Agent for the treatment and/or prophylaxis of an autoimmune disease and for the formation of regulatory T cells

Abstract

The present invention relates to an agent for the treatment and/or prophylaxis of an autoimmune disease, an agent for the formation of regulatory T cells (T.sub.Reg) in an organism and various methods in which the agents according to the invention are used.

Claims

1. A method for the treatment and/or prevention of the worsening of an autoimmune disease in an organism, the method comprising: (a) administering a mutein of human interleukin-2 (hIL-2 mutein) to the organism, wherein said hIL-2 mutein has an amino acid substitution in at least one of the positions 20, 88 or 126, numbered in accordance with the hIL-2 wild type sequence as set forth in SEQ ID NO: 1; and (b) if necessary, repeating step (a), wherein the autoimmune disease is type I diabetes, multiple sclerosis, or systemic lupus erythematosus, and wherein the administration induces regulatory T cells in the organism.

2. The method according to claim 1, wherein through the substitution at position 88 an asparagine is exchanged for an amino acid which is selected from the group consisting of: arginine (hIL-2-N88R), glycine (hIL-2-N88G), or isoleucine (hIL-2-N88I).

3. The method according to claim 1, wherein through the substitution at position 20 an aspartic acid is exchanged for an amino acid which is selected from the group consisting of: histidine (hIL-2-D20H), isoleucine (hIL-2-D20I), or tyrosine (hIL-2-D20Y).

4. The method according to claim 1, wherein through the substitution at position 126, a glutamine is exchanged for a leucine (hIL-2-Q126L).

5. The method according to claim 1, wherein the method further comprises administering to the organism an immunosuppressant.

6. The method according to claim 5, wherein the immunosuppressant is selected from the group consisting of: glucocorticoid, including decortin, prednisol; azathioprine; cyclosporin A; tacrolimus; an anti-T lymphocyte globulin; an anti-CD3 antibody; muromonab; an anti-CD25 antibody; basiliximab; daclizumab; an anti-TNF- antibody; infliximab; adalimumab; azathioprine; methotrexate; cyclosporin; sirolimus; everolimus; fingolimod; CELLCEPT (mycophenolate mofetil); myfortic; and cyclophosphamide.

7. The method according to claim 1, wherein the autoimmune disease is type I diabetes.

8. The method according to claim 1, wherein the autoimmune disease is multiple sclerosis.

9. The method according to claim 1, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

10. A method for the treatment and/or prevention of the worsening of an autoimmune disease in an organism, the method comprising: (a) administering a mutein of human interleukin-2 (hIL-2 mutein) to the organism, wherein said hIL-2 mutein has an amino acid substitution in at least one of the positions 20, 88 or 126, numbered in accordance with the hIL-2 wild type sequence as set forth in SEQ ID NO: 1; and (b) if necessary, repeating step (a), wherein the autoimmune disease is type I diabetes or multiple sclerosis.

11. The method according to claim 10, wherein through the substitution at position 88 an asparagine is exchanged for an amino acid which is selected from the group consisting of: arginine (hIL-2-N88R), glycine (hIL-2-N88G), or isoleucine (hIL-2-N88I).

12. The method according to claim 10, wherein through the substitution at position 20 an aspartic acid is exchanged for an amino acid which is selected from the group consisting of: histidine (hIL-2-D20H), isoleucine (hIL-2-D20I), or tyrosine (hIL-2-D20Y).

13. The method according to claim 10, wherein through the substitution at position 126, a glutamine is exchanged for a leucine (hIL-2-Q126L).

14. The method according to claim 10, wherein the method further comprises administering to the organism an immunosuppressant.

15. The method according to claim 14, wherein the immunosuppressant is selected from the group consisting of: glucocorticoid; decortin; prednisol; azathioprine; cyclosporin A; tacrolimus; an anti-T lymphocyte globulin; an anti-CD3 antibody; muromonab; an anti-CD25 antibody; basiliximab; daclizumab; an anti-TNF- antibody; infliximab; adalimumab; azathioprine; methotrexate; cyclosporin; sirolimus; everolimus; fingolimod; CELLCEPT (mycophenolate mofetil); myfortic; and cyclophosphamide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows that hIL-2-N88R in healthy subjects at equal or lower dosage in comparison to proleukin induces a greater increase in the regulatory CD4.sup.+CD25.sup.+Foxp3.sup.+ T cells.

(2) FIG. 2 shows that hIL-2-N88R in healthy subjects at equal or lower dosage in comparison to proleukin induces a greater increase in the regulatory CD4.sup.+CD25.sup.Foxp3.sup.+ T cells.

(3) FIG. 3 shows that hIL-2-N88R in melanoma patients at equal or lower dosage in comparison to proleukin induces a greater increase in the regulatory CD4.sup.+CD25.sup.+Foxp3.sup.+ T cells.

(4) FIG. 4 shows that hIL-2-N88R in melanoma patients at equal or lower dosage in comparison to proleukin induces a greater increase in the regulatory CD4.sup.+CD25.sup.Foxp3.sup.+ T cells.

(5) FIG. 5 shows that hIL-2-N88R in multiple sclerosis patients at equal or lower dosage in comparison to proleukin induces a greater increase in the regulatory CD4.sup.+CD25.sup.+Foxp3.sup.+ T cells.

(6) FIG. 6 shows that hIL-2-N88R in multiple sclerosis patients at equal or lower dosage in comparison to proleukin induces a greater increase in the regulatory CD4.sup.+CD25.sup.Foxp3.sup.+ T cells.

(7) FIG. 7 shows that hIL-2-N88R in multiple sclerosis patients at equal or higher dosage in comparison to proleukin induces a lower increase in the cytotoxic CFSElow/CD3.sup.+CD8.sup.+CD45RO.sup.+ T cells.

(8) FIG. 8 shows that hIL-2-N88R in healthy subjects at equal or higher dosage in comparison to proleukin induces a lower increase in the cytotoxic CFSElow/CD3.sup.+CD8.sup.+CD45RO.sup.+ T cells.

(9) FIG. 9 shows that hIL-2-N88R in the mouse type I diabetes model in comparison to hIL-2 wild type leads to a higher percentage increase in FoxP3.sup.+ cells within the CD4.sup.+ cells (A). In addition, these CD4.sup.+FoxP3.sup.+ cells exhibit higher expression of CD25 (B).

(10) FIG. 10 shows that in the mouse type I diabetes model, in contrast to hIL-2 wild type, hIL-2-N88R prevents the development of diabetes.

(11) EMBODIMENTS

(12) 1. Material and Methods

(13) 1.1 Separation of PBMCs from Whole Blood for Use In Vitro

(14) Peripheral mononuclear blood cells (PBMCs) from healthy subjects, melanoma patients or MS patients are separated from the blood by means of lymphocyte separation medium (Histopaque, Sigma Aldrich). For this, two tubes of blood (7 or 10 ml) from the same subject or patient are transferred into a sterile 50 ml tube and made up to 30 ml with RPMI 1640 (InVitrogen, #14190-69). Next, 30 ml of the diluted blood are layered onto 15 ml of a density gradient solution (density=1.077; Histopaque, Sigma Aldrich, #10771).

(15) After centrifugation at 400 g for 40 min at 20 C. without braking, two white blood cell rings are harvested and transferred into a sterile 50 ml tube and washed twice with phosphate buffered saline (PBS; InVitrogen #14190-169). In the event of contamination with red blood cells, an RBC (red blood cells) lysis is performed; 2 ml of RBC lysis solution are added to the cell pellet and incubation is performed for 2 min with gentle mixing at room temperature, followed by a washing procedure with a large volume of complete medium (RPMI 1640 with 10% fetal calf serum).

(16) The number of live leucocytes is determined by exclusion staining using trypan blue (InVitrogen #15250-061) and a haemocytometer (FisherBioblock A2759B).

(17) 1.2 CFSE Labelling

(18) After counting, the cells are washed twice in PBS and resuspended in PBS at a concentration of 110.sup.6 cells/ml. CFSE (InVitrogen # C1157) is added at a final concentration of 0.5 M. After 10-minute incubation in the dark at 37 C., the CFSE-labelled cells are washed three times with fresh complete medium at 4 C. and resuspended in complete medium at a concentration of 110.sup.6 cells/ml for plating out.

(19) 1.3 Stimulation of the PBMCs In Vitro

(20) The PBMCs either remain unstimulated or are stimulated with hIL-2 wild type (proleukin) or hIL-2-N88R (BAY 50-4798; Lot #PR312C008) with or without a pool of synthetic peptides, which are derived from the melanoma-specific proteins gp100, TRP-2, MART-1 and tyrosinase or from the protein MOG specific for multiple sclerosis (MS), each peptide being added in a final concentration of 2.5 M (melanoma peptide) or 30 g/ml (MS peptide).

(21) Stimulator and peptide are added under the following 23 conditions:

(22) TABLE-US-00001 TABLE 1 Conditions for stimulation of PBMCs Condition Stimulator Final concentration 1 hIL-2-N88R .sup.10.sup.11M no peptides (BAY 50-4798; #PR312C008) 2 hIL-2-N88R .sup.10.sup.11M peptides (BAY 50-4798; #PR312C008) 3 hIL-2-N88R 10.sup.9M no peptides (BAY 50-4798; #PR312C008) 4 hIL-2-N88R 10.sup.9M peptides (BAY 50-4798; #PR312C008) 5 hIL-2-N88R 10.sup.8M no peptides (BAY 50-4798; #PR312C008) 6 hIL-2-N88R 10.sup.8M peptides (BAY 50-4798; #PR312C008) 7 hIL-2-N88R 10.sup.7M no peptides (BAY 50-4798; #PR312C008) 8 hIL-2-N88R 10.sup.7M peptides (BAY 50-4798; #PR312C008) 9 hIL-2-N88R 10.sup.6M no peptides (BAY 50-4798; #PR312C008) 10 hIL-2-N88R 10.sup.6M peptides (BAY 50-4798; #PR312C008) 11 hIL-2 wild type .sup.10.sup.11M no peptides (proleukin) 12 hIL-2 wild type .sup.10.sup.11M peptides (proleukin) 13 hIL-2 wild type 10.sup.9M no peptides (proleukin) 14 hIL-2 wild type 10.sup.9M peptides (proleukin) 15 hIL-2 wild type 10.sup.8M no peptides (proleukin) 16 hIL-2 wild type 10.sup.8M peptides (proleukin) 17 hIL-2 wild type 10.sup.7M no peptides (proleukin) 18 hIL-2 wild type 10.sup.7M peptides (proleukin) 19 hIL-2 wild type 10.sup.6M no peptides (proleukin) 20 hIL-2 wild type 10.sup.6M peptides (proleukin) 21 PHA 5 g/ml 22 unstim. 23 peptide only peptides

(23) Next the cells were cultured for six days at 37 C. in an atmosphere with 5% CO.sub.2 content.

(24) 1.4 Proliferation Assay and Phenotyping on the FC500 Flow Cytometer

(25) The staining of the cells with fluorescence-labelled antibodies to cell surface molecules makes it possible to study the proliferation of a specific subgroup of lymphocytes (memory and activation markers, see Tab. 2). The immunostaining with fluorochrome-labelled (PE: phycoerythrin, ECD: PE-Texas Red, APC: allophycocyanin, PC7: PE-Cy7) antibodies is performed before and after six days of culturing with the stimulators.

(26) On the sixth day, the first two stainings (1 and liso) are performed with non-CFSE-labelled cells (CFSE: carboxyfluorescein diacetate succinimidyl ester); the other stainings are performed on CFSE-labelled cells.

(27) TABLE-US-00002 TABLE 2 Staining scheme for PBMCS PE ECD APC PC7 1 CD25 CD45 Foxp3 CD4 1iso CD25 CD45 Rat CD4 IgG2a 2 CD127 CD45 CD25 CD4 3 CD3 CD45 CD25 CD8 4 CD16 CD56 CD3 5 CCR7 CD3 CD45RA CD4 6 CCR7 CD3 CD45RA CD8 7 CD8 CD3 CD45RO CD4 1 CD25 CD3 Foxp3 CD4 1iso CD25 CD3 Rat CD4 IgG2a 2 CD8 CD3 CD25 CD4 3 CCR7 CD3 CD45RA CD4 4 CD8 CD3 CD45RO CD4

(28) CD25-PE, Foxp3-APC and rat IgG2a-APC are from ebiosciences; CD25-APC, CD45RA-APC and CD45RO-APC were purchased from BD Biosciences. All other antibodies are from Beckman-Coulter, France.

(29) 1.5 Type I Mouse Diabetes Model

(30) 12 week old NOD (non-obese diabetes) mice are treated daily with hIL-2 mutein or hIL-2 wild type. Negative control animals were analogously treated with physiological salt solution (saline). The treatment groups consisted of 3-5 animals. On day 0 to 15, a quantity of 5K or 25K-units of hIL-2 mutein or hIL-2 wild type was administered to the mice. From day 17, in the treatment groups with 5K units, this was increased to 100K-units (=6.112 g). The treatment of the animals with 25K-units was maintained unchanged. The last dosing was performed on day 31. In a parallel experiment, treatment was performed from day 0 to day 31 with a fixed dose of 25K-units. Diabetes was detected by monitoring of glucose levels in the urine. Blood samples were taken from the mice on day 17 and day 30. The samples were analyzed in the FACS using anti-CD4, anti-CD25 and anti-FoxP3 staining and the percentage of FoxP3.sup.+ cells among the CD4.sup.+ T cells and the mean fluorescence intensity (MFI) of the CD25 expression on CD4.sup.+FoxP3.sup.+ cells was thus determined.

(31) 2. Results

(32) 2.1 Induction of Regulatory T Cells by hIL-2-N88R

(33) As a suitable in-vitro system for testing the effect of the muteins according to the invention, peripheral mononuclear blood cells (PBMCs) were used. PBMCs consist of T cells (75% CD4- and CD8-positive) and B and NK cells (25% positive) and thus constitute a cell population well representing the immune system.

(34) PBMCs from six healthy subjects (10.sup.6 cells/ml) were stimulated with wild type IL-2 (proleukin) or IL-2-N88R [BAY 50-4798, Lot #PR312C008 (BAY#C008)] at concentrations which lay between 10.sup.11 and 10.sup.6 M, or in the positive control with the non-specific mitogen phytohaemagglutinin (PHA) at a concentration of 5 g/ml or with culture medium only (Med). On day 0 and on the sixth day after the stimulation, the content of the regulatory CD4.sup.+CD25.sup.+Foxp3.sup.+ T cells within the CD3.sup.+ lymphocytes was determined. The result is shown in FIG. 1 and the following Table 3.

(35) TABLE-US-00003 TABLE 3 Percentage of CD3.sup.+CD4.sup.+CD25.sup.+Foxp3.sup.+ T cells after stimulation; mean values from six healthy subjects; S.D.: standard deviation Conditions Mean value S.D. day 0 day 0 1.367 2.060 Med 0.683 0.741 hIL-2-N88R 10.sup.11M 1.483 1.017 (BAY#C008) 10.sup.9M 1.783 1.153 ##1C008) 10.sup.8M 3.267 1.596 10.sup.7M 6.483 2.642 10.sup.6M 5.200 2.375 hIL-2 wild type 10.sup.11M 2.183 1.030 (proleukin) 10.sup.9M 3.067 1.255 10.sup.8M 3.600 1.330 10.sup.7M 4.600 1.992 10.sup.6M 4.967 2.199 PHA 5 g/ml 1.400 1.081 peptide only 1.340 0.680

(36) From this experiment it follows that hIL-2-N88R at concentrations of 10.sup.7 M and 10.sup.6 M leads to marked induction of the subpopulation of the regulatory T cells CD4.sup.+CD25.sup.+Foxp3.sup.+. The induction here is markedly greater than with stimulation of the PBMCs by hIL-2 wild type.

(37) In a second preparation, the increase in the subpopulation of the regulatory T cells CD4.sup.+CD25.sup.Foxp3.sup.+ after stimulation with hIL-2-N88R in comparison to hIL-2 wild type was studied. The result is shown in FIG. 2 and the following Table 4.

(38) TABLE-US-00004 TABLE 4 Percentage of CD3.sup.+CD4.sup.+CD25.sup.Foxp3.sup.+ T cells after stimulation; mean values from six healthy subjects Conditions Mean Value S.D. day 0 day 0 0.317 0.402 day 6 Med 0.133 0.151 hIL-2-N88R 10.sup.11M 0.200 0.141 (BAY#C008) 10.sup.9M 0.320 0.179 #1C008) 10.sup.8M 0.517 0.354 10.sup.7M 0.917 0.601 10.sup.6M 5.250 3.141 hIL-2 wild type 10.sup.11M 1.000 1.864 (proleukin) 10.sup.9M 0.717 0.293 10.sup.8M 1.000 0.429 10.sup.7M 1.433 0.403 10.sup.6M 2.533 0.903 PHA 5 g/ml 0.700 1.715 peptide only 0.160 0.089

(39) Here too, it is seen that the stimulation with hIL-2-N88R leads to a marked increase in the subpopulation of the regulatory T cells CD4.sup.+CD25.sup.Foxp3.sup.+, which at concentrations of 10.sup.6 M is markedly greater than with stimulation with hIL-2 wild type.

(40) 2.2 hIL-2-N88R Induces Regulatory T Cells in Melanoma Patients

(41) Next, it was investigated whether the hIL-2 mutein according to the invention N88R also stimulates the antigen-specific activity of immune cells. For this, PBMCs (10.sup.6 cells/ml) from three melanoma patients were stimulated with hIL-2-N88R (BAY 50-4798, Lot #PR312C008) or hIL-2 wild type (proleukin) at concentrations which lay between 10.sup.11 and 10.sup.6 M, in the presence or absence of a melanoma-associated peptide pool, with 5 g/ml PHA or with culture medium only. Next, the subpopulations of the regulatory T cells CD4.sup.+CD25.sup.+Foxp3.sup.+ and CD4.sup.+CD25.sup.Foxp3.sup.+ respectively were determined. The result is shown in FIG. 3 and Table 5 and FIG. 4 and Table 6 respectively.

(42) TABLE-US-00005 TABLE 5 Percentage of CD3.sup.+CD4.sup.+CD25.sup.+Foxp3.sup.+ T cells after stimulation; mean values from three melanoma patients Conditions Mean Value S.D. day 0 day 0 2.800 2.052 day 6 Med 1.133 0.839 hIL-2-N88R 10.sup.11M 1.833 1.185 (BAY #C008) 10.sup.11M + Pept 2.467 0.666 10.sup.9M 3.000 1.015 10.sup.9M + Pept 3.033 0.379 10.sup.8M 3.600 0.819 10.sup.8M + Pept 5.567 2.499 10.sup.7M 6.100 0.458 10.sup.7M + Pept 6.233 0.058 10.sup.6M 7.533 2.413 10.sup.6M + Pept 8.533 3.225 hIL-2 wild type 10.sup.11M 2.767 0.751 (proleukin) 10.sup.11M + Pept 2.500 0.985 10.sup.9M 3.333 0.802 10.sup.9M + Pept 3.433 1.102 10.sup.8M 4.133 0.862 10.sup.8M + Pept 3.633 1.002 10.sup.7M 4.367 1.201 10.sup.7M + Pept 4.300 0.755 10.sup.6M 6.667 1.405 10.sup.6M + Pept 5.600 1.323 PHA 5 g/ml 1.400 0.346 peptide only 1.667 1.060

(43) TABLE-US-00006 TABLE 6 Percentage of CD3.sup.+CD4.sup.+CD25.sup.Foxp3.sup.+ T cells after stimulation; mean values from three melanoma patients Conditions Mean Value S.D. day 0 day 0 0.567 0.643 day 6 Med 0.133 0.231 hIL-2-N88R 10.sup.11M 0.267 0.462 (BAY #C008) 10.sup.11M + Pept 0.333 0.252 10.sup.9M 0.300 0.265 10.sup.9M + Pept 0.500 0.300 10.sup.8M 0.500 0.265 10.sup.8M + Pept 0.800 0.700 10.sup.7M 0.900 0.436 10.sup.7M + Pept 0.677 0.306 10.sup.6M 4.100 1.682 10.sup.6M + Pept 3.200 1.646 hIL-2 wild type 10.sup.11M 0.267 0.208 (proleukin) 10.sup.11M + Pept 0.200 0.100 10.sup.9M 0.967 0.603 10.sup.9M + Pept 0.967 0.451 10.sup.8M 1.633 1.002 10.sup.8M + Pept 1.400 0.624 10.sup.7M 1.400 0.656 10.sup.7M + Pept 1.533 0.702 10.sup.6M 2.733 1.861 10.sup.6M + Pept 2.600 1.473 PHA 5 g/ml 0.000 0.000 peptide only 0.200 0.100

(44) Here it was found that the administration of hIL-2-88R also leads to a marked increase in the regulatory T cells in melanoma patients. In the case of the subpopulation CD4.sup.+CD25.sup.+Foxp3.sup.+ at concentrations of 10.sup.7 M and 10.sup.6 M, and in the subpopulation CD4.sup.+CD25.sup.Foxp3.sup.+ at a concentration of 10.sup.6 M, this is markedly greater than with stimulation with corresponding concentrations of wild type-IL-2 (proleukin).

(45) 2.3 hIL-2-N88R Induces Regulatory T Cells in Patients with Multiple Sclerosis

(46) Next it was investigated whether the hIL-2 mutein according to the invention N88R also stimulates the antigen-specific activity of immune cells. For this, PBMCs (10.sup.6 cells/ml) from two multiple sclerosis patients were stimulated with hIL-2-N88R (BAY 50-4798, Lot #PR312C008) or hIL-2 wild type (proleukin) at concentrations which lay between 10.sup.11 and 10.sup.6 M, in the presence or absence of a multiple sclerosis-associated peptide, with 5 g/ml PHA or with culture medium only. Next, the subpopulations of the regulatory T cells CD4.sup.+CD25.sup.+Foxp3.sup.+ and CD4.sup.+CD25.sup.Foxp3.sup.+ respectively were determined. The result is shown in FIG. 5 and Table 7 and FIG. 6 and Table 8 respectively.

(47) TABLE-US-00007 TABLE 7 Percentage of CD3.sup.+CD4.sup.+CD25.sup.+Foxp3.sup.+ T cells after stimulation; mean values from two multiple sclerosis patients. Conditions Mean Value S.D. day 0 day 0 0.95 0.21 day 6 Med 3.55 1.91 hIL-2-N88R 10.sup.11M 3.8 0.99 (BAY #C008) 10.sup.11M + Pept 4.2 2.40 10.sup.9M 6.1 2.40 10.sup.9M + Pept 9.05 5.02 10.sup.8M 9.75 2.19 10.sup.8M + Pept 11.15 3.32 10.sup.7M 11.95 5.30 10.sup.7M + Pept 9.9 1.70 10.sup.6M 7.9 1.41 10.sup.6M + Pept 9.3 1.41 hIL-2 wild type 10.sup.11M 6.3 2.55 (proleukin) 10.sup.11M + Pept 8.8 4.10 10.sup.9M 8.25 0.49 10.sup.9M + Pept 8.45 1.20 10.sup.8M 8.15 2.47 10.sup.8M + Pept 9.55 3.04 10.sup.7M 8.8 3.39 10.sup.7M + Pept 8.8 3.25 10.sup.6M 9.45 0.78 10.sup.6M + Pept 7.6 1.84 PHA 5 g/ml 5.5 3.68 peptide only 3.95 2.19

(48) TABLE-US-00008 TABLE 8 Percentage of CD3.sup.+CD4.sup.+CD25.sup.Foxp3.sup.+ T cells after stimulation; mean values from two multiple sclerosis patients Conditions Mean Value S.D. day 0 day 0 0.35 0.35 day 6 Med 0.14 0.14 hIL-2-N88R 10.sup.11M 0.28 0.28 (BAY #C008) 10.sup.11M + Pept 0.14 0.14 10.sup.9M 0.07 0.07 10.sup.9M + Pept 0.07 0.07 10.sup.8M 0.28 0.28 10.sup.8M + Pept 0.00 0.00 10.sup.7M 0.14 0.14 10.sup.7M + Pept 0.14 0.14 10.sup.6M 4.10 4.10 10.sup.6M + Pept 2.90 2.90 hIL-2 wild type 10.sup.11M 0.00 0.00 (proleukin) 10.sup.11M + Pept 0.07 0.07 10.sup.9M 0.07 0.07 10.sup.9M + Pept 0.00 0.00 10.sup.8M 0.14 0.14 10.sup.8M + Pept 0.14 0.14 10.sup.7M 0.28 0.28 10.sup.7M + Pept 0.21 0.21 10.sup.6M 0.07 0.07 10.sup.6M + Pept 0.85 0.85 PHA 5 g/ml 0.07 0.07 peptide only 0.07 0.07

(49) It was found that the administration of hIL-2-N88R also leads to a marked increase in the T cells in multiple sclerosis patients. In the case of the subpopulation CD4.sup.+CD25.sup.+Foxp3.sup.+ at concentrations of 10.sup.8 M and 10.sup.7 M, and in the subpopulation CD4.sup.+CD25.sup.Foxp3.sup.+ at a concentration of 10.sup.6 M, this is markedly greater than with stimulation with corresponding concentrations of hIL-2 wild type (proleukin).

(50) 2.4 hIL-2-N88R Induces Only Minimal Proliferation of Cytotoxic CD8.sup.+ T Cells in Patients with Multiple Sclerosis and in Healthy Subjects

(51) Further, the stimulation of cytotoxic CD8+ central memory T cells was studied. For this, PBMCs from healthy subjects or multiple sclerosis patients were treated as described in 2.3. The percentage of CFSElow/CD3.sup.+CD8.sup.+CD45RO.sup.+ T cells was analysed. The result is shown in FIG. 7 and Table 9 and FIG. 8 and Table 10.

(52) TABLE-US-00009 TABLE 9 Percentage of CFSElow/CD3.sup.+CD8.sup.+CD45RO.sup.+ T cells after stimulation; mean values from two multiple sclerosis patients. Conditions Mean Value S.D. day 0 day 0 2.45 1.06 day 6 Med 0.95 0.21 hIL-2-N88R 10.sup.11M 1.3 0.57 (BAY #C008) 10.sup.11M + Pept 1.2 1.13 10.sup.9M 3.55 2.47 10.sup.9M + Pept 0.5 0.14 10.sup.8M 3.3 1.41 10.sup.8M + Pept 1.2 0.71 10.sup.7M 10.5 7.50 10.sup.7M + Pept 9.1 0.71 10.sup.6M 22.9 9.76 10.sup.6M + Pept 1 0.28 hIL-2 wild type 10.sup.11M 5.2 7.35 (proleukin) 10.sup.11M + Pept 3.1 0.28 10.sup.9M 14.3 15.56 10.sup.9M + Pept 19.45 2.90 10.sup.8M 36.5 1.98 10.sup.8M + Pept 41.85 2.47 10.sup.7M 54.8 8.49 10.sup.7M + Pept 54.85 12.94 10.sup.6M 58.05 1.06 10.sup.6M + Pept 98.2 0.85 PHA 5 g/ml 0.55 0.07 peptide only 2.45 1.06

(53) TABLE-US-00010 TABLE 10 Percentage of CFSElow/CD3.sup.+CD8.sup.+CD45RO.sup.+ T cells after stimulation; mean values from three healthy subjects Conditions Mean Value S.D. day 6 Med 0.23 0.23 hIL-2-N88R 10.sup.11M 0.27 0.06 (BAY #C008) 10.sup.11M + Pept 1.73 2.23 10.sup.9M 0.43 0.23 10.sup.9M + Pept 0.80 0.72 10.sup.8M 2.13 1.86 10.sup.8M + Pept 2.07 1.17 10.sup.7M 3.83 4.02 10.sup.7M + Pept 5.77 6.30 10.sup.6M 17.43 17.31 10.sup.6M + Pept 17.87 12.97 hIL-2 wild type 10.sup.11M 1.53 1.23 (proleukin) 10.sup.11M + Pept 2.37 2.57 10.sup.9M 18.20 23.35 10.sup.9M + Pept 16.93 10.96 10.sup.8M 43.30 36.72 10.sup.8M + Pept 37.80 20.80 10.sup.7M 38.53 25.53 10.sup.7M + Pept 32.37 20.90 10.sup.6M 37.93 27.66 10.sup.6M + Pept 30.83 21.51 PHA 5 g/ml 96.07 1.79 peptide only 1.83 1.59

(54) In contrast to hIL-2 wild type, in multiple sclerosis patients and also in healthy subjects, hIL-2-N88R leads to only a slight proliferation of central memory CD8.sup.+ T cells, and this at every concentration studied.

(55) 2.5 Treatment with hIL-2 Mutein Prevents the Development of Type I Diabetes in the Animal Model

(56) In comparison to hIL-2 wild type, the treatment of NOD mice with hIL-2-N88R leads to a higher percentage increase in FoxP3+ cells within the CD4+ cells (FIG. 9 (A)). In addition, these CD4+FoxP3+ positive cells exhibit higher expression of CD25 (FIG. 9 (B)). FIG. 10 shows that, in contrast to the hIL-2 wild type, hIL-2-N88R treatment in the mouse type I diabetes model prevents the development of the diabetes in all mice in the treatment group.

(57) 3. Conclusion

(58) The experiments performed by the inventors show clearly that owing to their potential for the induction of regulatory T cells (T.sub.Reg) the hIL-2 muteins according to the invention and sections thereof are substances which are suitable for the treatment and/or prophylaxis of an autoimmune disease or for the induction of T.sub.Reg in an organism and for the formation of T.sub.Reg in vitro. This is demonstrated by the inventors not only in vitro but also in vivo.