EP1294857A2

EP1294857A2 - Genetically modified t-cells, method for producing them and use thereof

Info

Publication number: EP1294857A2
Application number: EP01949257A
Authority: EP
Inventors: Thomas Ritter; Hans-Dieter Volk; Markus Hammer; Christine Brandt; Grit Schroeder; Manfred Lehmann; Alexander Fluegel
Original assignee: Universitatsklinikum Charite Berlin
Current assignee: Charite Universitaetsmedizin Berlin
Priority date: 2000-06-09
Filing date: 2001-06-08
Publication date: 2003-03-26
Also published as: WO2001094551A2; WO2001094551A3; DE10128980A1; CA2381459A1; DE10128980B4; US20020119571A1; JP2004523202A

Abstract

The invention relates to in-vitro genetically modified T-cells for preventing allogenic transplant rejection in vivo, to methods for producing them and to their use. T-cells of the transplant recipient are stimulated in vitro with cells of the transplant donor or with cells which express dominant MHC molecules and at the same time, transduced with immunomodulating genes by means of gene transfer. After the gene transfer, the transduced T-cells begin to express the immunomodulatory genes. The gene transfer can be carried out using retro viruses, other viral vector systems or liposomes. The conditions selected for the experiment, which result in the generation and expansion of allo-specific T-cells, ensure that the T-cells migrate into the allogenic transplant and into the draining lymph nodes specifically according to the in vivo application and can express the immunomodulating genes there. The invention provides an effective means of preventing the rejection of allogenic transplants (cells, tissues, organs) and is therefore an effective means of inducing and maintaining tolerance of allogenic transplants (cells, tissues, organs) in transplant medicine.

Description

Gene-modified T cells, processes for their production and their use

description

The invention relates to in vitro gene-modified T cells for preventing allogeneic graft rejection in vivo, methods for their production and their use. Here, T cells of the transplant recipient are stimulated in vitro by cells from the transplant donor or by cells which express dominant MHC molecules, and at the same time are transduced using gene transfer methods with immunomodulating genes. After gene transfer, the transduced T cells begin to express the immunomodulatory genes. The gene transfer can be carried out with the help of retroviruses or other, non-viral gene transfer techniques, for example liposome formulations. Due to the chosen experimental conditions, which lead to the generation and expansion of allo-specific T cells, the T cells migrate specifically after the in vivo application both into the allogeneic graft and into the draining lymph nodes and can express the immunomodulating genes there. The invention makes it possible to effectively prevent the rejection of allogeneic grafts (cells, tissues, organs) and thus represents an effective means both for inducing tolerance and for maintaining tolerance towards allogeneic grafts (cells, tissues, organs) in transplantation medicine The success of conventional immunosuppression with cyclosporin A, FK506, glucocorticoids or OKT3 (monoclonal antibody (mAb) against CD3) does not solve the problem of graft rejection satisfactorily. A lifelong drug immunosuppression almost always leads to serious side effects and can rarely completely inhibit chronic rejection. The goal of transplant research is to achieve lifelong acceptance of a foreign organ with short-term therapy. There are already some approaches in animal models that come close to this requirement. The basis for understanding these approaches is the precise analysis of the rejected or tolerated tissue. It always shows a massive infiltration of the tissue with granulocytes, monocytes and lymphocytes during the acute rejection. The fact that the depletion of CD3-positive cells by OKT3 protects the graft against rejection shows the critical role of the T lymphocytes (Ode-Hakim et al, 1996). Similarly, immunodeficient SCID mice (without B and T cells) are unable to reject a foreign organ. Within the T cell population, Th cells appear to be the initiators of the rejection. This was de shown in CD4 or CDS T cell depleted mice. While the CD8-deposed mice can reject the graft, the CD4-deputated cannot do so (Campos et al., 1995). If one starts from the Thl / Th2 paradigm (Mosmann et al., 1989), mainly Thl cells are involved in the early phase of acute rejection. An increase in characteristic Thl cytokines (IFN-γ, IL-2) in the graft was also shown in rat models using semiquantitative PCR (Siegling et al., 1994a). After in vitro restimulation, the CD4 cells obtained from the organ also predominantly produce Thl cytokines. The common goal of all treatment protocols is to inhibit the potentially damaging Thl cells in their formation or function. While conventional methods try to do this with global depletion or lymphocyte insertion, newer approaches intervene in the T cell activation process. Monoclonal antibodies against the CD4 receptor modify the TCR signal (Lehmann et al, 1992; Siegling et al., 1994b). CTLA4-Ig binds to the B7 molecules of the antigen-presenting cells (APC's) and thus blocks costimulatory signals (Sayegh et al, 1995). At the end of this short treatment (approx. 2 weeks), a stable tolerance develops in many models (Cobbold & Waldmann, 1998). Regulatory CD4 cells are likely to mediate this tolerance. It could be shown that transfer of these cells to syngeneic animals also creates tolerance. This phenomenon was first described in 1993 as "infectious tolerance" (Qin et al., 1993). However, the attempt was only successful in a weak "rejection model". With the help of a non-depleting AK against CD4 (RIB 5/2), this was also shown in a strong rejection model (Onodera et al, 1996a). With the help of semi-quantitative PCR, a greatly increased interleukin-4 (IL-4) mRNA level in the transplanted organs (even after several transfers) was detected. This indicates the importance of Th-2 cytokines, especially interleukin-4. In addition to IL-4, a number of cytokines are able to modulate Thl-mediated immune reactions. IL-4 induces the differentiation of naive CD4 ⁺ T cells into Th2 cells, is produced by them and strongly inhibits the secretion of IFN-γ. This suppresses the development of a Thl response (Banchereau, 1991). IL-10, which is primarily formed by monocytes / macrophages and T cells, has a whole range of anti-inflammatory properties. Among other things, it was shown that i) IX-10 inhibits MHC class II expression on monocytes, ii) inhibits the production of inflammatory cytokines such as IFN-γ, IL-1 and IL-8 and TNF-α and iii) proliferation suppressed allogeneically activated lymphocytes (de Waal Malefyt et al, 1991a; de Waal Malefyt et al., 1991b; Ralph et al., 1992; Cassatella et al, 1993; Qin et al, 1997). IL-12 has been described as an additional, T cell-independent differentiation factor for Thl cells. This cytokine is secreted by macrophages and B cells, leads to an increase in IFN-γ production by NK cells, CD4 ⁺ T cells and CD8 ⁺ T cells and thus induces the differentiation of naive CD4 ⁺ T cells to Thl cells (Hsieh et al, 1993: Germann et al., 1993; Kennedy et al 1994). IL-12 is a heterodimeric glycoprotein consisting of a 40kDa (ρ40) and a 35kDa (ρ35) subunit. The IL-12p40 subunit is able to specifically inhibit the effects of the heterodimer. According to Mattern et al. (1993) supernatants of mouse IL-12 p40 transfected COS cells inhibit various IL-12 effects in vitro. IL-12p40 inhibits the proliferation of PHA and IL-12 activated splenocytes.

In experimental and clinical studies, it was possible to show in allotransplantations that, in the phase of acute rejection, the expression of various cytokines takes place, the cellular origin of which is Thl cells, Th2 cells, cytotoxic CD8 ⁺ T cells, but also non-lymphocytic cells (Macrophages, endothelial cells, mast cells) can be assigned (Dallman et al., 1991). There is evidence that Thl cytokines play a key role in the pathogenesis of acute graft rejection. This is based on studies of the cytokine expression patterns in grafts from tolerant animals. Investigations on the effective tolerance induction with anti-CD4 monoclonal antibodies offer hints for this (Benjamin and Waldmann, 1988; Takeuchi, 1992; Siegling et al., 1994a). The mechanism is not entirely clear, especially since depletion of CD4 ⁺ T cells is not necessary for tolerance induction. Tolerance induction by anti-CD4 treatment is associated with a clear suppression of Thl cytokine expression in the graft. This led to the conclusion that Thl cytokines play an essential role in allograft rejection (Siegling et al., 1994a, Lehmann et al., 1997). The importance of Th2 cytokines is less clear. In tolerant animals, the Th2 response alone did not lead to graft rejection. The persistence of Th2 cytokines in animal grafts could be regarded as an epiphenomenon, but could also be decisive for inhibiting the Thl response and thus be a decisive criterion for the graft reaction. This assumption is confirmed by observations that a temporary Thl / Th2 cytokine imbalance of the T cell response can lead to a permanent imprint of the immune response immediately after antigen contact (Scott, 1991). Initial investigations in vitro show in the transplant model that the function of transplant-inflilating cells can also be influenced. Produce the frequency of IFN-γ- the cells could be reduced by 50-70% under the influence of recombinant IL-4 (Merville et al, 1993). From this, the hypothesis can be derived that temporary overexpression of IL-4 at the site of the allo response can lead to graft acceptance. The overexpression of IL-4 after ex vivo gene transfer with the help of recombinant adenoviruses led to a significant extension of the graft acceptance in the allogeneic kidney transplant model of the rat compared to untreated or treated with a reporter construct (Kato et al., 1999a). The role of IL-4 in the induction of tolerance to allogeneic grafts is controversial in the literature. It has been shown that local overproduction of IL-4 either by adenoviral transfected or IL-4 transgenic grafts does not lead to prolongation of graft acceptance (Smith et al., 1997; Mueller et al., 1997). On the other hand, it has been shown that transgenic, IL-4 producing grafts or the systemic application of IL-4 in combination with cyclosporin A can extend the survival time of allogeneic grafts (Takeuchi et al., 1997; Rabinovitch et al., 1997) , It should be noted that the work is often methodologically inadequate, so that it cannot be decided whether methodological problems have an impact on the result.

In contrast to IL-4, the importance of IL-10 for extending graft acceptance is less controversial. It has been shown that overexpression of TGF-ßl and vIL-10, an Epstein-Barr virus-coded homolog to human or murine IL-10, leads to prolongation of graft acceptance in various allogeneic heart transplantation models (Qin et al., 1995; Josien et al., 1998). Kato et al. were able to show that the co-application of IL-4 and vIL-10 with the help of recombinant adenoviruses significantly increased the survival of allogeneic kidney transplants in a strong rejection model (Kato et al., 1999b). Interestingly, the sole application of IL-4 in this model has no effect on extending graft acceptance.

Overexpression of IL-12p40 also appears to have positive effects on graft survival. It could be shown that the local application of IL-12p40 inhibited the Thl-mediated immune response and prevented the rejection of allogeneic myoblasts that were transfected with the cDNA for IL-12p40 (Kato et al, 1997). Similar results were obtained in the islet cell transplantation model in diabetic mice, where overexpression of IL-12p40 prevented Thl-mediated autoaggression (Rothe et al., 1997; Kato et al., 1998). The co-application of IL-4 and IL-12p40 with the help of recombi- adenoviruses significantly named the prolonged survival of allogeneic kidney transplants in a strong rejection model (Kato et al., 1999b).

The problem of many works on cytokine transfer is still the application of the cytokine. The systemic administration of a cyto ins can never create a local environment that corresponds to the physiological situation. In addition, cytokines in the serum have a very short half-life, so that the therapeutic protein would have to be supplied continuously in order to achieve a desired serum level (H.-D. Volk, personal communication). Adenovirus-mediated gene transfer from the donor organ can increase cytokine expression in the transplant, but it is usually only brief and in no way dependent on activation.

However, retrovirally transfected T cells are able to stably and permanently express a protein (Blaese et al., 1995). Particularly activated T cells increasingly express their transgene (Quinn et al., 1998; Hammer et al., 2000). Bromberg et al. describe in Transplantation, Vol. 59, 6, 809-816, 1995 a method for retroviral or adenoviral gene transfer directly into the graft. However, exposure of the patient to viruses cannot be avoided.

The invention has for its object to provide new ways to prevent allogeneic graft rejection, which eliminates the disadvantages of the known means and methods. The task was solved by providing in vitro gene-modified, alloreactive T cells that express a therapeutic gene.

The task of opening a new possibility for preventing allogeneic graft rejection was achieved in particular by stimulating T cells of the graft recipient in vitro by cells of the graft donor or by cells which express dominant MHC molecules and simultaneously or later by means of gene transfer of immunomodulating (therapeutic, for example viral IL-10, for example from EBV or CMV, IL-4, IL-12p40) genes are transduced. After the gene transfer, the transduced T cells begin to express the immunomodulatory genes. The gene transfer can take place either with the help of retroviruses or with non-viral methods (liposomes, gene cannons). The chosen test conditions lead to the generation and expansion of the allo-specific, transduced T cells in vitro. Because of their allo-specificity, the modified T cells have the property, after the in vivo application of the cells at the time of an allogeneic organ transplant, specifically in both the allogeneic graft and the drainage. immigrate to the lymph node and express the immunomodulating (therapeutic) genes there.

The invention makes it possible to effectively prevent the rejection of allogeneic grafts (cells, tissues, organs). The modified cells according to the invention therefore migrate into the transplant on account of their allo-specificity. It has been found that the production of IL-4, IL-10 and IL-12p40, depending on the degree of activation of the cells according to the invention, creates a local environment of Th2 cytokines or Thl antagonists directly at the point of Ag contact , It was thus possible to generate all-reactive T cells producing IL-4, EL-10 or IL-12p40 by retroviral gene transfer in vitro.

The focus is on the expression of the transgene in the transplanted tissue itself. So far, such (generated T cells) cells have mainly been used to fight tumors. Here, ex vivo generated tumor-specific T cells were transfected with pro-inflammatory cytokines (such as TNF-alpha), which "fight" the tumor and its metastases in the event of later infiltration.

Grafts stressed by ischemia / reperfusion, infection or rejection crises also express autologous stress proteins against which an immune response can be generated (e.g. heat shock protein HSP 70, specific autoreactive T cells). These cells can also be generated in vitro and used as a biological “drag delivery” system. Instead of directly alloreactive T cells (against donor MHC molecules), indirectly alloreactive (against donor MHC peptides presented by recipient MHC) can also be generated and used. Both approaches have the advantage of less reactivity to the graft compared to directly alloreactive T cells. The in vitro transduced, gene-modified T cells are obtained by co-culture or by co-incubation with cell culture supernatants from so-called amphotrophic cell lines, which e.g. which produce recombinant retroviruses with the therapeutic transgenes. The method according to the invention for producing the in vitro transduced, gene-modified T cells consists of the following steps:

Production of the corresponding packaging cell lines which produce the recombinant retroviruses which are capable of gene transfer and which code for therapeutic transgenes

(by transfection).

- Generation of the alloreactive T cells in vitro. The cell line that produces the retrovirus capable of gene transfer with the therapeutic transgene is grown in culture taken. In addition, the lymphocytes (donor T cells or cells that express dominant MHC molecules and recipient T cells) are isolated from whole blood. The donor T cells or the cell lines which express dominant MHC molecules must be irradiated in order to prevent proliferation of these cells). Afterwards, cocultivation consisting of the mixed lymphocyte culture (primary MLC) and the retrovirus-producing packaging cell line is carried out. The retroviral gene transfer can also take place in that only the virus supernatant of the packaging cell line is added to the culture of the lymphocytes (donor T cells or cells which express dominant MHC molecules and recipient T cells), so that cocultivation with the packaging cell line is given can be dispensed with.

- If non-viral gene transfer methods are used, there is also no co-culture of the mixed lymphocyte culture (donor T cells or cells that express dominant MHC molecules and recipient T cells) with the packaging cell line. The resulting or originated alloreactive T cells are transduced directly in vitro using non-viral gene transfer methods with plasmids which code for the therapeutic genes. The alloreactive T cells serve as "universal" carriers of therapeutic genes. Above all, these are:

Gene products that are removed from the cell and their immunoregulatory influence on other cells, e.g. exercise alloreactive or transplant-infiltrating cells (e.g. cytokines (IL-13, cytokines that are homologous to the IL-10 gene, e.g. the CMV IL-10)

- Gene products which are expressed on the cell surface of the regulatory T cells and which develop their immuno-regulatory action through interaction with other cells (alloreactive or transplant-infiltrating cells) (for example CTLA-4 or genes which belong to the family of the Notch -Ligands / receptors belong, such as hSerrate-1, hDeltal or Notchl-4)

Gene products that are expressed intracellularly and, due to their cell-protective effect, impart an extended survival time to the regulatory T cells (e.g. anti-apoptotic genes such as bcl-2, bcl-xl, bag-1)

- Cell-protective genes (eg anti-apoptotic genes, heat shock genes) IL-4, IL-10, vIL-10 and IL-2p40 are preferably used as therapeutic genes (transgenes). Hemoxygenase-1 can also be used. The transduced, gene-modified T cells can be used in different applications (iv, ip), different combinations thereof and / or different dosages at different times.

The in vitro transduced, gene-modified T cells are suitable for preventing allogeneic graft rejection in vivo and can be used in the transplantation of allogeneic cells, tissues and organs. Examples include the transplantation of stem cells, bone marrow, skin, kidney, heart, liver, lungs, cells of the central nervous system, or Langerhans islands. Here, T cells of the transplant recipient are stimulated in vitro by cells from the transplant donor or by cells which express dominant MHC molecules and simultaneously transduced by means of gene transfer, with immunomodulating genes being transferred. The result is the generation of T cells that can induce tolerance induction or maintenance of tolerance to allogeneic grafts.

The essence of the invention consists in a combination of known - amphotrophic Zeil lines, retroviral vectors, mixed lymphocyte culture - and new elements - co-culture from the mixed lymphocyte culture (primary MLC) and the therapeutic retrovirus-producing Zeil line - which leads to that gene-modified T cells are created that can express therapeutic genes and, due to the allo-specificity, can migrate into both the allograft and the draining lymph nodes. The success of the method is that the rejection of allogeneic grafts (cells, tissues, organs) is effectively prevented and thus an effective means in transplant medicine is made available. The use of therapeutic T cells according to the invention consists in the prevention of allogeneic graft rejection. The (in vitro) transduced, gene-modified T cells are used as agents for inducing tolerance and for maintaining tolerance to allogeneic grafts (cells, tissues, organs) and for stimulating the T cells of the transplant recipient.

The invention is to be explained in more detail on the basis of exemplary embodiments, without being limited to these examples. embodiments

Example 1: Production of the therapeutic T cell lines Generation of the Zeil lines

First, the corresponding Zeil lines are produced, which the recombinant retroviruses produce with the therapeutic transgenes. The NIH / 3T3-derived Zeil line PT67 (Retropack ™, Clontech) serves as the starting point for the production of infectious, replication-incompetent retrovirus. PT 67 contains the genes gag, pol and env (10A1 strain) of the Moloney Murine Leukemia Virus (MoMuLV). The cells are grown in DMEM 10% FCS, 4mM L-glutamine, 100U / ml penicillin and 100μg / ml streptomycin at 37 ° C in a 5% CO ₂ atmosphere. The transfection of this Zeil line with a retroviral vector, which does not contain the above-mentioned genes, but contains a therapeutic gene and a packaging signal, allows the production of replication-deficient retrovirus (ie the virus can infect its target cells, but cannot replicate in them and others) Infect cells) (Coffin et al., 1996; Ausubel et al, 1996). The PT67 cells are transfected by means of calcium phosphate transfection according to standard protocols (Maniatis). By selection with G 418 (0.5 mg / ml) clones and, in turn, derived from them Zeil lines are established which produce both the replication-deficient retrovirus and the therapeutic gene. In the case of IL-4 and IL-10, ELISA tests can be used to determine the Zeil lines with the highest production of the therapeutic gene, which is then used in all further experiments. In the case of IL-12p40, no ELISA test is available. Here the biological activity is determined directly in the bioassay (inhibition of IFN-γ production by activated spleen cells).

Example 2: Generation of the alloreactive T cells in vitro

One to two days before the mixed lymphocyte culture (cultivate irradiated donor T cells with recipient T cells), the Zeil line which produces the recombinant retrovirus with the therapeutic transgene is taken in culture (DMEM + 10% FCS + Selection antibiotic G 418 0.5 mg / ml final concentration). On day 1, the coculture, consisting of the mixed lymphocyte culture (primary MLC) and the retrovirus-producing Zeil line, is carried out: the cells of the Zeil line trypsinized, centrifuged for 5 minutes at 1200 rpm and taken up in T-cell medium (TCM) without FCS. Then the cells are counted and seeded to a density of 2xl0 ⁵ -2xl0 ⁶ cells / per 96 plate. First, the cells are allowed to grow in a CO ₂ incubator (5% CO) at 37 ° C. for 3-4 hours before the T cells are added. The T cells of the transplant recipient are previously isolated from the peripheral blood using a Fikoll gradient according to standard protocols. For antigen presentation, the cells of the transplant donor (T cells) are also isolated according to the standard protocol. If Zeil lines expressing dominant MHC epitopes are used, these are thawed 1-2 days beforehand and cultivated in the CO incubator until use. The cells used for antigen presentation (as stimulator cells for the T cells of the transplant recipient) must be irradiated at 30 gy for 10 minutes before addition to the mixed lymphocyte culture in order to inhibit this from the undesired proliferation. After irradiation of the antigen presenting cells, they are centrifuged and taken up in 20 ml TCM without FCS. Then the cell number is determined and 50μl TCM with 3.5 x 10 ⁵ - 4 x 10 ⁵ cells of the transplant recipient and the antigen-presenting cells in 3% autologous serum and 4μg / ml polybrene in 96well round-bottom plates (total volume 100μl) and plated Incubated 37 ° C in the CO ₂ incubator with complete rest. Day 4: On day five, the MLC is implemented from round floor slabs in flat floor slabs. For this purpose, about 50μl of the culture supernatant is pipetted off (is discarded) and the T cells are transferred into a 96-well flat-bottom plate by resuspending 2-3 times without air bubbles. In addition, 100 μl medium (+ hrιL-2, 25U / ml), which should be freshly prepared, will be added. The cells are cultivated for a further 48 hours at 37 ° C. under a CO ₂ atmosphere (5%). Day 6:

The G 418 selection takes place on day 6, i.e. all cells in the course of which the retroviral gene transfer was not transduced perish within the scope of the G 418 selection. Conversely, only the cells which have been transduced by the retrovirus survive the G 418 selection. From this stage on, the cells should always be cultivated under G 418 (0.4 mg / ml G-418 final concentration). The cells are cultivated in this medium for a further 48 hours. Day 8: Restimulation 2 "

The cells are restimulated on day eight after the first stimulation. As already described for the first stimulation, either the transplant donor's PBMC is used for this or Zeil lines expressing dominant MHC epitopes. The cells are again irradiated (10 min, 30 Gy), then centrifuged (1,200 rpm, 5 min) and then taken up in 20 ml TCM without FCS and the cell number is determined. For the restimulation, lOOμl are first removed from the 96well microtiter plate, which contains the MLC cells, and 6x10 ⁵ stimulator cells are added. It is also adjusted to 3% autologous serum and a G 418 concentration of 0.4 mg / ml. The cells are then incubated for a further two days. Day 10: On day 10, fresh TCM medium (+ hrIL-2, 25U / ml) is added (remove lOOμl and lOOμl TCM medium (+ hιTL-2, 25U / ml) + G 418, 0.4mg / ml). The cells are then incubated for a further two days. Day 12:

Proliferating cells (blasts) should be located in the cell culture plates, which can be expanded by further restimulation steps in order to have a sufficiently large number of cells available for application in the clinic. On day 14 there is also the option of cleaning and isolating the generated blasts using a Fikoll gradient (Ficoll 3000). The next restimulation (3 ") takes place 24 hours after the gradient. For stimulation, PBMC cells of the transplant donor or Zeil lines which express dominant MHC molecules or Zeil lines which are transfected with the genes for these molecules and these can be constitutive express (K. Wood) can be used.

Example 3: Alternatives to coculture

Instead of the coculture with the amphotrophic cell line, only the cell culture supernatant containing the retrovirus should be used for the transduction of the T cells.

Example 4: The bioassay: detection of the therapeutic gene in the supernatant can be carried out by:

IL-4, ELISA, MHC-II upregulation on spleen cells vIL-10, ELISA, inhibition of TNF-α production by macrophages, reduction of MHC-II expression on monocytes

IL-12p40, no ELISA, inhibition of IFN-γ production after stimulation of spleen cells.

Example 5: Immunoregulatory potential of the allospecific T _VIL - _IO lymphocytes The inhibition of naive T cell proliferation by vIL-10 transgenic lymphocytes was initially demonstrated in vitro in the mixed lymphocyte culture (MLC). This in vitro system is designed to mimic the situation of T cell reactivity after an allogeneic organ transplant. For this purpose, the naive recipient cells (supposed to imitate the T cells of the transplant recipient) were stained with the dye SNARF ™ on day 0. This dye has the property that it is passed on to the two daughter cells in equal parts during cell division, so that the fluorescence intensity decreases. The stained recipient cells were then sown with irradiated stimulator cells (supposed to imitate the transplant-specific cells) in a ratio of 1: 1 (3.5 x 10 ⁵ cells each) in 96-well flat soil. In order to investigate the influence of the therapeutic T _VJL - _IO T cells on the antigen-induced proliferation of the naive lymphocytes, these were added in a ratio of _1:20 (5%). A syngeneic control and allospecific T _EGFP lymphocytes (with an irrelevant control gene, enhanced green fluorescent protein, as a so-called therapeutic gene) were used as the control approach, which were used in the same ratio. The fluorescence intensity and decrease were measured by fluorescence flow cytometry (FACS) on days 1-4. In comparison with the allogeneic control without therapeutic T cells, an inhibition of the proliferation to approximately 70-80% on days 3 and 4 could be demonstrated in the batches with T _VIL - _IO lymphocytes (FIGS. 1 and 2).

Example 6: Inhibition of interferon-γ production in naive T-lymphocytes by T _VIL - _IO cells

The inhibition of interferon-γ production in naive T lymphocytes by T _VIMO cells was also demonstrated in the MLC. The same experimental approach was used. In this case, another agent (CFSE) was used as membrane dye, which glows in fluorescence channel 1 of the flow cytometer. IFN-γ was detected by means of a PE-labeled monoclonal intracellular antibody against IFN-γ in FACS on day 4. The decrease in IFN-γ production in naive T lymphocytes after cultivation with allospecific TvIL-10 lymphocytes is 50% on day 4 (Figure 3). A comparison of the transgenic T _V IL-IO cells with T _EGFP cells and non-transgenic allospecific lymphocytes at the protein and RNA level provides information as to whether the therapeutic cells differ in their cytokine expression patterns (rIL-2, rlFN-γ, rIL -10 etc.) from the other cells. Activation markers (rCD25), apoptotic (FasL) and antiapoptotic (Bag-1) gene expression patterns are also analyzed. These attempts Before they help to clearly characterize the therapeutic cells and to analyze possible mechanisms of action of these lymphocytes.

Example 7: Gene transfer to alloreactive T cells using non-viral methods: In addition to viral gene transfer, non-viral gene transfer to generate the alloreactive, gene-modified T cells is also to be investigated. For this purpose, the allospecific T cells produced using the mixed lymphocyte culture are e.g. incubated with certain liposome formulations containing the plasmid with the therapeutic gene or bombarded with a gene gun. Co-culture with the virus-producing packaging cell line is not necessary for this approach.

Example 8: Gene transfer to alloreactive T cells with other viral vector systems: In addition to retroviral gene transfer based on the murine Moloney Leukemia Virus (MoMuLV), the production of the alloreactive, gene-modified T cells is also to be protected with the aid of other viral vector systems. This is supposed to be gene transfer with lentiviral contracts (these are also retroviruses, but based on the human immunodeficiency virus (HIV), with contracts based on adeno-associated viruses (AAV) and constructs based on cytomegaloviruses (CMV) include.

literature

Bagley, J., Aboody-Guterman, K., Breakefield, X., Iacomini, J. Long-term expression of the gene encoding green fluorescent protein in murine hematopoetic cells using retroviral gene transfer. Transplantation 1998, 65, 1233-1240.

Blaese, RM, Culver, KW, Miller, D., Carter, C, Fleisher, T., Clerici, M., Shearer G., Chang, L., Chiang, Y., Tolsthev, P., Grennblatt, JJ, Rosenberg, SA, Klein, H., Berger, M., Müllen, CA., Ramsey, WJ, Muul, L., Morgan, RA, Anderson, WF, T-Lymphocyte-Directed Gene Therapy for ADASCID: Initial Trial Results After 4 Years. Science 1995, 270: 475-477

Cobbold, S. & Waldmann, H., How do monoclonal antibodies induce tolerance? A role for Infectious Tolerance? Annual Reviews Immunology 1998, 16: 619-644 Campos, L., Naji, A., Deli, BC, Kern, JH, Kim, JI, Barker, CF, Markmann JF, Survival of MHC-Deficient Mouse Heterotropic Cardiac Allografts. Transplantation 1995, 59: 187-191

Flügel, A., Willem, M., Berkowicz, T., Wekerle, H. Gene transfer into CD4 + T lymphocytes: Green fluorescent protein-engineered, encephalitogenic T cells illuminate brain autoimmune responses. Nature Med. 1999.7: 843-847

Hammer, M., Flügel, A., Seifert, M., Lehmann, M., Brandt, C, Volk, H.-D., Ritter, T. Potential of allspecific gene-engineered T cells in transplantation gene therapy : specific T-cell activation determines transgenic expression in vitro and in vivo. 1999. Human Gene Therapy, submitted

Lehmann, M., F. Sternkopf, F. Metz, J.Brock, W.-D. Docke, A. Plantikow, B. Kuttler, H.J. Hahn, B. Ringel, H.-D. People. A novel high-efficient anti-CD4 monoclonal antibody induces long-term survival of rat skin allografts. Transplantation 1992, 54: 959-962

Lehmann, M., Kuttler, B., Siegling, A., Fordalla, A., Riedel, H., Lacha, J., Hahn, H.-J., Brock, J., Volk, H.-D. Characterization of the anti-CD4-induced permanent acceptance of rat renal allografts. Transplant Proc. 1993, 25: 2859.

Lehmann M, Graser E, Risch K, Hancock WW, Muller A, Kuttler B, Hahn HJ, Kupiec-Weglinski JW, Brock J, Volk HD. Anti-CD4 monoclonal antibody-induced allograft tolerance in rats despite persistence of donor-reactive T cells. Transplantation 1997 Oct 27; 64 (8): 1181-7

Mosmann, T.R. & Coffmann, R.L., Thl and Th2 cells: different pattems of lymphokine secretion lead to a diffrent functional properties. Annual Reviews Immunology 1989.7: 145-173

Ode-Hakim, S., Docke, WD, Kern, F., Volk, HD, Reinke, P. DTH-like mechanisms in late acute rejection - role of CD 4+ T lymphocytes. Transplantation 1996, 61: 1233-1340 Onodera, K., Lehmann, M., Al <alin, E., Volk, H.-D., Sayegh, MH, Kupiec-Weglinski, JW Induction of "infectious ,, tolerance to MHC-incompatible cardiac allografts in CD4 monoclonal antibody-treated sensitized rat recipients. J. Immunol. 1996a, 157: 1944-1950

Onodera, K., Hanckock, W.W., Graser, E., Lehmann, M., Volk, H.-D. Sayegh, M.H., Strom, T. B., Kupiec-Weglinski, J.W. Th2-cytokines and the development of "infectious ,, tolerance in rat cardiac allograft recipients. J. Immunol, 1997, 159: 1572-1581

Qin, S., Cobbold., S.P., Pope, H., Elliott, J., Kioussis, D., Davies, J., Waldmann, H. "Infectious" transplantation tolerance. Science, 1993, 259: 974-977

Qin, L., Chavin, K., Ding, Y., Favaro, JP, Woodward, JE, Lin, J., Tahara, H., Robbins, P., Shaked, A., Ho, DY, Sapolsky, RM , Lotze, MT, Bromberg, JS Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Transplantation 1995, 59: 809-816

Quinn, E.R., Lum, L.G., Trevor, K.T., T cell activation modulates retrovirus-mediated gene expression. Human Gene Therapy 1998, 9: 1457-1467

Ritter, T., Risch, K., Schroeder, G., Kolls, J., Siegling, A., Graser, E., Reinke, P., Brock, J., Lehmann, M., Volk, H.D. Intragraft overexpression of IL-4 neither sufficient nor essential for tolerance induction to cardiac allografts in high-reponder strain combinations. Transplantation 1999, 15: 1427-1430.

Sayegh, M.H., Akalin, E., Hanckock, W.W., Rüssel, M.E., Carpenter, C.B., Turka, L.A. CD28 / B7 blockade after alloantigenic challenge in vivo inhibits Thl cytokines but spares Th2. 1995. J. Exp. Med. 178: 1801-06

Siegling, A., M. Lehmann, H. Riedel, C. Platzer, J. Brock, F. Emmrich, H.-D. People. A non-depleting anti-rat monoclonal antibody which suppresses T helper 1-like but not T helper 2- like intragraft lymphokine secretion induces long-term survival of renal allografts. Transplantation! 994a, 57: 464-467 Siegling, A., M. Lehmann, C. Platzer, F. Emmrich, H.-D. People. A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats. 1994b. J. Immunol. Meth. 177: 23-28

Suzuki, T., Taghara, H., Narula, S., Moore, K.W., Robbins, P.D., Lotze, M.T. Viral Interleukin 10 (IL-10), the Human Herpes Virus 4 Cellular IL-10 Homologue, Induces Local Anergy to Allogenic and Syngenic Tumors. J.Exp.Med. 1995, 182: 477-486

Takeuchi T., Ueki, T., Sunaga S., Ikuta K, Sasaki Y., Li B., Moriyama N., Miyazaki J., Kawabe K. Murine interleukin 4 transgenic heart allograft survival prolonged with down-regulation of the Thl cytokine mRNA in grafts. Transplantation 1997 Jul 15; 64 (l): 152-7

VanBuskirk, A.M., Wakely, M.E., Orosz, CG Transfusion of polarized TH2-like cell populations into SCID mouse cardiac allograft recipients results in acute allograft rejection. Transplantation 1996 Jul 27; 62 (2): 229-38

List of abbreviations

AAV Adeno-Associated Virus Ag Antigen

Ak antibody

APC antigen presenting cells

B cells B cells

B7 molecules surface marker on APC, important for the activation of T-cells bag-1 Bcl-2 associated athanogenic, anti-apoptosis gene, interacts with Bcl-2 bcl-2 B cell leukemia-2, anti-apoptosis gene bcl-xl Bcl- 2 Homolog, antiapoptosis gene cDNA complementary DNA, copy DNA

CD cluster of differentiation, nomenclature for surface molecules CD4 specific surface markers on T helper cells

CMN cytomegalovirus

(RIB 5/2) Term for a monocl against the CD4 molecule. antibody CD8 T-specific surface marker on cytotoxic T cells CMV IL-10 cytomegalovirus IL-10, homologous to human IL-10 or vIL-10 CTLA-4 cytotoxic T-cell late antigen CTLA4-Ig fusion protein, consisting of CTLA-4 ( cytotoxic T-cell late antigen) and the Fc part of the IgG antibody

DMEM Dulbeccos' modified Eagle's Medium DNA deoxyribonucleic acid (Deoxyribonucleic acid) EBV Epstein-Barr Virus EGFP Enhanced Green Fluorescent Protein, green glowing reporter gene ELISA Enzyme Linked Immunosorbent Assay gy Gray (Gy) hDeltal Homo sapiens delta (Drosophila der -family-like 1 ( the notch

Ligands) hSerrate-1 Homo sapiens serrate 1 (from the Notch ligand family) FKS fetal calf serum

IFN interferon

Ig immunoglobulin

IL Interleukin kDa Kilo-Dalton mAb monoclonal antibody

MHC Major Histocompatibility Complex

MLC Mixed Lymphocyte Culture mRNA Messenger Ribonucleic Acid

NIH / 3T3 mouse fibroblasts NK cells Natural killer cells

Notchl-4 Homo sapiens Notch (Drosophila) homolog 1-4 (Notch receptor)

OKT3 mAb against CD3

PBMC peripheral blood mononuclear cells

PCR polymerase chain reaction PHA phytohaemagglutinin

SCID mice immunodeficient mice that do not have B and T cells

TCM T cell medium

TCR signal T cell receptor TGF transforming growth factor

T lymphocytes Thymus-dependent or stemmed lymphocytes

T-cells derived from thymus T-lymphocytes

Thl cells T cells with T helper phenotype

Th2 T cells with T helper2 phenotype

TNF tumor necrosis factor vIL-10 viral interleukin-10, derived from Epstein-Barr virus, has high AS-

Homology to human IL-10

Claims

claims

1. (In vitro) gene-modified T cells obtained by stimulating T cells of a transplant recipient in vitro by cells from a transplant donor or by cells that express dominant MHC molecules, and simultaneously or later by means of gene transfer from immunomodulating therapeutic genes be transduced.

2. (In vitro) gene-modified T cells according to claim 1, characterized in that it is alloreactive T cells.

3. (In vitro) gene-modified T cells according to claims 1 and 2, obtained by: a) cultivating a cell line which produces a retrovirus capable of gene transfer with a therapeutic gene (packaging cell line) and b) lymphocytes are isolated from whole blood or spleen or lymph nodes (irradiated donor T cells or irradiated cells which express dominant MHC molecules and

Recipient T cells) and c) either cocultivation, consisting of the mixed lymphocyte culture (primary MLC) and the packaging cell line, is carried out or only retrovirus-containing cell culture supernatant is used for the transduction, so that there is cocultivation with the packaging cell line can be dispensed with.

4. (In vitro) gene-modified T cells according to claim 3, characterized in that a Moloney Murine leukemia virus or a lentivirus is used as the retrovirus.

5. (In vitro) gene-modified T cells according to approach 1 and 2, obtained by that

Lymphocytes are isolated from the whole blood or spleen or lymph nodes (irradiated donor T cells or irradiated cells that express dominant MHC molecules and recipient T cells) and the allospecific T cells with liposome formulations that are produced with the aid of this mixed lymphocyte culture Contains plasmid with the therapeutic gene, incubated or bombarded with a gene gun.

6. (In vitro) gene-modified T cells according to claim 1 to 5, characterized in that it is the therapeutic genes a) cytokines b) interleukins c) Notch ligands / receptors d) cell protective genes (eg antiapoptotic genes, heat shock genes).

7. (In vitro) transduced T cells according to claim 6, characterized in that the therapeutic genes are a) IL-4 or b) IL-10 or viral IL-10 (eg from EBV or CMV) c) IL-12p40 or d) IL-13 or e) Hemoxygenase-1 f) CTLA-4 or g) hSerrate-1 or h) hDelta-1 or i) Notch 1-4 or j) bcl-2 or k) bcl- xl or 1) bag-1.

8. A method for producing gene-modified T cells, characterized in that T cells of a transplant recipient are stimulated in vitro by cells from a transplant donor or by cells that express dominant MHC molecules and transduced simultaneously or later by gene transfer from immunomodulating therapeutic genes become.

9. The method according to claim 8, characterized in that it is all-reactive T cells.

10. The method according to claim 8 and 9, characterized in that: a) a cell line that produces a gene capable of transferring a retrovirus with a therapeutic gene is taken in culture (packaging cell line) and b) lymphocytes are isolated from whole blood (irradiated donor T cells or irradiated cells that express dominant MHC molecules and Recipient T cells and c) either a cocultivation consisting of the mixed lymphocyte culture (primary MLC) and the packaging cell line is carried out or only retrovirus-containing cell culture supernatant is used for the transduction, so that cocultivation with the Packaging cell line can be dispensed with.

11. The method according to claim 10, characterized in that a Moloney Murine leukemia virus or a lentivirus is used as the retrovirus.

12. The method according to claim 8 and 9, characterized in that lymphocytes are isolated from whole blood or spleen or lymph nodes (irradiated donor T cells or irradiated cells which express dominant MHC molecules and recipient T cells) and incubated with this mixed lymphocyte culture, allospecific T cells with liposome formulations containing the plasmid with the therapeutic gene, or bombarded with a gene gun.

13. The method according to spoke 8 to 12, characterized in that the therapeutic genes are a) cytokines b) interleukins c) Notch ligands / receptors d) cell-protective genes (e.g. antiapoptotic genes, heat shock genes).

14. The method according to claim 13, characterized in that the therapeutic genes are a) IL-4 or b) IX-10 or viral EL-10 (eg from EBV or CMV) c) IL-12p40 or d) IL -13 or e) Hemoxygenase-1 f) CTLA-4 or g) hSerrate-1 or h) hDelta-l or i) Notch 1-4 or j) bcl-2 or k) bcl-xl or l) bag-l.

15. Use of the (in vitro) gene-modified T cells according to claims 1 to 7 in transplant medicine.

16. Use according to claim 15 for preventing allogeneic graft rejection in vivo.

17. Use according to claim 15 and 16 as a means for inducing tolerance and for maintaining tolerance to allogeneic grafts (cells, tissues, organs).

18. Use according to claim 15 to 17 for stimulating the T cells of the transplant recipient.