Title:  Antibodies to cytokines in the prevention and treatment of inflammatory bowel disease

United States Patent:  6,663,864

Issued:  December 16, 2003

Inventors:  Kink; John A. (Madison, WI); Worledge; Katherine L. (Madison, WI); Stafford; Douglas C. (Madison, WI)

Assignee:  Promega Corp. (Madison, WI)

Appl. No.:  325825

Filed:  June 4, 1999

Abstract

Methods are described for treating inflammatory bowel disease in animals, including humans. Specific avian polyclonal antibodies directed to proinflammatory cytokines (such as IL-6 and TNF) are shown to have a beneficial effect in animal models predictive of human therapy for the treatment of colitis, including Crohn's disease.

SUMMARY OF THE INVENTION

The present invention relates to therapeutics for the prevention and treatment of IBD. Specifically, the present invention contemplates the prevention and treatment of IBD in humans as well as other animals through the use of antibody therapy.

It is not intended that the present invention be limited to a particular type of antibody. Polyclonal and monoclonal antibodies are contemplated in the context of the present invention. Such antibodies may be made in a variety of animals [e.g., rabbits, horses, cows (e.g., in the milk), and birds]. The present invention also contemplates human and "humanized" antibodies.

It is preferred that the antibodies not be complement fixing. More specifically, avian antibodies (e.g., chicken antibodies from eggs) are preferred. It is contemplated that the treatment with such antibodies will have the desired result of reducing the symptoms (as well as morbidity and mortality) caused by IBD.

In one embodiment, the present invention contemplates a method comprising the administration of antibodies which bind to inflammatory mediators such as TNF. Preferably, the antibody is reactive with TNF across species. Specifically, the present invention demonstrates that immunization with human TNF generates neutralizing antibody capable of reacting with endogenous murine TNF. Thus, the present invention provides anti-TNF antibody that will react with mammalian TNF generally.

It is not intended that the present invention be limited to antibodies to TNF.alpha.. As demonstrated herein, antibodies to mediators believed to be downstream in the inflammatory cascade from TNF, such as IL-6 or IL-12 are effective. Such antibodies can be used preventively or (as demonstrated in an experimental model of IBD) during the acute stage of pathogenesis. While some previous work described in the literature has suggested that the use of TNF antibodies in the acute phase of IBD is contraindicated, the data contained herein indicates otherwise.

In another embodiment, the antibodies are combined with other reagents including but not limited to other antibodies. In one such embodiment, therapy comprises administration of a formulation comprising antibodies to a first cytokine and a second cytokine (e.g. antibodies to both TNF and IL-6).

In another embodiment, the present invention contemplates a method of relieving symptoms of and rescuing mammals (including humans) from episodes of acute or chronic IBD utilizing anti-cytokine antibodies such as anti-TNF antibodies.

In another embodiment, the present invention contemplates a method of relieving symptoms of and rescuing-mammals (including humans) from episodes of acute or chronic IBD utilizing a combination comprising anti-TNF antibodies. The present invention contemplates a method of treatment, comprising: (a) providing i) a mammal for treatment; ii) a therapeutic preparation, comprising anti-TNF polyclonal antibodies and (b) administering said antibodies to the lumen of said mammal.

In another embodiment, the present invention also contemplates a method of treatment, comprising: a) providing: i) a human patient with symptoms of inflammatory bowel disease, ii) a therapeutic formulation comprising avian polyclonal antibodies directed to TNF, and; b) administering said formulation to said patient.

In another embodiment, the present invention also contemplates a method of treatment, comprising: a) providing: i) a human patient with symptoms of inflammatory bowel disease, ii) a therapeutic formulation comprising avian polyclonal antibodies directed to IL-6, and; b) administering said formulation to said patient.

In another embodiment, the present invention also contemplates a method of treatment, comprising: a) providing: i) a human patient with symptoms of inflammatory bowel disease, ii) a therapeutic formulation comprising avian polyclonal antibodies directed to IL-6 and TNF, and; b) administering said formulation to said patient.

In another embodiment, the present invention also contemplates a method of treatment, comprising: a) providing: i) a human patient with symptoms of Crohn's disease, ii) a therapeutic formulation comprising avian polyclonal antibodies directed to TNF, and; b) administering said formulation to said patient.

In another embodiment, the present invention also contemplates a method of treatment, comprising: a) providing: i) a human patient with symptoms of Crohn's disease, ii) a therapeutic formulation comprising avian polyclonal antibodies directed to IL-6, and; b) administering said formulation to said patient.

In another embodiment, the present invention also contemplates a method of treatment, comprising: a) providing: i) a human patient with symptoms of Crohn's disease, ii) a therapeutic formulation comprising avian polyclonal antibodies directed to IL-6 and TNF, and; b) administering said formulation to said patient.

In the above embodiments, it is preferred that said administering is done under conditions such that said symptoms of IBD (e.g. symptoms of Crohn's disease) are reduced.

It is not intended that the present invention be limited to specific preparations of antibodies. However, polyclonal antibodies are preferred. Most importantly, it is preferred that the antibodies not be complement fixing. More specifically, avian antibodies (e.g. chicken antibodies from eggs) are preferred.

The treatment with the antibodies has the unexpected result of reducing mortality rates in animals when administered after the onset of a chronic or acute IBD episode.

DESCRIPTION OF THE INVENTION

The present invention relates to therapeutic compositions and methods for the prevention treatment of IBD, and in particular the prevention and treatment of IBD in humans as well as other animals. The antibodies of the present invention have particular application to the prevention and treatment of Crohn's disease.

The present invention further teaches treatments comprising anti-cytokine antibody (and combinations of antibodies to cytokines) and compositions and methods used after the onset of symptoms of IBD. As noted above, the present invention also contemplates treatment comprising administering formulations comprising anti-cytokine antibody (e.g. anti-TNF, anti-IL-6, and/or anti-IL-12). In accordance with the present invention, such formulations are administered via intravenous, parenteral, rectal or oral route, although the present invention is not limited to these methods of administration.

It is not intended that the present invention be limited by the particular nature of a formulation or combination. The present invention contemplates combinations as simple mixtures as well as chemical hybrids. An example of the latter is where the receptor is covalently linked to a pharmaceutical such as a corticosteroid, or where two receptor types are covalently joined. Covalent binding can be accomplished by any one of many commercially available crosslinking compounds.

It is not intended that the present invention be limited by the particular nature of the therapeutic preparation. For example, such compositions can be provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and excipients.

These therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts. Unlike convention treatment approaches with drugs (e.g. steroids), treatment with the antibodies of the present invention do not run the risk (from a practical standpoint) of overdosing the patient. Excess antibody administered orally or rectally will simply pass through the treated subject.

Such compositions are typically prepared as sprays (e.g., intranasal aerosols) for topical use. However, they may also be prepared either as liquid solutions or suspensions, or in solid forms. Oral formulations (e.g., for gastrointestinal inflammation) usually include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain 1%-95% of active ingredient, preferably 2%-70%.

The compositions are also prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.

The antibodies of the present invention are often mixed with diluents or excipients which are physiological tolerable and compatible. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.

Additional formulations which are suitable for other modes of administration, such as topical administration, include salves, tinctures, creams, lotions, and, in some cases, suppositories. For salves and creams, traditional binders, carriers and excipients may include, for example, polyalkylene glycols or triglycerides.

In a preferred embodiment, enteric formulations are employed, including but not limited to encapsulated antibodies. The terms "encapsuled" or "encapsulating" refers to the covering of a solid (e.g., lyophilized) form of antibody. The covering may comprise an enteric coating or a capsule. The terms "enteric coating" or "enteric film" are used interchangeably and refer to a material or compound which is resistant to acid pH (i.e., an acid-resistant compound), such as that found in the stomach. An enteric coating when applied to a solid inhibits the dissolution of the solid in the stomach.

Standard techniques are known to the art for the encapsulation of solid compositions. These techniques include microencapsulation of a solid composition wherein an enteric coating is applied to the solid composition. The coated material may be delivered orally to a subject by suspending the microencapsulated particles in pharmaceutical suspension solutions known to the art.

When a solid antibody is to be encapsulated using an enteric coating, the enteric coating may be applied using a one step coating process in which the enteric film is directly applied to the solid antibody; the coated antibody is said to be overcoated with the enteric film. Alternatively, a two step coating process may be employed wherein the solid antibody is first used to overcoat a non-pariel (i.e., a sugar particle of about 40-60 mesh size) and then the antibody-coated non-pariel is overcoated with the enteric film. Desirable enteric coatings for the delivery of antibody include polymethacrylates such as Eudragit.RTM. L30D (Rohm Tech, Inc.)

Solid antibody may formulated for oral delivery by insertion of the desired quantity of antibody into a capsule; the capsule would preferable have the characteristic of being resistant to dissolution in the stomach and being capable of dissolving in the intestines. Numerous suitable capsule formulations are available to the art; in addition standard techniques are available for the filling of capsules including the use of inert filler materials to provide sufficient bulk of the filling of a capsule with a therapeutic composition in a solid form. In addition to the use of microencapsulated antibody, the solid antibody may be delivered orally in tablet or pill form. The solid antibody may be combined with inert materials to provide sufficient bulk for the pressing of the tablet or pill. Once formed, the tablet or pill may then be coated with an enteric film to prevent dissolution in the stomach and to enhance dissolution in the intestines.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

EXAMPLE 1

Production of Antibodies to TNF, IL-6, IL-8, and IL-12 Homodimer and Heterodimer in the Hen

This example involved (a) preparation of the immunogen and immunization, (b) purification of anti-TNF, anti-IL-6, and IL-8 and anti-IL-12 (homodimer and heterodimer) chicken antibodies from egg yolk (IgY), and (c) detection of specific antibodies in the purified IgY preparations.

(a) Preparation of the Immunogen and Immunization

Recombinant human (rH) Tumor Necrosis Factor Alpha, (TNF), recombinant human Interleukin 6, (IL-6), recombinant human Interleukin 8, (IL-8) and recombinant mouse Interleukin 12 homodimer or heterodimer was purchased (lyophilized-without bovine serum albumin (BSA) and designated carrier-free) from R&D Systems Inc., Minneapolis, Minn. and produced in E. coli. TNF and interleukins 6 and 12 are considered proinflammatory cytokines. Active TNF exists as three TNF molecules forming a trimer. The native form of IL-12 exists as a heterodimer containing 2 distinct subunits designated as p30 and p40. Reports have indicated that the p40 subunit is the binding domain of the heterodimer and antibodies against only this subunit may be required to neutralize the native IL-12. (Gillessen S, et al. Eur J Immunol 1995;25:200-206). The p40 subunit can form a dimer termed the IL-12 homodimer. Experiments using the p40 subunit were preformed because this form may be easier to prepare recombinantly compared to the heterodimer. Interleukin-8 is a class of chemotatic cytokines termed chemokines. The lyophilized cytokines and chemokines were reconstituted in phosphate-buffered saline pH 7.2-7.5 (PBS) at 50 ug/ml and from 2-10 ug of TNF was used to immunize each hen. Each hen received one 0.5 ml sub-cutaneous 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.

(b) Purification of Anti-cytokine Chicken Antibodies from Egg Yolk (IgY)

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 9,000xg 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 resuspended in PBS at approximately 1/6 the original yolk volume. 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." The concentration of the fractionated IgY's were estimated by measuring the absorbance at 280nm (an optical density at 280 nm of 1.3 equals 1 mg of IgY/ml. The antibody concentrations were about 20-30 mg/ml.

(c) Detection of Anti-cytokine Antibodies in the Purified IgY Preparations

In order to determine if anti-cytokine response was generated and to determine relative levels of the response, enzyme-linked immunosorbent assays (ELISA) were performed. Briefly, ninety-six well Falcon Pro-bind micro-titer plates were coated overnight at 40C with 100 ul/well with different cytokines (TNF, IL-6, IL-12 homodimer or heterodimer and IL-8) at 0.1-1.0 ug/ml PBS. The wells are then blocked with PBS containing 1% BSA and 0.05% Tween 20 and incubated for about 1 hour at 370 C. The blocking solution was removed and the immune or preimmune IgY was diluted in PBS containing BSA and the plates were incubated for 1 hour at 370 C. The plates were washed 3 times with PBS containing 0.05% Tween 20 and three times with PBS alone. Alkaline phosphatase-conjugated anti-chicken IgG was diluted 1:1000 in PBS containing 1% BSA and 0.05% Tween 20, added to the plates and incubated 1 hour at 370 C. The plates were washed as above and p-nitrophenyl phosphate at I mg/ml in 0.05 M Na2CO3, pH 9.5, 10 mM MgCl2 was added. The plates were read in a Dynatech plate reader at 410 nm about 30 minutes after substrate addition. Good antibody titers (reciprocal of the highest immune IgY generating a signal about 3-fold higher than that of preimmune) ranging from 10,000 to 50,000 was generated.

The level of antibody response in the hens against all the cytokines tested were very good. Given the low amounts of antigen used for immunization, indicates cytokines may be very immunogenic in the hens and the avian system is a well-suited method to generate anti-mammalian cytokine antibodies.

Claim 1 of 20 Claims

What is claimed is:

1. A method of treatment, comprising:

a) providing:

i) a mammal having a symptom of inflammatory bowel disease, wherein said symptoms are associated with diseases selected from the group consisting of ulcerative colitis, proctitis, and Crohn's disease,

ii) a therapeutic enteric formulation comprising avian polyclonal antibodies directed to TNF, and;

b) orally administering said formulation to said mammal under conditions such that said symptom is reduced.

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