USE OF POLYKLONALER ANTI-IL-2 ANTIBODIES FOR THE TREATMENT OF INSULIN-DEPENDENT DIABETES MELLITUS AND LUPUS ERYTHEMATOSUS

The present invention relates to therapeutics for the prevention and treatment of autoimmune disease, and in particular the prevention and treatment of autoimmune disease in humans through the use of luminally administered polyclonal antibody.

A progressive and maintained response by the immune system against self-components is termed autoimmunity. Normally self-tolerance mechanisms prevent the immune response from acting on self-components. However, all mechanisms have a risk of breakdown and occasionally the immune system turns on its host environment in an aggressive manner as to cause disease. This breakdown leads to the copious production of autoreactive B cells producing autoantibodies and/or autoreactive T cells leading to destructive autoimmune disease. The cellular mechanisms of autoimunity are the same as those involved in beneficial immune responses to foreign components which include antibody-dependent cell cytotoxicity, delayed-type hypersensitivity (DTH), and T-cell lympholysis.

Human autoimmune diseases can be divided into two categories: organ-specific and systemic. In organ-specific autoimmune disease, autoreactivity is directed to antigens unique to a single organ. In systemic autoimmune disease, autoreactivity is largely directed toward a broad range of antigens and involves a number of tissues. Disease in either type results from the generation of one or both autoreactive cell types (B or T cells). Autoreactive B cells leads to the generation of autoantibodies or immune complexes. Autoreactive T cells leads to the cellular DTH responses from T_DTH cells or cytotoxic responses from T_c cells. Some autoimmune diseases in humans and the immune response and antigen(s) involved are shown in Table 1.

The current view of the etiology of autoimmune diseases posulates that both autoreactive T and B cells exist normally in the body. Control of these cells involves immune surveillance mechanisms which can induce tolerance to these cells and/or the selective elimination of these cells. Factors which overcome immune surveillance are thought to be responsible for the proliferation of these autoreactive cells leading to autoimmune disease.

Immune surveillance can be circumvented in several proposed ways: (1) Autoreactive cells can be stimulated through molecular mimicry by cross-reactive microbial antigens. A number of viruses and bacteria have been shown to possess antigenic determinants that are identical to normal host-cell components. Thus, antibodies generated against these microbial antigens can also recognize and damage host cells. Cross-reacting antibodies to heart muscle developed after a Streptococcus infection, for example, is thought of be the cause of an rheumatic fever. (2) In some cases, foreign antigen can directly stimulate autoreactive cells. Lipopolysaccharides or viral antigens from Epstein-Barr virus (EBV) or cytomegalovirus causes the direct stimulation of certain B cells. During mononucleosis, a disease caused by EBV, a variety of autoantibodies reactive to self-components are generated. Specifically, EBV can activate B cells to produce autoantibodies to nuclear DNA and immune cells. (3) Release of antigens normally sequestered from the immune system is another example of the breakdown of immune surveillance leading to autoimmune disease. Experimentally, animals injected parenterally with basic myelin protein, an antigen primarily found in the brain, develop experimental autoimmune encephalomyelitis. (4) Expression of specific HLA alleles has been associated with autoimmune individuals. It is thought that cells expressing these HLA's may act as a prime target for autoreactive cells. Individuals with the B27 HLA allele has a 90% increased relative risk of developing ankyosing spondylitis.

Current therapies for autoimmune diseases are not cures, but are aimed at reducing symptoms to provide the patient with a acceptable quality of life. In organ-specific autoimmune disorders, the symptoms can be corrected by the removal of the organ if possible. In some autoimmune diseases such as myasthenia gravis some success have been achieved by removing the thymus. In addition, in organ-specific autoimmune disorders, symptoms can be corrected by metabolic control with biologically active compounds. For example, hypothroidism can be controlled by the administration of thryroxine or perrrnicious anemia can be treated with injections of vitamin B₁₂. Drugs used in most cases of autoimmune disease, especially systemic auto-immune disease, provide general nonspecific suppression of the immune system. For the most part these drugs do not distinguish between the pathological immune response and the protective immune response. Immunosuppressive drugs (e.g., corticosteroids, azathioprine, cyclophoshamide and cyclosporin) are often given to suppress the proliferation of autoreactive lymphocytes. Anti-inflammatory drugs also are prescribed to patients with rheumatoid arthritis. Unfortunately these drugs, besides not working in many patients, have very serious side-effects. The general suppression of the immune response puts the patient at greater risk to infection and cancer. Clearly there is a significant need for agents capable of preventing and treating autoimmune diseases. It would be desirable if such therapy could be administered in a cost-effective and timely fashion, with a minimum of adverse side effects.

WO-A-98/14209 discloses the oral administration of chicken yolk antibodies against TNF for the treatment of systemic manifestations of an infectious non-intestinal disease associated with pathogenic organisms or molecules in a mammal. WO-A-00/08139 and Eigler et al., IMMUNOLOGY TODAY, 1997, vol. 18, pages 487-492 disclose the use of anti-TNF-gamma antibodies as useful for therapeutic purposes.

WO-A-99/09055 discloses the use of anti-IFN-gamma antibodies to treat diseases wherein INF-gamma activity is pathogenic such as in autoimmune diseases. Jacob et al., J. EXP. MED., 1987, vol. 166, pages 798-803, Nicoletti et al., ENDOCRINOLOGY, 1997, vol. 138, pages 281-288 and EP-B-0 695 189 disclose the use of anti-IFN-gamma antibodies, respectively, for the treatment of lupus-like nephritis, autoimmune diabetes and inflammatory bowel disease. Froyen et al., BIOTHERAPY, 1997, vol. 10, pages 49-57 refers to the therapeutic use of anti-IFN-gamma antibodies in the treatment of diabetes.

Constantinescu et al., JOURNAL OF IMMUNOLOGY, 1998, vol. 161, pages 5097-5104 discloses the inhibition of experimental autoimmune encephalomyelitis by anti-IL-12 antibodies. WO-A-99/36073 discloses the treatment of experimental auto-immune encephalomyelitis and diabetes by anti-IL-12 antibodies. Nicoletti et al., IMMUNOLOGY, 1999, vol. 97, pages 367-370 discloses to the use of anti-IL-12 monoclonal antibodies for the treatment of autoimmune diabetogenesis. Marth et al., GASTROENTEROLOGY, 1996, vol. 110, page 958 discloses the administration of anti-IL-12 antibodies for the treatment of autoimmune diseases.

The present invention is defined in the claims and relates to therapeutics for the prevention and treatment of autoimmune disease. Specifically, the present invention contemplates the prevention and treatment of autoimmune disease in humans as well as other animals through the use of ligands directed to cytokines. The examples demonstrate the production of antibodies to the proinflammatory cytokine IL -2. The examples demonstrate a novel finding that ligands (such as antibodies) against pro-inflammatory cytokines such IL-2 administered luminally are effective (as demonstrated in experimental models of both T cell-mediated and B cell-mediated autoimmune disease) at delaying the onset and reducing the severity of autoimmune disease.

It is not intended that the present invention be limited by the formulation for administration. In one embodiment, the formulation is formulated for administration to the lumen of the intestine. In a preferred embodiment, said medicament is formulated for oral administration. However, said medicament can also be formulated for parenteral administration.

Where antibodies are the ligand employed, the present invention is not limited to the source of the anto-cytokine antibodies. In one embodiment, said antibodies are avian polyclonal antibodies (including but not limited to chicken antibodies). In one embodiment, said antibodies are purified antibodies. In the case of chicken antibodies, it is convenient that said chicken antibodies are purified from chicken eggs.

Members from the class of IL receptors or IL receptor analogues can be selectively employed in soluble form to treat autoimmune patients. Soluble tumor necrosis factor (TNF), receptors, or TNF receptor analogues can be used to treat autoimmune patients. Therapeutic preparation comprising, in combination, both soluble TNF and soluble IL receptors are useful in the treatment of autoimmune disease.

The existence of membrane receptors to cytokines is now well-established. Many of these receptors have now been cloned and expressed in high yield. See U.S. Patents Nos. 4, 968, 607, 5, 925, 351, 5,919,903, 5, 919, 456, 5, 965, 704, 5, 945, 511, 5,945,397, 5,925,735. Such receptors are useful as ligands for use in the treatment of autoimmune disease.

The present invention is defined in the claims and contemplates the use of antibodies directed to proinflammatory cytokines and inflammatory mediators administered to or at the lumen to treat autoimmune diseases. In one embodiment antibodies (raised in birds or mammals) against the offending inflammatory mediators are contemplated and these antibodies can be administered systemically, orally or mucosally either prophylatically or therapeutically to the patient. A variety of classes of inflammatory mediators are contemplated to be important to generate antibodies useful in the prevention and treatment of inflammatory diseases. Illustrative cytokines and inflammatory mediators are shown in Table 2. It is envisioned that ligands against these mediators would be used either singly or in combination to treat a specific disease. Combination therapies would consist of ligands to several mediators within a given pathway.

It is not intended that the present invention be limited to a particular mechanism. Indeed, and understanding of the precise mechanism by which the administration of ligands to cytokines achieves a therapeutic benefit is not necessary in order to successfully practice the present invention. While not limited to any particular mechanism, the inventors believe that cytokines play a major role in the initiation and regulation in immune response and that the dys-regulation of the cytokine network may also lead to the activation of autoreactive T cells leading to autoimmune. Preferential activation of a specific set of T cells, T_H1, is thought to play a central role in the pathogenesis of a number of autoimmune diseases. T cells with the CD4 phenotype are divided into subsets referred as T_H1 and T_H2 based on the nature of their immune reactivity and their cytokine secretion profile. T_H1 cells are associated cell-mediated inflammatory reactions and act as effector cells in infectious disease. Secreted cytokines that set the T_H1 subset apart are interferon gamma, tumor, necrosis factor (TNF) interleukin-2 (IL-2) and upon activation interleukin-12 (IL-12). T_H1 cytokines are referred to as proinflammatory cytokines because they activate cytotoxic, inflammatory and delayed hypersensitiviy reactions. In contrast, T_H2 cells are associated with helper cell function and antibody production. T_H2 cells upon activation secrete interleukins 4 (IL-4), 5 (IL-5), and 10 (IL-10). Cytokines from a T_H1 cells tend to inhibit the actions of the T_H2 cell and vice versa. Whether the characterization of such cells is correct or not, the data shows that the therapeutic methods (described in more detail below) result in a dramatic delay and/or reduction in autoimmune symptoms and disease.

The phrase "ligand directed to a cytokine" is herein defined as meaning any molecule having affinity for a cytokine. Ligands can be chemically synthesized or designed by molecular evolution. Alternatively, such ligands can be known biomolecules or portions thereof (e.g., receptors, antibodies). It is not intended that the present invention be limited to the mechanism by which ligands achieve a therapeutic benefit. Ligands may be "antagonists" in that they neutralize the biological impact of a secreted cytokine. On the other hand, ligands may simply block recognition of cytokines or interfere with cell function. For example, ligands may interact with a cell-surface cytokine so as to result in immune cells not participating in autoimmune phenomenon (e.g., the cells are caused to enter apoptosis, etc.).

The phrase "administered to or at the lumen" is herein defined as administration that preferentially delivers compound(s) to the space in the interior of the intestines at a concentration in excess of what is found in circulation. Such delivery can be achieved by a variety of routes (e.g., oral, rectal, etc.) in a variety of vehicles (e.g., tablet, suppository, etc.). By contrast, a parenteral administeration is not designed to preferentially deliver compounds to the lumen (although some incidental delivery can take place through normal biodistribution).

The phrase "symptoms of autoimmune disease" is herein defined as any abnormal symptoms than can be attributed to the generation of autoreactive B and/or T cells. For example, autoantibodies are a common symptom associated with autoimmune disease. Symptoms are "reduced" when the severity [as measured by frequency (e.g. the number of episodes over time), amount (e.g. the amount of autoantibody or other indicator) or other parameter] of one or more symptoms are reduced. All symptoms need not be reduced for symptoms to be "reduced." Moreover, symptoms need not be eliminated for symptoms to be "reduced."

The phrase "at risk for autoimmune disease" is herein defined as individuals with familial incidence of autoimmunity. For example, many autoimmune diseases are associated with genetic factors such as certain HLA specificities.

A "proinflammatory cytokine" is any cytokine that can activate cytotoxic, inflammatory or delayed hypersensitivity reactions. Examples of such cytokines are IL-2, TNF and INF-gamma. Examples of inflammatory mediators are found in Table 2.

The following examples serve to illustrate certain aspects of the present invention.

Production of antibodies proinflammatory mediators to IL-2 in the Hen.

This example involved (a) preparation of the immunogen and immunization (b) purification of anti-IL-2 chicken antibodies from egg yolk (1gY), and (c) detection of specific antibodies in the purified 1gY preparations.

Recombinant mouse Interleukin 2, (IL-2) was purchased (lyophilized without bovine serum albumin (BSA) and designated carrier-free) from R&D Systems Inc., Minneapolis, MN and produced in E. coli. The lyophilized cytokine was reconstituted in phosphate-buffered saline pH 7.2-7.5 (PBS) at least 50 ug/ml. From approximately 2 to 50 ug of the cytokine was used to immunize groups of hens. Each hen received one 0.5 ml subcutaneous injection containing the individual cytokine with 75 ug Quil A adjuvant (Superfos Biosector, Denmark, distributed by Accurate Chem., Westbury, N.Y.) in PBS. The hens were immunized every 2 weeks for at least 3 times then placed on a maintenance immunization schedule where the hens were immunized every 4-6 weeks.

Groups of eggs were collected per immunization group at least 3-5 days after the last booster immunization. The chicken yolk immunoglobulin (IgY) was extracted by a two-step polyethylene glycol (PEG) 8000 method performed according to a modification of the procedure of Polson et al., Immunol. Comm., 9:495 (1980). The yolks were separated from the whites and the yolks were placed in a graduated cylinder. The pooled yolks were blended with 4 volumes of PBS and PEG was added to a concentration of 3.5%. When the PEG was dissolved, the protein and lipid precipitates that formed were pelleted by centrifugation at 9,000 x g for 15 minutes. The supernatants were decanted and filtered through 4 layers of gauze to remove the floating particulates and a second PEG step was performed by adding PEG to a final concentration of 12% (the supernatants were assumed to contain 3.5% PEG). After a second centrifugation, the supernatants were discarded and the IgY pellets were typically resuspended in PBS or 0.1 M carbonate buffer pH 9.5 at approximately 1/6 -1/8 the original yolk volume. The concentration of the fractionated IgY's were estimated by measuring the absorbance at 280nm (an optical density at 280 nm of 1.1 to 1.3 equals 1 mg of IgY/ml. The antibody concentrations were about 20-40 mg/ml. The IgY's used for oral administration in the animal models were resuspended in high pH-carbonate buffer. This was done to to help increase the pH in the stomach and minimize the acid hydrolysis of the IgY. IgYs extracted from the eggs of immunized hens are designated as "immune IgY," while IgYs extracted from the eggs of unimmunized hens is designated "preimmune IgY."

In order to determine if an anti-cytokine response was generated and to determine relative levels of the response, enzyme-linked immunosorbent assays (EIA) were performed. Briefly, ninety-six well Falcon Pro-bind micro-titer plates were coated overnight at 40°C with 100 ul/well with the cell mediator (IL-2) at 0.1-1.0 ug/ml PBS. The wells are then blocked with PBS containing 3% BSA and 0.05% Tween 20 and incubated for about I hour at 37°C. The blocking solution was removed and the immune or preimmune IgY was diluted antibody diluent (1% BSA with 0.05% tween 20 in PBS). The samples at a beginning concentration of 20-40 mg/ml were diluted 1:30 to 1:100 in antibody diluent then added in duplicate wells of the microtiter plate. Typically the samples were then serially diluted 5-fold within the plate and incubated for about 1 hour at 37°C. The plates were then washed 3 times with PBS containing 0.05% Tween 20 then three times with PBS alone. Alkaline phosphatase-conjugated anti-chicken IgG (Kirkegaard and Perry Labs (KPL), Gaithersburg, MD), generally diluted 1:1000 in antibody diluent, was added to the plates and incubated about I hour at 37°C. The plates were washed again as above and substrate was added. Substrate was prepared either using p-nitrophenyl phosphate (104 phosphatase, Sigma Chemicals, St. Louis, MO) at 1 mg/ml in 0.05 M Na₂CO₃, pH 9.5, 10 mM MgCl₂ or using the Phosphatase Substrate System from KPL. The plates were read in a Dynatech plate reader at 410 nm from 10-30 minutes after substrate addition.

Very good antibody titers was detected by EIA against the mediator. Titers were defined as the reciprocal of the highest immune IgY generating a EIA signal about 3-fold higher than that of preimmune IgY. Titers values of the cellular mediator was approximately 10,000 or more. Repeated immunizations of hens using up to 10 ug of IL-1 per hen failed to generate a detectable IgY response in the hens. Overall, though the rest of the inflammatory mediators with adjuvant proved to be immunogenic in the hens. Good antibody titers were generated using small amounts of antigen. Thus the avian system appears to be a well-suited approach to produce high titer antibodies against mammalian inflammatory mediators.

This example involved the testing of the anti-IL-2 IgY neutralizing ability in a cell-based neutralization assay. The ability of anti-IL-2 IgY to neutralize the bioactivity of IL-2 was determined in a cell-based assay based on a protocol described in Current Protocols in Immunology, Vol. 1, Section 6.3.4, 1994. CTLL-2 cell line (ATCC) requiring rhIL-2 (R&D systems) as a growth factor at 4 ng/ml was grown in RPMI 1640 with 10 % fetal calf serum at 37°C at 5% CO₂. Avian anti-IL-2 IgY was diluted serially two-fold with culture media in a 96 well microtiter plate. Goat anti-rh IL-2 IgG (R&D Systems) and preimmune IgY at the same concentrations were also serial diluted and served as positive and negative controls, respectively. Recombinant human IL-2 at 0.2 ng/well was added and pre-incubated with antibody for 1 hour at 37°C. CTLL-2 cells at 10⁴/well was then added and incubated for 20 hours at 37°C, 5% CO₂ in a humidified chamber. Cell viability was measured using the chromogenic Cell Titre 96 Proliferation Assay (Promega Corporation, Madison, WI) recording the optical density at 490 nm. The results demonstrated that the anti-IL-2 IgY was effective at neutralizing rhTNF compared to preimmune IgY in the CTLL-2 cell-based assay. The ND 90 of anti-IL-2 IgY was approximately 500 ug/ml.

An autoimmune animal model of insulin-dependant diabetes mellitis (IDDM) was used to determine if luminally-administered anti-cytokine therapy may be effective at preventing or delaying the onset of disease. IDDM is an autoimmune disease that effects about 0.2 % of the population causes the destruction of the insulin-producing (beta) islet cells in the pancreas. This destruction involves the presence of pancreatic autoimmune antibodies and leukocytic infiltration (insulitis) of the pancreatic islet cells. These events effectively lower the amount of normal insulin needed to maintain normal glucose metabolism resulting in diabetes. Nonobese diabetic (NOD) mice spontaneously develop autoimmune T-cell mediated IDDM. Diabetes in the NOD mouse is similar to human IDDM in both genetics and autoimmune pathogenesis (C.J. Boitard et al., Autoimmunity 15 (Suppl):12-13, 1993 and A.A. Rossini et al., Annu. Rev. Immunol., 3:289-320, 1995). This mouse strain has provided an important model for dissecting the pathogenesis of autoimmune diabetes in humans. NOD mice spontaneously develop insulitis between 2-4 weeks of age and progresses to diabetes 10-20 weeks later in about 80% of the female mice and 20% of the male mice. Severe IDDM ensues that results in excessive urine production containing high levels of glucose. With time, severe IDDM eventually results in a death in a most of the NOD mice.

This example involves: a) description of establishment of the NOD model and treatment, b) Methods and results to determine oral anti-cytokine efficacy on the delay of onset of glycosuria (high levels of glucose in the urine and c) prevention of mortality from disease.

1. a) The NOD Model And Treatment. Six week old (about 20 gram) female NOD mice were purchased (The Jackson Laboratory, Bar Harbor ME) and maintained under specific pathogen free (SPF) conditions. The maintenance of the mice in SPF conditions was performed according to procedures described in Current Protocols in Immunology, (1997) 15.9.1-15.9.23 by Edward H. Leiter (John Wiley & Sons, Inc.). SPF conditions required the use of autoclaved cages and barrier protected isolator lids with autoclaved food and bedding. The drinking water was filtered and acidified to pH 2-3 with HCI to prevent the growth of Pseudomonas. Groups of NOD mice (7/group) were aseptically treated orally with antibodies to the proinflammatory mediator, IL-2. Treatment controls consisted of treating mice orally with either vehicle (0.1 M carbonate buffer pH 9.2-9.5) or preimmune IgY. The IgY's were diluted in 0.1 M carbonate buffer to minimize IgY hydrolysis in the stomach. Treatments were sterile filtered (0.4) before use. Approximately 0.2 mls of a 20-40 mg/ml (200-400 mg/kg/day) of IgY solution was orally administered using a 20 G, 3.5 cm long feeding needle (Popper & Sons, New Hyde, NY). Treatments were administered orally once per day, five days (weekdays) a week. Mid-way through the study, one mouse in the vehicle-treated group died from dosing unrelated to IDDM disease.
2. b) Efficacy: Delay Of Onset Of Glycosuria. The monitoring of the glycemic status began on shortly after arrival using reagent strips to semi-quantitatively measure urine glucose. Either Diastix (glucose) or Uristix (glucose and protein) reagent strips for urinalysis were used (Bayer Diagnostics, Elkhart, IN). The level of glycemia was determined colorimetrically from turquoise to dark brown with the strips representing glucose levels of 100, 250, 500, 1000, and 2000 mg of glucose/dl. Individual mice were placed in clean cages without bedding and a drop of urine was collected on the test area and read after 30 seconds. Mice are considered diabetic with glycosuria when urine glucose levels were > 250 mg/dl. Treatment results are shown in Table 4. The results indicate that oral anti-cytokine therapy using anti-IL-2 IgY could delay the onset of diabetes in the NOD mouse model. The onset of glycosuria first appeared when the mice were approximately 14-15 weeks old after about 8 weeks of treatment. Glycosuria first appeared at the same time in some mice treated with vehicle and preimmune. Initial onset of glycosuria in the anti-IL-2 treated mice was delayed. During treatments from approximately week 11 to week 16, 67 % and 43 % of vehicle treated and preimmune treated mice were glycosuric. In contrast, only 14 % of the anti-Il-2 treated mice, respectively were glycosuric. After 16 weeks of treatment nearly all the mice in the vehicle treated (100 %) and preimmune treated mice (83 %) had diabetes. In contrast, roughly half of the anti-cytokine treated group (42 % for anti-IL-2 were diabetic. Preimmune IgY treatment compared to vehicle treatment was found to have no therapeutic effect in terms of delay of diabetes. Results, however indicated that the onset of diabetes in NOD mice with was delayed approximately 6 weeks after treatment with oral anti-cytokine therapy.
3. c) Efficacy: Prevention Of Mortality From Disease. Results indicated that oral anti-cytokine therapy using anti-IL-2 IgY prevented death due to the complications of diabetes in the NOD mice model (Table 5). The time of death in the mice during treatment was monitored and closely coincided with several weeks of high glucose levels in the blood (2000 mg/dl). Death from diabetes started in both in the vehicle and preimmune treated mice at 17 weeks of age after about 11 weeks of treatment. Sixty-seven % of vehicle treated and 57 % of preimmune treated NOD mice were dead after 22 weeks of age. Significantly, none of the anti-IL-2 treated were dead after 22 weeks (Table 5). The results showed that oral anti-IL-2 therapy in NOD mice could prevent mortality from disease compared to the control treated mice.

An autoimmune animal model of Systemic Lupus Erythematosus (SLE) was used to determine if oral anti-cytokine therapy may be effective at preventing or delaying the onset of disease. SLE is a systemic autoimmune disease that usually appears in women from 20 and 40 years of age and is characterized by fever, weakness, joint pain, erythematous lesions, pleurisy and kidney disfunction. Affected individuals may produce autoantibodies to a vast array of self-components such as DNA, red blood cells, and platelets. Immune complexes of autoantibodies are formed which are deposited on blood vessels resulting in vasulitis and glomerulonephritis in the kidney. The New Zealand black x New Zealand white F, hybrid mouse (NZB/W¹ ) spontaneously develop severe autoimmune disease that closely resembles SLE in humans (A.N. Theofilopoulos and F.J. Dixon, Adv. Immunol., 37:269, 1985). This model in particular has been very useful in understanding the immunological defects involved in the development of SLE autoimmunity. NZB/W¹ mice develop immune complex-mediated glomerulonephritis, resulting in the excretion of high levels of protein in the urine and an anti-DNA IgG serum response. As true with SLE in humans, the incidence of autoimmunity in NZB/W¹ female mice is much higher than males. Mice eventually develop a fatal disease around 6-9 months in female animals.

This example involves a description of: a) the NZB model and treatment, b) methods and results to determine oral anti-cytokine efficacy on the delay of onset of proteinuria (high levels of protein in the urine, c) Reduction of an anti-double stranded (ds) DNA serum response and d) Prevention of mortality from disease.

1. a) The NZB/W' Model And Treatment. Six week old (about 20 gram) female NZB/W¹ mice were purchased (The Jackson Laboratory, Bar Harbor ME) and maintained under normal non-SPF conditions according to the breeder. Groups of NZB/W¹ mice (7/group) were treated orally with anti-IL-2 IgY diluted in 0.1 M carbonate buffer pH 9.2-9.5. Treatment control mice were treated orally with either vehicle (0.1 M carbonate buffer pH 9.2-9.5) or preimmune IgY. Approximately 0.2 mls of a 20-40 mg/ml (200-400 mg/kg/day) of IgY solution was orally administered using a 20 G, 3.5 cm long feeding needle (Popper & Sons, New Hyde, NY). Treatments were administered orally once per day, five days (week days) a week.
2. b) Efficacy: The Delay Of Onset Of Proteinuria. The presence of proteinuria in the NZB/W¹ mice was detected using reagent strips to semi-quantitatively measure urine protein. Uristix (glucose and protein) reagent strips for urinalysis were used (Bayer Diagnostics, Elkhart, IN). The presence of measurable amounts of protein in the urine by the reagent strips is considered abnormal and indicative of disease. The level of proteinuria was determined colorimetrically from light green (negative-trace) to dark green with the strips measuring protein levels of 30, 100, 300, and >2000 mg of protein/dl of urine. Individual mice were placed in clean cages without bedding and a drop was collected on the test area and read after 30 seconds. Mice are considered to have proteinuria when urine protein levels were > 30 mg/dl. Treatment results are shown in Table 6. The results indicate that oral anti-cytokine therapy with anti-IL-2 could delay the onset of diabetes in the NZB/W¹ mouse model. The onset of proteinuria first appeared in the preimmune and vehicle-treated mice approximately when the mice were 14 weeks old after about 7 weeks of treatment. During treatments from week 7 to week 28 the majority (71%-100%) of the vehicle-treated or Preimmune-treated mice had proteinuria (>30 mg/dl). Significantly, proteinuria in the NZB/W¹ mice treated orally with anti-IL-2 IgY was not detected until week 21 of treatment. After 28 weeks of treatment most of the vehicle-treated and preimmune treated mice were proteinuric (86 % (6/7)). In contrast, at week 28 of treatment, only 29 % (2/7) of the anti-IL-2 treated mice treated mice were proteinuric. These results indicate that oral anti-cytokine therapy to IL-2 could effectively delay the onset of proteinuria in NZB/W¹ mice compared to the control treated mice.
3. c) Reduction Of An Anti-Double Stranded (ds) DNA Serum Response. The presence of an anti-ds DNA response which is pathognomonic for SLE was determined using a commercial enzyme immunoasssay (EIA) kit designed to measure specific autoantibodies against ds DNA in human serum. Blood samples were taken at various times during treatment from each NZB/W¹ mouse from the tail vein. Serum was then collected after the blood was allowed to clot. The microtiter EIA assay (Bindazyme Anti-ds DNA EIA kit, The Binding Site, Birmingham, England) was performed on the serum samples according to manufacturers instructions except instead of using the antihuman peroxidase conjugate, a sheep anti-mouse IgG peroxidase (The Binding Site) was used. The sheep anti-mouse IgG peroxidase conjugate was diluted 1/10,000 in the assay as recommended by the manufacturer. Internal assay controls were run and serum samples were tested in duplicate. Tetramethylbenzidine substrate was used and after 10 minutes the plates were read at 450 nm. The higher the absorbance value the test serum at 450 nm, the higher the anti-ds DNA response. The average absorbance with the standard error of the mean of each treatment group at various times is shown in Table 7. The results shown a similar increase in an anti-ds DNA serum response in both the vehicle and preimmune treated mice. This response is roughly reduced by half in the anti-IL-2 treated NZB/W¹ mice throughout the treatment regimen. The anti-ds DNA serum response in the anti-cytokine treated mice is delayed approximately 11 weeks comparing the results from week 17 to week 28. Results indicated that oral anti-cytokine therapy using anti-IL-2 IgY could effectively reduce the anti-ds DNA response in NZB/W¹ mice compared to the control treated mice.
4. d) Prevention Of Mortality From Disease. Results indicated that oral anti-cytokine therapy using anti IL-2 and anti IL-2 could delay mortality due to the complications of SLE in the NZB/W¹ mice model (Table 8). The time of death in the mice during treatment was monitored and closely coincided with high anti-ds DNA levels in the serum. Death began to occur in the vehicle and preimmune-treated NZB/W¹ mice at 26 weeks of age (20 weeks of treatment) matching previous reports on this model. At a week 36, 57 % (4/7) of vehicle treated and 71 % (5/7) of preimmune-treated mice died from disease. In contrast, none of the mice in the anti-IL-2 have died and all still appeared healthy. The results showed that oral anti-IL-2 therapy in NZB/W¹ mice could prevent mortality from disease compared to the control treated mice.