DE10131971A1 - Preparation of hematopoietic stem cell preparations, useful in transplants for treating leukemia and viral infections, does not induce graft versus host disease - Google Patents

Abstract

Producing (M1) a hematopoietic stem cell preparation (A) that is suitable for allogenic transplantation and, after transplantation, does not induce graft versus host disease, is new. In M1, the material containing hematopoietic stem cells (HSC) is taken from a donor and used to prepare a cell suspension from which T cells are isolated and purified. These cells are treated for selective elimination or functional inactivation of a portion of them, then the remaining T cells combined with the original (T cell-free) cell suspension, either at the time of transplantation, or later in the context of a modified donor lymphocyte infusion. Independent claims are also included for: (1) isolating (M2) a T cell preparation free of alloreactive T cell; and (2) HSC preparation from which alloreactive T cells have been selectively eliminated or inactivated, produced by M1.

Description

The present invention relates to a method for the production of a hematopoietic stem cell Preparation for allogeneic transplantation is suitable and after transplantation into a Recipients no graft versus host disease causes a method for isolating one of alloreactive T cells largely freed T cell Preparation, a hematopoietic stem cell preparation and the use of this hematopoietic Stem cell preparation for the production of a graft for allogeneic transplantation.

Under the term leukaemias are different Diseases summarized by malignant Transformation of hematopoietic or lymphatic Cells emerge. Common feature of this Is the proliferation of leukemia cells in diseases Bone marrow as well as often in lymphatic Tissues and blood. The symptoms of these diseases result from repression and oppression normal hematopoiesis and impairment of the immune system.

The only curative therapy for the treatment of Leukemia is cytotoxic chemotherapy. Just with CNS involvement or for CNS prophylaxis in addition, radiation therapy was used. at younger patients (≤ 65 years) can for the therapy of leukemia diseases a bone marrow stem cell transplant (Bone marrow transplant) or peripheral Perform blood stem cells. In principle there are two Process available, namely the allogeneic Stem cell transplantation HLA-identical Siblings, other family members or relatives Foreign donors and the autologous transplantation of stem cells of the Patients.

In bone marrow transplantation, a Suspension of healthy bone marrow cells in one Transfused recipient with impaired blood formation. The Bone marrow contains hematopoietic stem cells, which settle in the bone marrow spaces and after proliferation and differentiation Replace recipient blood formation. Others too hematopoietic cells such as stem cells of peripheral blood, umbilical cord blood cells or fetal liver cells, can be used for transplantation be used.

Contain all suspensions of hematopoietic cells to varying degrees lymphocytes, too Immune reactions against organs of the immunogenetic foreign recipient are capable. This reaction that especially with allogeneic transplants occurs as a graft-versus-host response (GvH- Reaction; Graft-versus-host disease) designated.

The graft versus host response or graft versus Host disease is caused by an immune response from the non-autologous grafts against strangers Histocompatibility antigens caused by the recipient. When transplanting a graft from HLA The GvH reaction is against identical donors Minor histocompatibility antigens directed at other donors are also against HLA antigens (human leucocyte antigen), i.e. the Major histocompatibility complex (MHC) directed. In the healthy The transferred organism becomes the recipient organism quickly eliminated. For recipients where the Immune defense through radiation or immunosuppressive Treatment is suppressed, the GvH response lead to the so-called secondary reaction, one severe acute or chronic illness with Liver and spleen enlargement, atrophy of the lymphoid organs, diarrhea, skin changes and Cachexia. The outbreak of the graft-versus-host response can be lethal in some cases Can lead to diseases such as liver failure.

Acute graft-versus-host disease often breaks down in the first 2-3 weeks after Bone marrow transplant at a time when severe neutropenia and the patient through infections with bacteria and fungi endangered is. A chronic GvH reaction breaks down however, about 100 to 400 days after transplant. In the case of a chronic GvH reaction in particular recurrent sinubronchial infection problems It can be an "Asplenia syndrome" with Defense weakness against gram-positive pathogens, in particular Pneumococci. Due to the GvH Response is the allogeneic mortality rate Bone marrow transplants at an early stage very high. In a study by Barlogie et al. (Barlogie et al., Seminars in Hematology, 32 (1995), 31-44) received 268 patients Allografts. Died within a year about 50% of patients. Overall survived in a period of 4 years only 35% of all Patients.

Avoiding and Treating Graft-versus- Host response is therefore one of the main problems the bone marrow transplant.

To prevent the graft versus host response after transplantation, for example, immunosuppressive treated with methotrexate and ciclosporin A. Nevertheless, about 30 to 50% of the HLA-identical occurs transplanted patients GvH disease requiring treatment, which affects approximately 10 to 20% can become life threatening. Another The strategy to prevent the GvH reaction is the Removal of donor T lymphocytes from the Bone marrow before the transplant.

Numerous methods have been developed for the separation or inactivation of T lymphocytes. For example, procedures such as density gradient centrifugation, agglutination with soybean lectin or sheep erythrocyte rosette formation, but also treatments with cytotoxic drugs, corticosteroids or anti-T cell monoclonal antibodies and positive selection of CD34 ⁺ can be used to remove T cells from bone marrow transplants. Cells are performed (Champlin, Journal of Hematotherapy, 2 (1993), 27-42; Reisner et al., The Lancet, 2 (1981), 327-331; Waldmann et al., The Lancet, 2 81984), 483- 486; Antin et al., Blood, 78: 2139-2149 (1991); Soiffer et al., J. Clin. Oncol., 10 (1992), 1191-1200). A relatively efficient method for removing T cells from bone marrow is that of Reisner et al. (1981). These procedures first involve differential agglutination with soybean lectin, removing mature leukocytes including T cells, B cells, monocytes and granulocytes. The remaining T cells are then removed using the erythrocyte rosette method. Currently, T cells are removed very efficiently using the MACS method. This selection process, which is based on magnetic separation using microbeads against CD34, allows a very efficient depletion of the T cells and at the same time brings about a very good purity of the CD34 ⁺ stem cells.

By ex vivo removal of T cells from the Graft can indeed increase the frequency of graft versus-host response sometimes significantly reduced become. But it also turned out that the removal of T cells with an increased Risk of viral opportunistic infections (Li et al., Blood, 83 (1994), 1971-1979), one increased occurrence of lymphomas associated with the Epstein Barr virus related, one impairing ingrowth of the graft and thus increased graft rejection (Martin, J. Exp. Med., 178 (1993), 703-712) and the loss of the graft versus leukemia (GVL) effect goes hand in hand with the recurrence rate of leukemia increasing dramatically may increase (Goldman et al., Ann. Intern. Med., 108: 806-814 (1988).

That increased risk of graft rejection after removing the T lymphocyte is in several Publications have been documented (Beatty et al., N. Engl. J. Med., 315 (1985), 765-771; Hale et al., Transplantation, 45: 753-759 (1988); Patterson et al., Br. J. Hematol., 63: 221-230 (1986). at a mouse model for allogeneic Bone marrow transplant has been found to do that Only then did the transplant remain in the recipient, if having a strong immunosuppressive treatment radiation therapy was carried out, the T cells of the donor were eliminated (Lapidot et al., Blood, 73: 2025 (1989).

Due to the lack of mature T cells from the donor in the Graft occurs after a Bone marrow transplant with bone marrow transplants in which T cells were eliminated, a pronounced one Immunodeficiency. The primary immunodeficiency exists in the absence of mature T cells of the Donors who are a temporally adaptive Immune reconstitution, for example against viral infections take. One of the consequences of this severe Immune deficiency is the increased risk that in the first six months after transplant Epstein-Barr virus (EBV) caused lymphoproliferative disorders occur (Shapiro et al., Blood, 1988, 71: 1234-1243; Zutter et al., Blood, 72 (1988), 520-522). This lymphoproliferative Disruptions very often do not speak to the usual ones Therapies.

The risk of a higher tumor recurrence rate after ex Removal of T lymphocytes is performed on the Loss of the graft versus leukemia (GvL) effect recycled. The term "graft versus leukemia Effect "describes the phenomenon that in a allogeneic transplantation the recurrence rate is usually less than an autologous Transplantation. Direct evidence of a graft versus-leukemia effect is in patients with recurrent chronic myelogenous leukemia after a bone marrow transplant with bone marrow freed from T cells Infusion of mononuclear cells from peripheral blood the bone marrow donor without further chemotherapy or have been treated with radiotherapy. That infusion led to the killing of leukemia cells (Drobyski et al., Blood, 82: 2310-2318 (1993); Sullivan et al., N. Engl. J. Med., 320: 828-834 (1989). Although the Infusion of mononuclear cells from peripheral blood for a complete and long-term remission can result in such patients, such Therapy nonetheless with the graft versus host Disease (Bar et al., J. Clin. Oncol., 11 (1993), 513).

The role of T cells has been investigated by Molldrem et al. and Gao et al. more accurate broken down (Molldrem et al., Blood, 88 (7) (1996), 2450- 2457; Gao et al., Blood, 94 (9) (1999), 2999-3006; Gao et al., Blood, 95 (2000), 2198-2203). there allo major histocompatibility complex (MHC) - restricted cytotoxic T lymphocytes (CTL) that are specific for antigens that are very common in increased amounts on the surface of leukemia cells be expressed from a allo-restricted T cell population and cloned to Mediation of a graft versus leukemia reaction used. In summary, the above show Observations that the curative effect of a allogeneic bone marrow transplant at least partly on the graft versus leukemia effect (GvL) is due and that T cells are not just trigger the graft versus host response but also of enormous importance in the Mediation of both antiviral immunity and Have graft-versus-leukemia effect. Most recently New approaches were therefore developed that based on that T cells only after Transplantation must be eliminated before the outbreak the graft versus host response or during the Outbreak.

Tiberghien and co-workers developed one Method using T cells with a retroviral vector were transduced ex vivo, the Thymidine kinase gene encompassed by the herpes simplex virus. The T Cells can then together with hemotopoetic Stem cells are administered to a patient. If the patient's graft versus host Ganciclovir developed to the disease Patients are administered to the transduced T- To eliminate cells. (Tiberghien et al., Blood, 84: 1333-1341 (1994)).

WO 97/45142 describes a method for Treatment of patients with leukemia, this being Patients genetically modified T cells can be administered that contain a polynucleotide that contains a negative selective marker. After this the cells over a certain period of time Patients remain and have a therapeutic effect exercise becomes an interacting means for example ganciclovir, administered to the patient, taking the genetically modified T cells be killed and thus the development of GvH Disease is prevented.

Other methods of transferring such Suicide genes in donor T lymphocytes are for example by Cohen et al. (Blood, 89 (1997), 4636-4645) and Bonini et al. (Science, 276 (1997), 1719-1723) described.

The procedures using genetic engineering however, altered T cells are for several reasons disadvantageous. A patient who is genetically engineered received modified T cells must with Medicines or chemical agents are treated to to kill the T cells. Such drugs or chemical agents go with heavy on the one hand Side effects. In addition, if the Use of medication or funds too late takes place, at least a partial GvH Can develop reaction. Besides, has found that transduced with a suicide gene T lymphocytes often become resistant to Develop ganciclovir (Garin et al., Blood, 97 (2001), 122-129).

Also methods for modifying the T cell Reactions using cytokines (Yang et al., Blood, 90: 11: 4651-4660 (1997); Hill et al., J. Clin. Invest., 102 81998), 115-123), to Elimination of T cells using Immunotoxins against CD25 or via activation of the CD69 antigen (Koh et al., Bone Marrow Transpl., 23: 1071-1079 (1999); Montagna et al., Blood, 93 (10) (1999), 3550-3557) and zur Blocking costimulation signals leading to optimal T cell activation are required (Blazar et al., J. Clin. Invest. 1998, 102: 473-482; Guinan et al., New Engl. J. Med., 340 (1999), 1704-1714), are known. However, these procedures are therefore disadvantageous because it causes all T lymphocytes, regardless of their specificity, impaired or be eliminated.

The development of further improved processes and Means to prevent allogeneic Bone Marrow Transplant Associated Graft-versus- Host disease in a recipient, in particular to eliminate the the graft versus host Disease-triggering T cells is therefore urgent required.

The basis of the present invention So the technical problem is, procedures develop with the help of one transplantable containing hematopoietic stem cells Material targeted and selective only the T cells removed or inactivated to develop the graft versus host response in a recipient contribute, but without eliminating the T cells or inactivate the graft versus Leukemia effect and antiviral immunity mediate, as well as a transplantable stem cell Provide material after transplant in a recipient not to develop a Graft-versus-host disease, at an increased rate Tumor recurrence rate and an increased incidence leads to viral diseases but is able to the graft versus leukemia effect and the mediate antiviral immunity effects.

• a) die Gewinnung eines hämatopoetische Stammzellen umfassenden Materials aus einem Donor,
• b) die Herstellung einer Zellsuspension aus dem hämatopoetische Stammzellen umfassenden Material,
• c) die Isolierung und Aufreinigung einer T-Zell- Population aus der Zellsuspension,
• d) die Behandlung der T-Zell-Population, so dass ein Teil der T-Zell-Population selektiv eliminiert beziehungsweise funktionell inhibiert wird, und
• e) die Vereinigung des nicht eliminierten Teils der T-Zell-Population mit der hämatopoetische Stammzellen umfassenden, T-Zell-freien Zellsuspension zum Zeitpunkt der Transplantation oder zu einem späteren Zeitpunkt im Rahmen einer modifizierten Donor-Lymphozyten- Infusion (DLI).

The invention solves the technical problem on which it is based by providing a method for producing a hematopoietic stem cell preparation which is suitable for allogeneic transplantation and does not cause a graft-versus-host reaction after transplantation in a recipient, comprising:

• a) obtaining a material comprising hematopoietic stem cells from a donor,
• b) the production of a cell suspension from the material comprising hematopoietic stem cells,
• c) the isolation and purification of a T cell population from the cell suspension,
• d) the treatment of the T cell population so that a part of the T cell population is selectively eliminated or functionally inhibited, and
• e) the union of the non-eliminated part of the T cell population with the hematopoietic stem cell-comprising, T cell-free cell suspension at the time of the transplantation or at a later point in time as part of a modified donor lymphocyte infusion (DLI).

According to the invention, it is therefore provided after extraction of the stem cell to be transplanted Material from the donor from this material first isolate all T lymphocytes and purify. Then these T lymphocytes subjected to a special treatment ex vivo in order to specifically and selectively those T cells eliminate that in the recipient for the outbreak of the Graft versus host response are responsible while all other T lymphocytes, like the T- Cells in the recipient which are graft-versus- Leukemia effects and the antiviral Effects of immunity, not impaired or be eliminated. The invention makes it Take advantage of the knowledge that graft versus leukemia Effect (GvL effect) and the graft versus host Reaction (GvH reaction) only partially by the same host-specific donor T cells according to the current clinical and experimental results, however, by different Leukemia-specific alloreactive donor T cell Populations are evoked.

The special treatment according to the invention is therefore exclusively on the elimination of the alloreactive T cell subpopulation directed in The GvH response causes recipients, but not on the elimination of other T cells. To the T- Lymphocytes by treatment are not to be eliminated include the T lymphocytes, which are leukemia-restricted Minor histocompatibility Recognize antigens and leukemia antigens and thus accompanying the recipient in the graft versus Can mediate leukemia reaction. To the non- eliminated T-lymphocytes also include the T- Lymphocytes, the viruses, especially the Epstein Barr virus and the cytomegalovirus, recognize and associated with this in the recipient antiviral Mediate effects of immunity and the risk more serious lower viral infections.

After eliminating the GvH reaction mediating alloreactive T lymphocytes remaining T cells with the transplanting, hematopoietic stem cells comprehensive material combined and together with it in transplanted the recipient. Alternatively be the manipulated T cells after Transplant at a later date in the frame a modified donor lymphocyte infusion (DLI) reinfused. The so manufactured by alloreactive T lymphocytes freed hematopoietic Stem cell preparation leads in the host or recipient not to the onset of graft versus host disease, can still use the graft versus Mediate leukemia effect and protection against viral Infections.

According to the invention it is particularly provided that the selective elimination of the GvH reaction producing alloreactive T cells via a Activation-induced cell death (AICD) occurs. Studies by the inventors of the present Invention on a mouse model for graft versus host Diseases have surprisingly shown that especially that mediated by CD95 / CD95 ligand Activation-induced cell death the possibility offers activated alloreactive T lymphocytes, especially those responsible for the GvH reaction To selectively and specifically eliminate T lymphocytes, while bystander T cells, including the T cells, those in the recipient the GvL response and antiviral Cannot mediate effects of immunity be affected. For inducing AICD Alloreactive T cells according to the invention initially repeated with a host or Recipient-specific allo-antigen, preferably in Presence of interleukin 2 (IL-2), ex vivo before Transplant stimulated. At the same time there was a Treatment with an anti-CD95 T cell Receptor-directed agonistic antibodies or a CD95 ligand.

According to the invention it could be shown that the AICD induction during a primary MLC (mixed lymphocyte culture) proliferation into one significant elimination of the host Immune system responsive (alloreactive) T cells leads. The elimination of the alloreactive T cells antigen specific. Further Analyzes showed that treated by alloreactive T-cell preparations greatly freed T-cell preparations have decreased proliferative responses. In Cytolysis tests also showed that the ability the T cell exempt from alloreactive T cells Preparations for the lysis of allogeneic target cells strong was impaired. The manufactured according to the invention However, T cell preparations could kill target cells the other antigen than that for stimulation allo-antigen used. flow Cytometry analyzes of responder T cells related to different times during a mixed lymphocyte culture with of T cells liberated stimulator cells were shown that activated alloreactive T lymphocytes Expression of the activation markers CD69, CD25 and CD95 up-regulated depending on time and then at Cultivation with a CD95 agonistic monoclonal antibody (mAb) for AICD were accessible. The AICD induction after Activation of the T cells with a certain allo-antigen is specific and affects reactivity not significant against third allo-antigens.

Finally, using a mouse Model for GvH reactions are shown that at Transfer of donor T cell populations that were significantly freed from alloreactive T cells, in recipients these are no graft versus host Developed disease. Recipients who received untreated donor T cells, however, showed all clinical signs of acute GvH disease and died within a short time after transmission of the T cells. These findings have been made through Flow cytometry analyzes underpinned. This showed that alloreactive T cells in mice untreated T cells had been administered expanded strongly during the expansion alloreactive T cells in mice, those of alloreactive T cell-liberated T cell populations administered had been very low.

These experiments therefore show very impressively that the T cells responsible for the outbreak of GvH Response recipients are responsible under Use of AICD specifically and selectively can be removed. The selective elimination host-reactive T lymphocytes from one to one transplanting hemopoietic stem cells Containing material therefore provides an ideal process specific immunotherapy because all other T- Cell subpopulations, for example viruses, such as the Epstein-Barr virus or the cytomegalovirus, or leukemia-restricted Do not recognize minor histocompatibility antigens and leukemia antigens be affected.

In connection with the present invention under a "transplant" the transfer of Cells, tissues or organs of an individual another individual or to another Body area of the same individual understood. A "Autologous transplant" is when the Donor and recipient (recipient) are identical. In a "syngeneic transplant" the donor and recipient are genetically identical Individuals, for example identical twins or Animals of the same inbred strain. Under one "Allogeneic transplant" becomes a transplant understood genetically at the donor and recipient are different individuals, but they are the same Belong to species.

In connection with the present invention "haematopoietic stem cells" are all those undetermined and determined stem cells understood that for the education of each Blood cell lines, i.e. for erythropoiesis, granulopoiesis, Thrombopoiesis and lymphocytopoiesis, responsible are and the cell loss in the individual Blood formation storage through appropriate zeline formation can compensate. The undetermined Stem cells have the ability in all blood cell lines mature, their division activity very is low (sleeper cells). The determined ones Stem cells are only limited, that is, in Viable and direction towards a blood cell line fill the downstream proliferation stores on (feeder cells).

In connection with the present invention under a "hematopoietic stem cell preparation" a cell-containing cell obtained from a donor Understood material, especially hematopoietic Stem cells, but also contains lymphocytes. Hematopoietic stem cell preparations are especially for Intended for use as a transplant material.

According to the invention it is provided that hematopoietic stem cell material from one any mammalian organism obtained as a donor can be. In a preferred embodiment of the Invention is hematopoietic stem cells containing material obtained from humans. In Another preferred embodiment is material comprising hematopoietic stem cells won the mouse. According to the invention, however other mammals also serve as donors.

Containing the hematopoietic stem cells Material is particularly from the bone marrow, the peripheral blood and umbilical cord blood of the donor won. Peripheral blood contains compared to Bone marrow lower hematopoietic concentrations Stem cells, however, is the proportion of Lymphocytes higher. With G-CSF the frequency of the Stem cells in the blood are increased. mobilized Donors therefore have more stem cells in their blood. Umbilical cord blood is also rich in hematopoietic Stem cells that are suitable for transplantation. Umbilical cord cells in newborns are immunological less aggressive and the GvH reactions fall match weaker.

Obtaining a hematopoietic stem cell extensive material from a donor according to the invention using usually procedures used in the field. Bone marrow can follow through, for example, by aspiration Puncture of the medullary canal of plates in particular Bones using special hollow needles all as a sternal and iliac crest puncture, be removed. With the allogeneic blood stem cell The transplant must oppose the donor for bone marrow transplantation no anesthesia undergo injections for about 5 to 6 days hematopoietic growth factor G-CSF and donates his blood leukocytes to a blood cell separator. Umbilical cord blood can appear after the separation of the newborn child from the placenta cycle from the Placenta can be obtained, taking about 100 ml Residual blood can be obtained. This amount is usually enough for transplantation for a patient with weighing up to 50 kg.

According to the invention, the HLA Antigens between donor and recipient largely match. The HLA (human leucocyte antigen) system is the Main histocompatibility complex (MHC) of a mammal and is found in humans of at least four gene loci from the HLA gene region coded. In a preferred embodiment of the The present invention provides that material comprising hematopoietic stem cells is isolated from a sibling whose HLA system is identical to that of the recipient. In a another preferred embodiment of the invention will encompass the hematopoietic stem cells Material isolated from an unrelated donor, its HLA system with that of the recipient matches. In another preferred Embodiment is the hematopoietic stem cell extensive material isolated from a family donor, where the 1 locus of its HLA system matches that of the Recipients do not match.

According to the invention, after obtaining a material comprising hematopoietic stem cells the donated cells in physiological solutions or media suspended, whereby a cell suspension from the hematopoietic Stem-comprising material is produced. to Production of the cell suspension can all be done on the Usually used in the field physiological solutions or media are used. In a preferred embodiment of the invention provided the cell suspension using the Dulbecco modified Eagles medium (DMEM) manufacture. Of course you can too other media, for example RPM1 1640 medium and similar are used. That to make the Cell suspension used can be serum and medium Antibiotics and, if necessary, other additives, such as Buffer substances, salts and / or amino acids, contain. Fetal is the preferred serum Calf serum (FCS) or horse serum is used. In a preferred embodiment of the invention provided that human cells in serum-free physiological solutions or media suspended become. An antibiotic is preferably a approved antibiotic used. In a preferred embodiment of the invention is a Antimycotic solution (GibcoBRL, Ichinnan, Great Britain), which contains streptomycin and penicillin, used. Of course you can too other suitable antibiotics are used.

According to the invention, T cell populations, for example CD90 ⁺ , CD3 ⁺ , CD4 ⁺ or CD8 ⁺ T cell populations, are isolated and purified after the production of a cell suspension from the cell suspension. To isolate the T cell populations, all of the methods commonly used in the art can be used according to the invention. In a preferred embodiment of the invention, the isolation and purification of the T cell population is carried out using a magnetic cell separation system. For this purpose, the cells are incubated with microbeads to which suitable antibodies are conjugated and, after washing, separated from the cell suspension with the aid of a magnetic field. For example, a murine CD90 ⁺ T cell population can be isolated and purified using microbeads conjugated with antibodies directed against murine CD90. A human CD4 ⁺ T cell population can be obtained using microbeads conjugated to antibodies directed against human CD4. Microbeads to which antibodies directed against human CD8 are bound can be used to isolate and purify a human CD8 ⁺ T cell population. The T cells bound to the microbeads via a suitable antibody can then be washed off the beads with a suitable buffer and transferred to suitable media or solutions.

In a preferred embodiment of the present invention, it is particularly provided that, in particular, CD3 ⁺ T cells are isolated and purified from the cell suspension of a material comprising hematopoietic stem cells isolated from a human donor. In a further preferred embodiment of the invention it is provided that CD4 ⁺ T cells are isolated and purified from a material comprising human hematopoietic stem cells. In a further preferred embodiment of the invention it is provided that CD8 ⁺ T cells are isolated and purified from a material comprising human hematopoietic stem cells. In further particularly preferred embodiments of the invention it is provided to isolate and purify CD4 ⁺ T cells, CD8 ⁺ T cells or CD90 ⁺ T cells from murine materials comprising hematopoietic stem cells.

According to the invention it is provided that the so isolated T cell population then ex vivo one subjected to special treatment for selective and targeted elimination respectively functional inactivation of only a part of the T cell Population, namely the alloreactive T cells, leads. In a preferred embodiment of the Invention is carried out ex vivo alloreactive T cells over a Activation-induced cell death (AICD) or propriocidal Cell death (PCD). In a particularly preferred Embodiment provides that the ex vivo Elimination of the alloreactive T cells via the CD95 / CD95 ligand-mediated Activation-induced cell death occurs.

The CD95 / CD95 ligand-mediated Activation-induced cell death represents an important one physiological pathway to control the clonal Expansion of antigen-activated T cells in the down phase the immune response (Lenardo et al., Annu. Rev. Immunol., 17: 221-253 (1999). The AICD also plays an important role in maintaining the peripheral T cell tolerance (Barrett et al., Bone Marrow Transpl., 21: 543-551 (1998); Van Parjis et al., Immunity, 4 81996), 321-329). Mature T cells become susceptible to AICD (Lenardo et al., Annu. Rev. Immunol., 17 (1999), 221-253; Klas et al., Int. Immunology, 5 81993), 625-630). This process requires one repeated T cell receptor (TCR) engagement under Use of an antigen or antibodies against the TCR / CD3 complex and is dependent on Cell cycle caused by interleukin 2 (IL-2) (Lenardo et al., Nature, 353 (1991), 858-862).

The principle of the induced according to the invention CD95 / CD95 ligand-mediated AICD consists of that donor T cells with one from the recipient derived allo-antigen in the presence of interleukin 2 (IL 2) are repeatedly stimulated and activated, the expression of the CD95 receptor is upregulated, and therefore receptive to AICD become. The donor T cells activated in this way are simultaneously with the CD95 ligand or against treated the CD95 receptor-directed antibody. Both the CD95 ligand (CD95L) and the counter Antibodies directed to the CD95 receptor lead to Cross-linking of the CD95 receptor on the surface of the T donor cells and thus induce one selective apoptosis. Donor T cells that are not specific for which are host specific allo antigens, are not subject to the CD95-mediated AICD Induction and therefore can survive and then be isolated. So that's it possible to specifically enrich and close T cells isolate that in the recipient, for example malignant cells or viruses react. The so T cells isolated at the time of Transplant or later in the year Framework of a so-called "DLI" (donor lymphocyte infusion) together with the hematopoietic stem cells containing cell suspension from which the T cells were originally isolated in the recipient be transferred.

In a preferred embodiment of the invention are therefore alloreactive T cells repeated contacting of the isolated T cell Population with an antigen, preferably one allo-antigen originating from the recipient, activated and simultaneously with one against one Apoptosis-inducing T cell receptor-directed Antibodies, for example an agonistic anti CD95 antibody, or one against one Apoptosis-inducing T cell receptor-directed ligands treated to be in the alloreactive T cells To induce apoptosis, for example AICD.

In a preferred embodiment of the invention it is envisaged that the T cell population will have Cells whose surface contains allo-antigens are brought into contact. According to the invention provided that it is the allo-antigen containing cells are preferably cells, from the recipient, a suitable one Cell culture or other suitable source isolated were. It is particularly preferred to antigen presenting cells such as for example spleen cells or mature ones derived from monocytes dendritic cells. In another preferred Embodiment can be used for activation alloreactive T cells genetically modified cells are used, especially those that Express antigen intensely. In another preferred embodiment, the T cell Population also with isolated and purified ones allo-antigens brought into contact and activated become. These can be antigen molecules act out of cells, tissues or organs of the recipient were isolated. According to the invention to activate alloreactive T cells too Antigen molecules are used using chemical synthesis process or by means of genetic engineering processes were produced. In a particularly preferred embodiment, the Activation of alloreactive T cells using Fragments of the antigen molecule, in particular Peptide fragments.

In a preferred embodiment of the invention the T cell population with Antigen-presenting cells, especially genetically engineered altered DC, the ligands for receptors of the Express "Death Receptor" family in contact brought to AICD in alloreactive T cells to induce.

The contacting preferably takes place isolated T lymphocytes with the allo-antigen or the cells containing the allo-antigen in the presence of interleukin 2 (IL-2). Offer cytokines such as IL-2 differentiated intervention options in the Regulation of hematopoietic cells and are also used Induction of graft versus leukemia reactions used.

After repeated contact or during the Contacting the isolated T cell population with the allo-antigen according to the invention Treatment with an anti-apoptosis inducer T cell receptor-directed ligand or a against an apoptosis-inducing T cell receptor targeted agonistic antibodies, in particular an agonistic monoclonal antibody to in activated alloreactive donor T lymphocytes To induce apoptosis, for example AICD.

In a preferred embodiment of the invention the activated is treated alloreactive T lymphocytes with the CD95 ligand (CD95L). According to the invention it is particularly provided that the activated alloreactive T cells with a isolated from natural sources and purified CD95L molecule are treated. According to the invention the CD95L molecule can also be a using genetic engineering techniques act produced molecule. The one activated for treatment alloreactive T cells used CD95L molecule can also be made using chemical synthesis Process be made. According to the invention also the possibility of only a fragment of the CD95L molecule to use. Especially preferred embodiment is the CD95L Molecule around a using recombinant DNA technology produced molecule, in particular around the Commercially available human CD95 ligands "rec. Human super FasL ". With the recombinant human ligand "rec. Human super FasL". it is a stable homotrimer that has no further crosslinking required to trimerize the CD95 receptor.

In a further particularly preferred embodiment of the invention, the activated alloreactive T cells are treated with an agonistic antibody (anti-CD95 mAb) directed against the CD95 receptor. In connection with the present invention, an “antibody” is understood to mean a polypeptide which is encoded by one or more immunoglobulin genes and which recognizes specific structures on an antigen, in particular on the CD95 receptor, and can specifically bind to them. The term "antibody" encompasses not only a complete molecule, but also a number of fragments that can be obtained by cleavage with various peptidases, for example fragments such as Fab, F (ab ') ₂ or Fvm, or that using recombinant DNA techniques which are able to recognize and bind epitopes on the CD95 receptor. The term "antibody" also includes modified antibodies such as oligomeric, reduced, oxidized and labeled antibodies.

In a particularly preferred embodiment the treatment of activated alloreactive T- Lymphocytes with an agonistic monoclonal Antibodies against the CD95 receptor. According to the invention the use of the is particularly preferred agonistic monoclonal antibody Jo2.

According to the invention, by inducing the CD95 mediated AICD in the activated alloreactive T cell population at least 60%, preferably at least 70%, particularly preferably at least 80% and most preferably at least 85%, 90%, 95% or more of the alloreactive T lymphocytes that are in the Recipients for the outbreak of graft versus host Response are eliminated.

After activation-induced cell death alloreactive T lymphocytes can be the leftover T cells that were not accessible to AICD and therefore have not been eliminated from the T cell Population are isolated. Then you can these isolated T cells with the hematopoietic Cell suspension comprising stem cells from which originally isolated the T cell population and were cleaned up, are united. That through Stem cell preparation obtained is for allogeneic Transplantation in recipients suitable and leads Transfer to recipient not to break out of graft-versus-host disease, however, can occur in Recipient and the graft versus leukemia effect mediate antiviral immunity effects.

In a preferred embodiment of the invention it is envisaged that the hematopoietic Stem cell containing material from a human Is obtained and used to produce an organism human hematopoietic stem cell preparation is used. In another preferred Embodiment of the present invention provided that the hematopoietic material from a Mouse won and to produce a mouse hematopoietic stem cell preparation used becomes.

• a) die Gewinnung eines hämatopoetische Stammzellen umfassenden Materials aus einem Donor,
• b) die Herstellung einer Zellsuspension aus dem hämatopoetische Stammzellen umfassenden Material,
• c) die Isolierung und Aufreinigung einer T-Zell- Population aus der Zellsuspension,
• d) die Behandlung der T-Zell-Population durch wiederholtes Inkontaktbringen mit einem allo- Antigen,
• e) die Behandlung der mit dem allo-Antigen in Kontakt gebrachten T-Zell-Population mit einem gegen einen Apoptose-induzierenden T- Zell-Rezeptor gerichteten Antikörper oder einem gegen einen Apoptose-induzierenden T- Zell-Rezeptor gerichteten Liganden, und
• f) die Gewinnung der nicht mit dem Antikörper oder dem Liganden reagierenden T-Zellen.

The present invention therefore also relates to a method for isolating a T cell preparation freed from alloreactive T cells, comprising:

• a) obtaining a material comprising hematopoietic stem cells from a donor,
• b) the production of a cell suspension from the material comprising hematopoietic stem cells,
• c) the isolation and purification of a T cell population from the cell suspension,
• d) treating the T cell population by repeated contact with an allo-antigen,
• e) treating the T cell population in contact with the allo-antigen with an antibody directed against an apoptosis-inducing T cell receptor or with a ligand directed against an apoptosis inducing T cell receptor, and
• f) obtaining the T cells which do not react with the antibody or the ligand.

The present invention also relates to a hematopoietic stem cell preparation used for allogeneic transplantation is suitable and that especially from the alloreactive T cell population is exempted when transplanting in one Causes a graft versus host response, which, however, still contains the T cells that are in the Recipient and the graft versus leukemia effect mediate antiviral immunity effects, after one of the methods of the invention has been.

In a particular embodiment, the present invention the use of this hematopoietic Stem cell preparation for manufacturing of a stem cell transplant that is used for allogeneic Transplantation into a recipient is suitable but none in the recipient after transplantation Graft-versus-host disease is caused.

• a) die Gewinnung eines hämatopoetische Stammzellen umfassenden Materials aus einem Donor,
• b) die Herstellung einer Zellsuspension aus dem hämatopoetische Stammzellen umfassenden Material,
• c) die Isolierung und Aufreinigung einer T-Zell- Population aus der Zellsuspension,
• d) die Eliminierung alloreaktiver T-Zellen aus der isolierten T-Zell-Population durch Induzierung des Aktivierungs-induzierten Zelltodes, insbesondere durch Inkontaktbringen der T-Zell-Population mit einem allo-Antigen und gleichzeitiges Behandeln mit einem gegen einen Apoptose-induzierenden T-Zell-Rezeptor gerichteten Antikörper oder einem gegen einen Apoptose-induzierenden T-Zell-Rezeptor gerichteten Liganden,
• e) die Isolierung der nicht eliminierten T- Zellen,
• f) die Verabreichung der von alloreaktiven T- Zellen befreiten, hämatopoetische Stammzellen umfassenden Zellsuspension an den Rezipienten, und
• g) die Verabreichung der signifikant von alloreaktiven T-Zellen befreiten allogenen T- Zellen an den Rezipienten im Rahmen einer modifizierter Donor-Lymphozyten-Infusion.

The present invention also relates to a method for preventing graft-versus-host disease in a recipient, which is treated in particular with regard to recurrent or persistent leukemia, comprising:

• a) obtaining a material comprising hematopoietic stem cells from a donor,
• b) the production of a cell suspension from the material comprising hematopoietic stem cells,
• c) the isolation and purification of a T cell population from the cell suspension,
• d) the elimination of alloreactive T cells from the isolated T cell population by inducing the activation-induced cell death, in particular by contacting the T cell population with an allo-antigen and simultaneous treatment with an anti-apoptosis-inducing T Cell receptor-directed antibody or a ligand directed against an apoptosis-inducing T cell receptor,
• e) isolation of the non-eliminated T cells,
• f) the administration of the cell suspension freed from alloreactive T cells and comprising hematopoietic stem cells to the recipient, and
• g) the administration of the allogeneic T cells which have been significantly freed from alloreactive T cells to the recipient as part of a modified donor lymphocyte infusion.

The present invention is accomplished by the following Figures and examples clarified. The figures demonstrate:

Fig. 1 shows that alloreactive after induction of AICD by means of an agonistic monoclonal antibody (mAb) against the CD95 receptor T cells have greatly reduced proliferative responses, wherein A) the proliferative responses of allogeneic CD4 ⁺ T cells shows and B) the proliferative responses of allogeneic CD8 ⁺ T cells. The reduction in the alloreactivity of CD4 ⁺ and CD8 ⁺ T responder cells after induction of the CD95-mediated AICD and subsequent treatment with an agonistic mAb proceeds in a dose-dependent manner.

CD4 ⁺ and CD8 ⁺ subpopulations were isolated from an inactivated T cell population of C3H / He (H-2 ^k ) mice by means of immunomagnetic separation. The isolated subpopulations were treated with irradiated allogeneic spleen cells from Balb / c (H-2 ^d ) mice in a mixed lymphocyte culture (MLC) over a period of 6 to 7 days in the absence of the agonistic anti-CD65 mAb Jo-2 ( filled squares) or in its presence (1 µg / ml (filled circles) or 5 ng / ml (filled triangles)). Proliferation of T cells was determined by 3H thymidine uptake analysis in the past 12 hours. The addition of an isotype-matched hamster Ig antibody (open circle) during the culture served as a control of the specificity. Syngeneic controls (open squares) are shown without AICD induction. A total of at least 5 experiments were carried out, but only the results of one experiment are shown. The results are reported as the mean ± standard deviation of three microtiter wells.

FIG. 2 shows that both T-lymphocytes at rest and preactivated T-lymphocytes respond to AICD induction. After AICD induction, T responder cells showed a greatly reduced proliferation, regardless of whether the alloreactive T cells were subjected to the anti-CD95 mAb treatment during the first or second stimulation. Small resting CD4 ⁺ T cells (H-2 ^k ) or CD4 ⁺ T cells which had been preactivated by crosslinking the T cell receptor using a CD3-directed monoclonal antibody (145-2C11), were stimulated with irradiated allogeneic spleen stimulator cells (H-2 ^d ) in the presence of an agonistic anti-CD95 mAb for five days. The remaining T cells were then obtained from the cultures and tested for their alloreactivity with regard to the allo-antigen H-2 ^d in a 5-day mixed lymphocyte reaction (MLR). The results of one of a total of three experiments are shown as the mean ± standard deviation of three microtiter wells. (A) shows the alloreactivity of T cells that had been primarily stimulated either only with H-2 ^d alone (filled squares) or in the presence of anti-CD95 mAb (filled circles). (B) shows the alloreactivity of T cells which had been preactivated with the anti-CD3 antibody after restimulation with H-2d alone (filled squares) or in the presence of an anti-CD95 mAb (filled circles). Syngeneic controls are shown without (unfilled squares) or with (unfilled circles) AICD induction.

FIG. 3 shows that the removal of alloreactive T cells by means of AICD using the allo antigen H-2 ^{d is} antigen-specific. Purified CD90 ⁺ T cells (H-2 ^k ) were analyzed in the absence (dotted bars) or in the presence of a soluble anti-CD95 mAb (1 µg / ml) (hatched bars) or an anti-CD95 mAb that was labeled with a coated anti-hamster Ig (black bars) was cross-linked, stimulated with the allo-antigen H-2 ^d in MLC for 6 days. The remaining allogeneic T lymphocytes were then stimulated with H-2 ^d or the allo-antigen H-2 ^b . The stimulation indices of a secondary MLR on the third day are shown. (A) shows AICD induction during the primary MLR, which resulted in reduced proliferation of the T cells responding to H-2 ^d after restimulation compared to untreated controls. (B) shows that responder cells, which were obtained after elimination of H-2 ^d -alloreactive T lymphocytes with soluble anti-CD95-MAK or with cross-linked anti-CD95-MAK, could react to the allo-antigen H2 ^b . As a control, the proliferation of untreated cells was examined. The results from two independent experiments are shown.

Fig. 4 shows the cytolytic response of alloreactive T cells to AICD-inducing. Shown are the results of a typical experiment, the impaired ability of allogeneic cytotoxic T lymphocytes (CTL) ^d for the lysis of H-2 - expressing A20 target cells after elimination of H-2 ^d -alloreaktiver T cells by AICD (filled circles) Comparison to untreated activated allogeneic control cells (filled squares) shows. The A20 target cells were lysed in an antigen-specific manner, since H-2 ^b- containing EL-4 target cells from untreated alloreactive CTL (open squares) or CTL that had been treated with an antibody directed against CD95 (open circles) were not were recognized. The cytotoxic T lymphocytes were generated by allogeneic stimulation of purified CD90 ⁺ T cells (H-2 ^k ) with spleen stimulator cells (H-2 ^d in the presence of an ant-CD95 mAb over a period of five days. The cells obtained were incubated with chromium-labeled A20 or EL-4 target cells for five hours and then analyzed for their lysis ability.

Fig. Shows that the elimination of alloreactive T cells by AICD prevents the onset of an acute GVH disease in a mouse P → F ₁ -GvH-Model 5. The survival curves of three independent experiments are shown, F ₁ recipients (n = 10) 2.5 × 10 ⁶ purified viable parental donor T lymphocytes being administered. T cells from C3H / He mice (H-2 ^k ), which had previously been treated with the allo-antigen H-2 ^d in vivo, were restimulated with H-2 ^d -expressing Balb / c stimulator cells and by means of AICD freed from alloreactive T cells. After transferring the treated T lymphocytes to irradiated (6.5 Gy) female F ₁ recipients, the animals did not develop acute GvH disease. In contrast, acute GvH disease developed in mice injected with untreated allogeneic control cells. (A) shows that the elimination of host-reactive antigens in CD4 ⁺ T cells by AICD induction in F ₁ recipients prevents the development of an acute GvH disease (filled squares). In contrast, the transfer of activated untreated control cells (filled triangles) or of donor T lymphocytes in which alloreactive responder cells by means of the soluble anti-CD95 mAb (filled circles) leads to the development of a lethal GvH within 20 days after transfer. Illness. In controls, an equivalent number of syngeneic spleen cells were transferred (open squares). (B) shows the results of a second experiment carried out with allogeneic CD8 ⁺ T cells.

FIG. 6 shows that donor T cells which have been freed from allogeneic T cells by means of AICD have a reduced expression in vivo. The results of flow cytometry analyzes are shown. Ten days after transfer to F ₁ recipients, the populations of untreated allogeneic CD4 ⁺ or CD8 ⁺ T cells (H-2 ^k ) had increased significantly (A), while allogeneic CD4 ⁺ or CD8 ⁺ T cells ( H-2 ^k ), which had been freed from alloreactive responder cells, showed very little expansion (B). Expansion analysis was performed on CD4 ⁺ and CD8 ⁺ T cells isolated from spleen and lymph node suspension cultures using immunomagnetic separation and labeled for H-2k ^k and H-2D ^d to distinguish them from the donor-derived T cells (H-2 ^k ) and the F ₁ -specific T lymphocytes (H-2 ^kxd ).

Materials and methods Mouse strains and tumor cell lines

C3H / HeJ (H-2 ^k ) mice, Balb / cN (H-2 ^d ) mice, C57BL / 6J (H-2 ^b ) mice and (C3H / He × Balb / c) -F ₁ (H - 2 ^kxd ) hybrid mice were bred and kept in the central animal husbandry of the University of Mainz. All mice were free of Sendai virus, mouse pneumonia virus, mouse hepatitis virus, etc. The animals were kept in sterilized microcages with filtering devices, giving them free access to autoclaved feed and filtered drinking water. Female F ₁ mice 8-14 weeks old were used as recipients to transfer T lymphocytes.

The A20 tumor cell line is a B cell lymphoma cell line derived from Balb / c (H-2 ^d ) mice. The thymoma cell line EL4 comes from C57BL / 6 (H-2 ^b ) mice.

Media and reagents

The tumor cell lines A20 and EL4 were in RPMI 1640 medium (Biomed; Berlin, Germany) cultured with 5% fetal calf serum (FCS; GibcoBRL, Ichinnan, UK), 5 mmol / l L- Glutamine, 0.1 mM β-mercaptoethanol, a 1% Antibiotic solution (Antimycotic®; GibcoBRL; Ichinnan, UK) solution consisting of Penicillin (10,000 IU), streptomycin (10 mg / ml) and Amphotericin B (25 µg / ml), 1% sodium bicarbonate, 1 mmol / l Sodium pyruvate and 0.8% non-essential Amino acids had been enriched. to Production of cell suspensions of the ex vivo isolated Cells was modified according to Dulbecco's Eagles Medium (DMEM, Biomed) used which is 2% fetal Calf serum or horse serum (GibcoBRL; Ichinnan, Great Britain) and 1% Antimycotic® (GibcoBRL; Ichinnan, Great Britain)). That for mixed lymphocyte reactions used Culture medium consisted of RPMI 1640 medium (Biomed), which with 10% fetal calf serum (GibcoBRL; Ichinnan, Great Britain), 5% NCTC (GibcoBRL; Ichinnan, Great Britain), 5 mmol / l L-glutamine, 0.1 mM β- Mercaptoethanol, a 1 percent Antimycotic® Solution (GibcoBRL), 1% sodium bicarbonate, 1 mmol / l Sodium pyruvate and 0.8% non-essential Amino acids was enriched.

Biotin-conjugated antibodies against H-2 ^d and H-2 ^k , FITC-labeled antibodies against CD3 and against CD4, phycoerytherin (PE) -conjugated anti-CD8 antibodies as well as suitable isotype controls were developed by BD Biosciences (Mountain View, California). FITC and PE labeled streptavidin were purchased from Jackson ImmunoResearch (West Grove, Pennsylvania). CD4 and CD8 microbeads used for the immunomagnetic separation of T cells were obtained from Miltenyi (Bergisch Gladbach, Germany). Antibody Jo-2 directed against CD95 was obtained from BD Biosciences (San Diego, California). The isotype matching hamster immunoglobulin (Ig) and polyclonal goat anti-hamster Ig were purchased from Serotec (Oxford, UK).

AICD induction in mixed tests Lymphocyte cultures (MLC)

Single cell suspensions of spleen cells or mixtures of spleen cells and lymph node cells were prepared in complete DMEM medium. The lymph node cells were obtained from groin, armpit and submandibular lymph nodes. The red blood cells were then lysed using NH ₄ Cl treatment. CD90 ⁺ , CD4 ⁺ and CD8 ⁺ T cells from C3H / He mice were purified using a magnetic cell separation system (MAOS) according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were coated with microbeads coated with either an anti-CD90, anti-CD4 or anti-CD8 antibody in MACS buffer (PBS containing 0.5% fetal calf serum) for 15 to 20 minutes at 4 ° C incubated. After removing excess beads by washing, the cell suspension was placed on a pre-washed VS ⁺ separation column and then separated by means of a magnetic field. The column was then washed twice with MACS buffer. The cells retained on the column were rinsed with MACS buffer and transferred to MLC medium. T cell preparations were labeled with anti-CD90, anti-CD4 or anti-CD8 antibodies at regular intervals and then analyzed by flow cytometry. The enriched T cells were more than 95% pure.

The purified, non-activated CD4 ⁺ , CD8 ⁺ or CD90 ⁺ -T responder cells (H-2 ^k ) were mixed with irradiated (30 Gy) spleen stimulator cells from Balb / c mice (H-2 ^d ), concentrations of 1 × 10 ⁶ cells / ml and 5 × 10 ⁶ cells / ml were obtained. The cells were then cultured for 5 to 6 days in MLR medium on 96-well microtiter plates in the absence or presence of different concentrations of the soluble agonistic anti-CD95 mAb Jo-2. To increase the pro-apoptosis effect, the wells were coated with anti-hamster Ig in a concentration of 5 μg / ml in PBS before adding the anti-CD95 antibody in order to enable crosslinking. In the case of a secondary MLC, responder T cells were either pre-stimulated in vitro, the isolated cells being treated with irradiated allogeneic spleen cells or immobilized anti-CD3 antibody for 3 days, or pre-stimulated in vivo, with donor mice 10 to 14 days before Isolation of T cells 5 × 10 ⁶ irradiated Balb / c spleen cells were injected intraperitoneally. The T cells were then isolated from the culture or from spleen and lymph node suspension cultures by means of immunomagnetic separation. The T cells were then suspended in a concentration of 5 × 10 ⁵ responder cells / ml in microtiter plate wells which contained 5 × 10 ⁶ spleen stimulator cells / ml. The cells were then cultivated for 5 days in the presence of the monoclonal antibody Jo2 in different concentrations. Depending on whether it was a primary or secondary MLC, the cells were harvested 2, 3, 4, 5 and 6 days after the start of the culture. Before cell harvesting, overnight labeling was carried out with ³ H-thymidine (0.037 MBq (1 µCi) per well). The radioactivity built into the DNA was determined by means of a beta liquid scintillation counter (Wallace, Turku, Finland).

Cytolytic tests

A total of 2 × 10 ⁶ A20 (H-2 ^d ) cells or EL-4 (H-2 ^b ) tumor cells were labeled with ⁵¹ chromium (100 μCi) for 4 hours. After washing three times in PBS buffer, the labeled target cells were plated at a density of 1 × 10 ⁴ cells / well into the wells of microtiter plates with a U-shaped bottom (NUNC, Wiesbaden, Germany). Allogeneic CD90 ⁺ responder T cells (H-2 ^k ) ^{that had been} preactivated in a primary MLC using H-2 ^d spleen cells for 5 days were washed three times and then in different effector / target cell ratios in three Wells were distributed and then incubated for 5 hours. The maximum release and the spontaneous release were determined by incubating the target cells with Triton-100-Sigma, Deisenhofen, Germany) or adding media. The supernatants were collected 5 hours later and the ⁵¹ Cr activity contained therein was determined in an auto-gamma counter (Packard Instruments, Dreieich, Germany). Lysis was reported as a percentage of the maximum release.

Investigation model for inducing an acute lethal GvH disease

A classic P → F ₁ model was used to investigate acute, lethal GvH reactions, with C3H / HeJ mice (H-2 ^k ) as donors of T cells and (C3H / He × Balb / c) -F ₁ mice (H-2 ^kxd ) were used as recipients. In a mixed lymphocyte culture, alloreactive T cells were eliminated in the presence of an agonistic anti-CD95 antibody over a period of 5 to 6 days. The remaining T cells were purified by means of immunomagnetic separation on a MACS column. Single cell suspensions of the highly purified, in vitro activated, viable (Trypan Blue Test) CD4 ⁺ or CD8 ⁺ T cells were injected into the recipient vein in different concentrations (F ₁ mice), the recipients 4 to 5 hours before transmission of the T cells had been subletally irradiated with ¹³⁷ Cs (6.5 Gy). As a control, F ₁ mice were used which corresponded in age and sex to the F ₁ mice to which T cells were injected. The controls were injected with syngeneic spleen cells or untreated allogeneic T cells. The recipient mice were continuously monitored for clinical signs of GvH disease such as diarrhea, hair loss and crooked posture. Her weight loss was also determined. Each study group comprised at least 4 animals. The transfer of the T cells was repeated at least three times.

Flow cytometry analysis

In general, 1 × 10 ⁶ viable T cells which had been isolated from spleen and lymph node preparations by means of immunomagnetic separation using CD4, CD8 or CD90-coated microbeads (Miltenyl, Bergisch Gladbach, Germany) were isolated per sample, Incubated for 15 minutes at 4 ° C with the specified antibodies. The antibodies were either directly conjugated or biotinylated, incubated with streptavidin-conjugated FITC or phycoerythrin (PE) as the second reagent. It was then washed twice with PBS containing 2.5% fetal calf serum and 1% sodium azide. Viable lymphocytes were determined using forward or side scattering. A two-color fluorescence analysis was performed using 10,000 cells on an Epics-Altra-FACS sorting device (Beckman-Coulter, Stanford, California).

example 1 Alloreactive T cells show after AICD induction by means of the agonistic anti-CD95-MAK Jo2 strong decreased proliferative responses

Since both CD4 ⁺ and CD8 ⁺ T cells show apoptosis after activation with CD95, it was investigated whether AICD induction is suitable as a method for the selective elimination of alloreactive T cells in an allogeneic mixed lymphocyte reaction (allo-MLR).

Unactivated T cells were isolated ex vivo from C3H / He (H-2 ^k ) mice and separated into CD4 ⁺ or CD8 ⁺ T cell subpopulations using immunomagnetic separation using the MACS method. The isolated T cell subpopulations were then cultured together with irradiated unfractionated spleen stimulator cells from Balb-c (H-2 ^d ) mice in the presence of increasing concentrations of the soluble agonistic anti-CD95-mAb Jo2 for 5 to 6 days. After stimulation with the allo-antigen H-2 ^d , the proliferative reactions of the alloreactive CD4 ⁺ or CD8 ⁺ T lymphocytes in the presence of the anti-CD95 mAb were reduced as a function of the dose by up to 90% ( FIGS. 1a and 1b). The reduction in proliferative reactions was specific for the anti-CD95-mediated apoptosis of T cells. Responder cells stimulated in the presence of hamster immunoglobulin (Ha-Ig), the isotype of which corresponded to that of the anti-CD95 mAb, gave results which corresponded to those obtained with untreated allogeneic controls ( FIGS. 1A and 1B). ,

During the 6-day stimulation with allogeneic stimulators (H-2 ^d ) freed from T cells, CD90 ⁺ responder cells were regularly isolated by means of immunomagnetic separation and analyzed by means of flow cytometric analysis. After staining with Annexin V and 7-AAD it was found that within the first 36 to 48 hours after activation the CD95 expression was increased and after treatment with the agonistic anti-CD95 mAb the proportion of apoptotic CD4 ⁺ and CD8 ⁺ -T Lymphocytes had increased significantly.

Example 2 Inactive and preactivated T lymphocytes are equally vulnerable to AICD

There are reports that according to T cells primary activation initially resistant to Are AICD. However, in a period of Continuously in contact with CD95 for 4 to 5 days brought, they show an increased susceptibility to a CD95-mediated apoptosis induction (Lenardo et al., Annu. Rev. Immunol., 17: 221-253 (1999); Klas et al., Int. Immunology, 5 (1993), 625-630). Therefore, the AICD activation with inactive T- Lymphocytes examined and with the preactivated T cells compared.

Both CD4 ⁺ T cells and freshly isolated, resting CD4 ⁺ T lymphocytes were analyzed after 3 days of polyclonal in vitro activation by means of TCR crosslinking using the anti-CD3 mAb (145-2C11) with the stimulated allo-antigen H-2 ^d in a mixed lymphocyte culture in the presence of an agonistic monoclonal anti-CD95 antibody for 5 days. The response of the remaining T cells to stimulation with the allo-antigen was then tested in a mixed lymphocyte reaction (MLR). After AICD induction, the ability to proliferate was significantly reduced in both the primary stimulated T cells ( FIG. 2A) and in the restimulated T cells ( FIG. 2B). Compared to untreated controls, proliferation decreased by 85% on the third day (compare FIGS. 2A and 2B), although the response to the antigen started earlier in the case of the responder cells preactivated by means of anti-CD3 mAb.

These results show that AICD induction after repeated stimulation of T lymphocytes with MHC allo antigens not from Preactivation status of alloreactive T cells dependent is.

Example 3 The AICD induction in alloreactive T cells is specifically with regard to the stimulating allo- antigen

To confirm that the elimination of alloreactive T cells is specific for a particular haplotype, the CD90 ⁺ T cells that were isolated after a primary allogeneic mixed lymphocyte reaction using the allo-antigen H-2 ^d in the presence of the anti-CD95 mAK remained, isolated and contacted with spleen stimulator cells expressing the third antigen H-2 ^b derived from C57BL / 6 mice. As expected from the results obtained above, restimulation with the allo-antigen H-2 ^d resulted in the proliferative response of the CD90 ⁺ T cells to controls being greatly reduced ( Fig. 3A). Interestingly, AICD induction was increased after cross-linking the ant-CD95 mAb using an anti-Ha-Ig bound to the microtiter plate. The crosslinking presumably leads to a more efficient trimerization of the CD95 receptor. In contrast, allogeneic T cells (H-2 ^k ) that had been treated with an anti-CD95 mAb showed no reduced proliferation when restimulated with the third allo-antigen H-2 ^b involved ( FIG. 3B).

These results suggest that the AICD Induction using the against CD95 directed agonistic monoclonal antibody Jo2 leads to the elimination of alloreactive T cells that are specific for a particular allo-antigen. It also shows that the networking of the monoclonal antibody to induce apoptosis strengthened.

Example 4 After AICD induction, alloreactive T cells show no cytolytic reactions

Another experiment tested whether the AICD induction using the anti-CD95 mAb also a decrease in cytolytic Alloreactive T cell responses.

CD90 ⁺ T responder cells were purified from C3H / He mice and stimulated with allogeneic spleen stimulator cells from irradiated Balb / c mice in the presence of the anti-CD95 monoclonal antibody (1 µg / ml) in a mixed lymphocyte culture for 5 days. After the treatment, the remaining T lymphocytes were obtained from the culture and tested in a cytolysis test using chromium-labeled A20 cells as target cells. At an effector / target cell ratio of 3: 1, untreated alloreactive control T lymphocytes showed a 50% -60% specific lysis. In comparison, the ability of the remaining cytotoxic T lymphocytes (CTL) to kill the A20 target cells was reduced by at least 80% ( FIG. 4). This reduction in alloreactive cytotoxicity after AICD induction was again specific for the allo-antigen H-2 ^d , since no significant lysis of EL-4 target cells expressing the antigen H-2 ^b could be determined ( FIG. 4 ). In summary, these results and the results described above show that the induction of allo-antigen-dependent apoptosis in an activated allogeneic T cell population by administration of an anti-CD95 agonistic monoclonal antibody to a clear elimination of allo-antigen-specific alloreactive T- Leads cells. However, T cells that were not previously activated as a function of an allo-antigen are not affected.

Example 5 Recipients of allogeneic T cells, which by means of AICD from host-reactive T cell subpopulations were freed from developing GvH disease

In order to confirm the effectiveness of in vitro elimination of alloreactive T cells by means of AICD, the ability of the unlimited T cells remaining after AICD induction to trigger graft-versus-host disease was tested. A classic parent C3H / He (H-2 ^k ) → F ₁ (Balb / c × C3H / He, H-2 ^dk ) -GvHD model was used. Unactivated allogeneic CD90 ⁺ donor T lymphocytes or CD90 ⁺ T cells which had been treated with H-2 ^d -Balb / c spleen cells 14 days before isolation from donor mice were treated with spleen stimulator cells cultured from irradiated Balb / c spleen mice in a mixed lymphocyte culture (MLC) in the presence of the monoclonal antibody Jo2 for 5 to 6 days. The remaining viable responder cells were then isolated. From this, CD4 ⁺ or CD8 ⁺ donor T cells were obtained by immunomagnetic separation and injected into the tail vein of F ₁ recipients who had been irradiated 6 to 8 hours before the T cells were transferred (6.5 Gy ). During the preparation of the T responder cells, these were washed thoroughly in order to remove the anti-CD95 mAb which was present in excess. Without exception, all F ₁ mice administered in vivo stimulated and in vitro restimulated donor T lymphocytes developed an acute graft-versus-host response 9 to 13 days after transmission of the T lymphocytes. The GvH response was clinically characterized by rapid weight loss, severe diarrhea, and crooked posture. All mice died within 20 days of T cell transmission ( Fig. 5). Surprisingly, the transfer of donor T lymphocytes, in which alloreactive donor cells were eliminated only with the soluble monoclonal antibody against CD95, also led to the development of a lethal GvH reaction ( FIG. 5). In contrast, the elimination of activated alloreactive T cells using the anti-CD95 mAb, which preceded the transfer of the purified allogeneic CD4 ⁺ ( Fig. 5A) or CD8 ⁺ T cells ( Fig. 5B) using the polyclonal anti-Ha-Ig had been cross-linked, to a significant improvement of the acute GvH disease in the F ₁ recipients. In this group, all recipients survived and showed no clinical signs of GvH disease. The condition of the animals remained stable and the animals developed no GvH reaction until day 120, the end of the examination. No control GvH disease developed in control mice given syngeneic spleen cells.

After the transfer of allo-antigen-activated T cells into F ₁ host mice, not only the development of GvH reactions was examined, but also the expansion of the transferred donor T cells in vivo. Splenic and lymph nodes were removed from F ₁ recipients at various times after transfer of the T lymphocytes. CD90 ⁺ cells were isolated from cell suspensions produced therefrom by means of immunomagnetic separation. The expansion of H-2 ^k positive T cells in the CD90 ⁺ cell population was analyzed by means of two-color flow cytometry and compared with the expansion of the T lymphocytes (H-2 ^kxd ) originating from the recipient ( FIG. 6). Ten days after the transfer of 2.5 × 10 ⁶ cells, the proliferation of allogeneic T cells (H-2 ^k ), which had been freed from alloreactive responder cells in vitro by means of AICD, was very low in the F ₁ recipients. A 6.4% proliferation was determined for CD4 ⁺ cells. In CD8 ⁺ cells, the proliferation was 4.5% ( Fig. 6B). In contrast, a significant expansion of untreated allogeneic T lymphocytes from C3H / He mice was found ( Fig. 6A; 33.4% for CD4 ⁺ and 22.8% for CD8 ⁺ cells).

Claims (26)

1. A method for producing a hematopoietic stem cell preparation which is suitable for allogeneic transplantation and does not cause graft-versus-host disease after transplantation into a recipient, comprising: a) obtaining a material comprising hematopoietic stem cells from a donor, b) the production of a cell suspension from the material comprising hematopoietic stem cells, c) the isolation and purification of a T cell population from the cell suspension, d) the treatment of the T cell population so that a part of the T cell population is selectively eliminated or functionally inhibited, and e) the union of the non-eliminated part of the T cell population with the hematopoietic stem cell-comprising, T cell-free cell suspension at the time of the transplantation or at a later point in time as part of a modified donor lymphocyte infusion (DLI). 2. The method of claim 1, wherein the selective Elimination or functional Inhibition of part of the T cell population by Activation-induced cell death occurs. 3. The method according to claim 1 or 2, wherein the material comprising hematopoietic stem cells is isolated from a sibling whose HLA system is identical to that of the recipient. 4. The method according to claim 1 or 2, wherein the material comprising hematopoietic stem cells isolating an unrelated donor, the HLA system matches that of the recipient. 5. The method according to claim 1 or 2, wherein the material comprising hematopoietic stem cells a family donor, the 1- Locus of its HLA system with that of the recipient does not match. 6. The method according to any one of claims 1 to 5, wherein CD3 ⁺ T cells are isolated and purified from the material comprising hematopoietic stem cells. 7. The method according to any one of claims 1 to 5, wherein CD4 ⁺ T cells are isolated and purified from the material comprising hematopoietic stem cells. 8. The method according to any one of claims 1 to 5, wherein CD8 ⁺ T cells are isolated and purified from the material comprising hematopoietic stem cells. 9. The method according to any one of claims 1 to 8, the isolated T cell population being treated in this way is that alloreactive T cells are eliminated or functionally inhibited. 10. The method according to any one of claims 1 to 9, being the elimination or functional Inhibition of the alloreactive T cells by repeated contacting of the isolated T- Cell population with an antigen and simultaneous Treat with one against one Apoptosis-inducing T cell receptor-directed Antibody or one against an apoptosis-inducing T cell receptor-directed ligand occurs. 11. The method of claim 10, wherein the T cell Population repeated with from the recipient originating allo-antigens is brought into contact. 12. The method of claim 11, wherein the T cell Population with cells on whose surface allo- Antigens are included. 13. The method of claim 12, wherein the T-cell Population is brought into contact with spleen cells. 14. The method of claim 12, wherein the T-cell Population with mature cells derived from monocytes dentritic cells is brought into contact. 15. The method of claim 11, wherein the T-cell Population with isolated and purified allo- Antigen is brought into contact. 16. The method of claim 15, wherein the T cell Population with recombinant allo-antigens in Is brought into contact. 17. The method according to any one of claims 10 to 16, wherein contacting the T cell population with the antigen in the presence of interleukin 2 (IL- 2) is done. 18. The method according to any one of claims 10 to 17, the T cell population during the repeated contact with the allo-antigen or then with one directed against the CD95 receptor monoclonal antibody is treated. 19. The method of claim 18, wherein the T cell Population during the repeated Bring into contact with the allo-antigen or afterwards with the against the CD95 receptor-directed monoclonal Antibody Jo2 is treated. 20. The method according to any one of claims 10 to 17, the T cell population during the repeated contact with the allo-antigen or then treated with the ligand of the CD95 receptor becomes. 21. The method of claim 18, wherein the T cell Population during the repeated Bring in contact with the allo-antigen or afterwards with one recombinant human CD95 ligand is treated. 22. The method according to any one of claims 1 to 21, wherein the hematopoietic material from a human organism obtained and for production of a human hematopoietic stem cell Preparation is used. 23. The method according to any one of claims 1 to 21, taking the hematopoietic material from a mouse obtained and used to produce a hematopoietic Stem cell preparation of the mouse is used. 24. A method for isolating an alloreactive T-cell preparation, comprising: a) obtaining a material comprising stem cells from a donor, b) the production of a cell suspension from the material comprising stem cells, c) the isolation and purification of a T cell population from the cell suspension, d) treatment of the T cell population by repeated contact with an allo-antigen, e) treating the T cell population contacted with the allo-antigen with an antibody directed against an apoptosis-inducing T cell receptor or with a ligand directed against an apoptosis inducing T cell receptor, and f) obtaining the T cells which do not react with the antibody or the ligand. 25. Hematopoietic stem cell preparation in which the alloreactive T cell population selectively eliminated or is functionally inhibited and that is suitable for allogeneic transplantation, whereby no graft versus host in a recipient Disease is caused, can be produced after one of claims 1 to 23. 26. Use of a hematopoietic stem cell Preparation according to claim 25 for the production of a Stem cell graft for allogeneic Transplant into a recipient, being in the recipient no graft versus host after transplant Disease is caused.