Title:  Materials and methods for treating autoimmune disease

United States Patent:  6,403,562

Assignee:  University of Florida (Gainesville, FL)

Filed:  July 27, 1999

The subject invention pertains to novel methods for treating autoimmune-related diseases, such as Multiple Sclerosis (MS). One embodiment of the method of the invention comprises administering interleukin-10 (IL-10) and transforming growth factor-beta (TGF-.beta.), in combination, to a person afflicted with or predisposed to an autoimmune disease. When administered in combination, these cytokines act in a synergistic manner as suppressor factors to inhibit the activation of self-reactive T cells that are involved in autoimmune disease.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns novel therapeutic and prophylactic methods for treating autoimmune-related diseases, such as Multiple Sclerosis (MS). In one embodiment of the subject method, an effective amount of the cytokines IL-10 and TGF-.beta. are administered to a person afflicted with, or predisposed to, an autoimmune disease. Preferably, IL-10 and TGF-.beta. are administered to a patient in combination or in a therapeutically effective order. The methods of the invention can induce stable remission of disease in a patient. These cytokines function in a synergistic manner to suppress autoimmune related immune responses.

The subject invention also concerns methods for inhibiting T cell responses associated with autoimmune diseases. In one embodiment, T cell responses, such as T cell activation, proliferation or cytokine production, can be inhibited in a synergistic manner by administering an effective amount of IL-10 and TGF-.beta. to an animal. As exemplified herein, MBP specific T cell responses, such as activation and proliferation, are inhibited in an animal by administering IL-10 and TGF-.beta.. Thus, the methods of the subject invention can be used to inhibit pathological T cell responses in an animal having an autoimmune disorder.

Treatment of a patient with IL-10 and TGF-.beta. according to the methods of the present invention can also be used to inhibit the humoral arm of the immune system. Thus, the methods of the subject invention can also be used to inhibit B cell responses, such as antigen specific B cell activation and antibody production, in autoimmune diseases. For example, the present invention can be used to inhibit anti-MBP antibody and MBP-specific B cell effects in EAE in mice and other animals, and in multiple sclerosis in humans.

The subject invention can also be used to prevent or reduce activation of microglia and lymphocytic infiltration into the central nervous system that can be associated with certain autoimmune diseases, such as multiple sclerosis.

It has been determined that IFN.tau. downregulates the expression of MHC class II molecules on lymphocytes. Accordingly, in another embodiment of the present method, an effective amount of IL-10 and TGF-.beta. is administered along with IFN.tau. in a patient.

The IL-10, TGF-.beta. and IFN.tau. used with in the methods of the present invention can be produced either by natural or recombinant means. These cytokines can be of mammalian origin. Preferably, the cytokines are human IL-10, TGF-.beta. and IFN.tau.. Biologically active muteins (mutated proteins) of the cytokines, as well as other molecules, such as fragments, peptides and variants, that possess substantially the same bioactivity as the subject cytokines, are contemplated within the scope of the subject methods. For example, IL-10, TGF-.beta., and IFN.tau. polypeptides that contain amino acid substitutions, insertions, or deletions that do not substantially decrease the biological activity and function of the mutant polypeptide in comparison to native polypeptide is within the scope of the present invention.

Therapeutic application of the subject cytokines and compositions containing them can be accomplished by any suitable therapeutic method and technique presently or prospectively known to those skilled in the art. The cytokines can be administered by any suitable route known in the art including, for example, oral, parenteral, subcutaneous, or intravenous routes of administration. Administration of the cytokines of the invention can be continuous or at distinct intervals as can be determined by a person skilled in the art.

The compounds of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E. W. Martin describes formulations which can be used in connection with the subject invention. In general, the compositions of the subject invention will be formulated such that an effective amount of the bioactive cytokine(s) is combined with a suitable carrier in order to facilitate effective administration of the composition.

The compositions used in present method may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.

The compounds of the subject invention may also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time.

Examples of carriers or diluents for use with the subject cytokines include ethanol, dimethyl sulfoxide, glycerol, silica, alumina, starch, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired therapeutic treatment, new pharmaceutical compositions of the invention will advantageously comprise between about 0.1% and 45%, and especially, 1 and 15% by weight of the total of one or more of the cytokines based on the weight of the total composition including carrier or diluent.

As specifically exemplified herein, IFN.tau. induces CD4 T cells to become suppressor cells in NZW mice by oral administration or intraperitoneal injection of IFN.tau., and by treatment of mouse spleen cells with IFN.tau. in tissue culture. The suppressor cells inhibit MBP stimulation of spleen cells from MBP-immunized mice, and protect mice against induction of EAE. In addition, the CD4 T suppressor cells produce both IL-10 and TGF-.beta., which act synergistically to inhibit MBP-specific T cell proliferation. Induction of suppressor cells can be blocked by pretreatment, but not post-treatment, of IFN.tau. with neutralizing antibodies, thus establishing that induction of suppressor cells is specific for IFN.tau., but that the inducer of suppressor cells is not itself IFN.tau.. Therefore, IFN.tau. inhibition of EAE appears to occur via induction of suppressor cells and their suppressor factors.

The induction of suppressor cells is not unique to IFN.tau., as IFN.beta. also induced suppressor cells in spleen cell cultures. Further, the dose response curves for the two IFNs were similar. Also, these suppressor cells produce suppressor factors that inhibit MBP stimulation of EAE spleen cells. Thus, type I IFNs may, in general, protect against autoimmune diseases such as MS by induction of suppressor cells and suppressor factors.

As indicated above, IFN.tau. protected mice against EAE when administered orally even though relatively little IFN was found in the circulation. The gut is lined with over half of the cells of the immune system. The suppressor cells induced by oral IFN.tau. administration must be mobile, since the autoreactive MBP-specific T cells that are inhibited are themselves mobile, and in fact migrate to the central nervous system to cause EAE in the absence of IFN.tau. treatment. IFN.tau.-treated mice that are immunized with MBP show little or no lymphocyte infiltration of the CNS.

The CD4 T suppressor cell induced is most likely the Th2 type based on the detection of TGF-.beta. and IL-10 in suppressor cell supernatants. Further, since this suppressor cell is induced by IFN.tau. and probably also by other type I IFNs in the absence of MBP, it is most likely to be antigen-nonspecific in its effect. In fact, preliminary data suggest that suppressor cell supernatant inhibits mitogen stimulation of mouse spleen cells, and superantigen induced effects were similarly suppressed by CD4 T suppressor cells and their supernatant via IL-10 and TGF-.beta.. There was no evidence that non-CD4 T cells, including CD8 cells, possessed suppressor cell activity. This observation is in contrast to some other studies on suppressor cells (Nouri et al., 1991; Mukasa et al., 1994; Blank et al., 1995; Castedo et al., 1993). Other studies have also shown that the immune response is suppressed by antigen-specific CD4 Th2 cells (Karpus and Swanborg, 1991; Nabozny et al., 1991; Martinotti et al., 1995; Smith et al., 1991).

One aspect of the subject invention pertains to the discovery that CD4 suppressor T cells produce both IL-10 and TGF-.beta. that acted synergistically to inhibit MBP stimulation of spleen cells from EAE mice. IL-10 was detected in sera of mice which received prolonged i.p. injections or prolonged oral feeding of IFN.tau.. Also demonstrated herein is the discovery that IFN.tau.-induced suppressor cells produce IL-10 and TGF-.beta. to synergistically inhibit MBP-specific T cell proliferation.

The subject invention also pertains to methods for inhibiting B cell responses, such as B cell activation or B cell antibody production, by contacting a B cell with an effective amount of IL-10 and TGF-.beta.. The invention also concerns methods for inhibiting a T cell response, such as antigen specific T cell proliferation or activation, by contacting a T cell with an effective amount of IL-10 and TGF-.beta..

The subject invention also concerns novel compositions comprising IL-10 and TGF-.beta., or biologically active muteins, fragments or variants thereof. Preferably, a composition of the invention comprises purified IL-10 and TGF-.beta.. More preferably, a purified composition is provided in a pharmaceutically acceptable carrier or excipient.

The subject invention also concerns kits comprising IL-10 and TGF-.beta. in one or more compartments. The kits can be used in practicing the methods of the invention. Preferably, the kits comprise purified IL-10 and TGF-.beta. compositions in a pharmaceutically acceptable carrier or excipient.

As used herein, the term "TGF-.beta." includes all types of TGF-.beta. including TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3.

All references cited herein are hereby incorporated by reference.

Materials and Methods

IFNs.

The ovine IFN.tau. (IFN.tau.) gene was expressed in Pichia pastoris using a synthetic gene construct (Heeke et al., 1996). IFN.tau. was secreted into the medium and was purified by successive DEAE-cellulose and hydroxylapatite chromatography to electrophoretic homogeneity as determined by SDS-PAGE and silver staining analysis. The purified protein had a specific activity of 2.9 to 4.4x10⁷ U/mg protein as measured by antiviral activity using a standard viral microplaque reduction assay on MDBK cells (Pontzer et al., 1991). MUIFN.beta. (specific activity 4.1x10⁷ U/mg) was obtained from Lee Biomolecular (San Diego, Calif.).

Antibodies and Cytokines.

Monoclonal rat anti-mouse IL-10, recombinant mouse IL-10, and monoclonal mouse anti-TGF-p.beta.₁, anti-TGF-.beta.₂, and anti-TGF-.beta.₃ were obtained from Genzyme, Cambridge, Mass. Ultrapure natural human TGF-.beta.₁, which shows cross-reactivity in most mammalian cell types, was also obtained from Genzyme. A 1:10 dilution of HL100, a monoclonal antibody specific for IFN.tau., was used to neutralize 5000 U/ml of IFN.tau. prior to usage. All antibodies and cytokines were used in proliferation assays as described herein.

Interferon Induction of Suppressor Cells.

Suppressor cells were induced both in vitro and in vivo. For in vitro induction, NZW mouse spleen cells (5.0x10⁷ /ml) were incubated with 5000 U/ml of IFN.tau. for 24 h at 37^oC., after which the cells were washed twice prior to use. In vivo induction of suppressor cells in naive NZW mice involved administration of a single dose of IFN.tau. (10⁵ U) either intraperitoneally (i.p.) or by oral feeding with PBS used as the vehicle for administration. After 24 h, mice were sacrificed and the spleens removed. Spleen cells were washed and resuspended in RPMI 1640 medium supplemented with 2% fetal bovine serum and used as described below.

Induction of EAE.

For induction of EAE, 300 .mu.g of bovine MBP (MBP) were emulsified in complete Freund's adjuvant (CFA) containing 8 mg/ml H37Ra (Mycobacterium tuberculosis, Difco, Detroit, Mich.) and injected into two sites at the base of the tails of NZW mice. On the day of immunization and 48 h later, 400 ng of pertussis toxin (List Biologicals, Campbell, Calif.) were also injected. Mice were clinically examined daily for signs of EAE, and severity of disease was graded using the following scale: 1) loss of tail tone; 2) hind limb weakness, 3) paraparesis, 4) paraplegia; 5) moribund/death.

Adoptive Transfer of Suppressor Cells.

Suppressor cells were induced in vitro with IFN.tau. as described above and resuspended in phosphate buffered saline (PBS). NZW mice were injected intraperitoneally with 100 .mu.l of PBS containing 5x10⁶ suppressor cells 48 h before, on the day of, and 48 h after immunization with MBP for induction of EAE. Mice were examined daily for signs of EAE, and the severity of disease was graded as noted above.

CD4 T Cell Isolation and Depletion.

CD4 T cells effects were examined using both positive and negative CD4 T cell selection processes. The Cellectplus mouse CD4 kit (Biotex Laboratories, Inc., Alberta, Canada), an immunoaffinity column, was used to isolate CD4 cells from NZW mouse spleen lymphocyte cultures treated with media or IFN.tau.. Depletion of CD4 T cells from mouse spleen lymphocyte cultures treated with IFN.tau. or media was carried out using rat anti-mouse L3/T4 CD4 monoclonal antibody (Biosource International, Camarillo, Calif.) and Low-Toxic-M rabbit complement (Accurate Chemical and Scientific Corporation, Westbury, N.Y.). Lymphocytes from NZW mouse spleen were resuspended at 10⁷ cells/ml in RPMI 1640 medium and incubated with 1:10 dilution of anti-mouse L3/T4 CD4 antibody for 1 h at 4^oC. Cells were then centrifuged and resuspended in 1:10 dilution of rabbit complement in RPMI 1640 medium for 1 h at 37^oC. The cultures were washed and used for further experimentation.

Production of Suppressor Factor.

Suppressor cells were generated in vitro by incubating spleen cells with 5000 U/ml of IFN.tau. for 24 h at 37^oC. as described above. Cells were then washed and resuspended at 10⁸ cells/ml in fresh culture medium. After incubating for an additional 2 h at 37^oC., clarified supernatants were collected and tested for suppressor activity.

Proliferation Assay.

Spleen cells from MBP-immunized NZW mice (2.5-5.0x10⁵ cells/well) were co-cultured with IFN.tau.- or IFN.beta.-induced suppressor cells (1.0-5.0x10⁵ /well), suppressor cell supernatants, or IL-10 and TGF-.beta. in the presence of 30 or 100 .mu.g/ml of MBP. Suppressor cell supernatants were also pretreated for 2 h with either anti-IL10 antibody (25 .mu.g/ml) or anti-TGF-.beta. antibody (25 .mu.g/ml ) and then cultured with MBP-specific cells in the presence of MBP. Cultures were incubated for 96 h at 37^oC. The cultures were then pulsed with [³H]-thymidine (1.0 .mu.Ci/well; Amersham, Indianapolis, Ind.) 18 h before harvesting onto filter paper discs using a cell harvester. Cell-associated radioactivity was quantified using a .beta.-scintillation counter. Stimulation index was determined by dividing experimental CPM by control (unstimulated) CPM.

Claim 1 of 6 Claims

We claim:

1. A method for treating an autoimmune disease that is characterized by a self-reactive T cell response in a patient, comprising administering a synergistically effective amount of IL-10 and TGF-.beta., or a biologically active fragment of either both of said IL-10 and TGF-.beta., to said patient.

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