HK1118220A - Use of anti-madcam antibodies for the treatment of coeliac disease and tropical sprue - Google Patents

Description

Use of anti-MAdCAM antibodies for the treatment of coeliac disease and tropical sprue

Technical Field

The present invention relates to the use of an anti-MAdCAM antibody for the manufacture of a medicament for the treatment of coeliac disease and/or tropical sprue.

Prior Art

The cell adhesion molecule (MAdCAM) of Mucosal addressin cell adhesion is a member of the immunoglobulin superfamily of cell adhesion receptors. It is one of the adhesion molecules that participates in the recruitment (if necessary) of lymphocytes to tissues by interacting with integrin molecules on the surface of lymphocytes.

Has been shown to inhibit the binding of MAdCAM to its integrin binding partner (alpha)₄β₇) Conjugated antibodies, such as anti-MAdCAM antibodies (e.g., MECA-367; U.S. Pat. No. 5403919, U.S. Pat. No. 5538724) or anti-alpha₄β₇Antibodies (e.g., Act-1; U.S. Pat. No. 6551593), inhibit leukocyte extravasation into the inflammatory bowel, and are therefore useful in the treatment of Inflammatory Bowel Disease (IBD).

However, anti-MAdCAM antibodies such as MECA-367 have no therapeutic use in human patients; MECA-367 binds mouse MAdCAM, but does not show much affinity for human MAdCAM molecules. Furthermore, as a rat antibody, it elicits an immune response in human patients and is therefore not suitable for therapeutic use. Mouse monoclonal antibodies against human MAdCAM have been described (WO 96/24673), but these may also be immunogenic in humans. Recently, fully human anti-human MAdCAM antibodies with strong specificity and affinity for human and primate MAdCAM that are useful in therapy have been developed and described in WO 2005/067620.

MAdCAM and alpha have been proposed₄β₇Integrin interactions(e.g., blocking anti-MAdCAM antibody or anti-alpha)₄β₇Integrin antibodies, such as MLN02, which is a humanized Act-1, described in WO 01/078779) are useful for the treatment of Inflammatory Bowel Disease (IBD). However, it has now been found that inhibitors of this interaction, including MAdCAM blocking antibodies, can also be used to treat coeliac disease and tropical sprue.

Coeliac disease (also known as gluten-sensitive enteropathy or sprue) is a small bowel disorder. In the uk, europe and usa, 1 out of 300 people are affected by this condition. Coeliac disease is a common condition and affects anyone of any age. It is thought to be more common in men, but is likely to occur equally in men and women.

Gluten is a mixture of two proteins (gliadin and glutenin) and is found in wheat, barley and rye. It reacts with the small intestine and causes damage by activating the immune system to attack the delicate intestinal membranes responsible for the absorption of nutrients and vitamins. Although the condition can be diagnosed at any age, it is often diagnosed in children who have introduced grain into their diets after weaning. The symptoms can be subtle and the patient can experience no cause of discomfort for a certain period of time before a diagnosis is made.

Initial symptoms typically include becoming irritated and feeling painful, with poor appetite and an inability to gain weight. The stool (defecation) becomes weak, loose and unpleasant. Some children begin to vomit and diarrhea and are therefore often misdiagnosed as "gastroenteritis". The stomach may become distended and the muscles of the arms and legs are thinned and thinned. In adults, symptoms may be similar, including weight loss with pale complexion, severe diarrhea, or constipation and abdominal distension with "gas discharge". Half of the adults with coeliac disease do not have any symptoms from the intestines. It often visits the doctor for the following reasons: extreme fatigue, psychological problems such as depression, bone pain and even sometimes fractures (due to thinning of the bone), canker sores or blisters, pruritic rashes mainly occurring in the elbows and knees (called dermatitis herpetiformis).

Some women with abdominal disorders are difficult to conceive and will therefore be diagnosed. Recurrent abortion (spontaneous abortion) is sometimes associated with abdominal diseases. Some women are diagnosed during pregnancy because their intestines do not absorb enough iron and vitamins to meet pregnancy needs, thus making them severely anemic. Mothers with abdominal disorders grow more frequently with infants of small intrauterine age (retarded intrauterine growth).

Left untreated, abdominal disease causes anemia, bone disease and in less cases some forms of cancer. The most important treatment at present is to avoid all gluten-containing foods. This often results in improved or even no loss of intestinal membrane damage. However, if gluten is reintroduced into the diet, the damage is caused anew.

While abdominal disease is not preventable, adherence to a gluten-free diet reverses damage to the small intestine. This requires considerable autonomy. There is a need to find a drug with low risk side effects as follows: can be administered to patients with normal diet and can avoid mineral and vitamin deficiency and other diseases related to abdominal diseases.

Tropical sprue is a digestive problem that occurs in tropical and subtropical regions. People with tropical sprue do not absorb nutrients well, especially vitamin B12 and folic acid. Diarrhea is a major symptom of tropical sprue; people who consume large amounts of fatty foods experience more severe diarrhea than people who consume low-fat diets. Other symptoms include abdominal cramps, nausea, weight loss, exhaustion and dyspepsia.

Approximately 1 in every 1000000 population is affected by heat-streaked inflammatory diarrhea and occurs between about equatorial north latitude 30 ° to equatorial south latitude 30 °. It is more common in some countries, including india, maritime, cuba, puerto rico, and the dominican republic. It is unusual or absent in africa, the bahama isles and jamaica. The condition causes pain to residents and visitors in affected countries, but generally affects only visitors who stay for 6 months or more.

The cause of tropical sprue has not been established, but is likely due to a combination of factors including infection and malnutrition, which together cause damage to the intestinal membranes, which in turn prevents them from absorbing nutrients.

The diagnosis of tropical sprue can be complicated by the fact that many disorders have similar symptoms. Stool and blood tests were performed to rule out other causes of diarrhea. If these tests are negative and the patient has been living in the tropics for a long time, tropical sprue may be the cause of the disease. A biopsy may be performed to examine the villi to identify the typical smoothness of the small intestine villi.

Certain blood tests may also aid in the diagnosis of tropical sprue. Since the disease blocks the absorption of certain vitamins and minerals, low levels of albumin, calcium or vitamins D, A, K and E are observed. Patients also develop anemia due to vitamin B12 and folate deficiency. In addition, stool samples may exhibit excessive fat.

The treatment will typically be 3 to 6 months supplementation with antibiotics and folic acid (folic acid or folate). People lacking vitamin B12 will also receive vitamin supplements.

Disclosure of Invention

One aspect of the invention is the use of an antibody that specifically binds MAdCAM in the manufacture of a medicament for the treatment of coeliac disease and/or tropical sprue. Another aspect of the invention is a method of treating coeliac disease and/or tropical sprue (preferably coeliac disease) using a therapeutically effective amount of an anti-MAdCAM antibody.

Another aspect of the invention is anti-alpha₄β₇Use of an integrin antibody for the manufacture of a medicament for the treatment of coeliac disease and/or tropical sprue, preferably coeliac disease. Preference is given toThe anti-alpha is₄β₇The integrin antibody is humanized Act-1, also known as MLN 02. Another aspect of the invention is the use of a therapeutically effective amount of anti-alpha₄β₇A method of treating coeliac disease and/or tropical sprue (preferably coeliac disease) with an antibody (preferably MLN 02).

Another aspect of the invention is MAdCAM-alpha₄β₇Use of an inhibitor of integrin-mediated adhesion for the manufacture of a medicament for the treatment of coeliac disease and/or tropical sprue. Another aspect of the invention is the use of a therapeutically effective amount of MAdCAM-alpha₄β₇A method of treating coeliac disease and/or tropical sprue with an inhibitor of integrin-mediated adhesion.

Preferably, the anti-MAdCAM antibody or antigen-binding portion thereof for use in the present invention can specifically bind MAdCAM. More preferably, at least the CDR sequences of the antibody are human CDR sequences or antigen-binding portions of human antibodies. Preferably, the antibody is a human antibody, further preferably a human monoclonal antibody or an antigen-binding portion thereof, more preferably an antibody or an antigen-binding portion thereof that is an antagonist of MAdCAM.

Preferably, the antibody or portion thereof has at least one of the following properties:

(a) binding to human cells;

(b) at least 100-fold more selective for MAdCAM than VCAM or fibronectin;

(c) at 3X 10^-10K of M or less_dBinds to human MAdCAM; or

(d) Inhibition of alpha₄β₇Binding of the expressing cells to human MAdCAM,

(e) inhibiting the recruitment of lymphocytes to gastrointestinal lymphoid tissue.

Preferably, the antibody or antigen-binding portion inhibits the human MAdCAM to α₄β₇And has at least one of the following properties:

(a) cross-competes for binding to MAdCAM with a reference antibody;

(b) competes for binding to MAdCAM with a reference antibody;

(c) binds to the same MAdCAM epitope as the reference antibody;

(d) with substantially the same K as the reference antibody_dBinding to MAdCAM;

(e) binds MAdCAM at approximately the same off rate as the reference antibody;

wherein the reference antibody is selected from: monoclonal antibody 1.7.2, monoclonal antibody 1.8.2, monoclonal antibody 6.14.2, monoclonal antibody 6.22.2, monoclonal antibody 6.34.2, monoclonal antibody 6.67.1, monoclonal antibody 6.73.2, monoclonal antibody 6.77.1, monoclonal antibody 7.16.6, monoclonal antibody 7.20.5, monoclonal antibody 7.26.4, monoclonal antibody 9.8.2, monoclonal antibody 6.22.2-mod, monoclonal antibody 6.34.2-mod, monoclonal antibody 6.67.1-mod, monoclonal antibody 6.77.1-mod and monoclonal antibody 7.26.4-mod.

In another aspect of the invention, the heavy chain variable region, the light chain variable region, or both of the anti-MAdCAM antibody has at least 90% identity in amino acid sequence to the corresponding regions of a monoclonal antibody selected from the group consisting of: monoclonal antibody 1.7.2, monoclonal antibody 1.8.2, monoclonal antibody 6.14.2, monoclonal antibody 6.22.2, monoclonal antibody 6.34.2, monoclonal antibody 6.67.1, monoclonal antibody 6.73.2, monoclonal antibody 6.77.1, monoclonal antibody 7.16.6, monoclonal antibody 7.20.5, monoclonal antibody 7.26.4, monoclonal antibody 9.8.2, monoclonal antibody 6.22.2-mod, monoclonal antibody 6.34.2-mod, monoclonal antibody 6.67.1-mod, monoclonal antibody 6.77.1-mod and monoclonal antibody 7.26.4-mod.

Preferably, the antibody is selected from:

(a) contains a polypeptide shown in SEQ ID NO: 2 and SEQ ID NO: 4 and does not contain a signal sequence;

(b) contains a polypeptide shown in SEQ ID NO: 6 and SEQ ID NO: 8 and no signal sequence;

(c) contains a polypeptide shown in SEQ ID NO: 10 and SEQ ID NO: 12 and no signal sequence;

(d) contains a polypeptide shown in SEQ ID NO: 14 and SEQ ID NO: 16 and no signal sequence;

(e) contains a polypeptide shown in SEQ ID NO: 18 and SEQ ID NO: 20 and no signal sequence;

(f) contains a polypeptide shown in SEQ ID NO: 22 and SEQ ID NO: 24 and no signal sequence;

(g) contains a polypeptide shown in SEQ ID NO: 26 and SEQ ID NO: 28 and no signal sequence;

(h) contains a polypeptide shown in SEQ ID NO: 30 and SEQ ID NO: 32 and not containing a signal sequence;

(i) contains a polypeptide shown in SEQ ID NO: 34 and SEQ ID NO: 36 and no signal sequence;

(j) contains a polypeptide shown in SEQ ID NO: 38 and SEQ ID NO: 40 and no signal sequence;

(k) contains a polypeptide shown in SEQ ID NO: 42 and SEQ ID NO: 44 and no signal sequence;

(l) Contains a polypeptide shown in SEQ ID NO: 46 and SEQ ID NO: 48 and no signal sequence;

(m) a polypeptide comprising the sequence shown in SEQ ID NO: 52 and SEQ ID NO: 54 and does not contain a signal sequence;

(n) a polypeptide comprising the sequence shown in SEQ ID NO: 56 and SEQ ID NO: 58 and no signal sequence;

(o) a polypeptide comprising the sequence shown in SEQ ID NO: 60 and SEQ ID NO: 62 and no signal sequence;

(p) a polypeptide comprising the sequence shown in SEQ ID NO: 64 and SEQ ID NO: 66 and no signal sequence; and

(q) a polypeptide comprising the sequence shown in SEQ ID NO: 42 and SEQ ID NO: 68 and does not contain a signal sequence.

In another aspect of the invention, the monoclonal antibody, or an antigen-binding portion thereof, is selected from the group consisting of:

(a) the heavy chain comprises the amino acid sequences of the heavy chain CDRs 1, CDR2 and CDR3 of a reference antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod

(b) The light chain comprises the amino acid sequences of the light chain CDRs 1, CDR2, and CDR3 of a reference antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod

(c) The antibody comprises a heavy chain of (a) and a light chain of (b); and

(d) the antibody of (c), wherein the heavy and light chain CDR amino acid sequences are selected from the same reference antibody.

In another aspect of the invention, the monoclonal antibody or antigen binding portion comprises:

(a) a heavy chain comprising an antibody heavy chain variable region amino acid sequence selected from the group consisting of: 1.7.2(SEQ ID NO: 2); 1.8.2(SEQ ID NO: 6); 6.14.2(SEQ ID NO: 10); 6.22.2(SEQ ID NO: 14); 6.34.2(SEQ ID NO: 18); 6.67.1(SEQ ID NO: 22); 6.73.2(SEQ ID NO: 26); 6.77.1(SEQ ID NO: 30); 7.16.6(SEQ ID NO: 34); 7.20.5(SEQ ID NO: 38); 7.26.4(SEQ ID NO: 42); and 9.8.2(SEQ ID NO: 46); 6.22.2-mod (SEQ ID NO: 52); 6.34.2-mod (SEQ ID NO: 56); 6.67.1-mod (SEQ ID NO: 60); 6.77.1-mod (SEQ ID NO: 64); and 7.26.4-mod (SEQ ID NO: 42);

(b) a light chain comprising the variable light chain amino acid sequence of an antibody selected from the group consisting of: 1.7.2(SEQ ID NO: 4); 1.8.2(SEQ ID NO: 8); 6.14.2(SEQ ID NO: 12); 6.22.2(SEQ ID NO: 16); 6.34.2(SEQ ID NO: 20); 6.67.1(SEQ ID NO: 24); 6.73.2(SEQ ID NO: 28); 6.77.1(SEQ ID NO: 32); 7.16.6(SEQ ID NO: 36); 7.20.5(SEQ ID NO: 40); 7.26.4(SEQ ID NO: 44); and 9.8.2(SEQ ID NO: 48); 6.22.2-mod (SEQ ID NO: 54); 6.34.2-mod (SEQ ID NO: 58); 6.67.1-mod (SEQ ID NO: 62), 6.77.1-mod (SEQ ID NO: 66); and 7.26.4-mod (SEQ ID NO: 68); or

(c) The heavy chain of (a) and the light chain of (b).

Another aspect of the invention is the use of the heavy and/or light chain of the anti-MAdCAM antibody or variable regions or other antigen-binding portions thereof or a nucleic acid molecule encoding any of the foregoing and a pharmaceutically acceptable carrier. This aspect of the invention includes fragments of any of the above antibodies (including but not limited to Fab fragments, F (ab')₂Fragments, single chain fv (scFv) fragments).

Preferably, the anti-MAdCAM antibody is a human inhibitory anti-MAdCAM antibody selected from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod as described in WO 2005/067620. Preferably, the anti-MAdCAM antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66 or 68 (with or without a signal sequence) or a variable region of any of the amino acid sequences or one or more CDRs from the amino acid sequences. The anti-MAdCAM antibody preferably comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, or 64 (with or without a signal sequence) or a variable region amino acid sequence or one or more CDR amino acid sequences derived from said amino acid sequence. The anti-MAdCAM antibody is preferably a human anti-MAdCAM antibody having an amino acid sequence from the beginning of CDR1 to the end of CDR3 of any of the above sequences. The anti-MAdCAM antibody used in the present invention may also be an anti-MAdCAM antibody containing one or more FR regions of any one of the above-mentioned sequences.

The anti-MAdCAM antibody used in the present invention may also include an anti-MAdCAM antibody containing one of the above-described amino acid sequences in which one or more modifications have been made. For example, a cysteine that is chemically reactive in the antibody may be substituted with another residue (including but not limited to alanine or serine). The substitution may occur at atypical cysteines or at typical cysteines. Such substitutions may be made in the CDRs or framework regions or in the constant domains of the antibody variable domains.

Amino acid substitutions may also be made to eliminate potential proteolytic sites in the antibody. Such sites may be present within the CDRs or framework regions or constant domains of the antibody variable domains. Substitution of cysteine residues and removal of proteolytic sites reduces heterogeneity in antibody products. Asparagine-glycine pairs that can form potential deamidation sites can be eliminated by altering one or both of the two residues. Amino acid substitutions may be made for the addition or removal of potential glycosylation sites in the variable region of the antibodies of the invention.

The C-terminal lysine of the heavy chain of the anti-MAdCAM antibody used in the present invention can be cleaved off. The heavy and light chains of the anti-MAdCAM antibody may optionally include signal sequences.

The 12 preferred inhibitory human anti-MAdCAM monoclonal antibodies (1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2) for use in the present invention are described in detail in WO2005/067620 (the entire contents of which are incorporated by reference in this patent specification).

Detailed Description

Classes and subclasses of anti-MAdCAM antibodies

The antibody may be an IgG, IgM, IgE, IgA or IgD molecule. Preferably, the antibody belongs to the IgG class and to IgG₁、1gG₂、1gG₃Or 1gG₄Sub-classes. More preferably, the anti-MAdCAM antibody is 1gG₂Or 1gG₄Sub-classes. More preferably, the anti-MAdCAM antibody is conjugated to a monoclonal antibody which is 1gG₂1.7.2, 1.8.2, 7.16.6, 7.20.5, 7.26.4, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod or as 1gG₄6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1 or 9.8.2 (as described in WO 2005/067620) belonging to the same class and subclass.

The class and subclass of anti-MAdCAM antibodies can be determined by any method known in the art. In general, the class and subclass of an antibody can be determined using antibodies specific for a particular class and subclass of antibody. Such antibodies are commercially available. ELISA, Western blot analysis, and other techniques can determine the class and subclass. Alternatively, the class and subclass can be determined as follows: determining the sequence of all or a portion of the heavy and/or light chain constant domains of the antibody; comparing the amino acid sequence thereof with known amino acid sequences of immunoglobulins of various classes and subclasses; and the class and subclass of the antibody was identified as the class showing the highest sequence identity.

Species selectivity and molecular selectivity

The anti-MAdCAM antibodies used in the present invention exhibit both species and molecular selectivity. The anti-MAdCAM antibody can bind MAdCAM in human, cynomolgus or dog. Other anti-MAdCAM antibodies useful in the present invention are not able to bind to new world monkey species such as marmosets. One can determine the species selectivity of anti-MAdCAM antibodies using methods well known in the art. For example, one can use Western blot analysis, FACS, ELISA, or immunohistochemistry to determine species selectivity. In a preferred embodiment, one can use immunohistochemistry to determine species selectivity.

anti-MAdCAM antibodies that specifically bind MAdCAM for use in the invention are at least 10-fold more selective for MAdCAM than VCAM, fibronectin or any other antigen, preferably at least 20, 30, 40, 50, 60, 70, 80 or 90-fold more, most preferably at least 100-fold more. Preferably, the anti-MAdCAM antibody does not exhibit any significant binding to VCAM, fibronectin or any other antigen other than MAdCAM. One can determine the selectivity of anti-MAdCAM antibodies for MAdCAM using methods well known in the art in light of the teachings of the present specification. For example, one can determine this selectivity using Western blot analysis, FACS, ELISA, or immunohistochemistry.

anti-MAdCAM antibodies bind MAdCAM affinity

The anti-MAdCAM antibody used in the present invention preferably can specifically bind MAdCAM with high affinity. anti-MAdCAM antibodies for use in the invention are administered at 3X 10^-8K of M or less_dSpecifically binds MAdCAM as measured by surface plasmon resonance (such as BIAcore). Preferably, the antibody is at 1 × 10^-8Or lower or 1X 10^-9M or lower Kd specifically binds MAdCAM. More preferably, the antibody is expressed at 1 × 10^-10K of M or less_dSpecifically binds MAdCAM. The antibody used in the invention is 2.66X 10^-10M or less, 2.35X 10^-11M or less or 9X 10^-12K of M or less_dSpecifically binds MAdCAM. Preferably, the antibody is expressed at 1 × 10^-11K of M or less_dSpecifically binds MAdCAM. Preferably, the antibody has substantially the same K as an antibody selected from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod as described in WO2005/067620_dSpecifically binds MAdCAM.

Having "substantially the same K" as a reference antibody_d"has an antibody of. + -.100 pM, preferablyK of. + -.50 pM, more preferably. + -.20 pM, further preferably. + -.10 pM,. + -.5 pM or. + -.2 pM_d(K with reference antibody in the same experiment_dBy comparison). Preferably, the antibody has substantially the same K as an antibody comprising one or more variable domains or one or more CDRs as follows_dBinding MAdCAM, the variable domain or CDR being from an antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod as described in WO 2005/067620. Preferably, the antibody has a K substantially identical to an antibody or variable domain thereof comprising an amino acid sequence_dBinds MAdCAM, the amino acid sequence being selected from one of the following: SEQ ID NO as described in WO 2005/067620: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58, 62, 64, 66, or 68 (with or without signal sequence). Preferably, the antibody has substantially the same K as an antibody comprising one or more of the following CDRs_dBinding MAdCAM, the CDR being from an antibody comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58, 62, 64, 66, or 68.

The binding affinity of an anti-MAdCAM antibody to MAdCAM can be determined by any method known in the art. In one embodiment, the binding affinity can be measured by competitive ELISA, RIA, or surface plasmon resonance (such as BIAcore). In a more preferred embodiment, the binding affinity is measured by surface plasmon resonance. In a further preferred embodiment, the binding affinity and off-rate are measured using BIAcore. Examples of determining binding affinity can be found in WO 2005/067620.

Half-life of anti-MAdCAM antibodies

The anti-MAdCAM antibodies used in the present invention have a half-life of at least 1 day in vitro or in vivo. Preferably, the antibody or portion thereof has a half-life of at least 3 days. More preferably, the antibody or portion thereof has a half-life of 4 days or more. Further preferably, the antibody or portion thereof has a half-life of 8 days or more. The antibodies or antigen binding portions thereof used in the present invention may also be derivatized or modified to have a longer half-life, as discussed below. In another preferred embodiment, the antibody may contain point mutations to increase serum half-life, as described in WO 00/09560.

Antibody half-life can be measured by any means known to those skilled in the art. For example, antibody half-life can be measured by Western blot analysis, ELISA, or RIA over a suitable period of time. Antibody half-life can be measured in any suitable animal such as a primate (e.g., cynomolgus monkey, or human).

Identification of MAdCAM epitopes recognized by anti-MAdCAM antibodies

The invention also provides the use of human anti-MAdCAM antibodies that bind to the same antigen or epitope as the human anti-MAdCAM antibodies provided in this patent. In addition, the invention provides the use of human anti-MAdCAM antibodies that compete or cross-compete with human anti-MAdCAM antibodies. Preferably, the human anti-MAdCAM antibody is 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod as disclosed in WO 2005/067620. Preferably, the human anti-MAdCAM antibody comprises one or more variable domains or one or more CDRs from an antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Preferably, the human anti-MAdCAM antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58, 62, 64, 66 or 68 (with or without a signal sequence) or one of their variable domains. Preferably, the human anti-MAdCAM antibody comprises one or more amino acid sequences derived from SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58, 62, 64, 66, or 68, or a pharmaceutically acceptable salt thereof.

One can use various methods known in the art to determine whether an anti-MAdCAM antibody binds to the same antigen as another anti-MAdCAM antibody. For example, one can capture an antigen using a known anti-MAdCAM antibody, elute the antigen off the anti-MAdCAM antibody, and then determine whether the test antibody will bind to the eluted antigen. One can determine whether an antibody competes with another anti-MAdCAM antibody as follows: the anti-MAdCAM antibody is allowed to bind to MAdCAM under saturating conditions, and the ability of the test antibody to bind MAdCAM is then measured. If the test antibody is capable of binding MAdCAM simultaneously with the anti-MAdCAM antibody, the test antibody may bind an epitope different from the anti-MAdCAM antibody. However, if the antibody is not able to bind MAdCAM simultaneously, the test antibody can compete with the human anti-MAdCAM antibody. This experiment can be performed using ELISA or surface plasmon resonance or preferably BIAcore. To test whether an anti-MAdCAM antibody cross competes with another anti-MAdCAM antibody, one can use the competition method described above, i.e., determine whether a known antibody blocks a test antibody, and conversely, determine whether the test antibody blocks the known antibody, in both directions.

Hydroxyl chain and heavy chain gene use

The invention also provides the use of anti-MAdCAM antibodies comprising a light chain variable region encoded by the human kappa gene. Preferably, the light chain variable region is encoded by the human vka 2, A3, a26, B3, O12 or O18 gene family. Preferably, the light chain contains no more than 11, no more than 6, or no more than 3 amino acid substitutions relative to a sequence in which the species is human vka 2, A3, a26, B3, O12, or O18. Preferably, the amino acid substitution is a conservative substitution.

Preferably, the VL of the anti-MAdCAM antibody contains a mutation compared to the germline (germline) amino acid sequence identical to any one or more of the VLs of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod described in WO 2005/067620. The invention includes the use of anti-MAdCAM antibodies employing the same human vk and human jk genes as the exemplified antibodies. The antibody may contain one or more of the same mutations as compared to the germline as one or more of the exemplified antibodies, or the antibody may comprise different substitutions at one or more of the same positions as one or more of the listed antibodies. For example, the VL of the anti-MAdCAM antibody may comprise one or more of the same amino acid substitutions as those present in antibody 7.16.6 and another amino acid substitution that is the same as antibody 7.26.4. In this way, one can mix and match different characteristics of antibody binding to alter, for example, the affinity of the antibody for MAdCAM or its rate of dissociation from the antigen. Mutations can be made at the same positions as present in any one or more of the VLs of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, or 7.26.4-mod, but conservative amino acid substitutions are not made with the same amino acid. For example, one may conservatively substitute aspartic acid if one of the amino acid substitutions in one of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, or 7.26.4-mod is glutamic acid as compared to the germline. Similarly, if the amino acid substitution is serine, one may conservatively substitute threonine.

The light chain of an anti-MAdCAM antibody may contain an amino acid sequence identical to the VL amino acid sequence of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. The light chain preferably comprises the same amino acid sequence as the light chain CDR region of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. The light chain can contain an amino acid sequence having at least one light chain CDR region of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, or 7.26.4-mod. The light chain may contain amino acid sequences having CDRs from different light chains using the same vk and jk genes. Preferably, the CDRs from the different light chains are derived from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Preferably, the light chain comprises SEQ ID NO: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 64, 66 or 68 (with or without a signal sequence). Preferably, the light chain comprises a heavy chain consisting of SEQ ID NO: 3. 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67 (with or without a signal sequence) or a nucleotide sequence encoding an amino acid sequence having an insertion, deletion or substitution of 1 to 11 amino acids therein. Preferably, the amino acid substitution is a conservative amino acid substitution. The antibody or portion thereof may comprise a lambda light chain.

The invention also provides the use of an anti-MAdCAM antibody or a portion thereof comprising a human VH gene sequence or a sequence derived from a human VH gene. The heavy chain amino acid sequence may be derived from the human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33 or 4-4 gene family. Preferably, the heavy chain contains no more than 15, no more than 6 or no more than 3 amino acid changes relative to the sequence of a gene whose species is human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33 or 4-4.

SEQ ID NO disclosed in WO 2005/067620: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42 and 46 provide the amino acid sequences of the full-length heavy chains of the 12 anti-MAdCAM antibodies used in the present invention. All SEQ ID Nos referred to in the present description refer to the sequences actually disclosed in WO 2005/067620.

Preferably, the VH of the anti-MAdCAM antibody contains the same mutation compared to the germline amino acid sequence as any one or more of the VH of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Similar to the above, the antibody and one or more of the exemplified antibodies contain one or more of the same mutations compared to the germline. The antibodies may also contain different substitutions at the same position or positions as one or more of the exemplified antibodies. For example, the VH of the anti-MAdCAM antibody may contain one or more amino acid substitutions identical to the amino acid present in antibody 7.16.6, and additional amino acid substitutions identical to antibody 7.26.4. In this manner, one can mix and match different characteristics of antibody binding to alter, for example, the affinity of the antibody for MAdCAM or its dissociation rate from the antigen. Amino acid substitutions compared to germline can be made at the same position as substitutions compared to germline present in any one or more of the VH's of the reference antibody 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod, but that position can be substituted with a different residue, which is a conservative substitution compared to the reference antibody.

Preferably, the heavy chain of an anti-MAdCAM antibody for use in the invention comprises an amino acid sequence identical to the amino acid sequence of the VH of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. More preferably, the heavy chain comprises the same amino acid sequence as the heavy chain CDR region of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Preferably, the heavy chain comprises an amino acid sequence from at least one CDR region of a heavy chain from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.4, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod, or the heavy chain may comprise an amino acid sequence with CDRs from a different heavy chain. Preferably, the CDRs from the different heavy chains are derived from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Preferably, the heavy chain comprises SEQ ID NO: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, or 64 (with or without a signal sequence). The heavy chain may also comprise a heavy chain consisting of SEQ ID NO: 1. 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, or 63, or a nucleotide sequence encoding an amino acid sequence having an insertion, deletion, or substitution of 1 to 15 amino acids therein. The substitution is preferably a conservative amino acid substitution.

Nucleic acid, vector, host cell and recombinant method for preparing antibody

Nucleic acids, vectors, host cells and recombinant methods for making such antibodies are described in WO 2005/067620.

Derivatized and labeled antibodies

An antibody or portion of an antibody of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein). In general, the antibody or portions thereof can be derivatized such that MAdCAM binding is not adversely affected by the derivatization or labeling. Thus, antibodies and antibody portions useful in the invention are intended to include both intact and modified forms of the human anti-MAdCAM antibodies described herein. For example, an antibody or antibody moiety for use in the present invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent bond association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide (e.g., a streptavidin core region or a polyhistidine tag) that can mediate the association of the antibody or antibody moiety with another molecule.

A derivatized antibody may be prepared by cross-linking two or more antibodies (of the same or different type, e.g., to produce a bispecific antibody). Suitable crosslinking agents include these heterobifunctional reagents (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional reagents (e.g., disuccinimidyl suberate) having two different reactive groups separated by a suitable spacer. These cross-linking agents are commercially available from Pierce Chemical company, Rockford, III.

Another type of derivatized antibody is a labeled antibody. Useful detection agents for derivatizing the antibodies or antibody moieties of the invention include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. The antibody may also be labeled with an enzyme useful for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibody is labeled with a detectable enzyme, it is detected by the addition of additional reagents that are available to the enzyme to produce a detectable reaction product. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine can produce a colored reaction product that can be detected. Detection can also be achieved by labeling the antibody with biotin and by indirectly measuring avidin or streptavidin binding. The antibody may be labeled with a magnetic agent such as gadolinium. Antibodies can also be labeled with a predetermined polypeptide epitope that is recognized by a secondary reporter (e.g., leucine zipper pair sequence, secondary antibody binding site, metal binding domain, epitope tag). In certain embodiments, the labels are linked by spacer arms of various lengths to reduce potential steric hindrance.

anti-MAdCAM antibodies can also be labeled with radiolabeled amino acids. Radiolabels are useful for both diagnostic and therapeutic purposes. For example, radiolabels may be used to detect tissues expressing MAdCAM by x-ray or other diagnostic techniques. In addition, radiolabelling may be usedIs used as toxin for treating affected tissue or tumor expressing MAdCAM. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionuclide-³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹TC、¹¹¹In、¹²⁵I、¹³¹I。

anti-MAdCAM antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological properties of the antibody, for example, to increase serum half-life or to increase tissue binding. This method can also be applied to any antigen-binding fragment or antibody version of anti-MAdCAM antibody.

Pharmaceutical composition and kit

In another aspect, the invention provides compositions comprising inhibitory human anti-MAdCAM antibodies and methods of treating a subject with such compositions. In some embodiments, the subject treated is a human. In other embodiments, the subject is a domestic animal subject. In some embodiments, the livestock subject is a dog or a non-human primate.

Treatment may involve administration of one or more inhibitory anti-MAdCAM monoclonal antibodies or antigen-binding fragments thereof, alone or in combination with a pharmaceutically acceptable carrier. The inhibitory anti-MAdCAM antibodies and compositions comprising the same may be administered in combination with one or more other therapeutic, diagnostic or prophylactic agents. Additional therapeutic agents include anti-inflammatory or immunomodulatory agents. These agents include, but are not limited to, topical and oral corticosteroids, such as prednisolone (prednisolone), methylprednisolone, NCX-1015, or budesonide; aminosalicylates such as mesalazine (mesalazine), olsalazine (olsalazine), balsalazide (balsalazide) or NCX-456; immunomodulators such as azathioprine (azathioprine), 6-mercaptopurine (6-mercaptoprine), methotrexate (methotrexate), cyclosporine (cyclosporine), FK506, IL-10 (Ilodexin), IL-11 (Oprelvkin), IL-12, MIF/CD 4 antagonists, CD40 antagonists such as TNX-100/5-D12, OX40L antagonists, GM-CSF, pimecrolimus (pimecrolimus), or rapamycin (rapamycin); anti-TNF α agents such as infliximab (infliximab), adalimumab (adalimumab), CDP-870, onacept (onercept), etanercept (etanerrcept); anti-inflammatory agents such as PDE-4 inhibitors (roflumilast, etc.), TACE inhibitors (DPC-333, RDP-58, etc.), and ICE inhibitors (VX-740, etc.), and IL-2 receptor antagonists such as daclizumab; selecting a class of adhesion molecule antagonists, such as natalizumab (natalizumab), MLN-02, or Alicaifu (aliforsen); analgesics, including, but not limited to, COX-2 inhibitors, such as rofecoxib (rofecoxib), valdecoxib (valdecoxib), celecoxib (celecoxib), P/Q-type voltage sensitive channel (α 2 δ) modulators, such as gabapentin (gabapentin) and pregabalin (pregabalin), NK-1 receptor antagonists, cannabinoid receptor modulators, and δ opioid receptor agonists; and antineoplastic, antiangiogenic or chemotherapeutic agents. Such additional agents may be included in the same composition or administered separately.

As used herein, "pharmaceutically acceptable carrier" means any and all physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption enhancing or absorption delaying agents, and the like. Examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, acetate buffer containing sodium chloride, dextrose, glycerol, polyethylene glycol, ethanol, and the like and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride in the composition. Other examples of pharmaceutically acceptable substances are surfactants, wetting agents, or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which can enhance antibody shelf life or potency.

The compositions for use in the present invention may take a variety of forms, such as, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, lyophilized cakes, dry powders, liposomes, and suppositories. The preferred form depends on the desired mode of administration and therapeutic application. Typically preferred compositions are in the form of injectable or infusible solutions, e.g., similar to compositions used for passive immunization of humans. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intradermal). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular, intradermal, or subcutaneous injection. If desired, the antibody may be administered by means of a pump, enema, suppository or indwelling reservoir or the like.

In general, therapeutic compositions must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, lyophilized cake, dry powder, microemulsion, dispersion, liposome, or other desired structure suitable for high drug concentrations. Sterile injectable solutions can be prepared by: the desired amount of anti-MAdCAM antibody is incorporated into a suitable solvent containing one or a combination of ingredients as desired, followed by aseptic processing. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, whereby powders of the active ingredient plus any additional ingredient desired are prepared from previously sterile-treated solutions thereof. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. For example, the desired properties of the solution can be maintained by the surfactant and the desired particle size (in the case of a dispersion, by the surfactant, phospholipid and polymer). Prolonged absorption of the injectable compositions can be brought about by incorporating into the compositions agents which delay absorption, for example, monostearate salts, polymeric materials, oils and gelatin.

The antibodies of the invention can be administered by a variety of methods known in the art, but for many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, intradermal, or intravenous infusion. Those skilled in the art will appreciate that the route and/or manner of administration will vary depending on the desired result.

In certain embodiments, the antibody can be prepared with carriers that prevent rapid release of the antibody, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Various methods for preparing such formulations have been patented or are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems (J.R. Robinson, eds., Marcel Dekker, N.Y. (1978)).

In certain embodiments, the anti-MAdCAM antibodies of the invention can be administered orally, for example, via an inert diluent or an absorbable edible carrier. The compounds (and other ingredients, if desired) can also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or added directly to the subject's diet. For oral therapeutic administration, the anti-MAdCAM antibody may be incorporated with excipients and used in the form of ingestible lozenges, buccal tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer the compounds of the present invention by a route other than parenteral, it may be necessary to coat the compound with a material or co-administer it with the compound to prevent its inactivation.

The compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding portion of the invention. A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic effect at the required dosage over the required period of time. The therapeutically effective amount of an antibody or antibody portion will vary depending on the following factors: such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which the therapeutically beneficial effect of the antibody or antibody portion is greater than any toxic or detrimental effect thereof. A "prophylactically effective amount" is an amount effective to achieve the desired prophylactic effect at the desired dosage over the desired period of time. Typically, a prophylactically effective amount can be less than a therapeutically effective amount, as a prophylactic dose is used in a subject prior to or early in the disease.

Dosage regimens can be adjusted to provide the best desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over a period of time, or the doses may be proportionally reduced or increased as dictated by the exigencies of the therapeutic condition. Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and achieving dosage consistency. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the mammalian subjects to be treated; each unit comprising a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit form of the invention depend on and directly depend on the following factors: (a) the unique properties of anti-MAdCAM antibodies or portions thereof and the particular therapeutic or prophylactic effect that is desired, and (b) the inherent limitations in formulating such antibodies to treat individual sensitivity.

Exemplary, non-limiting ranges for a therapeutically or prophylactically effective amount of an antibody or portion of an antibody of the invention are from 0.025 to 50 mg/kg, more preferably from 0.1 to 25, from 0.1 to 10, or from 0.1 to 3 mg/kg. In some embodiments, the modulator contains 5 mg/ml of antibody in a buffer containing 20mM sodium acetate, pH 5.5, 140mM NaCl, and 0.2 mg/ml polysorbate 80. In other embodiments, for intravenous use, for example, the formulation contains 10 mg/ml of the antibody in 2.73 mg/ml sodium acetate trihydrate, 45 mg/ml mannitol, 0.02 mg/ml disodium EDTA dihydrate, 0.2 mg/ml polysorbate 80 (adjusted to pH 5.5 with glacial acetic acid). In other embodiments, for subcutaneous or intradermal use, for example, the formulation contains 50 mg/ml antibody, 2.73 mg/ml sodium acetate trihydrate, 45 mg/ml mannitol, 0.02 mg/ml disodium EDTA dihydrate, 0.4 mg/ml polysorbate 80 (adjusted to pH 5.5 with glacial acetic acid). It is noted that dosage values will vary with the type and severity of the condition to be alleviated. It should be further understood that: the particular dosage regimen for any particular subject should be adjusted at any time according to the individual need and the professional judgment of the individual administering the composition or supervising the administration of the composition, and the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or application of the claimed composition.

In one embodiment, the antibody is administered as a formulation in the form of a sterile aqueous solution having a pH between about 5.0 to about 6.5 and containing about 1 mg/ml to about 200 mg/ml of antibody, about 1 mmol to about 100 mmol histidine buffer, about 0.01 mg/ml to about 10 mg/ml polysorbate 80, about 100 mmol to about 400 mmol trehalose, and about 0.01 mmol to about 1.0 mmol disodium EDTA dihydrate.

Another aspect of the invention provides a kit comprising an anti-MAdCAM antibody or antibody portion of the invention or a composition comprising the antibody. The kit may comprise a diagnostic or therapeutic agent in addition to the antibody or composition. The kit may also contain instructions for use in a diagnostic or therapeutic method. In a preferred embodiment, the kit comprises an antibody or a composition comprising the antibody and a diagnostic agent that can be used in the methods described below. In another preferred embodiment, the kit comprises the antibody or a composition comprising the antibody and one or more therapeutic agents that can be used in the methods described below.

Gene therapy

The antibodies for use in the present invention may be administered to a patient in need thereof via gene therapy. The therapy may be performed in vivo or ex vivo. In a preferred embodiment, nucleic acid molecules encoding both the heavy and light chains are administered to a patient. In a more preferred embodiment, the nucleic acid molecule is administered so that it stably integrates into the chromosome of the B cell, since the cell can specifically produce antibodies. In a preferred embodiment, precursor B cells are transfected or infected ex vivo and reimplanted in a patient in need thereof. In another embodiment, precursor B cells or other cells are infected in vivo using recombinant viruses known to infect cell types of interest. Typical vectors for gene therapy include liposomes, plasmids and viral vectors. Exemplary viral vectors are retroviruses, adenoviruses and adeno-associated viruses. Following infection in vivo or ex vivo, antibody expression levels can be monitored by sampling from treated patients and using immunoassays as known in the art or described herein.

In a preferred embodiment, gene therapy comprises the steps of administering an isolated nucleic acid molecule encoding an anti-MAdCAM antibody heavy chain or antigen-binding portion thereof and expressing the nucleic acid molecule. In another embodiment, the gene therapy comprises the steps of administering an isolated nucleic acid molecule encoding an anti-MAdCAM antibody light chain or an antigen-binding portion thereof and expressing the nucleic acid molecule. In a more preferred method, the gene therapy comprises the steps of administering and expressing an isolated nucleic acid molecule encoding an anti-MAdCAM antibody heavy chain or antigen-binding portion thereof of the invention and an isolated nucleic acid molecule encoding an anti-MAdCAM antibody light chain or antigen-binding portion thereof of the invention. The gene therapy may also include the step of administering another anti-inflammatory or immunomodulatory agent.

anti-MAdCAM antibody vs₄β₇Inhibition of MAdCAM-dependent adhesion:

the invention also provides a method for binding MAdCAM and inhibiting the activity of alpha₄β₇Use of anti-MAdCAM antibodies for the binding and adhesion of cells of integrins to MAdCAM or to MAdCAM by other cognate ligands (such as L-selectin). In a preferred embodiment, the MAdCAM is human MAdCAM and is in soluble form or expressed on the cell surface. In another preferred embodiment, the anti-MAdCAM antibody is a human antibody. In another embodiment, the antibody or portion thereof inhibits α₄β₇And MAdCAM, wherein IC₅₀Values of no more than 50 nM. In a preferred embodiment, the IC₅₀The value did not exceed 5 nM. In a more preferred embodiment, the IC₅₀Values were less than 5 nM. In a more preferred embodiment, the IC₅₀A value of less than 0.05. mu.g/ml,0.04 micrograms/ml or 0.03 micrograms/ml. In another preferred embodiment, the IC₅₀Values less than 0.5, 0.4, or 0.3. mu.g/ml. The IC₅₀The values may be measured by any method known in the art. In general, ICs₅₀Values can be measured by ELISA or adhesion analysis. In a preferred embodiment, the IC₅₀Values are measured by adhesion assays using cells or tissues that naturally express MAdCAM or cells or tissues that have been constructed to express MAdCAM.

Unless defined otherwise herein, scientific and technical terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. Also, unless the context requires otherwise, a single term shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques used in the present specification for cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention can be generally practiced according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al, molecular cloning: a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al, Current Protocols in Molecular Biology, Greene publishing associates (1992), and Harlow and Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, or as is conventional in the art, or as described herein. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients.

The term "polypeptide" encompasses natural or synthetic proteins, protein fragments, and polypeptide analogs of a protein sequence. The polypeptide may be monomeric or polymeric.

The term "isolated protein" or "isolated polypeptide" is based on its origin or derived from a protein or polypeptide: (1) not associated with the native binding component that it originally accompanies in its native state, (2) does not contain other proteins from the same species; (3) may be expressed by cells from different species; or (4) is not present in the native state. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which the polypeptide naturally originates is "isolated" from its natural binding component. Proteins can also be made substantially free of naturally associated components by separation using protein purification techniques well known in the art.

A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. Substantially pure polypeptide or protein will typically constitute about 50%, 60%, 70%, 80% or 90% W/W of the protein sample, more typically about 95%, and preferably will be more than 99% pure. Protein purity or homogeneity can be demonstrated by a variety of means well known in the art, such as performing polyacrylamide gel electrophoresis of a protein sample, followed by visualization of individual polypeptide bands upon staining the gel with a stain well known in the art. For some purposes, purification can be performed by HPLC or other methods well known in the art to provide higher resolution.

The term "polypeptide fragment" as used in this specification refers to a polypeptide having an amino-terminal deletion and/or a carboxy-terminal deletion, but wherein the remaining amino acid sequence is identical to the corresponding position in the naturally occurring sequence. In some embodiments, a fragment is at least 5, 6, 8, or 10 amino acids long. In other embodiments, the fragment is at least 14 amino acids long, more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70, 80, 90, 100, 150 or 200 amino acids long.

The term "polypeptide analog" as used herein refers to a polypeptide having an amino acid sequence with a portion of the amino acid sequenceA polypeptide comprising a fragment of at least 25 amino acids that is substantially identical and has at least one of the following properties: (1) can specifically bind to MAdCAM under conditions suitable for binding; (2) inhibition of alpha₄β₇The ability of integrins and/or L-selectin to bind MAdCAM; or (3) the ability to decrease cell surface expression of MAdCAM in vitro or in vivo. Typically, the polypeptide analogs contain a conservative amino acid substitution (or insertion or deletion) relative to the naturally-occurring sequence. Analogs are generally at least 20 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids long or longer, and are often as long as a full-length naturally occurring polypeptide.

An "immunoglobulin" is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50 to 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgA and IgE, respectively. In both light and heavy chains, the variable and constant regions are connected by a "J" region comprising about 12 or more amino acids, wherein the heavy chain also includes a "D" region comprising about 10 or more amino acids. See generally, fundamentals immunology ch.7(Paul, w. editor, second edition, Raven Press, n.y. (1989)) which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair form antibody binding sites so that the immunoglobulin has two binding sites.

Immunoglobulin chains display the same general structure with relatively conserved Framework Regions (FRs) connected by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from both chains of each pair are aligned by the framework regions to form an epitope-specific binding site. From N-terminus to C-terminus, both light and heavy chains comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The assignment of amino acids in each domain corresponds to Sequences of proteins of Immunological Interest (National Institutes of health, Bethesda, Md. (1987 and 1991)) by Kabat or Chothia & leave j.mol.biol.196: 901-917 (1987); chothia et al, Nature, 342: 878-883 (1989).

"antibody" refers to an intact immunoglobulin or to an antigen-binding portion thereof that can compete with intact antibody for specific binding. In some embodiments, the antibody is an antigen-binding portion thereof. Antigen-binding fragments are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include, inter alia, Fab ', F (ab')₂Fv, dAb and Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding capability on the polypeptide. A monovalent fragment of the Fab fragment consisting of the VL, VH, CL and CH1 domains; f (ab)₂The fragment contains a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; the Fd fragment consists of the VH and CH1 domains; the Fv fragment consists of the VL and VH domains of a single arm of an antibody; while dAb fragments (Ward et al, Nature, 341: 544-546(1989)) are composed of VH domains.

The antibody referred to as (for example) 1.7.2, 1.8.2, 6.14.2, 6.34.2, 6.67.1, 6.77.2, 7.16.6, 7.20.5, 7.26.4 or 9.8.2 used in the present specification is a monoclonal antibody produced by a hybridoma having the same name. For example, antibody 1.7.2 is produced by hybridoma 1.7.2. The antibodies designated 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod are monoclonal antibodies whose sequences have been modified from their respective parents by site-directed mutagenesis.

Single chain antibodies (scFv) are antibodies whose VL and VH regions are paired via a synthetic linker that enables them to be a single protein chain to form monovalent molecules (Bird et al, Science, 242: 423-. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing such domains to pair with the complementary domains of the other chain and form two antigen binding sites (see, e.g., Holliger, P. et al, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak, R.J. et al, Structure, 2: 1121-1123 (1994)). One or more CDRs from an antibody of the invention can be introduced covalently or non-covalently into a molecule to make it an immunoadhesin that specifically binds MAdCAM. Immunoadhesins can incorporate CDRs as part of a larger polypeptide chain; the CDRs can be covalently linked to become another polypeptide chain; or the CDRs may be introduced in a non-covalent manner. The CDRs can allow the immunoadhesin to specifically bind to a particular antigen of interest.

The antibody may have one or more binding sites. If more than one binding site is present, the binding sites may be the same or different from each other. For example, a naturally occurring immunoglobulin has two identical binding sites, a single chain antibody or Fab fragment has one binding site, while a "bispecific" or "bifunctional" antibody (diabody) has two different binding sites.

An "isolated antibody" is an antibody as follows: (1) does not bind to naturally-occurring binding components (including other naturally-bound antibodies) that would otherwise accompany it in its natural state, (2) does not contain other proteins from the same species; (3) may be expressed by cells from different species; or (4) is not present in the native state. Examples of isolated antibodies include anti-MAdCAM antibodies that have been affinity purified using MAdCAM, anti-MAdCAM antibodies that have been produced in vitro by hybridomas or other cell lines, and human anti-MAdCAM antibodies derived from transgenic mammals or plants.

The term "human antibody" as used herein means an antibody in which the variable and constant region sequence antibodies are human sequences. The term encompasses antibodies having sequences derived from human genes that have been altered, for example, to reduce potential immunogenicity, increase affinity, eliminate cysteine or glycosylation sites that may cause undesirable folding, and the like. The term encompasses such antibodies recombinantly produced in non-human cells (which may confer glycosylation not characteristic of human cells). The term also encompasses antibodies that have been raised in transgenic mice containing some or all of the human immunoglobulin heavy and light chain loci.

In one aspect, the invention provides humanized antibodies. In some embodiments, the humanized antibody is derived from non-human species of antibodies, the heavy and light chain of the antibody framework and constant domain of certain amino acids have been subjected to mutations in order to avoid or eliminate immune responses in humans. In some embodiments, humanized antibodies can be produced by fusing constant domains from human antibodies to variable domains from non-human species. Examples of how to form humanized antibodies can be found in U.S. Pat. nos. 6,054,297, 5,886,152 and 5,877,293. In some embodiments, the humanized anti-MAdCAM antibodies of the invention comprise the amino acid sequence of one or more framework regions of one or more human anti-MAdCAM antibodies of the invention.

In another aspect, the invention includes the use of a "chimeric antibody". In some embodiments, the chimeric antibody refers to an antibody that contains one or more regions from one antibody and contains one or more regions from one or more other antibodies. In a preferred embodiment, one or more CDRs are derived from a human anti-MAdCAM antibody of the invention. In a more preferred embodiment, all CDRs are derived from a human anti-MAdCAM antibody of the invention. In another preferred embodiment, the CDRs from more than one human anti-MAdCAM antibody of the invention are mixed and paired in a chimeric antibody. For example, a chimeric antibody may contain a CDR1 from a first human anti-MAdCAM antibody light chain, this CDR1 may be combined with a CDR2 and a CDR3 from a second human anti-MAdCAM antibody light chain, and the CDR of the heavy chain may be derived from a third anti-MAdCAM antibody. Furthermore, the framework regions may be derived from the framework regions of the same anti-MAdCAM antibody, one or more different antibodies (such as human antibodies), or humanized antibodies.

A "neutralizing antibody," "inhibitory antibody," or antagonist antibody is suppressibleAt least about 20% of alpha₄β₇Or alpha₄β₇An antibody that binds MAdCAM by the expressing cell or any other cognate ligand or cognate ligand expressing cell. In a preferred embodiment, the antibody is directed to alpha₄β₇Integrins or alpha₄β₇The inhibition of binding of the expressing cells to MAdCAM is at least 40%, more preferably 60%, even more preferably 80%, 85%, 90%, 95% or 100%. The decrease in binding can be measured by any method known to those skilled in the art, for example, measuring α as measured by an in vitro competitive binding assay₄β₇Examples of reduced outcome of MAdCAM by expressing cells are shown in example 1.

Antibody fragments or analogs will be readily prepared by those skilled in the art following the teachings of this specification. The preferred amino-and carboxy-termini of the fragments or analogs occur near the border region of the functional domain. The nucleotide and/or amino acid sequence data can be compared to published or proprietary all sequence databases to thereby determine structural and functional domains. Preferably, computerized comparison methods are used to determine sequence motifs or expected protein conformation domains that occur in other proteins of known structure and/or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known (Bowie et al, Science, 253: 164 (1991)).

The term "k_off"refers to the dissociation rate constant used to characterize the dissociation of an antibody from an antibody/antigen complex.

The term "K_d"refers to the dissociation constant of a particular antibody-antigen interaction. Antibodies are said to bind to antigens with dissociation constants ≦ 1 μ M, preferably ≦ 100nM, and most preferably ≦ 10 nM.

The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopic determinants are typically composed of chemically active surface groups of the molecule (e.g., amino acids or carbohydrate side chains) and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. The An epitope can be in "linear" or "conformational" form. In a linear epitope, all points of interaction between a protein and an interacting molecule (such as an antibody) occur in a linear fashion along the protein's major amino acid sequence. In conformational epitopes, interaction point crossings occur at amino acid residues that are separated from each other on the protein.

As used herein, the 20 well-known amino acids and their abbreviations follow well-known usage. See Immunology-A Synthesis (2 nd edition, edited by E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated by reference into this patent specification. Stereoisomers of 20 well-known amino acids (e.g., D-amino acids), unnatural amino acids (e.g., α -disubstituted amino acids, N-alkyl amino acids), lactic acid, and other unknown amino acids are also suitable for use in the components of the polypeptides of the invention. Examples of non-known amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formyl methionine, 3-methylhistidine, 5-hydroxylysine, s-N-methyl arginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used in the present specification, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

The term "polynucleotide" as referred to in this specification means a polymeric form of nucleotides (either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide) having a length of at least 10 bases. The term includes single-stranded as well as double-stranded forms of DNA.

The term "isolated polynucleotide" as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, depending on its source, which "isolated polynucleotide": (1) not associated with all or a portion of the polynucleotide in which the "isolated polynucleotide" is found in nature, (2) operably linked to a polynucleotide to which it is not linked in nature, or (3) not present in nature as part of a larger sequence.

The term "oligonucleotide" as referred to herein includes naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides, which typically have a length of 200 bases or less. The oligonucleotide is preferably 10 to 60 bases long and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases long. Oligonucleotides are typically single stranded, e.g., for probes; however, the oligonucleotides may also be double-stranded, for example in the case of construction of gene mutants. The oligonucleotide of the present invention may be a sense strand oligonucleotide or an antisense strand oligonucleotide.

The term "naturally occurring nucleotide" as referred to in the present specification includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" as referred to in the present specification includes nucleotides with modified or substituted sugar groups and the like. Reference herein to the term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroamidite, phosphoroamidate, and the like. See, e.g., LaPlanche et al, nucleic acids sres.14: 9081 (1986); stec et al, j.am.chem.soc.106: 6077 (1984); stein et al, nucleic acids res.16: 3209 (1988); zon et al, Anti-cancer Design 6: 539 (1991); zon et al, Oligonucleotides and nanoparticles: a Practical Approach, pages 87-108 (F. Eckstein eds., Oxford University Press, Oxford England (1991)); stec et al, U.S. patent No. 5,151,510; uhlmann and Peyman, Chemical Reviews, 90: 543(1990), the disclosures of which are incorporated herein by reference. The oligonucleotide may optionally include a label for detection.

An "operably linked" sequence includes both expression control sequences adjacent to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. The term "expression control sequence" as used herein refers to a polynucleotide sequence that is essential for effecting the expression and processing of the coding sequence to which it is ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance transcription efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of the control sequences will vary depending on the host organism; in prokaryotes, the control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, the control sequences generally include promoters and transcription termination sequences. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences.

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which the vector is incorporated. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon incorporation into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors for recombinant DNA techniques are usually in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It is understood that the term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Since certain changes may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "selectively hybridize" as referred to herein means to detectably and specifically bind. The polynucleotides, oligonucleotides, and fragments thereof of the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize the amount of detectable binding to non-specific nucleic acids that can be assessed. As is known in the art and discussed in this specification, selective hybridization conditions can be achieved using "high stringency" or "high stringency" conditions. An example of "high stringency" or "high stringency" conditions is a method of incubating a polynucleotide with another polynucleotide, wherein a polynucleotide may be immobilized on a solid (such as a membrane) surface, incubated in hybridization buffer containing 6X SSPE or SSC, 50% formamide, 5X Denhardt's reagent, 0.5% SDS, 100 micrograms/ml denatured fragmented salmon sperm DNA at a hybridization temperature of 42 ℃ for 12 to 16 hours, and then washed twice with wash buffer containing 1X SSC, 0.5% SDS at 55 ℃. See Sambrook et al, supra, pages 9.50-9.55.

The term "percent sequence identity" with respect to a range of nucleotide sequences refers to the residues in the two sequences that are the same when aligned for maximum identity. The length of the sequence identity comparison may exceed that of a nucleic acid sequence containing at least about 9 nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, FASTA, GaD or Bestfit (a program in Wisconsin Package Version 10.3, Accelrys, San Diego, Calif.) can be used to compare polynucleotide sequences. FASTA, including, for example, programs FASTA2 and FASTA3, provides alignment and percent sequence identity of the best overlapping regions between query and search sequences (Pearson, Methods enzymol.183: 63-98 (1990); Pearson, Methods Mol, biol.132: 185-219 (2000); Pearson, Methods enzymol.266: 227-258 (1996); Pearson, J.mol.biol.276: 71-84 (1998); incorporated by reference in this patent specification). Unless otherwise specified, default parameters for a particular program or algorithm may be used. For example, percent sequence identity between nucleotide sequences can be determined using FASTA with its default parameters (using a word width of 6 and NOPAM factors for the scoring matrix) or using the default parameters provided by Gap with its Wisconsin Package Version 10.3 (incorporated by reference in this patent specification).

Unless otherwise indicated, reference to a nucleotide sequence also includes the complementary portion thereof. It will therefore be appreciated that reference to a nucleic acid molecule having a particular sequence also includes the complementary strand thereof having the sequence complementary thereto.

Within the molecular biology industry, researchers may use the terms "percent sequence identity", "percent sequence similarity", and "percent sequence homology" interchangeably. In the present application, these terms have the same meaning only for nucleotide sequences.

The term "substantial similarity" or "substantial sequence similarity", when referring to a nucleic acid or fragment thereof, means that there is nucleotide sequence identity in at least about 85%, preferably at least about 90% and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, when optimally aligned with an appropriate nucleotide insertion or deletion to another nucleic acid (or its complementary strand), as determined by any of the well-known sequence identity algorithms, such as FASTA, BLAST or Gap, as described above.

The term "substantial identity" when applied to polypeptides means that two peptide sequences have at least 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 98% or 99% sequence identity when they are most aligned, for example by the programs GAP or BESTFIT using default GAP weighting. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, e.g., Pearson, Methods mol. biol. 24: 307-31(1994), which is incorporated by reference into this patent specification. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chain: serine and threonine; 3) amide-containing side chain: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; and 6) sulfur containing side chains: cysteine and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine.

Alternatively, conservative substitutions are described in Gonnet et al, Science, 256: 1443-45(1992) (incorporated by reference in this patent specification) has any change in the PAM250 log-similarity matrix with a positive value. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-similarity matrix.

Sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG contains programs such as "Gap" and "Bestfit" which can be used to determine by default the sequence homology or sequence identity between closely related polypeptides (such as polypeptides from organisms of different species) or between a wild-type protein and a mutein thereof. See, for example, Wisconsin package Version 10.3. FASTA (a program in Wisconsin package Version 10.3) may also be used to compare polypeptide sequences with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) can provide alignment and percent sequence identity of the best overlapping regions between query and search sequences (Pearson (1990); Pearson (2000)). Another preferred algorithm when comparing the sequences of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, in particular blastp or tblastn (using default parameters). See, e.g., Altschul et al, j.mol.biol.215: 403-; altschul et al, Nucleic Acids Res.25: 3389-402 (1997); this document is incorporated by reference into this patent specification.

The length of polypeptide sequences over which homology comparisons are made will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues and preferably more than about 35 residues. When searching databases containing sequences from a large number of different organisms, it is preferred to compare amino acid sequences.

The term "label" or "labeled" as used herein refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable label, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide having a biotinyl moiety detectable by labeled avidin (e.g., streptavidin comprising a fluorescent label or having enzymatic activity detectable by optical or colorimetric methods). In another embodiment, the label or marker may be a therapeutic agent, for example, a drug conjugate or toxin. Multiple markersMethods for polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: a radioisotope or radionuclide (e.g.,³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹ln、¹²⁵l、¹³¹l), fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., peroxidase, β -galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotin groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin (pertussis toxin), taxol, cytochalasin b, gramicidin d, (micGrancidin d), ethidium bromide (ethidium bromide), emetine (emetine), mitomycin (mitomycin), epidophyllotoxin (etoposide), podophyllotoxin (tenioside), vincristine (vinblastine), colchicine (colchicine), doxorubicin (bidoxorubicin), bidolicin (bidoxorubicin), and the like, Daunorubicin (daunorubicin), dihydroxyanthraquinone (dihydroxyanthridin dione), mitoxantrone (mitoxantrone), mithramycin (mithramycin), actinomycin D (actinomycin D), 1-dehydrotestosterone (1-dehydrotestosterone), glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol (propranolol), and puromycin (puromycin) and analogs or homologs thereof. In certain embodiments, labels are added through spacer arms of various lengths to reduce potential steric hindrance.

Claims (11)

1. Use of an anti-MAdCAM antibody, or an antigen-binding portion thereof, for the manufacture of a medicament for the treatment of coeliac disease and/or tropical sprue.

2. The use according to claim 1, wherein the medicament is for the treatment of coeliac disease.

3. The use of claim 1 or claim 2, wherein the anti-MAdCAM antibody is a human monoclonal antibody.

4. The use of claim 3, wherein the antibody or portion has at least one of the following properties:

(a) binding to human cells;

(b) at least 100-fold more selective for MAdCAM than VCAM or fibronectin;

(c) at 3X 10^-10K of M or less_dBinds to human MAdCAM; or

(d) Inhibition of alpha₄β₇Binding of the expressing cells to human MAdCAM;

(e) inhibiting the recruitment of lymphocytes to gastrointestinal lymphoid tissue.

5. The use of any one of claims 1 to 4, wherein the antibody or antigen-binding portion inhibits the alpha-pairing of human MAdCAM₄β₇And wherein the antibody or portion thereof has at least one of the following properties:

(a) cross-competes for binding to MAdCAM with a reference antibody;

(b) competes for binding to MAdCAM with a reference antibody;

(c) binds to the same MAdCAM epitope as the reference antibody;

(d) with substantially the same K as the reference antibody_dBinding to MAdCAM;

(e) binds MAdCAM at approximately the same off rate as the reference antibody;

wherein the reference antibody is selected from: monoclonal antibody 1.7.2, monoclonal antibody 1.8.2, monoclonal antibody 6.14.2, monoclonal antibody 6.22.2, monoclonal antibody 6.34.2, monoclonal antibody 6.67.1, monoclonal antibody 6.73.2, monoclonal antibody 6.77.1, monoclonal antibody 7.16.6, monoclonal antibody 7.20.5, monoclonal antibody 7.26.4, monoclonal antibody 9.8.2, monoclonal antibody 6.22.2-mod, monoclonal antibody 6.34.2-mod, monoclonal antibody 6.67.1-mod, monoclonal antibody 6.77.1-mod and monoclonal antibody 7.26.4-mod.

6. The use of any one of claims 1 to 5, wherein the heavy chain variable region, the light chain variable region, or both of the anti-MAdCAM antibody has at least 90% identity in amino acid sequence to the corresponding regions of a monoclonal antibody selected from the group consisting of: monoclonal antibody 1.7.2, monoclonal antibody 1.8.2, monoclonal antibody 6.14.2, monoclonal antibody 6.22.2, monoclonal antibody 6.34.2, monoclonal antibody 6.67.1, monoclonal antibody 6.73.2, monoclonal antibody 6.77.1, monoclonal antibody 7.16.6, monoclonal antibody 7.20.5, monoclonal antibody 7.26.4, monoclonal antibody 9.8.2, monoclonal antibody 6.22.2-mod, monoclonal antibody 6.34.2-mod, monoclonal antibody 6.67.1-mod, monoclonal antibody 6.77.1-mod and monoclonal antibody 7.26.4-mod.

7. The use of any one of claims 1 to 6, wherein the antibody is selected from the group consisting of:

(a) contains a polypeptide shown in SEQ ID NO: 2 and SEQ ID NO: 4 but not the signal sequence;

(b) contains a polypeptide shown in SEQ ID NO: 6 and SEQ ID NO: 8 without a signal sequence;

(c) contains a polypeptide shown in SEQ ID NO: 10 and SEQ ID NO: 12 but not a signal sequence;

(d) contains a polypeptide shown in SEQ ID NO: 14 and SEQ ID NO: 16 but not the signal sequence;

(e) contains a polypeptide shown in SEQ ID NO: 18 and SEQ ID NO: 20 but does not contain a signal sequence;

(f) contains a polypeptide shown in SEQ ID NO: 22 and SEQ ID NO: 24 but not a signal sequence;

(g) contains a polypeptide shown in SEQ ID NO: 26 and SEQ ID NO: 28 but not a signal sequence;

(h) contains a polypeptide shown in SEQ ID NO: 30 and SEQ ID NO: 32 but not a signal sequence;

(i) contains a polypeptide shown in SEQ ID NO: 34 and SEQ ID NO: 36 but not a signal sequence;

(j) contains a polypeptide shown in SEQ ID NO: 38 and SEQ ID NO: 40 without a signal sequence;

(k) contains a polypeptide shown in SEQ ID NO: 42 and SEQ ID NO: 44 without a signal sequence;

(l) Contains a polypeptide shown in SEQ ID NO: 46 and SEQ ID NO: 48 without a signal sequence;

(m) a polypeptide comprising the sequence shown in SEQ ID NO: 52 and SEQ ID NO: 54 without a signal sequence;

(n) a polypeptide comprising the sequence shown in SEQ ID NO: 56 and SEQ ID NO: 58 but not a signal sequence;

(o) a polypeptide comprising the sequence shown in SEQ ID NO: 60 and SEQ ID NO: 62 without a signal sequence;

(p) a polypeptide comprising the sequence shown in SEQ ID NO: 64 and SEQ ID NO: 66 without a signal sequence; and

(q) a polypeptide comprising the sequence shown in SEQ ID NO: 42 and SEQ ID NO: 68 but does not contain a signal sequence.

8. The use of any one of claims 1 to 7, wherein the heavy chain C-terminal lysine is cleaved from the anti-MAdCAM antibody.

9. The use of any one of claims 1 to 8, wherein the monoclonal antibody or antigen binding portion thereof is selected from the group consisting of:

(a) the heavy chain comprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences of a reference antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod

(b) The light chain comprises the light chain CDR1, CDR2, and CDR3 amino acid sequences of a reference antibody selected from: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod

(c) The antibody comprises a heavy chain of (a) and a light chain of (b); and

(d) the antibody of (c), wherein the heavy and light chain CDR amino acid sequences are selected from the same reference antibody.

10. The use of any one of claims 1 to 9, wherein the monoclonal antibody or antigen binding portion comprises:

(a) a heavy chain comprising the heavy chain variable region amino acid sequence of an antibody selected from the group consisting of: 1.7.2(SEQ ID NO: 2); 1.8.2(SEQ ID NO: 6); 6.14.2(SEQ ID NO: 10); 6.22.2(SEQ ID NO: 14); 6.34.2(SEQ ID NO: 18); 6.67.1(SEQ ID NO: 22); 6.73.2(SEQ ID NO: 26); 6.77.1(SEQ ID NO: 30); 7.16.6(SEQ ID NO: 34); 7.20.5(SEQ ID NO: 38); 7.26.4(SEQ ID NO: 42); and 9.8.2(SEQ ID NO: 46); 6.22.2-mod (SEQ ID NO: 52); 6.34.2-mod (SEQ ID NO: 56); 6.67.1-mod (SEQ ID NO: 60); 6.77.1-mod (SEQ ID NO: 64); and 7.26.4-mod (SEQ ID NO: 42);

(b) a light chain comprising the light chain variable region amino acid sequence of an antibody selected from the group consisting of: 1.7.2(SEQ ID NO: 4); 1.8.2(SEQ ID NO: 8); 6.14.2(SEQ ID NO: 12); 6.22.2(SEQ ID NO: 16); 6.34.2(SEQ ID NO: 20); 6.67.1(SEQ ID NO: 24); 6.73.2(SEQ ID NO: 28); 6.77.1(SEQ ID NO: 32); 7.16.6(SEQ ID NO: 36); 7.20.5(SEQ ID NO: 40); 7.26.4(SEQ ID NO: 44); and 98.2(SEQ ID NO: 48); 6.22.2-mod (SEQ ID NO: 54); 6.34.2-mod (SEQ ID NO: 58); 6.67.1-mod (SEQ ID NO: 62); 6.77.1-mod (SEQ ID NO: 66); and 7.26.4-mod (SEQ ID NO: 68); or

(c) The heavy chain of (a) and the light chain of (b).

11. Anti-alpha₄β₇Use of an integrin antibody, or an antigen-binding portion thereof, for the manufacture of a medicament for the treatment of coeliac disease and/or tropical sprue.