[0002] Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM) is nowadays considered to be an autoimmune disease (Endocr. Rev. 1994, 15 (4), 516-542), characterised by the presence of anti-beta cell antibodies and by its sensitivity to immunosuppressive therapy. In both man and in the nonobese diabetic (NOD) mouse, IDDM results from a predominantly cellular type immune response, the humoral response being characterised by secretion of anti-membrane antibodies and secretary beta cell anti-products (N. Engl. J. Med. 1981, 304, 1454-1465, N. Engl. J. Med., 1992, 327, 302). The cellular type immune response is characterised by histological lesions or insulitis caused by infiltration of macrophage and B and T lymphocyte type inflammatory and immune cells into the islets of Langerhans of the pancreas (Diabetologia, 1989, 32, 282-289; Insulitis and type 1 diabetes, Academic Press Tokyo, 1986, 35-50).
[0003] The NOD mouse is a spontaneous model of autoimmune diabetes or type 1 diabetes. Converging arguments indicate that the onset of the disease is under the control of CD4
[0004] It has been indirectly suggested and demonstrated that, because of their ability to produce interleukin-4 (IL-4), T helper 2 (Th2) lymphocyte cells are the immunoregulatory CD4
[0005] However, a further study has shown that while diabetologically Th2-like cells discovered infiltrated into the islets do not cause the disease to appear, they do not afford significant protection (Science, 1995, 268 (5214), 1185-1188). Thus while it has been indirectly suggested and shown that the onset of diabetes in the NOD mouse is under the control of Th2 cells, no explanation has been given or suggested regarding the character of the anomaly present in the NOD mouse of the physiological process which occurs which is the origin of the emergence of an anti-Langerhans islets autoimmunity at the origin of diabetes in such animals. It has also recently been suggested that Th2 cell differentiation could be controlled by a T cell sub-type characterised by the TCR-αβ, CD4
[0006] It has also been shown that this sub-type can be restricted by class I molecules of the major histocompatibility complex (MHC) and preferably uses the Vβ8 gene for the T receptor (J. Exp. Med., 1993, 178, 901-908), a sub-type proliferation of which is specifically induced by interleukin-7 (J. Exp. Med., 1994, 180 (2), 653-661).
[0007] Mammalian interleukin-7 proteins (cytokine IL-7s), in particular in man and in the mouse, the corresponding DNA, expression vectors coding for the IL-7s, and processes for their production, including recombinant systems, have been described (U.S. Pat. No. 4,965,195).
[0008] Mammalian interleukin-7 proteins will hereinafter be designated “IL-7”. IL-7 is a lymphopoietic growth factor which can stimulate the development and proliferation of bone marrow cells (WO 89/03884). Stimulation of platelet production. (WO 90/09194) by induction and proliferation of megacaryocytes was one of the first applications of IL-7. Other IL-7 applications have also been described such as cancer treatment or a treatment of a viral infection by immunotherapy from modified cells producing IL-7, either by direct injection of modified cells in vivo, or by a prior in vitro treatment phase before injection (WO 92/01459). The use of IL-7 for proliferating specific anti-HIV human T lymphocytes as a potential AIDS therapy (J. of Leucocyte Biology, 1995, 58 (6), 623-633), for treating malignant melanoma in dermatology (Hautarzt, 1995, 46 (10), 676-682), and as a potentialiser for a vaccine to prevent infection (microbial and viral) and tumours (WO 94/22473) have also been described. The use of an anti-IL-7 antibody for studying and researching physiological and pathological processes, in particular regarding differentiation and proliferation of lymphocytes (WO 94/28160), has also been described. Other applications combining the use of IL-7 with other cytokines have been described, such as the combination with IL-4 for in vitro induction of pre-B cell differentiation (WO 94/04658) or with IL-3 to treat leucopenia (WO 92/04465) and to prevent bone marrow disorders after cancer therapy or bone marrow grafts (WO 93/03061).
[0009] The authors of the present invention have shown here, on the basis of a phenotype study using flow cytometry using HSA, CD4, CD8, CD44 and Vβ8 markers, that a sub-population of thymocytes with phenotype CD44
[0010] Thus in a first aspect, the present invention provides for the use of IL-7 or T lymphocytes incubated in the presence of IL-7 or modified lymphocytes producing IL-7 for the preparation of drugs or pharmaceutical compositions for treating autoimmune diseases, in particular autoimmune diseases generated by a failure in immunoregulation by CD4
[0011] Preferably, the autoimmune diseases are autoimmune diseases generated by a failure in the production of IL-4 by Th2 cells.
[0012] Particularly preferably, the autoimmune diseases are autoimmune diseases generated by a failure in the production of IL-4 connected with a quantitative and functional deficiency of a T cell sub-type with phenotype HSA
[0013] In addition to insulin-dependent diabetes mellitus, other autoimmune diseases, autoimmune encephalo-myelitis, autoimmune rheumatoid arthritis, polyarthritis, autoimmune type 2 hepatitis, autoimmune gastritis, autoimmune sclerosis, sialadenitis, adrenalitis, oophoritis, glomerulonephritis, and autoimmune thyroiditis, can preferably be treated by said drugs or compositions, as can any autoimmune type pathogenic mechanism in a therapy associated with treatment of AIDS.
[0014] The autoimmune disease is preferably insulin-dependent diabetes mellitus.
[0015] Advantageously, the T lymphocytes previously incubated in the presence of IL-7, used in the present invention, are autological or syngeneic cells from cells of the patients for whom the compositions comprising them are intended.
[0016] The invention also encompasses pharmaceutical compositions offering a novel approach for treating autoimmune diseases generated by a failure in immunoregulation by CD4+ T cells.
[0017] In particular, the invention encompasses pharmaceutical compositions offering a novel approach for treating autoimmune diseases generated by a failure in the production of IL-4 by Th2 cells, particularly autoimmune diseases generated by a failure in IL-4 production connected with a quantitative and functional deficiency of a T cell sub-type with phenotype HSA
[0018] Such compositions comprise IL-7 as the active principle, preferably in its soluble form, and/or autologous or syngeneic T lymphocytes from cells of a patient for whom the pharmaceutical composition is intended, said T lymphocytes having previously been incubated in the presence of IL-7. They can also be in the form of combinations with other active principles, for example other immunomodulating agents.
[0019] These different compositions can be administered in a number of different ways, as the skilled person will be able to determine, depending on the type of composition concerned.
[0020] Compositions comprising IL-7 as the active principle can be administered systemically, for example, preferably intravenously, intramuscularly, intradermally or orally.
[0021] Compositions comprising T lymphocytes as the active principle are preferably administered intravenously or intraperitoneally.
[0022] Preferred modes of administration, also dosages and optimum galenical forms, can be determined using the criteria which are generally considered in establishing a therapeutic treatment which is tailored to a patient, for example age or body weight of the patient, the seriousness of their general condition, tolerance to treatment and any known side effects.
[0023] The present invention also relates to a process for the production of a drug or pharmaceutical composition for treating autoimmune diseases, in particular autoimmune diseases generated by a failure in immunoregulation by CD4
[0024] In particular, the invention concerns a process for the production of a drug or pharmaceutical composition for treating autoimmune diseases generated by a failure in IL-4 production by Th2 cells, in particular autoimmune diseases generated by a failure in IL-4 production connected with a quantitative and functional deficiency of a T cell sub-type with phenotype HSA
[0025] The present invention concerns a therapeutic treatment method characterised in that a therapeutically effective dose of IL-7 or T lymphocytes which have previously been incubated in the presence of IL-7 is administered to a patient with an autoimmune disease, in particular an autoimmune disease generated by a failure in immunoregulation by CD4
[0026] Advantageously, the method of the invention is applicable to treating insulin-dependent diabetes mellitus.
[0027] The present invention is illustrated by the accompanying
[0028]
[0029] In the experiment shown, freshly harvested HSA
[0030] Other features and advantages of the invention are apparent from the remaining description including the Examples and Tables summarising the results of the experiments.
[0031]
[0032] In the experiment shown, mice of different strains aged from 8 to 12 weeks received daily sub-cutaneous injections of 2 μg of IL-7 or bovine serum albumin (excipient) over 7 consecutive days. On the eight day, the mice were treated with anti-CD3 antibody then the cultivated splenocytes (5×10
[0033] Female NOD and C57BL/6 mice were kept under non-pathogenic specific environmental conditions. All of the NOD mice were verified as manifesting no evident sign of diabetes (no glycosuria or hyperglycemia). The thymus of about 5 to 15 exsanguine mice were carefully removed and mixed. The mature double negative (DN) and CD4
[0034] Marking was carried out as described in J. Exp. Med. 1994, 180, 653-661. The antibodies used were produced using commercially available hybridomas. Anti-TCR-αβ (H57-597 clone, Pharmigen) or anti-Vβ8 antibodies (F23.1 clone described in J. Immunol. 143, 3994-4000), biotinylated or labelled with fluorescein (FITC), were used combined with anti-CD4 antibodies (RM 4.5 clone, Pharmigen) labelled with phycoerythrin (PE) and/or with anti-CD44 antibodies (IM 7.8 clone, Pharmigen) labelled with FITC.
[0035] For 3-colour labelling, after incubation in the presence of biotinylated antibodies, the cells were then. incubated with the appropriate monoclonal antibodies labelled with FITC and PE and conjugated with streptavidin-Tricolor (SAv-Tri, Caltag).
[0036] The non-specific labelling reference was monitored in parallel. The apparatus used was a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif.). The minimum sample was about 1×10
[0037] Cytokine production was measured from cells cultured in a medium as described in J. of Immunol., 1995, 155 (10), 4544-4550 and J. Exp. Med., 1994, 180, 653-661. About 10
[0038] The IL-4 was measured using sandwich type ELISA, as described in J. of Immunol. 1995, 155 (10), 4544-4550.
[0039] The results obtained and shown in Table 1 below show that the appearance of the sub-population of thymocytes with phenotype TCR-αβTABLE 1 % of CD44 AGE in population of STRAIN (WEEKS) HSA C57BL/6 3 *25.1 ± 0.8 8 27.9 ± 10.1 NOD 3 12.5 ± 0.3 8 25.3 ± 7.0
[0040] Table 2 below shows the results obtained for double negative thymocyte populations (CD4TABLE 2 * % of CD44 STRAIN CD4 CD4 NOD 23 ± 8 34 ± 1 C57BL/6 26 ± 10 *59 ± 7
[0041] It was also verified that thymocyte cells characterised by the phenotype HSA
[0042] Similarly, it has been shown (
[0043] While it has already been shown that IL-7 specifically induces proliferation of the sub-population of HSA
[0044] The two experiments described below show that lymphoid cells originating from the thymus gland of the 3-week old NOD mouse can protect against the onset of diabetes in a co-transfer model. More precisely, 50 million total thymocytes from 3-week old NOD mice simultaneously injected with 5 to 10 million splenic cells from 10-week old diabetic NOD mice with irradiated NOD receptors prevented the onset of diabetes normally observed when splenic cells alone are injected. Injecting less than 10 million thymocyte cells had no protective effect. However, when the thymocytes had previously been incubated in vitro in the presence of IL-7 for 60 hours at 37° C., one million thymocytes afforded protection in the co-transfer model described above.
[0045] Further, it is known that one injection of cyclophosphamide (200 mg/kg) could cause the onset of diabetes in 8-week old-male NOD mice in a much shorter time period (10 to 20 days) than that of spontaneous onset of diabetes (2 to 4 months). In vivo treatment with IL-7 consisting of 2 injections of IL-7, administered the day before and the day after injecting cyclophosphamide, protected against the onset of diabetes induced by cyclophosphamide.
[0046] These two series of experiments indicate that IL-7 plays a protective role against the onset of diabetes in genetically predisposed mice.
[0047] 3.1 Protection Against Diabetes by Thymocytes Incubated in the Presence of Interleukin-7 in a Co-Transfer Model
[0048] Thymocytes were prepared from the thymus of 3-week old female NOD mice. These thymocytes were incubated in vitro for 60 to 72 hours at 37° C. in an RPMI medium containing 10% of foetal calf serum with added IL-7 (500-1000 U/ml). Following this incubation, 4, 2 or 1 million of these thymocytes and 5 or 10 million splenic cells from NOD mice which had recently become diabetic were simultaneously injected into 10-week old male NOD mice irradiated with a dose of 700 rad. Onset of diabetes was monitored three times a week by looking for glycosuria and confirmed when glycosuria was observed by evidence of hyperglycemia.
[0049] The results shown in Table 3 below show that thymocytes incubated in the presence of IL-7 protect against diabetes in doses which are not protective when thymocytes are used which are not incubated in the presence of IL-7.
TABLE 3 Protective effect of total thymocytes treated with IL-7 in a model of diabetes induced by passive transfer of cells originating from diabetic mice Number of diabetic mice as a function of number of weeks Transferred Number of after transfer Experiment thymocytes animals 1 w.* 4 w.* 6 w.* 8 w.* No 1 — 6 0 4 5 5 untreated 6 0 3 4 6 treated 6 0 0 0 1 with IL-7 No 2 — 5 0 0 4 4 treated 8 0 0 0 0 with IL-7 No 3 — 8 0 6 6 NT treated 6 0 0 0 NT with IL-7
[0050] 3.2 Protection Against Diabetes Induced by Injecting Cyclophosphamide, Using Injections of Interleukin-7
[0051] 6 to 7-week old female NOD mice received one injection of 200 mg/kg of cyclophosphamide, framed on days −1 and +1 by an intravenous injection of 1 μg of IL-7. Onset of diabetes was detected by looking for glycosuria and hyperglycemia as described in Example 1.
[0052] The results shown in Table 4 indicate that treatment with IL-7 significantly prevented the onset of diabetes compared with mice which received cyclophosphamide with no IL-7.
TABLE 4 Protective effect of IL-7 in the cyclophosphamide induced diabetic model Number of diabetic mice after 2 weeks following injection of Treatment Number of mice cyclophosphamide — 10 8 IL-7 10 2
[0053] These results show that NOD mice have a premature deficit of a sub-population of thymocytary HSA
[0054] The results indicate that the anomaly of this deficit in NOD mice, affecting both the sub-populations of CD4
[0055] Further, the in vivo experiments, whether by injecting cells which had previously been incubated in the presence of IL-7 or by direct injection of an effective quantity of IL-7 into the mouse, demonstrated that the use of IL-7 had a protective effect against the onset of an autoimmune disease, in particular diabetes.
[0056] In vivo treatment with IL-7 potentialises the production of IL-4 by splenic T cells (
[0057] The sub-cutaneous IL-7 injection protocol comprised two doses of 2 μg per day for 4 to 7 consecutive days. Control mice were treated with an identical volume of excipient solution (bovine serum albumin). IL-4 production was raised after a single intravenous injection (1.33 μg) of an antibody directed specifically against the murine CD3 molecule (anti-CD3, clone 145-2C11, hamster IgG). The animals were sacrificed 90 minutes following injection of the anti-CD3 antibody; the spleens were immediately removed and splenic cells cultured for 90 minutes in the absence of any stimulation (Yoshimoto et al., J. Exp. Med., 179, 1285-1295). The RPMI medium, containing decomplemented foetal calf serum (10%), β-mercaptoethanol (0.05 mM) and antibiotics (penicillin, 100 Ul/ml and streptomycin, 100 μg/ml), was used for all of the cell culture experiments. The supernatants were removed and their IL-4 content determined using the CT.4S biological test, a line dependent on IL-4 (Hu-Li et al., J. Immunol., 142, 800-807). The IL-4 concentration was expressed as U/ml (one unit corresponded to approximately 1 pg) following a standard curve established with serial dilutions of recombinant murine IL-4. The sensitivity limit of the test was about 10 U/ml.
[0058] The absence of peripheral IL-4 production by T NK1
[0059] These results, which constitute the first demonstration of an in vivo pharmacological effect of IL-7 on the production of IL-4 by T NK1