The present invention encompasses methods and compositions useful in diagnosing and treating hepatic disorders, especially those characterized by inflammation. The method comprises administration of an agent which prevents the interaction of MAdCAM with a MAdCAM binding partner or ligand. These compositions are useful in treating diseases or disorders involving .alpha.4.beta.7/MAdCAM blockade, as well as inhibiting a primary event in the inflammatory response such as blocking interactions between intercellular adhesion molecules and their ligands. Disorders treatable using the methods disclosed herein include infections, especially viral infections, iatrogenic disorders, cholestatic disorders, hereditary disorders, sarcoidosis, organ transplant, and the like. The diagnostic methods of the invention can be employed to detect the presence of a disorder or to monitor the course of therapy used to treat the disorder.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention encompasses methods and compositions useful in the diagnosis, prognosis and treatment of hepatic disorders. The methods and compositions of the invention can be employed in the diagnosis, prognosis and treatment of a variety of hepatic disorders, especially those characterized by MAdCAM-1 associated leukocyte recruitment to the liver. Hepatic disorders within the present invention include any systemic disease or disorder characterized by MAdCAM-1 expression including infections, especially viral infections, autoimmune disorders, iatrogenic disorders, hereditary disorders, cholestatic disorders, sarcoidosis, organ transplant and graft versus host disease after bone marrow transplant. The invention is preferably used to treat hepatitis, especially viral hepatitis, and autoimmune hepatitis, cholestatic disorders such as primary biliary cirrhosis and primary sclerosing cholangitis and allograft rejection.

The methods of treatment encompassed within the present invention comprise administration to a host in need thereof of an agent which prevents the interaction of MAdCAM with a MAdCAM binding partner or ligand such as the .alpha.4.beta.7 integrin or which inhibits the expression of MAdCAM in the liver. The methods are useful in preventing MAdCAM-1 associated leukocyte recruitment to the liver as well as inhibiting a primary event in the inflammatory response such as blocking interactions between intercellular adhesion molecules and their ligands. In preferred embodiments, the methods of the present invention are employed to reduce or prevent the infiltration of .alpha.4.beta.7 bearing leukocytes into liver thereby decreasing the severity of inflammation and the degree of tissue injury in the hepatic disease or disorder treated.

The invention includes compositions, including pharmaceutical compositions comprising agents such as antibodies for the treatment of hepatic disorders as well as kits and articles of manufacture. Kits and articles of manufacture preferably include: (a) a container; (b) a label on said container; and (c) a composition comprising an active agent contained within said container; wherein the composition is effective for treating a hepatic disorder, the label on said container indicates that the composition can be used for treating a hepatic disorder, and the active agent in said composition comprises an agent which prevents the interaction of MAdCAM with a ligand therefor. The kits optionally include accessory components such as a second container comprising a pharmaceutically-acceptable buffer and instructions for using the composition to treat a hepatic disorder.

Also disclosed are methods useful in the prognosis and diagnosis of hepatic disorders, especially those characterized by the expression of MAdCAM-1 in hepatic tissue. The diseases or disorders for prognosis or diagnosis under the present invention include those diseases and disorders treatable within the context of the present invention. The diagnostic methods can be employed to detect the presence of MAdCAM-1 in a sample, especially a liver biopsy or the presence of infiltrating leukocytes bearing a ligand for MAdCAM-1 in the sample. The methods can be employed to detect the disorder or to monitor, stage or predict the course of the disease or the therapy used to treat the disorder.

MODES FOR CARRYING OUT THE INVENTION

Hepatic Diseases and Disorders

The methods of the invention are useful in the diagnosis, prognosis and treatment of a variety of hepatic disorders, in particular those characterized by the presence of MAdCAM bearing cells. Therefore, according to the present invention, a hepatic disorder is any liver disease or disorder accompanied by the expression of MAdCAM-1 in the liver and surrounding vasculature. For example, the methods of the invention are useful in the diagnosis, prognosis and treatment of variety of hepatic disorders including those resulting from infection, iatrogenic disorders, hereditary disorders, autoimmmune disorders, cholestatic syndromes, sarcoidosis, organ transplantation, and the like so long as the disorder is characterized by the presence of MAdCAM-1 bearing cell types.

Diseases or disorders within the scope of the present invention include but are not limited to the diseases and disorders detailed in Table I (see Original Patent).

Particularly preferred disorders within the context of the invention are chronic hepatitis particularly hepatitis resulting from infection, particularly viral infection. Included in this category are the established serological categories of chronic hepatitis, including viral (HBV, HDV, HCV), autoimmune hepatitis (classic lupoid type and subtypes), autoimmune overlap syndromes, drug induced (for example nitrofurantoin, alpha methyldopa, isoniazid) and so-called "cryptogenic" hepatitis. In this regard the skilled artisan will make reference to chapter 9, and especially Tables 9.2 and 9.3 in Pathology of the Liver, 3rd. Edition, (Macseen, Anthony, Scheuer, Burt and Portman, eds.) Churchill Livingstone (1994) the disclosure of which is incorporated in its entirety herein by reference. As the skilled artisan will recognize, some chronic liver diseases not included within the definition of chronic hepatitis may have histological features of chronic hepatitis (for example, piecemeal necrosis). These disorders such as, for example, diseases of intra or extrahepatic bile ducts, are included within the definition herein. Infection with a number of viruses is known to result in serious inflammation of the liver including the hepatitis viruses, hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV, delta agent) hepatitis E, hepatitis F and other viruses such as Epstein-Barr virus, cytomegalovirus, adenovirus, paramyovirus, and the like. At least seven types of hepatitis virus (designated A-G) have been identified to date. Of these, one of the most devastating is hepatitis C virus (HCV, also called non-A, non-B). An estimated 3.9 million people in the US are currently infected with HCV, and an estimated 8,000-10,000 deaths each year result from HCV-associated chronic liver disease. Current therapies include .gamma.-interferon, emphasize B and ribivirin, each of which have limited efficacy and serious side effects. Current therapy also includes transplantation, however, since the infected individual remains infected with the virus, post-transplant immunosuppressed patients exhibit increased viral RNA levels and often rapidly progress to liver disease with the new liver.

Chronic cholestatic syndromes are characterized by progressive inflammatory destruction of intrahepatic bile ducts resulting in hepatic dysfunction, fibrosis and cirrhosis. Examples of this type of disorder include primary biliary cirrhosis, primary sclerosing cholangitis and adult idiopathic ductopenia.

Hereditary disorders treatable by the methods disclosed herein include those inflammatory disorders associated with a gene-linked trait. Examples include Wilson's disease, .alpha.1-antitrypsin deficiency and inherited metabolic disorders such as galactosemia and tyrosineanemia.

Diagnosing and Prognosing a Hepatic Disorder

Hepatic disorders for prognosis and diagnosis within the context of the present invention are described above and are characterized by the presence of MAdCAM in a sample, for example a sample of hepatic tissue or surprisingly in a cell free sample such as serum. Therefore, one embodiment of the present invention is directed to the detection and/or measurement of MAdCAM in a sample and the use of such detection or measurement in the diagnosis, staging, determination of severity, and prognosis in general of the hepatic disease or disorder. Further, since the expression of MAdCAM has been shown to correlate with the presence of lymphocytes bearing the .alpha.4.beta.7 integrin, prognosis and diagnosis of hepatic disorders within the context of the present invention encompass the measurement or detection of the presence of lymphocytes bearing .alpha.4.beta.7 integrin.

A. Detecting Soluble or Cell-Free MAdCAM

The present invention includes a method for diagnosis and prognosis of diseases and disorders not limited to hepatic diseases and disorders but appropriately used therefor, based upon the discovery that MAdCAM can be detected in the serum of a subject. Therefore the present invention includes methods of diagnosis and prognosis of diseases or disorders characterized by the expression of MAdCAM bearing cell types in general and which include but are not limited to the hepatic diseases or disorders listed above.

According to this aspect of the present invention, a sample which is subjected to testing is a sample derived from a subject such as a human and includes, but is not limited to, any biological fluid, preferably a bodily fluid. Especially preferred are cell-free samples, the term cell-free being used herein to indicate that the sample is substantially devoid of cells or that the sample is substantially free of cell types bearing MAdCAM. Examples of bodily fluids include, but are not limited to, whole blood, serum, plasma, urine, synovial fluid, cranial or spinal fluid, saliva, tissue infiltrate, cervical or vaginal exudate, tissue infiltrate, pleural effusions, bronchoalveolar lavage fluid, gastric lavage fluid, small or large bowel contents, fecal preparations, and the like. In another embodiment, the biological fluid may be a cell culture medium or supernatant of cultured cells. Preferably the sample is a blood sample and especially a serum sample. The methods provided by the present invention overcome many of the limitations of prior art methods of measuring or detecting MAdCAM, which heretofore required samples comprising cells followed by immunohistochemical techniques or direct or indirect immunofluorescence analysis by microscopy or flow cytometry. Limitations of the prior art procedures include the requirement for: (1) fairly rare tissue samples comprising a large number of cells, (2) extensive preparation time, and (3) expensive equipment, such as a flow cytometer. The methods provided herein overcome these limitations.

Any procedure known in the art for the measurement of analytes can be used in the practice of the measurement of MAdCAM in a sample. Such procedures include but are not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, enzyme immunoassays (EIA), preferably the enzyme linked immunosorbent assay (ELISA), "sandwich" immunoassays, precipitin reactions, gel diffusion reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays, to name but a few. For examples of preferred immunoassay methods, see U.S. Pat. No. 4,845,026 (Jul. 4, 1989) and U.S. Pat. No. 5,006,459 (Apr. 9, 1991).

For diagnostic and prognostic applications, a MAdCAM binding partner, typically an antibody will be labeled with a detectable moiety and used to detect MAdCAM in a sample as described above. Numerous labels are available which can be preferably grouped into the following categories:

(a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I, .sup.3H, and .sup.131I. The MAdCAM binding partner such as an antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed., Wiley-Interscience, New York, N.Y., Pubs., (1991) for example and radioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available. The fluorescent labels can be conjugated to the MAdCAM binding partner such as an antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme preferably catalyses a chemical alteration of the chromogenic substrate which can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g. orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and

(iii) .beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl-.beta.-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-.beta.-D-galactosidase.

Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.

In the assays of the present invention, a MAdCAM binding partner such as an antibody is preferably bound to a solid phase support or carrier. By "solid phase support or carrier" is intended any support capable of binding an antigen or antibodies. Well-known supports, or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

In a preferred embodiment, an antibody-MAdCAM-antibody sandwich immunoassay is done, i.e., MAdCAM is detected or measured by a method comprising binding of a first antibody to the MAdCAM antigen, and binding of a second antibody to the MAdCAM, and detecting or measuring MAdCAM immunospecifically bound by both the first and second antibody. In a specific embodiment, the first and second antibodies are monoclonal antibodies. In this embodiment, the second monoclonal antibody preferably binds to a site different from that of the first antibody (as reflected e.g., by the lack of competitive inhibition between the two antibodies for binding to the antigen). In another specific embodiment, the first or second antibody is a polyclonal antibody. In yet another specific embodiment, both the first and second antibodies are polyclonal antibodies.

In a preferred embodiment, a "forward" sandwich enzyme immunoassay is used, as described schematically below. An antibody (capture antibody, Ab1) directed against the MAdCAM is attached to a solid phase matrix, preferably a microplate. The sample is brought in contact with the Ab1-coated matrix and such that any MAdCAM in the sample to which Ab1 is specific binds to the solid-phase Ab1. Unbound sample components are removed by washing. An enzyme-conjugated second antibody (detection antibody, Ab2) directed against a second epitope of the MAdCAM binds to the antigen captured by Ab1 and completes the sandwich. After removal of unbound Ab2 by washing, a chromogenic substrate for the enzyme is added, and a colored product is formed in proportion to the amount of enzyme present in the sandwich, which reflects the amount of MAdCAM in the sample. The reaction is terminated by addition of stop solution. The color is measured as absorbance at an appropriate wavelength using a spectrophotometer. A standard curve is prepared from known concentrations of the MAdCAM, from which unknown sample values can be determined.

Other types of "sandwich" assays are the so-called "simultaneous" and "reverse" assays. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncompleted labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.

B. Diagnostic and Prognostic Methods in General

The methods of the present invention can be used alone or in conjunction with other diagnostic tests for the diagnosis and detection of a hepatic disorder. Viral infections can be detected using techniques known in the art. Hepatitis C infection, for example, can be detected using commercially available serologic assays which detect anti-HCV antibodies or molecular assays which detect HCV RNA genomes within an infected patient. The methods of present invention can be used alone or in conjunction with these routine tests as an aid in diagnosis. As another example, in many cases the specific cause of a liver disorder is identified on the basis of elevated liver function tests or an enlarged liver. The diagnostic methods of the present invention can be used alone or in conjunction with these tests to diagnose a disease or disorder within the context of the present invention. As but a further example, blood tests and a liver biopsy are routinely used to diagnose or confirm a diagnosis and as well to determine the amount, extent and severity of damage to the liver. The diagnostic methods of the present invention can be used alone or in conjunction with these tests in determining the amount, extent, or severity of damage to the liver.

Somewhat more particularly, a diagnostic tests performed on for example a serum sample, an in vivo sample or a liver biopsy can, with the present invention, be extended to the detection of MAdCAM expression in the sample. Further, the detection of MAdCAM in the sample can be used to monitor the course or progression of the disease as well as the course of or effectiveness of a therapeutic treatment.

In a particular embodiment, the diagnostic techniques described can be used to follow the progress of therapy. In a subject undergoing therapeutic treatment that results in an increase or a decrease in the amount of trafficking lymphocytes, the amount of lymphocyte trafficking may serve as a useful measure for the success or failure of the treatment. Thus, the present invention provides a method for monitoring the effect of a therapeutic treatment in a subject which comprises measuring at suitable time intervals the amount of MAdCAM expressed in a sample of liver tissue or conversely the amount or number of lymphocytes in the sample. The total amount of MAdCAM or .alpha.4.beta.7 is compared to a "baseline" or "control" value which depending on the disease, and the treatment, may be the amount of MAdCAM in a similar sample from a normal subject, from the patient prior to disease onset or during remission of disease, or from the patient prior to the initiation of therapy. One of ordinary skill in the art will readily discern the appropriate baseline value to use in a particular situation without undue experimentation.

A preferred subject for the methods of the present invention is a vertebrate, including but not limited to a mammal, fish, amphibian, reptile, bird, marsupial, and most preferably, a human either fetal or adult human liver. Thus the methods and kits of this invention are applicable to human clinical and veterinary uses.

According to a particular aspect of the present invention, a sample, for example a liver biopsy sample is derived from a subject by methods routine to those skilled in the art. The most common way a liver sample is obtained is by liver biopsy, a procedure used to obtain a small amount of liver tissue which can be subsequently examined employing routine immunohistochemical techniques in conjunction with the methods of the present invention. For example liver sample can be obtained by needle biopsy directly into the liver of subject, or for example by guiding a needle into the liver of the subject through the abdomen or chest using various imaging techniques known to the skilled artisan. Less commonly, samples are obtained using techniques such as laproscopy, transvenous or transjugular liver biopsy and surgical liver biopsy.

As noted above any procedure known in the art for the measurement of analytes can be used in the practice of the instant invention to detect the presence of MAdCAM or a ligand therefor, such as .alpha.4.beta.7. Such procedures include but are not limited to immunohistochemical techniques known to those skilled in the art, competitive and noncompetitive assay systems using techniques such as radioimmunoassays, enzyme immunoassays (EIA), preferably the enzyme linked immunosorbent assay (ELISA), "sandwich" immunoassays, precipitin reactions, gel diffusion reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays, to name but a few.

Kits comprising one or more containers or vials containing components for carrying out the assays of the present invention are also within the scope of the invention For instance, such a kit can comprise reagents required for the immunohistochemical analysis of a sample such as a liver biopsy. Reagents may include one or more binding partners, e.g., an antibody or antibodies, to antigen, for example a leukocyte integrin or other MAdCAM binding partner, or MAdCAM itself. For histological assays the kit contains the chromogenic substrate as well as a reagent for stopping the enzymatic reaction when color development has occurred. The substrate included in the kit is one appropriate for the enzyme conjugated to one of the antibody preparations such as an anti-human MAdCAM antibody. These are well-known in the art. The kit can optionally also comprise a standard; i.e., a known amount of purified MAdCAM.

In another embodiment, a kit can comprise more than one set of reagents. For example a kit can comprise a pair of antibodies or other binding partners, each pair directed against a different target molecule, thus allowing the detection or measurement of a plurality of such target molecules in a sample, for example, MAdCAM and a liver cell-specific surface protein or MAdCAM and .alpha.4.beta.7.

Compositions

Compositions useful in the therapeutic and the diagnostic methods of the present invention, are available to the skilled artisan and can be identified based upon their ability to prevent, block or suppress MAdCAM mediated cell adhesion. The compositions are useful in the treatment and diagnosis of hepatic disorders associated with that adhesion, such as inflammation and immune reactions.

It will be understood that appropriate agents able to prevent, block or suppress MAdCAM mediated cell adhesion may accomplish this effect in various ways. Without limitation to a particular theory, one class of agents will bind to MAdCAM-1 with sufficient affinity and specificity to prevent interaction with lymphocytes expressing a naturally occurring ligand for MAdCAM such as the lymphocyte integrin .alpha.4.beta.7. Another class of agents will bind to a naturally occurring leukocyte ligand for MAdCAM such as the lymphocyte integrin .alpha.4.beta.7 and thereby prevent its interaction with MAdCAM.

Exemplary agents are antibodies preferably a monoclonal, chimeric and or humanized antibody or an antigen binding fragment thereof which inhibits adhesion of leukocytes to MAdCAM. A further exemplary agent is a soluble MAdCAM molecule or a molecule based upon MAdCAM such as a soluble form of MAdCAM comprising the integrin binding site of MAdCAM or a MAdCAM immunoadhesin comprising, for example, the extracellular domain of MAdCAM fused to an immunoglobulin constant domain.

As a further example of an agent, a peptide or a molecule based upon a peptide sequence present in MAdCAM and required for integrin binding can be used as an agent within the context of the present invention. The amino acid motif GLDTSL conserved and present in Ig-like adhesion receptors including human MAdCAM can be used to design appropriate agents. International Publication Number WO 97/25351 provides molecules of this type which mimic the conserved amino acid motif LDTSL of MAdCAM. Alternatively, it has been shown that integrins can selectively bind a variety of Arg-Gly-Asp (RGD) containing ligands. RGD-based peptide inhibitors with different structures can be prepared which are effective agents within the context of the present invention (Jackson et al., (1997) J. Med. Chem. 40:3359-3368).

A further agent is an antisense nucleic acid, which is complementary, in whole or in part, to a target molecule comprising a sense strand, and can hybridize to the target molecule. When introduced into a cell antisense nucleic acid can inhibit the expression of the gene encoded by the sense strand. Antisense nucleic acid in whole or in part complimentary to the nucleic acid sequence of MAdCAM such as those described in International Publication No. WO 96/24673 can be produced for this purpose.

In a preferred embodiment the agent is an antibody, which antibody has the desirable properties of binding to MAdCAM-1 and preventing its interaction with the leukocyte associated ligand. Useful antibodies are available to the skilled artisan such as those described herein or those described by Podolsky et al., (1993) J. Clin. Invest. 92(1):372-380; Picarella et al., (1997) J. Immunol. 158:2099-2106; and Hesterberg et al., (1996) Gastroenterology 111:1373-1380. The following techniques can, without limitation, be employed in identifying and isolating appropriate agents useful in blocking or preventing the interaction between MAdCAM-1 and a MAdCAM binding partner. The compositions of the invention can be assayed by techniques known in the art in order to demonstrate their activity. Such assays include, but are not limited to, the following in vitro tests for the ability to interact with MAdCAM proteins, to inhibit MAdCAM related activity, or to selectively inhibit the generation of MAdCAM derived peptides. In vitro methods include a protein based assay such as that described in Berlin et al., (1993) Cell 74:185-195 where purified MAdCAM is applied to glass slides for binding assays. Agents within the context of the present invention inhibit the binding of normal lymphocytes to the immobilized MAdCAM.

In another appropriate assay purified .alpha.4.beta.7 is immobilized on a solid support such as a glass slide or a plastic plate pre-incubated with an antibody to .alpha.4 subunit that does not block interaction of the integrin with MAdCAM. In this assay MAdCAM or preferably a MAdCAM-immunoglobulin chimera is incubated with the immobilized integrin in the presence or absence of a suspected agent. The binding or absence of binding of MAdCAM in the presence of the agent being tested can then be measured with a detecting agent such as an anti-MAdCAM or anti-Ig antibody.

Alternatively a cell based assay employing a cell transfected with the .alpha.4.beta.7 integrin subunits and which expresses the intact integrin can be used in a cell based assay for identification of appropriate agents. For instance, the ability of a monoclonal antibody to inhibit adhesion of the natural cellular ligands to the cells expressing MAdCAM or the .alpha.4.beta.7 integrin can be used. Typically, the agent of the invention is incubated with the MAdCAM/.alpha.4.beta.7-bearing cells in the presence of the natural receptor/ligand-bearing cells, wherein the MAdCAM-bearing cells have been immobilized on a solid support. Inhibition of the cellular adhesion is then assessed by either calculating the amount of the bound mAb or assessing the displaced cells.

Agents effective in blocking or preventing the association of MAdCAM with lymphocytes may be identified by in vivo assays such as a model utilizing cotton-top tamarins. Cotton-top tamarins (CTT's; Saguinus oedipus) are a New World nonhuman primate species that, in captivity, develop spontaneous and often chronic colitis that clinically and histologically resembles ulcerative colitis in humans (Madera et al., (1985) Gastroenterology 88:13-19). This model has been used to show that the murine anti-human monoclonal antibody ACT-1 (Lazarovits et al., (1984) J. Immunol. 1331857) to .alpha.4.beta.7 cross reacts with CTT .alpha.4.beta.7 and reduces leukocyte cell density in inflamed colonic mucosa, attenuates histological inflammatory activity, and rapidly resolves clinical disease. Thus it will be useful in identifying additional substances that interfere with the interaction between hepatic-expressed MAdCAM and a MAdCAM ligand.

A lymphocyte homing assay such as that associated with the murine tri-nitro-benzene sulfonic acid (TNBS) induced colitis model and described in Viney et al., (1996) J. Immunol. 157:2488-2497 may be appropriate for the identification of agents and compositions.

The specificity or discrimination between two or more competing substrates is determined by the ratios of bound to unbound. For example, according to a cellular assay such as those described herein, radiolabeled or flourescent labeled .alpha.4.beta.7 is incubated with immobilized MAdCAM-1 receptor-immunoglobulin chimeras in varying concentration of unlabeled candidate compound. Increasing concentrations of successful candidate molecule effectively prevent binding of labeled .alpha.4.beta.7 to immobilized receptor chimeras. The concentration of unlabeled agent at which 50% maximal .alpha.4.beta.7 is displaced is referred to as the EC50 and reflects the receptor binding affinity. Therefore a candidate compound with an EC50 of 100 nM displays a substantially weaker interaction with a receptor than candidate agent with an EC50 of 10 nM. This discrimination in substrate specificity indicates that the preferred agent or antagonist has utility in, for example, preventing or blocking the interaction of MAdCAM with leukocyte surface antigens and especially .alpha.4.beta.7 in a setting where both the natural ligand and the so-called agent or antagonist are present.

An exemplary agent is a monoclonal antibody reactive with .alpha.4.beta.7 or MAdCAM. Antibodies are assessed by affinity constants. Affinity constants are a measure of the interaction between a particular ligand and its cognate receptor. The "binding affinity" or the measure of the strength of association between a particular receptor ligand interaction is generally measured by affinity constants for the equilibrium concentrations of associated and dissociated configurations of the ligand and its receptor. The present invention contemplates such an interaction between an agent or composition and the endothelial cell adhesion molecule MAdCAM-1. In general, the dissociation constants of ligand/integrin interactions in solution are relatively weak and range from low micromolar to high nanomolar. Additivity of multiple adhesive interactions at a cell surface, or the "avidity," provides the necessary binding energy to anchor leukocytes to the vascular endothelium. Therefore, in general, a useful composition or agent has a higher affinity for the integrin receptor than its native ligand. Such an antagonist blocks or prevents a high percentage of the cell surface interactions involved in cellular adhesion mediated by the .alpha.4.beta.7/MAdCAM-1 interaction. Preferably the binding of the agent or antagonist should occur at an affinity of about k.sub.a=10.sup.-4M or greater to be useful for the present invention, with greater than about 10.sup.-8M being more preferable, and most preferably between about 10.sup.-8M and about 10.sup.-10M.

As additional criteria, those forms of the molecule that are readily absorbed by tissues, that are protected from rapid metabolism and/or that provide for prolonged half life, are preferentially selected in producing the compositions of the invention. One skilled in the art may also effect modifications of the protein formulation, to effect absorption. These modifications include, but are not limited to, use of a pro-drug and chemical modification of the primary structure (Wearley, L. L., 1991, Crit. Rev. in Ther. Drug Carrier Systems, 8(4):333). In minimizing metabolism of the protein and thereby increasing the effective amount of protein, such modifications include but are not limited to chemical modifications and covalent attachment to a polymer (Wearley, L. L., 1991, supra).

Therapeutic Methods and Pharmaceutical Compositions

While not intending to be limited by a mechanism of action, it is believed that migration of activated leukocytes from the blood stream into the liver tissue is dependent on the interaction of the lymphocytes with the MAdCAM in the liver tissue. Leukocyte traffic across the vessel walls to extravascular vascular tissue is necessary for host defense against microbial organisms or foreign antigens and repair of tissue damage. Under some circumstances, however, leukocyte-endothelial interactions may have deleterious consequences for the host. During the process of adherence and transendothelial migration, leukocytes may release products such as oxidants, proteases, or cytokines that directly damage endothelium or cause endothelial damage by releasing a variety of inflammatory mediators. The interaction of MAdCAM-1 with leukocyte surface molecules, such as .alpha.4.beta.7, facilitates leukocyte migration and contributes to the destructive effects of the inflammatory process.

Therefore, according to the present invention, agents that prevent the interaction between hepatically expressed MAdCAM and ligands such as .alpha.4.beta.7 can be employed to treat these types of disorders in the liver.

Additionally, the pharmaceutical compositions of the present invention can be used to eliminate or block the injury occurring in transplanted livers.

The preclinical and clinical therapeutic use of the present invention in the treatment of diseases or disorders associated with MAdCAM will be best accomplished by those of skill, employing accepted principles of diagnosis and treatment. Such principles are known in the art, and are set forth, for example, in Braunwald et al., eds., Harrison's Principles or International Medicine, 11th Ed., McGraw-Hill, H.Y. (1987).

The most effective mode of administration and dosage regimen of agent will depend on the type of disease to be treated, the severity and course of the disease, whether the agents are administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the agents such as antibodies, and the discretion of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments.

For most therapeutic applications, the agents may be administered to a mammal, preferably a patient, in a pharmaceutically acceptable dosage form, including those that may be administered to a patient intravenously as a bolus or by continuous infusion over a period of minutes, hours, days, weeks, or months, intramuscularly, subcutaneously, intra-articularly, intrasynovially, intrathecally, or periostally, or by oral, topical, or inhalation routes.

A dose of agent may be administered to the patient in one or more single administrations, continuous infusion, or bolus injection. For example, an initial dose of the agent is administered to the patient by injection or infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. According to another embodiment of the invention, the effectiveness of the agent may be improved by administering the agent serially or in combination with another agent that is effective for this purpose (for example, interferon .gamma.).

The compositions of the present invention may be part of a delivery system such as liposomes. Delivery systems involving liposomes are discussed in International Patent Publication No. WO 91/02805 and International Patent Publication No. WO 91/19501, as well as U.S. Pat. No. 4,880,635 to Janoff et al. These publications and patents provide useful descriptions of techniques for liposome drug delivery.

The compositions of the invention can be administered to a subject in need thereof to treat the subject by either prophylactically preventing a disease state or relieving it after it has begun. The pharmaceutical compositions of the invention may be administered in any suitable manner, including parental, topical, oral, or local (such as aerosol or transdermal) or any combination thereof. The compositions are preferably administered with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, oral administration, usually using a solid carrier and I.V. administration a liquid salt solution carrier.

The compositions of the present invention include pharmaceutically acceptable components that are compatible with the patient and the protein and carbohydrate moieties of the compositions of the invention. These generally include suspensions, solutions and elixirs, and most especially biological buffers, such as phosphate buffered saline, saline, Dulbecco's Media, and the like. Aerosols may also be used, or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like (in the case of oral solid preparations, such as powders, capsules, and tablets).

As used herein, the term "pharmaceutically acceptable" preferably means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The formulation of choice can be accomplished using a variety of the aforementioned buffers, or even excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin cellulose, magnesium carbonate, and the like. "Peglation" of the compositions may be achieved using techniques known to the art (see for example International Patent Publication No. WO92/16555, U.S. Pat. No. 5,122,614 to Enzon, and International Patent Publication No. WO92/00748). Oral compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders.

A sufficient amount of the compositions of the invention should be administered to the patient to ensure that a substantial amount of the interaction between MAdCAM and a binding partner is inhibited. In this way, hepatic inflammation can either be prevented or ameliorated. The selection of compositions, frequency of administration, and amount of composition so administered will be in accordance with the particular disease being treated and its severity, the type of agent employed, the method of administration, the overall condition of the patient, and the judgment of the treating physician. Typical dosing regions will be analogous to treatment of these disease states by the use of antibodies and other biologicals. Typically, the compositions of the instant invention will contain from about 1% to about 95% of the active ingredient, preferably about 10% to about 50%. Preferably, the dosing will be between about 1-100 mg/kg. About 1 mg to about 50 mg will be administered to a child, and between about 25 mg and about 1000 mg will be administered to an adult. Other effective dosages can be readily determined by one of the ordinary skill in the art through routine trials establishing dose response curves.

In determining the dosage of compositions to be administered, it must be kept in mind that one may not wish to completely block all of the MAdCAM molecules, or may wish to completely block such receptors for only a limited amount of time. In order for a normal healing process to proceed, at least some of the white blood cells or neutrophils must be brought into the tissue in the areas where the wound, infection or disease state is occurring. Thus, the dose of the composition administered as a blocking agent must be adjusted carefully based on the particular needs of the patient while taking into consideration a variety of factors such as the type of disease that is being treated.

Thus, an effective amount of a composition in accordance with the present invention is an amount effective to inhibit the interaction between MAdCAM and a MAdCAM-binding partner.
 

Claim 1 of 13 Claims

1. A monoclonal antibody produced by a hybridoma having ATCC accession number CRL-12530.
 

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