on the cell surface in the context of the MHC-I complex. The cell nuclei required are obtained using classic methods Enucleization processes (e.g. with LyseS, with various surfactants and their combinations with the help of NH4CI or Saponin etc.).

An interesting application of combined cell nuclei: According to the invention, 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. In this way, a donor-specific tolerance can be achieved in an elegant manner. Another interesting special case is the fusion of the donor cell nucleus with the recipient stem cell, whereby the donor cell nucleus can be taken from a donor stem cell or somatic (haematopoietic) cell. Here too, the same principle of the "Trojan horse" prevents early rejection of the foreign (donor) cell. In all HTT-AIT techniques, including their modifications, 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).

(13) 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. 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. 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).

In the 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.

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. (on macrophages) or CD83 or T6 (on dendritic cells) 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. With all-allergen-specific antibodies of other, non-IgE subclasses, e.g. of the IgG, IgM and / or IgA subclass, 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.

In the special case of allergy, it is recommended according to the invention to use the 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. 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. It was also observed that the same fusogenic polymers (PEG, PVP and combinations) show better cryobiological properties than the generally recommended but toxic DMSO for freezing cells. Both properties, immune stimulation and cell protection when the cells freeze, are more pronounced in high molecular weight polymers (PEG, PVP) than in low molecular weight representatives. The use of the polymers mentioned (PEG, PVP and combinations) (a) for immunostimulation and (b) for cryoprotection is the subject of the present application. Further improvements of the 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

Hybridoma / trioma / quadroma formation at suboptimal concentrations of fusogens (e.g. PEG, PVP, PEG / PVP combinations).

Since the fusion of cells can take place both by chemical means and by physical means and various methods are available to the person skilled in the art for both chemical and physical fusion, it is obvious to the person skilled in the art that whenever a chemical fusion is described a physical fusion can also be carried out, e.g. with electrofusion / electroporation.

Instead of or in addition to cytokeratin-recognizing antibodies, 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. In this case and in the case of an even more selective, clonotypic hybridoma formation in which the antigen-specific B cell clones are recognized, the splenocytes of the immunized animal must first be preactivated in vitro in the presence of the specific antigen. In this clonotypic fusion, the microtitration plates must first be coated directly or indirectly (via poly-L-lysine treatment) with the specific antigen. Alternatively, all other selection methods used in conventional Mab extraction can be used.

If 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.

(14) Another point is the stimulation of immunocytes by fusogens in subfusogenic concentrations, whereby selectivity can also be achieved according to the invention by means of bifunctional and / or hybrid antibodies by approaching the interacting immunocompetent cells.

(1 5) A few more points should be mentioned:

(a) In the case of bone marrow transplants (BMT, SCT, PCT), the resistance to viral and / or bacterial infections can be specifically transferred to the recipient by using donors (one or more), which has been achieved clinically several times.

(b) According to the invention, 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.

(c) 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 (HTT) 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.

Since, according to the latest findings, temporary damage to the mitochondrial membrane enables the release of H0 ₂ and H ₂ 0 ₂ into the cytosol and indirectly into the intranuclear space, it is recommended to use Fc and partly Cu salts in combination with ascorbic acid and various org , Acids (via Fenton and Haber / Weiss reaction) to provoke this transient damage to the mitochondria - and indirectly the nuclear membrane. According to the latest findings, this leads to cell activation, even more so to type 1 (Th1) cell pre-programming. Another aspect is the direct reaction of Fe ³⁺ with H0 ₂ , which leads to a reduction in the H0 ₂ / H ₂ 0 ₂ quotient and thus to type 1 (Th1) differentiation at the expense of proliferation, i.e. switching the cell function from proliferation to differentiation leads.

Since such different clinical pictures as tumor, chronic viral and bacterial infections (including AIDS), allergies and even atherosclerosis appear to be associated with an increased Th2 / Th1 quotient, the following procedure is recommended to correct this quotient: First, mature, dysregulated T cells must be used of the patient on a humoral plus cellular level, ie with Mabs (plus Mab-saving cytostatics) and MIS effectors, can be radically eliminated without damaging the immature progenitor cells. With 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. This one-sided 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. In addition, the transfected DCs (APCs), with and without post-fusion, with hybridoma or trioma cells, 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). A previous cell activation has a favorable effect.

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.

With 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. Examples: Galagin, Morin, Catechin, Epicatechin. With other flavonoids, which inhibit both the synthesis of prostaglandins and that of leukotrienes, an anti-inflammatory effect can be achieved. Examples: quercetin, fisetin, luteolin, camphor oil, taxifolin, eryodictiol, 3 '4' - dihydroxyflavon.

It is known from animal experiments with bone marrow chimeras that the best results can be achieved in the presence of donor and recipient T cells plus APCs in the recipient animal. This can be achieved clinically as follows: (a) First, with a 1: 1 mixture and mitomycin C- pretreated (MIS effectors) or untreated donor-plus recipient T cells (or PBLs) in the myeloablative-pretreated recipient ( Patients), the mutual tolerance are induced, (b) Then the 1: 1 mixture of immature bone marrow or stem cells or T-depleted PBLs of donor and recipient is infused. (c) Now mature T cells and APCs from donor and recipient can be post-transfused. Note: This procedure can be simplified by transfusing only mature donor T cells as the last step.

As is also known from the animal model, 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. 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.

An important finding from our laboratory and animal experiments is that 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. According to the invention, it is therefore recommended to inject relatively small numbers of tailor-made mature T cells into a freshly in vivo T-depleted recipient. 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).

Our laboratory and animal experiments showed that when mature donor and recipient T cells (PBLs, spleen cells) are mixed, but better the MIS effector cells derived from them, there is a mutual tolerance, which also does not include the outbreak of GvHD in KMT Can prevent T cell depleted donor marrow in advance. For this reason, it is recommended according to the invention (a) to treat the recipient with a 1: 1 mixture of mature donor and recipient T cells (PBLs) or advantageously from corresponding MIS effector cells before the actual KMT or HSCT, and (b) instead of the donor KM or stem cells to provide their 1: 1 mixture with corresponding recipient cells.

This GvHD-free technique is recommended according to the invention for the therapy of all those genetic diseases that are referred to as “correctable” or “treatable” for KMT, but which in practice have only rarely been treated with KMT, since classic KMT has considerable treatment risks connected is. This situation could now "change" seriously due to the improved technique described above. The desired replacement of the classic KMT associated with myeloablative patient conditioning by simply supplementing pathological T cell subclones with those of the healthy donor, which takes place under non-ablative conditions goes in the same direction. Since these new techniques, both of which are the subject of the present application, do not pose a lethal outcome risk, they could, as the better alternative, replace gene therapy with many additional problems in these genetic diseases.

An important further development of cell fusion or cell hybrid technology (HTT) is the "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.

In addition to tumor therapy, 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.

Another new development, the so-called "clonotypic" anti-idiotype vaccine (HTT / AIT-CLO) goes even further in the selectivity and thus freedom from side effects of the vaccination. Here, according to the invention, 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. This enrichment technique is also suitable for the "T-subset" -specific vaccine discussed above. The areas of application of the "clonotypical" vaccine correspond to those of the "T-subset" -specific vaccine (see above). However, the prerequisite is that the specific antigens ( Autoantigens, allergens) are known or are commercially available.

According to the invention, 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). 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. In the first strategy, 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. In the second strategy, the recipient first receives cells from a histocompatible donor and then (1-3 weeks later) cells from a histocompatible second donor. By using both new strategies, T cell depletion from the donor market can be eliminated. In a third variant, the recipient first receives histocompatible bone marrow and then a more incompatible graft from a second donor; Here 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.

In the specialist literature, there is often talk of "mixed allogeneic chimerism" as the highest goal in allogeneic bone marrow and stem-line transplants (KMT or HSCT). According to our knowledge, such a common chimerism is more likely in the 1st phase of KMT or HSCT counterproductive, because it brings the desired GVM (GVL / GVT) effect to a standstill before it can develop its anti-tumor effect.Therefore, according to the invention, it is recommended to proceed in two steps: a) first, by a time-limited fully allogeneic (donor type ) Chimerism the attack on residual tumor cells is started, in order to be replaced later by a real "mixed allogeneic chimerism" after the residual tumor cells have been eliminated; the latter serves to stabilize the state of tolerance between the donor and recipient. Since MIS effector cells are recommended for both forms of chimerism according to the invention of unmanipulated mature T cells (PBLs; DL effectors; spleen cells in an animal model), GVH or HVG complications are to be expected in this system.

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.

According to the invention, further ways of inducing tolerance are injecting 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. The basic rule here is that in vitro (e.g. in the MLC approach) pre-generated, mature Th2 (Ts) cells must be transfused into immature T cell precursor cells in the recipient.

In gene therapy approaches to tumor treatment, 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. According to the invention, 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. To increase mutual tolerance ("mixed allogeneic chimerism"), immunosuppression (glucocorticoids, CSA etc.) can be combined.

An additional way to get the dysregulated (pathological) T-cell subclones under control is their 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), with or without fusion with autologous or allogeneic APCs, followed by their reinjection in the patient.

Additional security in the elimination of any 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.

Of particular interest in this post-MIT / MIS and post-HTT stimulation are all cAMPi-reducing (e.g. propranolol) and Th2 / Ts inhibiting substances (e.g. ca antagonists).

The 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. Even more, with the use of inactivated (5000 - 10 OOOcgy) or fused with MHC II + cells (DCS, macrophages) 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.

The poor results of DLI (donor lymphocyte infusion) in ALL patients, sometimes 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). However, it should be mentioned that in this case 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). In this way, the quality and efficiency of the patient's tumoral CTLs to be selected are significantly increased. A further increase in efficiency of the in vitro post-expanded tumor-lytic CTLs can be achieved if the patient is pretreated with additional modern methods such as MIS / MIT before the preparation of his tumor-specific CTL. This could also be formulated differently by recommending the combination of MIS / MIT (+ post-MIS / MIT stimulation with BRMs), HTT and post-expansion of the CTLs, which are qualitatively improved by MIS and HTT.

In all immunosuppressive situations, including allergic diseases, which are characterized by an increased Th2 / Th1 ratio, it is recommended to reduce the intracellular cAMPi (e.g. with Gs-coupled receptors with antagonists) and at the same time to reduce NO synthesis (by inhibitors of iNOS and the cNOS), since the NO stimulates PG synthesis and inhibits 5-lipoxygenase.

After the results in high-dose chemotherapy, in which the patient's immunocytes are replaced by own or foreign (allogeneic) stem cells, are disappointing, it is recommended according to the invention to add the critical immunocompetent phase to the stem cells and bone marrow cells by adding MIS effector cells bridged. The z. Currently very current DLI / DLT technology can be significantly improved or its main complication, the z. T. 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 μg / 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.

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.

An interesting development is to first eliminate the remaining tumor and (premicro) metastatic cells in vivo under milder, non-ablative conditions, using cyclophosphamide and Mab or, preferably, only Mab, with MIS effector cells. Whether this is direct or indirect, i.e. unblocking the patient's immune system is irrelevant. After giving these MIS effector cells 2-10 days to perform this task, they are replaced by immature donor bone marrow or stem cells after preprogrammed cell death. Alternatively, a 1: 1 mixture of similar cells from the donor and recipient can be used. 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.

A similar basic idea lies in the recommendation according to the invention, firstly using MIS effector cells to eliminate tumor cells and the Ts (Th2) cells protecting them in a radical manner, and then using the MIT effectors to reprogram the APCs from the suppressor monocytes into the to accomplish tumoricidal effector function. The main difference between MIS and MIT effector cells ligated in the concentration of the mitomycin C used: while the cytolytic MIS effectors are treated with mitomycin C concentration 10ug / ml / 1,106 cells, the non-cytolytic MIT effectors contain mitomycin C concentration - 1 5 or more - 20ug / ml / 1 .106 cells.

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, cγclosporin 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 the encounter of immature (donor and / or recipient) cells on pre-formed mature allotolerant T cells.

In contrast to solid tumors, 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. In this case, 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. Among other things, 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. It is sufficient if they are metabolically active but cannot proliferate; these latter properties are important because the cell constructs or the cell suspension from which these cell constructs originated should be pretreated with mitomycin C and other substances that pre-program cell death in order to dispel any concerns about the re-injection of malignant cells into the patient. 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. Subsequent treatment of the tumor cells with xenogenizing haptens and / or viruses enhances the effect. In addition, with Mabs (a) against Th2 and / or Ts cells (anti-CD4 and / or anti CD8 Mabs) or (b) against Th2 cytokines (IL-4, IL13, IL-10) the premature Formation of counteracting, TSA (tumor vaccine) or pathogen-specific Th2 or TS cells is prevented and thus the yield of Th1 or TC memory cells is increased.

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.

The technique of xenogenization with strong haptens (eg DNCB) or viruses (eg NCD virus) can also find practical application in another situation, namely in the “pre-marking” of the MIS effectors in order to avoid the “development” of non- or To prevent insufficiently inactivated (cell death preprogrammed) MIS effectors by subsequent immunization of the MIS recipient with precisely these strong haptens or viruses.

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.

In the case of bone marrow and stem cell transplantation (BMT; HSCT), it is recommended according to the invention to replace the classic transfusion of donor cells in the mostly myeloablatively pretreated recipient (patient) with a 1: 1 mixture of donor and recipient cells. After it has been shown that the best results in bone marrow chimeras are obtained by transferring (a) F1 - hybrid cells in the P1 - or P2 - parental recipient and (b) the mixture of P1 - P2 - plus P2 - P1 - According to the invention, 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.

For the in vitro elimination of residual tumor cells, it is recommended to precede the method just described with the transfusion of donor MIS effector cells alone, with the detection and elimination of the remaining tumor cells being given 3-10 days. The efficiency of these MIS effector cells increases with the degree of histo-incompatibility between D and R and the in vitro preallunization of the MIS effectors against the recipient. Note: (a) The 1: 1 (D: R) MIS effector mixture is optimally produced by D: R two ways MLC in vitro, followed by mitomycin C treatment.

(b) T-cell-depleted D and R-PBLs can also be used as immature cells.

(c) 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.

(d) The particular advantage of the method described above is the later, i.e. Post-transplant cooperation between T cells and APCs, which in (a) classic KMT and HSCT (b) in DLI / DLT and (c) in the scheme proposed by the Seattle group based on antologous transplantation (after cytoreductive treatment), followed by allogeneic transplantation is not given.

An important new finding is the significant improvement in MIS / MIT and HTT technology by combining with BRMs in general (and their combinations). Examples include: poly r /: rC, bacterial lysate, levamisole and Th1 cytokines such as IL-2, IFNgamma, TNFalpha, IL-12 and IL-1. Especially to we recommend the combination of IFNalpha (3x106 U / d sc, 5 x weekly over 4 weeks) plus rLL-2 (3-6 x 106 IU / m2 daily, 5 x weekly, 4 weeks long).

Since chemotherapy and radiation damage mature and immature cells in an unknown manner, it is recommended according to the invention to eliminate the first part of the patient's leukocytes by leukapheresis and the remainder by Mabs. The leukocytes separated after treatment, e.g. B. can be reinjected with 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). Among other things, this brings the advantage that the regeneration time for the patient's immune defense is shortened and the fatal immune incompetence phase is bridged or at least significantly reduced.

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.

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). The "quality" of the HTT effectors generated in this way could be further increased by combining both HTT modifications, i.e. by simultaneously fusing cytokine (Th1 type) and TSA-transfected tumor cells with the APCs (DCs).

After it could be shown in our laboratory that 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. (in pure PBS) and that, on the other hand, ROS withdrawal in the medium (through ROS-degrading enzymes such as catalase, peroxidase and / or Mn-Cu / ZnSOD) leads to cell suppression, cell stimulation by ROS / ROI and all mediators of its generation on the one hand and cell suppression by all ROS scavengers (enzymatic and chemical) should be patented. In addition, the potentiation of rapid cell stimulation in two steps, ie by pretreating the cells (eg Th1 or Tc) with Catalase ,. (GSH) -peroxidase and / or Mn- or Cu / Zn-SOD in the 1st step, washing out and aftertreatment by XOD / xanthine-hypoxanthine and / or GOD / glucose (alternatively: H202) in the 2nd step. ROS / ROI can be combined with other, receptor-independent activation mechanisms, eg via canonophore or Ca ionomycin and / or phorbol esters. With ROS and RNS-generating and modulating systems, 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.

After our animal experiments have shown that a temporary elimination of (a) T4 cells (b) APCs (macrophages) and (c) B cells in the critical early phase of exposure to the tumor inoculum (or pathogen) increases vaccination protection According to the invention, it is recommended to switch off the leococyte subsets mentioned temporarily using appropriate Mabs (between days 3 and 10 after vaccination).

The role of arginase, an exoenzyme secreted by macrophages, may lie. more painful in starving tumor cells than tumor-specific in preventing NO generation. T cells, which could lead to Th1 polarization and partial Th2: Th1 (Ts: Tc subconversion. Therefore the use of arginase for Th2: Th1 and Ts: Tc interconversion is recommended according to the invention.

Since irritation or partial damage to the membrane structure generally leads to an increase in ROI, this principle is recommended according to the invention (a) for cell stimulation and (b) for Th2: Th1 or Ts: Tc interconversion. Examples are low-dose surfactants such as sodium salt of bile acids, sub-denaturing concentration of SDS, Brij or Teepol or Tween and digitonin / saponin.

After our experiments have shown that Nh3 leads to cell stimulation due to its membrane permeability and binding of the intracellular H + ion, it is recommended according to the invention to use NH3 and NH3-releasing systems (eg glutaminase / glutamine, asparginase / aspargin) alone or in combination with others, 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.

Since the constant struggle with various pathogens, but also with autoantigens, etc. to the increase of some. wrong, i.e. disease-promoting or disease-promoting immunocyte (T-cell) subsets, it is recommended according to the invention to deplete the set of mature T cells from time to time by combining T cell-specific Mabs plus MIS / MIT effector cells in vivo and by maturing Have T cells replaced. In view of the minimal to no side effects of this type of treatment, this seems realistic.

A treatment with a small number of allogeneic PBLs or MIT effector cells derived therefrom without previous Mab treatment would also be conceivable, because this would result in a "change" from immunosuppressive to immunoreactive immune readiness (via reprogramming of the accessory cells). Tolerance breakthrough was only possible by adding allogeneic PBLs. Of particular interest in increasing the basic reactivity of the immune system is the low-dose administration of various antagonists of Gs-coupled receptors or receptor subtypes, such as propranolol or cimetidine (ranitidine) or without subdosed Ca antagonists, such as nifedipine After the approval of the iNOS or cNOS inhibitors, the combination with subdosed representatives of this substance class could further expand the spectrum of these subdosed immune deblockers.

Because of considerable difficulties in transfecting the target cells with viral vectors (low yield of transfected cells, limited expression time of the transfected genes, unknown location of the transduced genes, problems in controlling complex genes, for example thalassemia or sickle cell anemia, immune response against viral structures on transfected cells) the hybridization (fusion) of deficient cells with proficient cells or wild-type cells is alternatively recommended according to the invention. Here, all forms of hybridization, i.e. Zeil: cell, core: Zeil and core: core mergers within the scope of the invention.

All genetic diseases that can be "corrected" with bone marrow transplantation come into question, whereby there are basically two solutions according to the invention: (a) Through improved bone marrow and stem cell transplantation, as has been described in detail in this and in the previous application, the elimination of the potentially lethal risk paves the way to a broad therapy of all these genetic diseases via improved bone marrow and stem cell - Transplant opened. Moreover, the relative independence of the MHC identity between donor and recipient essentially expands the circle of potential donors. (B) By fusing the defective mutant (patient cell) with a professional or wild-type cell, the defect in the cell can be remedied, at least in the heterozygous sense , For gene defects that affect only one organ, fused hybrid cells with other cells of the patient that do not have this defect are provided. In the case of a generalized genetic defect, allogeneic partner cells must be used. In order to prevent rejection of such cell constructs, tolerance towards the donor must be established beforehand or allogeneic partner cells that do not express MHC must be provided. Examples of such nucleated, MHC-negative partner cells are reticulocytes and K562 cells (human erathroleukaemic cell line). Alternatively, 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. However, 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. OOOcgy or with Mitomycin C (20-30) µg / ml; Incubation time: 18 h) can be carried out at the stage of the tumor cells used or of the hybrid cells formed.

Since with "mixed allogeneic chimerism" the donor APCs also settle in the thymus and assume the role of thymus epithelial cells, we recommend that in the final phase of MIS (more precisely: MIS-BM / SC) treatment immature donor bone marrow or inject stem cells into the recipient in order to achieve the ideal constellation, ie D- plus R-specific MHC restriction of the maturing T cells, which can then cooperate optimally with D- and R-derived APCs.

It is known from animal experiments that pre-irradiated thymus cells take on their function in the thymus after being injected into the recipient. Since, in addition, organ-specific homing of allogeneic thymus cells can be expected from various observations, it is recommended, after establishing the donor-specific tolerance (eg by “mixed allogeneic chimerism” or by a method described in this application) donor thymus cells in the To inject recipients, whereby future generations of maturing T cells acquire D- and R-specific MHC restrictions at the same time, thereby guaranteeing optimal cooperation between D- and R-derived T cells and APCs.

An important principle is the rapid recruitment of "tailor-made" immunocytes (T cells), if the mature T cells (B cells) are pre-eliminated in vivo with Mabs plus MIS effector cells and then "tailor-made" mature T cells are injected (iv ) become. Even a small number of T cells with the desired properties (e.g. Th1, Th2, Tc, or Ts subset with or without antigen specificity) that are activated shortly before the injection are sufficient to quickly generate large populations to generate "tailor-made" mature T cells. "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.

Since we were able to show in animal experiments that in the early phase of the immune response against the antigen (eg TSA) the elimination of macrophages and B cells prevents the premature generation of the counteracting Th2 and Tc cells, 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). On the one hand, 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.

Note: (a) Separation methods based on FACS and MACS can be used to separate the T cells from the APCs and B cells. In a simplified version, only (plastic) adherent cells (APCs) are separated and later injected. (b) The same principle, i.e. the advantageous absence of APCs and B cells in a certain phase of the antigen- or alloantigen-specific immunization can be used to increase the efficiency of MIS / MIT preparation.

In the BMT and HSCT, a further technique for achieving the desired “mixed allogeneic chimerism” is proposed according to the invention, in which both (a) the donor bone marrow, stem cell or PBL / PBMCs suspension as also (b) the recipient (in vivo) is depleted from mature T cells. Two variants are provided here: In the first, only anti-CD3, anti-CD4 and / or anti-CD8 or, in addition to anti-CD3, other pan-T-specific Mabs are used both in the T-depletion of the donor cell Suspension and, if permitted, used in the in vivo pretreatment of the recipient. In the second variant, the Mabs are combined with MIS / MIT effectors. Note: Polaclonal antibodies such as ATG, ALS etc. can be used instead of or in addition to Mabs. To save costs, the combination of AK with some cytostatics, primarily cyclophosphamide, can be considered. Optimally, the "mixed allogeneic chimerism" is "stabilized" with a 1: 1 mixture of (mature) donor plus recipient MIS effectors.

In generalized immunosuppressive positions it is recommended according to the invention (a) with H0 ₂ (0 ₂ ^" ) and / or H ₂ 0 ₂ (0 ₂ ^{2 '} ) -generating or increasing systems (for example XOD / xanthine, XOD / hypoxanthine, GOD / Glucose etc.) (b) with antagonists of Gs-coupled receptors or receptor subtypes (eg propranolol) (c) with Th2 / Ts-suppressing Ca antagonists and / or iNOS / cNOS inhibitors (d) with Gi agonists and (e) using BRMs (eg type 1 cytokines) to convert the immunosuppressive base layer into an immunoresponsive one, in which the agonists or antagonists have to be administered in very low (sub-pharmacological) doses. Another way of “correcting” the Th2 / Th1 - And the T4 / T8 quotient is the use of enzymatic and chemical NO, sometimes H0 ₂ scavengers. The fact that we were able to clinically observe a rapid correction of both mentioned quotients when combining T-depleting Mabs with MIS / MIT effectors led to the recommendation according to the invention for the use of these T-depleting techniques alone or in combination with the above systems to remedy the immunosuppressive Location of the patient.

According to our latest knowledge, 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 ^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

Corresponding G protein subtypes (Go, Gp, G;) or phospholipase subtypes (PLCPLA2), and (c) an increased DAG or PKC quotient,

PLA PKG where DAG stands for diacylglycerol, PKC for protein kinaseC and PKG for 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.

The pre-establishment of "mixed allogeneic chimerism" by injecting a 1: 1 mixture of D and R-MIS effectors into the T-depleted recipient is not only for allogeneic BMT and HSCT, but also for all organ transplants and later discussed

Micro (single cell) transplantation, the so-called ECT (engineered cell transplantation) is recommended.

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. various Gs antagonists and NOS inhibitors) to break the cAMP: NO / PEG2 cycle that is building up and ( c) Vaccinate with the HTT-AIT technique against allergen-specific T cells (using hybrid cells consisting of allergen-specific T cells plus autologous and / or allogeneic MHC II-positive cells (eg DCs or macrophages). In a simplified version only the last step, the HTT-AIT vaccine, is used and possibly combined with the 1st step (T depletion in vivo). To further increase efficiency, fusion proteins consisting of allergen plus IL-12 (or a other Th1 lymphokines) is recommended. Incidentally, 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.

As described elsewhere in this application, Ag-specific T cells (for example allergen or 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.

In the case of allergies, the binding of 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. In addition, allergy-promoting cytokines (eg IL-4, IL-5, IL-1 3, sometimes IL-10) can be obtained by appropriate Mabs be neutralized. These 2 steps can also be combined with the so-called clonotypic (HTT-CL, see later!) HTT modification.

The fact that with ConA + Gs agonists (ie agonists of Gs-coupled receptors, e.g. isoprenaline or adrenaline or adenosine) one can generate Ts (and Th2) in vitro after 48 hours, which is also 1: 1 after treatment with mitomycin C - Mixing with naive PBLs (T cells) from the same donor, both in the PHA proliferation approach and in MLC, forms the basis for the proposed clinical use of mitomycin C- pretreated donor Ts or th2 cells. 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.

An interesting new technique is to use 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. In the event of signs of a GvHD outbreak, R-specific alloreactive MIS / MIT effectors of the donor would be used, whereas in HvGR the D-specific alloreactive effectors of the recipient would be used. After each of these treatments, the i.v. Injection of a 1: 1 mixture of D and R MIS / MIT effectors provided.

Since in animal experiments 50ug / mouse of a pan-T-specific Mab (eg Anti-Thy1 .2-Mab) decides whether the donor bone marrow or the 1: 1 mixture of donor bone marrow and spleen cells is activated or rejected, It is recommended according to the invention that in the case of non-ablatively prepared patients with variation of the pan-T-specific Ab (mono- or polyclonal Ab) on the one hand with a still-to-be-found dose of Ab, which would probably remain constant by the individual, the "mixed allogeneic chimerism" On the other hand, with these Aks, you would get a tool that would predict in advance the predominance of the donor vs. recipient or vice versa.If combined with cell death preprogrammed D or R-MIS / MIT effectors, 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 ". Here, 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. Similar to DLI after post-BMT recurrences for quick If the "mixed" is replaced by the "donor-type" allogeneic chimerism, an excess of R-MIS effectors, especially the all-pre-immunized ones, can rapidly deplete or inactivate the GvHD-maintaining D-CTL.

Instead of or in addition to the MIS / MIT effectors, 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.

Although a post-BMR relapse prescribes a "wrong" order, ie "mixed" before (instead of after) "donot-type" allogeneic chimerism, the resulting impaired T-cell: APC cooperation can be canceled the process of finally establishing "donor-type" chimerism with an excess of pralloimmunized R-MIS effectors, followed by injection of the 1: 1 mixture of D + R-MIT effectors and the 1: 1 mixture of immature Correct D + R cells. Alternatively, untreated prealloimmunized R-PBLs (+ immature R cells) can be used before establishing the fully allogeneic, i.e. 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.

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.

After we have found out that H0 ₂ promotes proliferation and 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).

Just as a "mixed allogeneic chimerism" induced by BMT enables the use of mature D-lymphocytes, the use of MIS effectors allows 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. Similarly, 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. Here the immature patient cells have to be replaced by BMT or HSCT; For unrestricted post-BMT / HSCT cooperation between T cells and APCs, a 1: 1 mixture of D + R cells (instead of "pure" D cells!) is recommended, as discussed above.

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.

As mentioned above, it is generally recommended for 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.

In order to prevent lymphoproliferative diseases as a result of the lost control of the B cells (plasma cells) by (eliminated) T cells, it is recommended to deplete the B cells simultaneously with T cells or to use ALS instead of ATG or ATS ,

In order to further increase the efficiency of HTT or HTT-AIT effectors, it is recommended to use the autologous or allogeneic APCs as fusion partners of the tumor (HTT) or idiotype-expressing T cell (e.g. Ts / Th2 in tumor patients) to stimulate the expression of MHC II and costimulator structures (e.g. B7.1 /B7.2, CD40 etc.) before the fusion. 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. Instead of unchanged allogeneic secretor cells, autologous gene-deficient cells can also be fused with similar allogeneic (proficient or Wildtγp-) 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. 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.) can be assumed or else immortalized cells are subsequently transduced with the desired cytokines etc. In all these cell constructs, cell death can be preprogrammed, for example, with mitomycin C pretreatment.

Since the supernatant can even prevent the formation of Ts after 1 d MLC, it is recommended to activate R-PBLs in a 1-3 day MLC against inactivated D-PBLs in vitro in a D-specific manner, to inject them into the patient and after maturing patient T cells - after previous in vivo T depletion - by pregenerated Th1 cells in the direction of new Th1 / Tc cells. The simultaneous injection of inactivated D-PBLs is sufficient for this. The anti-tumor effect is based on allo-induced reprogramming of patient APCs.

The simple generation of the Th1 cells using a 1-3 day MLC approach can be combined with a pre-programming of cell death within 1-3 days. In the case of tumor treatment, 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. In an 8-1 2 day (instead of 1 -3 day) MLC, 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. The additional introduction of the allogeneic element by cofusion with allogeneic cells, optimally allogeneic APCs, is advantageous.

The often described ex vivo enrichment and clonal expansion of 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. In addition, 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.

Since 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.

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 +.

On the basis of the latest knowledge about the "clonotypic" vaccination we have introduced, rejection reactions in organ transplantations, but also GvHD and HvGR in BMT and HSCT can be prevented in an elegant manner with almost no side effects in advance. In organ transplantation, the recipients with hybrid cells from autologous and / or allogeneic accessory cells plus donor T cells or PBLs, which have been pre-immunized in the MLC against recipient T cells or PBLs, are sc or id vaccinated. With BMT and HSCT, the recipients must both against hybrid cells from allopa-preactivated donor cells. T cells (or PBLs) plus APCs (DCs) and 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. Here, the T cells of the type 1 diabetic (IDDM patients) with or without preselection of the autoantigen-specific CD4 + and / or CD8 + cytotoxic T cells (using modern FACS or MACS enrichment methods) with autologous and / or allogeneic MHC JI + Cells merged.

An important step towards standardizing the process is the use of 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. In addition, the spectrum of these target cells should be expanded to include representatives of the MHC II + cells (DCs, macrophages) or these target cells should be partially or totally replaced by accessory cells as transfection target cells.

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.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, optimally in combination with MIS / MIT treatment. 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. To achieve a further increase in efficiency, 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.

The procedure described above for the treatment of type 1 diabetics (IDDM patients) with few side effects applies in principle to all AID (autoimmune diseases). If the autoantigen is known, the clonotype modification of the HTT or HTT / AIT technique, i.e. the so-called HTT-CL or HTT / AIT-CL are used; otherwise the HTT effectors can be prepared from the panT or T subset fraction of the patient leukocytes. Hair loss or various forms of allopecia can also be regarded as a special case of AID and can therefore be treated according to the same principle that is protected by patent law.

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. Through homing, 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: With 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. Another possibility for 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.

The efficiency of 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. After vaccination with this modified HTT hybrid cell, e.g. by post-treatment of the tumor surface (melanoma, bladder carcinoma) with 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.

As discussed elsewhere in this application, the efficiency of MIS / MIT and HTT effectors is partially enhanced by simultaneous stimulation with BRMs, cytokines, etc. increased dramatically. 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. In this way, i.a. A particularly consistent way of completely eliminating tumor-protective Ts and / or Th2 cells by humoral and cellular means, as has not yet been considered by other groups, is protected by patent. This is important when you consider that in our animal experiments a consistent Ts / Th2 depletion was more important than the eradication of the last tumor cells for the final healing of tumor-inoculated animals.

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. The particular advantage of Mab treatment, leukapheresis and their combination is the prevention of damage to mature and immature immunocytes, which is of particular importance in combating residual tumor in view of the crucial role of deblocked patient immunocytes, primarily tumor-specific CTLs. As a cost-saving step, leukapheresis can also be used in all variants of humoral Ts / Th2 elimination (see also EP 705 1 1 1).

The therapy introduced by 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.

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.

Conversely, 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. Our latest studies show that with Th1 / Tc generation, NH3 and NH3 generators (e.g. glutaminase / glutamine) and triethanolamine can increase efficiency, while RNS (NO and NO-releasing systems) conversely can increase Th2 / Ts formation promotes. 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.

Note: 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. Similarly, 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).

Several observations in our laboratory indicate that adenosine (including 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.

For all types of vaccines, i.e. of the "classic" type against bacterial and (retro) viral pathogens or against tumor antigen, but also with the newest HTT and HTT / AIT vaccines is generally recommended, not only against single immune cells (e.g. Th2, Ts etc.) in the critical post-vaccination phase (5-1 2d after vaccination) to actively intervene (in vivo), but additionally through cAMPi-reducing (eg Gs receptor antagonists) or pre-existing cAMPi-degrading PDE modulators and cGMPi-increasing substances of pathogen- or TSA-specific Th2 and / or Ts. The injection of Th1 cytokines (in low concentrations) in the first days (1-6 days) after vaccination also has a favorable effect.

As is known, the immunization against all (soluble) antigens except for the alloantigens in vitro only succeeds after previous in vivo immunization. With a trick, according to the invention, 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. This principle was tested (a) by fusion of BSA-linked keratin (from wool), (b) from the peptide from plant viruses (also BSA-linked) with NS1 myeloma cells, whereby the fused antigen was surprisingly found in 100% of the cells let (The fusion was carried out by the Bio-Rad Gene Pulzer).

If these are cell membrane antigens, for example TSTA / TSA, 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. According to the invention, 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.

When these karyoplasts are incubated with cytoplasts (other cells) or whole cells in the presence of PEG, fusion occurs. The following are to be protected under patent law: (a) cell, (b) nucleus: cell, (c) nucleus: nucleus, (d) chromosome: cell, (e) chromoson: nucleus,

(f) 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.

It is also proposed to introduce cell nuclei (karyoplasts), chromosomes and chromosome fragments into the cell in addition to DANN in the device “Gene gun” already introduced. This means that no further cDNA characterization and isolation is required.

A highly topical application could be used for genetic diseases

Introduction of the missing or mutated chromosome or

Chromosome fragment can be provided by appropriate material from efficient or wild-type cells.

The 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.

It is self-evident for the person skilled in the art that in all of the fusion or hybridization processes mentioned in this patent application and also in the fusion or hybridization processes mentioned in EP 374 207 and EP 705 1 1 1, all known embodiments known to the person skilled in the art, e.g. can be applied chemically, as with PEG, physically, as with electrofusion / electroporation, viral etc.

One of the most elegant ways to induce tolerance would still be to insert the donor thymic epithelial cell into the recipient thymus. After it has been known from the treatment of immunodeficiency with fetal thymus transplants (George's syndrome) or with cultivated thymic epithelial cells (various T cell deficits) that organ-specific "homing" of injected thymus cells works, it is recommended (a) to establish the D-thymic epithelial cells in the R-thymus according to the temporary D-specific tolerance, (b) to insert the D-specific MHC into the thymus through nucleus: cell or nucleus: cytoplast fusion; (c) the “mixed allogeneic Chimerism "for permanent simultaneous colonization of the R thymus by R and D-derived cells (Sykes, Sachs et al.). Since the establishment of mixed chimerism in several places of this application partly treated in conjunction with BMT and HSCT, this latter route appears to be the most practical.

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). To achieve the donor-type allogeneic chimerism, the anti CD3 (or the anti-CD4 plus anti-CD8) Mabs are used instead of anti-CD4.

Mixed allogeneic chimerism can also be achieved according to the invention by T-cell depletion of the donor PBLs (in vitro) and the recipient PBLs (in vivo). Corresponding Mab-derived immunotoxins can be used instead of the Mabs.

Similar to the. Organ transplantation simultaneous bone marrow transplantation (BMT) is recommended, the use of allogeneic, genetically modified cells can be leveled by previous BMT or HSCT in the standardized gene therapy, which is independent of the individual patient material. Since the conventional BMT or HSCT associated with myeloablative preparation can be replaced by the milder MIS / MIT-BM / SC technique (described in the present application), this novel, low-side-effect combination is highly topical. In order to further minimize the side effects, a new technique, the so-called "clonotypical" HTT-CL or HTT / AIT-CL technique is recommended. It became possible after new techniques for the isolation of antigen-specific T cells were developed In the presence of the antigen of interest, 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. By fusion with autologous and / or allogeneic MHC 11 + cells (eg DCs) are formed Cell hybrids which, after sc or id vaccination, lead to the elimination of the antigen-specific T cells.

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.

When treating tumors, 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. Even more, with 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.

In the case of hematological tumors, the fusion with MHC 11+ cells (eg DCs or macrophages) 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. In the case of organ transplants, 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. In organ transplant tones in vitro, 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. Cells are embossed and, after fusion with autologous and / or allogeneic APCs (eg DCs), are used for recipient immunization against his own, foreign organ-repellent T-subsets. In contrast, 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. With this double HTT vaccination, BMT and HSCT are pre-immunized against both GvHD and GvGR.

The vaccines in general (including HIV vaccines) 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.

An important new finding concerns the replacement of conventional BMT and HSCT, which are associated with myeloablative recipient preparation, with the much less stressful transfer of patients from T-depleted PBMCs, combined with non-ablative recipient preparation.

By using antibodies against certain precursors of mature T cells, the undesired premature re-induction of tolerance to remaining tumor and metastatic cells can be prevented according to the invention. 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.

Also in the case of polyclonal AKs (ATG, ALG, ALS etc.) there are those who "grasp" or "overlook" these preliminary stages, which is the case with the humoral component of patient preparation (eg for MIS / MIT or HTT treatment) must also be taken into account.

In the case of autologous BMT and HSCT, the main problem, the inefficient removal ("purging") 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.).

From previous experience in animal models and partly According to the invention, the following multi-stage therapy scheme for tumor treatment can be established in the clinic:

(a) First, pre-existing, tumor-protective Ts and / or Th2 cells have to be eliminated radically in vivo, whereby animal experiments clearly show that consistent Ts / Th2 elimination is more important for tumor regression than the unattainable target of detection and elimination of the last residual tumor cell. The radical Ts / Th2 elimination can only be achieved by combining the humoral (with Mabs) plus cellular (with MIS / MIT effectors) Ts / Th2 depletion.

(b) (b) This is followed by the phase of APC reprogramming, which succeeds with MIT effectors and / or pharmacologically (through cAMP / cGMP-reducing and Ts Th2 unilaterally inhibiting agonists or antagonists of certain immunological and non-typical immunological receptors ( described in the finest details in the previous application of the same applicant!).

(c) Now vaccinate with two types of HTT cell constructs simultaneously or in succession, namely those based on tumor cells and those based on Ts / Th2: APC hybrids. It is important that the patient macrophages are also kept in the Th1 activation state by allospecific signals during the two types of HTT vaccination. This can be done through the simultaneous presence of MIT effectors, which are optimally replaced every 3-6 days by new MTT effectors from other donors (D1, D2, D3 etc.); in such a case the APC fusion partner cells can be of autologous origin. Nevertheless, it is advisable to provide cell constructs with autologous pluss allogeneic MHC II + cells, or the combination of hybrid cells based on autologous APC plus those based on allogeneic APCs for the HTT vaccine.

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. As stated above, 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". In the aforementioned in vivo depletion of patient T Cells with anti-CD3 Mabs should additionally be mentioned that the humoral in vivo T-depletion can be carried out with all Mabs and Mab-derived immunotoxins and Mab-based fusion proteins, as described and protected in detail in one of the previous applications of the same applicant The combination with immunostimulants, BRMs, cytokines etc. recommended in the same application is also fully valid in the context of the multi-step method described here.

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.

Since there is often only one ApoE molecule per vesicle in the formation of the lipoprotein particles, ApoE could become a scarce commodity in dietary or otherwise caused hyperlipidemia. The fact that ApoE and others is secreted by macrophages and that the cholesterol is loaded with ApoE-loaded from the macrophages, speaks for a special position of this apoprotein in the stabilization of the plasma lipids. It is therefore recommended that the ApoE-encoding cDNA be transduced into autologous or allogeneic cells, in the latter case after pre-induced tolerance to donor (the genetically pre-manipulated secretor cell).

With 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.

According to the present invention, in addition to the pharmaceuticals mentioned in EP 705 1 1 1 for modeling second messengers and thus regulatory immunocytes for various diseases, which have been described in detail in EP 705 1 1 1, modulators of various cAMPs - and cGMP-specific phosphodiesterases (PDEs) 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). In this way, 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. On the other hand, the above-mentioned patent-protected pharmaceutical list is supplemented by okadaic acid (okadaic acid), which could contribute to the further increase in efficiency of the MIS / MIT effectors by inhibiting the type and II phosphatases.

Since laboratory tests have shown that ß-AR antagonists such as propranolol prevent IL-10 secretion by macrophages and thus block IL-12 synthesis and Th1 activation, this and other antagonists of cAMPi-increasing, Gs-coupled apply Special attention to receptors, especially in the unstable phase after tumor excision or chemo (radio) therapy (APC reprogramming phase).

A significant improvement in conventional therapies for hematological tumors (lymphomas / leukaemias) implies the following steps according to the invention:

(a) Gradual replacement of chemo (radio) therapy by using mono- and polyclonal antibodies alone or in combination with leukapheresis (both procedures may have to be repeated several times).

(b) Additional use of panT (CD3) and / or anti-CD4 + anti-CD8-Mabs or corresponding polyclonal Ab (ATG, ATGAM, ALG etc.), whereby combination with 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.

(c) Then 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.

(c) There may be further efficiency-increasing steps, as specified in the treatment of solid tumors (see above).

In the case of non-myeloablative preparation of the recipient (patient), for example with chemotherapy or radiotherapy, 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.

The particular advantage of the sequence proposed here: "temporary donor-type allogeneic chimerism" (for eradicating the residual tumor and metastatic cells), followed by "mixed allogeneic chimerism" (for establishing the D: R tolerance) compared to conventional, clinically practiced Post-BMT "mixed allogeneic chimerism" DLI treatment is (a) GvHD prevention and (b) optimal T (and B) cell: APC cooperation.

In order to set the 1: 1 mixture of D and R-MIS effectors used to establish "mixed chimerism" even more precisely, 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.

Since 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.

An efficient tool against budding GvHD or HvGR according to BMT and HSCT would be suspensions (frozen!) From R-PBLs or R-MIS effectors, which had previously been pre-immunized against D, and those from D-PBLs or D-MIS -Effectors that have been previously pre-immunized against R all. In addition, 3rd party PBLs that have been (all) pre-immunized (a) against R (b) against D could be kept on standby.

All genetic diseases that can be "corrected" with (allogeneic) bone marrow or stem cell transplantation, but which, due to the potentially lethal complication that can be associated with conventional BMT and HSCT in general, cannot be practiced (with a few exceptions) After the elaboration of the BMT and HSCT replacement procedures with few side effects and combined with non-ablative patient preparation, some of which are the subject of the present application, can be tackled clinically without any risk and without risk. The list of these genetic diseases can be special monographs, but also immunology textbooks such as those from Stites and Stobo can be removed. The combination of organ transplantation and bone marrow transplantation (of the same donor), which has been recognized as meaningful several times and which could not clinically assert itself due to the potentially lethal outcome of classic KMT, is gaining new relevance due to the low-side-effect alternative methods to conventional KMT.

If available, the cDNA or the so-called “naked DNA” encoding the antigen of interest (eg tumor antigen, autoantigen, pathogen, allergen etc.) 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 (inducing antigen, here: Clamydia pneumoniae) 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.

In general, when producing the hybrid cells from tumor cells plus APC, in addition to the mixing ratio 1: 1, e.g. 3: 1 and on the other hand can provide 1: 3, which can bring additional advantages. However, 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.

Clinical experience shows that repeated injection of MIS / MIT effectors of different origins (DonorA, B, C etc.) 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.

All embodiments of the HTT and / or MIS effectors can also be combined with cell constructs based on immortalized specialized subclasses (e.g. Th1, Th2, etc.).

By increasing the H ₂ 0 ₂ ^' and / or reducing the NO concentration, according to the invention (a) the tolerance breakthrough (b) the Th2: Th1 or Ts: Tc interconversion, and (c) an improvement in the Ag specific "priming" and "boosting" can be achieved. That is why all corresponding H202 and NO modulators are of interest.

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).

The fusion of mature, specialized cells (e.g. Thl, Th2, macrophages, NK cells, B cells etc.) with (a) embryonic (b) hematopoietic stem cells results in long-lived but not transformed cells. The same applies to the fusion with some long-lived but not transformed fibroblasts (cell lines). The fusion of 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.

It is recommended according to the invention to replace the transfection of individual cytokines into the target cell (tumor cell, TIL, fibroblasts) by fusing this target cell with specialized T cell subsets (primarily Th1 cells). The cytokine-secreting Th1 partner cells should be activated beforehand, e.g. with inactivated allogeneic PBLs.

Conventional gene therapy can be improved if costimulatory structures such as B7.1 /B7.2, CD40 etc. are combined with MHC II coexpression. It is also advantageous to combine the transfection with cytokines with the transduction of costimulatory cDNA (including the MHC II expression). The transduction achieved by means of viral vectors can partly replaced by core: cell or cell: cell fusion.

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).

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) in 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.

According to a special variant, 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). Therefore, every form of immune stimulation (e.g. with BRMs, cytokines etc.) as well as 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.) are always combined with radical 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 effects and (b) patients are made accessible to therapy in a later (terminal) phase of the disease.

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).

In the case of hematological tumors, 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. The therapeutic effect can be further enhanced by (prealloimmunized MIS / MIT effectors and various HTT vaccines).

The fact that 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.

Since 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. With this restimulation, 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.

Diseases related to bone loss (osteoporosis, periodontosis, etc.) lead 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. Therefore, for remineralization in medical and stomatological diseases, the use (a) of MIT effectors (after previous T cell depletion in vivo) (b ) of 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.

As already mentioned in an earlier application by the applicant (EP 374 207), not only is the receptor-subtype-specific increase in "second messengers" (cAMP, cGMP, CAI etc.), but also a general decrease in several "background signals" "recommended by blocking several receptors together with all receptor subtypes. This "cutting out" or "filtering" of unspecific "background signals" can be combined with targeted suppression or activation of desired immune-relevant and non-immunological receptors or receptor subtypes. 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. One variant provides for the 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.

The fact that the crossing of ras-transgenic mice with myc-transgenic mice leads to the expression of both oncogenes in the offspring leads to the conclusion that the fusion of two cells, each with a typical (dominant) characteristic, results in hybrid cells, both of which are desired Express characteristics. According to the invention, this fact enables the avoidance of more complex transfections (e.g. with 3 or more cytokine cDNAs) by hybridizing two cells, pretransduced with 1 -2 cytokines each, or the otherwise unrealizable introduction of more than 4-5 cytokine cDNAs. In this way e.g. the costimulatory structures are transduced together with MHC II into the autologous tumor cell or constructs are formed from partner cells that express cytokine-secreting plus costimulatory structures, which were previously not possible due to technical problems.

A particularly rapid, receptor-independent stimulation of immune cells with partial Th2: 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.

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.

Our group's new insights into the role of the H02 / H202 ratio in proliferation vs. Differentiation of the cell makes it possible for the first time to generate cells that have increased proliferation but are not tumorigenic. This goal is achieved by a pre-programmed (constitutive) slight increase in H02 synthesis and / or reduced H202 generation, which could be achieved by genetic engineering manipulation of the enzymes MnSOD, CuZnSOD, catalase and / or GSH peroxidase. This controlled increase in the proliferation rate could generally keep cells alive longer in vitro and specialized cells (Th1, Th2, Tc, Ts) could be "stabilized" and thereby made more efficient.

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.

By transfecting the genetically modified allogeneic effector or secretor cells with Th2 cytokines, e.g. IL-10, tolerance to individual transmitted cells can be achieved. Conversely, 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.

The tumor regression observed by Coley in some patients who were also suffering from tuberculosis could not be reproduced in our understanding and therefore improved later with BCG and other bacterial preparations, since the simultaneously induced Ts cells disrupted the treatment. According to the invention, 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).

It can be deduced from animal experiments that a continuous, uninterrupted conflict between the immune system and the antigen leads to the development of tolerance against this antigen. It is therefore recommended that this contact of the immune cells e.g. with the tumor antigen artificially by in vitro cultivation of the patient leukocytes in the absence of the tumor antigen. to interrupt and the "recovered" immunocompetent cells in the T-depleted patients reinject. Another effect plays a positive role here, namely the fact that only preactivated Ts or Th2 cells lead to further recruitment of further Ts or Th2 cells, while resting (memory) Ts or Th2 cells when reactivated the patient's immunocytes only become active after the patient's Th1 and Tc memory cells.

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. A similar effect can be achieved with 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 fact that the pre-existence of donor-specific or cross-reacting antibodies and / or T (and B) cells can lead to rapid rejection of the graft is now reversed and practically used to prevent this acute rejection reaction. For this purpose, 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.

An elegant way to generate 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. In contrast, Th2 generation is achieved through mitogenic stimulation in the presence of Gs agonists and / or cAMP-PDE blockers.

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).

Another possibility of Th1 and Tc generation is treatment (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.

Partial Th2: Th1 or Ts: Tc reprogramming can also be achieved by combining ROS-releasing systems plus cAMP-PDE stimulators or cGMP-PDE inhibitors.

Various important clinical pictures such as allergy, atherosclerosis or haematological disorders are often associated with the shift of the Th1 / Th2 quotient in favor of the Th2 subset. Therefore, efforts must be made to correct this quotient, either by in vivo depletion of the undesired T cells and their replacement by autologous or allogeneic T cells of autologous T cells from T cell precursors, or by the direct in vivo Conversion of the Th2 into the Th1 cells.

An elegant new way is vaccination (a) against the Th2 subset or (b) against the idiotype of the tumor-specific Ts and / or Th2 cells.

Some aspects of the present invention are illustrated by the accompanying figures. Show in it

1 shows the strong activation of PBLs by sub-toxic doses of ammonia

Figure 2 shows the strong activation of lymphocytes by subtoxic doses of

triethanolamine

Figure 3 shows the activation of PBLs by adding XOD / xanthine to the medium

Figure 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 and

XOD / Xanthin '

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

XOD / xanthine, ammonia and GSH can be achieved 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

ammonia

10 shows the stimulation of PBLs with an iNOS / cNOS inhibitor

Figure 1 1 the stimulation of lymphocytes with a subtoxic dose of

ammonia

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

PBL stimulation

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 and

Figure 23 shows the influence of the GOD / glucose system on the activity of PBLs.

Claims

Expectations

1 . Cell construct from activated T cells of a patient with the patient's own and / or foreign MHC II-positive cells for use as a drug.

2. Vaccine for combating activated T cells in a mammal comprising cell constructs which have arisen from the fusion of MHC II-positive cells of the mammal to be treated and / or MHC II-positive cells of a donor with an activated T cell.

3. Cell construct according to claim 1 or vaccine according to claim 2, characterized in that the MHC II-positive cells are dendritic cells, macrophages and / or B cells.

4. Cell construct according to claim 1 or vaccine according to claim 2, characterized in that the activated T cells are autoagressive cells, alloreactive cells, allergic cells or suppressor cells.

5. Cell construct according to claim 4, characterized in that the alloreactive cells are directed against a graft or a GvH reaction triggering cells.

6. Process for the production of cell constructs in which activated T cells of a patient are fused with the patient's own and / or foreign MHC II positive cells.

7. The method according to claim 6, wherein one uses as activated T cells autoagressive cells, alloreactive cells, allergic cells or suppressor cells.

8. The method according to claim 7, in which cells are used as alloreactive cells directed against a graft or triggering a GvH reaction.

9. The method according to any one of claims 6 to 8, in which the fusion of the cells is carried out by contacting activated T cells and MHC II-positive cells with bifunctional antibodies which have selectable markers, the bifunctional antibodies being a T cell-specific binding site and an MHC II-specific binding site, which separates the antibody loaded with the specifically bound cells via the selectable marker and then causes the fusion.

10. The method according to claim 6, in which hybrid antibodies are used as bifunctional antibodies.

1 1. Method according to one of claims 6 to 10, wherein the fusion is effected by physical, in particular by electroporation, chemically, in particular with PEG, or virally.

12. A method of treating cancer or virus comprising the following stages:

I. Removal of tumor- or virus-specific suppressor cells

II. If necessary, removal of activated suppressor cells

III. Treatment with cells fused from MHC II positive cells and tumor cells and / or cells fused from MHC II positive cells and suppressor cells

IV. Optionally treatment with activated patient lymphocytes, which are obtained by lymphocytes being obtained from a blood sample of the patient, incubated with tumor cells or virus cells, the activated cells are selected and multiplied in vitro and then infused back into the patient.

13. The method according to claim 12, wherein in stage IV the lymphocytes are incubated with mitogenic antibodies and interleukins.

14. The method according to claim 12, in which during and / or after stage IV, effector cells are activated independently of the receptor by treating them with an agent that uses the quotient ROI / RNI.

1 5. The method of claim 14, are used in the means for increasing the quotient ROI / RNI xanthine oxidase and their substrate.

1 6. The method of claim 15, wherein xanthine or hypoxanthine is used as the substrate.

1 7. The method of claim 12, in which dendritic cells, macrophages and / or B cells are used as MHC ll-positive cells.

18. The method according to claim 12, in which both the patient's own MHC II positive cells and foreign MHC II cells are used.

19. The method of claim 12, wherein the removal of the patient's T cells is accomplished using monoclonal antibodies directed against T cells.

20. The method of claim 17, wherein the T cells are removed by anti-CD3 antibodies.

21. The method of claim 12, wherein activated T cells are removed using donor lymphocytes with predetermined cell death.

22. The method according to claim 19, in which donor lymphocytes are pretreated with mitomycin C in a dose such that they die after 3 to 15 divisions.

23. A method for removing disregulated cells from a mammalian organism, in which MHC II-positive cells of the patient and / or MHC II-positive cells of a donor are fused with the disregulated cell and the fusion, optionally after amplification, is injected into the patient ,

24. Combination of xanthine oxidase, a substrate for the xanthine oxidase and optionally catalase and / or peroxidase for use as a medicament.

25. The composition according to claim 24, characterized in that the substrate is xanthine and / or hypoxanthine.

26. The composition according to claim 24 or claim 25, characterized in that a combination of xanthine oxidase and xanthine is used.

27. The composition according to any one of claims 23 to 26, characterized in that an agent which promotes superoxide formation is additionally used.