U.S. patent application number 10/118600 was filed with the patent office on 2002-11-21 for treatment of inflammatory bowel disease by inhibiting binding and/or signalling through alpha4beta7 and its ligands and madcam.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Newman, Walter, Picarella, Dominic, Ringler, Douglas J..
Application Number | 20020172679 10/118600 |
Document ID | / |
Family ID | 23527358 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020172679 |
Kind Code |
A1 |
Ringler, Douglas J. ; et
al. |
November 21, 2002 |
Treatment of inflammatory bowel disease by inhibiting binding
and/or signalling through alpha4beta7 and its ligands and
MAdCAM
Abstract
The invention relates to the treatment of individuals suffering
from a disease associated with leukocyte recruitment to the
gastrointestinal tract or other tissues as a result of binding of
leukocytes to gut-associated endothelium expressing the molecule
MAdCAM (such as inflammatory bowel disease), comprising
administering to the individual an effective amount of an antibody
which inhibits the binding of leukocytes to endothelial MAdCAM.
Inventors: |
Ringler, Douglas J.;
(Revere, MA) ; Picarella, Dominic; (Boston,
MA) ; Newman, Walter; (Boston, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
23527358 |
Appl. No.: |
10/118600 |
Filed: |
April 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10118600 |
Apr 8, 2002 |
|
|
|
08386857 |
Feb 10, 1995 |
|
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Current U.S.
Class: |
424/146.1 |
Current CPC
Class: |
A61P 1/04 20180101; G01N
33/6893 20130101; A61P 29/00 20180101; A61P 43/00 20180101; C07K
2319/00 20130101; Y10S 530/867 20130101; G01N 2500/00 20130101;
A61K 38/00 20130101; Y10S 424/81 20130101; Y10S 424/801 20130101;
C07K 14/70503 20130101; Y10S 530/866 20130101; Y10S 530/868
20130101 |
Class at
Publication: |
424/146.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method for treating an individual having a disease associated
with leukocyte recruitment to the gastrointestinal tract or other
tissues as a result of binding of leukocytes to gut-associated
endothelium expressing the molecule MAdCAM, comprising:
administering to the individual an effective amount of an antibody
which inhibits the binding of leukocytes to endothelial MAdCAM.
2. The method of claim 1 wherein antibody is a monoclonal antibody
or an antigen binding fragment thereof.
3. The method of claim 2 wherein the monoclonal antibody or antigen
binding fragment thereof inhibits adhesion of leukocytes expressing
an integrin containing the .beta.7 chain and endothelium expressing
MAdCAM.
4. The method of claim 3 wherein the monoclonal antibody or antigen
binding fragment thereof binds .alpha.4.beta.7 integrin.
5. The method of claim 4 wherein the monoclonal antibody or antigen
binding fragment thereof binds .beta.7.
6. The method of claim 3 wherein the monoclonal antibody or antigen
binding fragment thereof binds MAdCAM.
7. The method of claim 3 wherein the monoclonal antibody or antigen
binding fragment thereof has the antigenic specificity of a
monoclonal antibody selected from the group consisting of FIB 21,
FIB 30, FIB 504 and ACT-1.
8. The method of claim 7 wherein the monoclonal antibody or antigen
binding fragment thereof is selected from the group consisting of
FIB 21, FIB 30, FIB 504 and ACT-1 or antigen binding fragments
thereof.
9. The method of claim 8 wherein the monoclonal antibody is
ACT-1.
10. The method of claim 3 wherein the monoclonal antibody is
selected from the group consisting of a chimeric antibody and a
humanized antibody.
11. The method of claim 3 wherein the leukocytes are
lymphocytes.
12. The method of claim 3 wherein the leukocytes are monocytes.
13. The method of claim 3 wherein the disease is inflammatory bowel
disease.
14. The method of claim 13 wherein the disease is ulcerative
colitis.
15. The method of claim 13 wherein the disease is Crohn's
disease.
16. The method of claim 13 wherein the disease is Celiac disease,
enteropathy associated with seronegative arthropathies, microscopic
or collagenous colitis, eosinophilic gastroenteritis, or
pouchitis.
17. The method of claim 13 wherein the monoclonal antibody or
antigen binding fragment thereof binds .alpha.4.beta.7.
18. The method of claim 13 wherein the monoclonal antibody or
antigen binding fragment thereof binds MAdCAM.
19. The method of claim 13 wherein the monoclonal antibody or
antigen binding fragment thereof has the antigenic specificity of a
monoclonal antibody selected from the group consisting of FIB 21,
FIB30, FIB 504 and ACT-1.
20. The method of claim 19 wherein the monoclonal antibody or
antigen binding fragment thereof is selected from the group
consisting of FIB 21, FIB30, FIB 504 and ACT-1 or antigen binding
fragments thereof.
21. The method of claim 20 wherein the monoclonal antibody is
ACT-1.
22. The method of claim 13 wherein the monoclonal antibody is
selected from the group consisting of a chimeric antibody and a
humanized antibody.
23. The method of claim 13 wherein more than one monoclonal
antibody which inhibits the binding of leukocytes to endothelial
MAdCAM is administered.
24. The method of claim 13 wherein more than one monoclonal
antibody which inhibits the binding of leukocytes to endothelial
ligands is administered.
25. The method of claim 24 wherein at least one monoclonal antibody
inhibits the binding of leukocytes to an endothelial ligand other
than MAdCAM.
26. A method for treating inflammatory bowel disease in an
individual comprising administering to the individual an effective
amount of an antibody which binds endothelial MAdCAM or the
.alpha.4.beta.7 integrin.
27. The method of claim 26 wherein antibody is a monoclonal
antibody or an antigen binding fragment thereof.
28. The method of claim 27 wherein the monoclonal antibody or
antigen binding fragment thereof binds .alpha.4.beta.7
integrin.
29. The method of claim 28 wherein the monoclonal antibody or
antigen binding fragment thereof binds .beta.7.
30. The method of claim 27 wherein the monoclonal antibody or
antigen binding fragment thereof binds MAdCAM.
31. The method of claim 27 wherein the monoclonal antibody or
antigen binding fragment thereof has the antigenic specificity of a
monoclonal antibody selected from the group consisting of FIB 21,
FIB 30, FIB 504 and ACT-1.
32. The method of claim 31 wherein the monoclonal antibody or
antigen binding fragment thereof is selected from the group
consisting of FIB 21, FIB 30, FIB 504 and ACT-1 or antigen binding
fragments thereof.
33. The method of claim 32 wherein the monoclonal antibody is
ACT-1.
34. The method of claim 27 wherein the monoclonal antibody is
selected from the group consisting of a chimeric antibody and a
humanized antibody.
35. The method of claim 27 wherein the disease is ulcerative
colitis.
36. The method of claim 27 wherein the disease is Crohn's
disease.
37. The method of claim 27 wherein the disease is Celiac disease,
enteropathy associated with seronegative arthropathies, microscopic
or collagenous colitis, eosinophilic gastroenteritis, or pouchitis.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 08/386,857, filed Feb. 10, 1995. The entire teachings of the
above application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Inflammatory bowel disease (IBD), such as ulcerative colitis
and Crohn's disease, for example, can be a debilitating and
progressive disease involving inflammation of the gastrointestinal
tract affecting an estimated two million people in the United
States. Symptoms include abdominal pain, cramping, diarrhea and
rectal bleeding. IBD treatments have included anti-inflammatory
drugs (such as, corticosteroids and sulfasalazine),
immunosuppressive drugs (such as, 6-mercaptopurine, cyclosporine
and azathioprine) and surgery (such as, colectomy). Podolsky, The
New England Journal of Medicine, 325:928-937 (1991) and Podolsky,
The New England Journal of Medicine, 325:1008-1016 (1991).
[0003] Some studies have suggested that the cell adhesion molecule,
ICAM-1, mediates leukocyte recruitment to inflammatory sites
through adhesion to leukocyte surface ligands, i.e. Mac-1, LFA-1 or
.alpha.4.beta.2 (Springer, Nature, 346:425-434 (1990)). In
addition, vascular cell adhesion molecule-1 (VCAM-1), recognizing
the .alpha.4.beta.1 integrin (VLA-4), has been reported to play a
role in in vivo leukocyte recruitment as well (Silber et al., J.
Clin. Invest. 93:1554-1563 (1994)). It has been proposed that IBD
can be treated by blocking the interaction of ICAM-1 with LFA-1 or
Mac-1 or VCAM-1 with .alpha.4.beta.1 (e.g., WO 93/15764). However,
these therapeutic targets are likely involved in inflammatory
processes in multiple organs, and a functional blockade would
likely result in systemic immune dysfunction.
[0004] Mucosal addressin MAdCAM, a mucosal vascular adhesion
molecule, is a 58-66K glycoprotein adhesion receptor for
lymphocytes which is distinct from VCAM-1 and ICAM-1 (Briskin et
al., Nature, 363:461-463 (1993)). In contrast to VCAM-1 and ICAM-
1, MAdCAM is preferentially expressed in the gastrointestinal
tract, binds the .alpha.4.beta.7 integrin (also called LPAM-1 and
CD49d/CD.sup.-) found on lymphocytes, and participates in the
homing of these cells to mucosal sites, such as Peyer's patches in
the intestinal wall (Hamann et al., Journal of Immunology,
152:3282-3293 (1994)). The use of inhibitors to the binding of
MAdCAM to the receptor, .alpha.4.beta.7, in the treatment of
diseases such as IBD has not been suggested.
SUMMARY OF THE INVENTION
[0005] The invention relates to the treatment of individuals
suffering from a disease associated with leukocyte recruitment to
the gastrointestinal tract as a result of binding of leukocytes to
gut-associated endothelium expressing the molecule MAdCAM,
comprising administering to the individual an effective amount of a
compound, such as an antibody, which inhibits the binding of
leukocytes to endothelial MAdCAM. The antibody is preferably
monoclonal, chimeric and/or humanized or an antigen binding
fragment thereof, and inhibits adhesion of leukocytes expressing an
integrin containing the .beta.7 chain (such as .alpha.4.beta.7) to
endothelium expressing MAdCAM. In one embodiment, the monoclonal
antibody or antigen binding fragment thereof has the antigenic
specificity of a monoclonal antibody selected from the group
consisting of FIB 21, FIB 30, FIB 504 and ACT-1. Inflammatory bowel
diseases, such as but not limited to ulcerative colitis, Crohn's
disease, Pouchitis, celiac disease, microscopic or collagenous
colitis, and eosinophilic gastroenteritis can be treated according
to the claimed method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIGS. 1a and 1b are graphic illustrations of histologic
scores of inflammatory activity and epithelial injury from left
(descending) and right (ascending) colon of mice exposed to 10 days
of DSS in their drinking water. Three groups of mice are shown,
consisting of groups receiving an irrelevant rat IgG2a antibody,
FIB21, or FIB30 antibodies.
[0007] FIG. 2 is a graph of .gamma. counts per minute (cpm) (.+-.1
SEM) as a percentage of input from mice given DSS in the drinking
water for 10 days. Six groups consisted of negative controls given
water alone, positive controls given DSS alone, test groups given
irrelevant rat IgG2a antibody, FIB21, MECA-367, and FIB21 with
MECA-367.
[0008] FIG. 3 is a graph depicting the histologic scores (.+-.1
SEM) for villus fusion obtained from jejunal biopsy samples of
common marmosets before and on the 14th day of treatment with 2
mg/kg/day of ACT-1 monoclonal antibody.
[0009] FIG. 4 is a graph depicting the histologic scores (.+-.1
SEM) for villus atrophy obtained from jejunal biopsy samples of
common marmosets before and on the 14th day of treatment with 2
mg/kg/day of ACT-1 monoclonal antibody.
[0010] FIGS. 5 and 6 are graphic illustrations of the stool
consistency and inflammatory activity in colitic animals
(cotton-top tamarins) treated with ACT-1 antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The invention relates to the discovery that diseases
associated with leukocyte recruitment to the gastrointestinal
tract, such as IBD, or other mucosal tissues can be treated by
inhibiting MAdCAM binding to the .alpha.4.beta.7 integrin or
triggering of .alpha.4.beta.7-mediated cellular responses.
Compounds which inhibit binding include antibodies or antigen
binding fragments thereof which bind MAdCAM and/or the
.alpha.4.beta.7 integrin. Antibodies which can be used in the
method include recombinant or non-recombinant polyclonal,
monoclonal, chimeric, humanized and/or anti-idiotypic
antibodies.
[0012] Monoclonal antibodies that bind MAdCAM or .alpha.4.beta.7
have been described. For example, MECA 367 is an anti-MAdCAM
antibody of the IgG2a subtype and is described in Gallatin et al.,
Nature, 304:30 (1983) and Michie et al., Am. J. Pathol.
143:1688-1698 (1993). ACT-1 is a monoclonal antibody which binds
the .alpha.4.beta.7 integrin (Lazarovits et al., Journal of
Immunology, 133:1857 (1984) and Schweighoffer et al., Journal of
Immunology, 151:717-729 (1993)). FIB 21 binds the .beta.7 chain is
described and characterized in Berlin et al., Cell 74:184-195
(1993); Andrew, D. P. et al., J. Immunol. 153:3847-3861 (1994)).
Other monoclonal antibodies, such as antibodies which bind to the
same or similar epitopes as the antibodies described above, can be
made according to methods known in the art, such as Kohler et al.,
Nature, 256:495-497 (1975), Harlow et al., 1988, Antibodies: A
Laboratory Manual, (Cold Spring Harbor, N.Y.) or Current Protocols
in Molecular Biology, Vol. 2 (Supplemental 27, Summer '94), Ausubel
et al., Eds. (John Wiley & Sons: New York, N.Y.), Chapter 11
(1991). For example, antibodies can be raised against an
appropriate immunogen in a suitable mammal. Immunogens include, for
example, MAdCAM .alpha.4.beta.7 or immunogenic fragments thereof.
The mammal can be a mouse, rat, rabbit or sheep, for example. The
antibody-producing cell (e.g., a lymphocyte) can be isolated from,
for example, the lymph nodes or spleen of the mammal. The cell can
then be fused to a suitable immortalized cell (e.g., a myeloma cell
line), thereby forming a hybridoma. The fused cells can be isolated
employing selective culturing techniques. Cells which produce
antibodies with the desired specificity can be selected by a
suitable assay (e.g., ELISA).
[0013] In one embodiment, the immunogen can be an antibody which
binds, for example, MAdCAM .alpha.4.beta.7 or immunogenic fragments
thereof. The antibody raised thereby can be an anti-idiotypic
antibody, which can also be used in the present invention.
[0014] Single chain antibodies, and chimeric, humanized or
primatized (CDR-grafted or resurfaced, such as, according to EP
592406, Apr. 13, 1994) antibodies, as well as chimeric or
CDR-grafted single chain antibodies, comprising portions derived
from different species, can also be used in the invention. The
various portions of these antibodies can be joined together
chemically by conventional techniques, or can be prepared as a
contiguous protein using genetic engineering techniques. For
example, nucleic acids encoding a chimeric or humanized chain can
be expressed to produce a contiguous protein. See, e.g., Cabilly et
al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.
0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,
European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO
86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276
B1; Winter, U.S. Pat. No. 5,225,539; and Winter, European Patent
No. 0,239,400 B1. See also, Newman, R. et al., BioTechnology,
10:1455-1460 (1992), regarding primatized antibody, and Ladner et
al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science,
242:423-426 (1988)) regarding single chain antibodies.
[0015] In addition, functional fragments of antibodies, including
fragments of chimeric, humanized, primatized or single chain
antibodies, can also be produced. Functional fragments of the
foregoing antibodies retain at least one binding function of the
full-length antibody from which they are derived and, preferably,
retains the ability to inhibit interaction. For example, antibody
fragments capable of binding to the .alpha.4.beta.7 integrin;
MAdCAM or portion thereof include, but are not limited to, Fv, Fab,
Fab' and F(ab').sub.2 fragments. Such fragments can be produced by
enzymatic cleavage or by recombinant techniques. For instance,
papain or pepsin cleavage can generate Fab or F(ab').sub.2
fragments, respectively. Alternatively, antibodies can be produced
in a variety of truncated forms using antibody genes in which one
or more stop codons has been introduced upstream of the natural
stop site. For example, a chimeric gene encoding a F(ab').sub.2
heavy chain portion can be designed to include DNA sequences
encoding the CH.sub.1 domain and hinge region of the heavy
chain.
[0016] Antibodies and antigen binding fragments thereof which can
be used in the claimed method include antibodies which bind to
MAdCAM and/or .alpha.4.beta.7, such as the .beta.7 chain. For
example, antibodies from the group including FIB 21, FIB 30, FIB
504 and ACT-1 and mixtures thereof can be administered.
Alternatively or in addition, antigen fragments of these antibodies
can be administered.
[0017] Compounds which inhibit the binding of MAdCAM and the
.alpha.4.beta.7 integrin can be administered according to the
claimed method in the treatment of diseases which are associated
with leukocyte (such as lymphocyte or monocyte) recruitment to the
gastrointestinal tract or other tissues as a result of binding of
leukocytes to gut-associated endothelium expressing the molecule
MAdCAM. Diseases which can be treated accordingly include
inflammatory bowel disease, such as ulcerative colitis, Crohn's
disease, Celiac disease (nontropical Sprue), enteropathy associated
with seronegative arthropathies, microscopic or collagenous
colitis, eosinophilic gastroenteritis, or pouchitis resulting after
proctocolectomy and ileoanal anastomosis. In one embodiment, more
than one monoclonal antibody which inhibits the binding of
leukocytes to endothelial MAdCAM is administered. Alternatively, a
monoclonal antibody which inhibits the binding of leukocytes to
endothelial ligands is administered in addition to an anti-MAdCAM
or anti-.beta.7 antibody. For example, an antibody that inhibits
the binding of leukocytes to an endothelial ligand other than
MAdCAM, such as an anti-ICAM-1 or anti-VCAM-1 antibody can also be
administered. In another embodiment, an additional
pharmacologically active ingredient (such as a steroid) can be
administered in conjunction with the antibody of the present
invention.
[0018] A variety of routes of administration are possible
including, but not necessarily limited to parenteral (e.g.,
intravenous, intraarterial, intramuscular, subcutaneous injection),
oral (e.g., dietary), topical, inhalation (e.g., intrabronchial,
intranasal or oral inhalation, intranasal drops), or rectal,
depending on the disease or condition to be treated. Parenteral
administration is a preferred mode of administration.
[0019] Formulation of a compound to be administered will vary
according to the route of administration selected (e.g., solution,
emulsion, capsule). An appropriate composition comprising the
compound to be administered can be prepared in a physiologically
acceptable vehicle or carrier. For solutions or emulsions, suitable
carriers include, for example, aqueous or alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles can include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's
or fixed oils. Intravenous vehicles can include various additives,
preservatives, or fluid, nutrient or electrolyte replenishers (See,
generally, Remington's Pharmaceutical Science, 16th Edition, Mack,
Ed. 1980). For inhalation, the compound is solubilized and loaded
into a suitable dispenser for administration (e.g., an atomizer,
nebulizer or pressurized aerosol dispenser).
[0020] The compound is administered in an amount which will inhibit
binding of MAdCAM to the .alpha.4.beta.7 integrin. The compounds
can be administered in a single dose or multiple doses. The dosage
can be determined by methods known in the art and is dependent, for
example, upon the individual's age, sensitivity, tolerance and
overall well-being. Suitable dosages can be from 0.1-1.0 mg/kg body
weight per treatment.
[0021] The subject invention will now be illustrated by the
following examples, which are not intended to be limiting in any
way.
EXEMPLIFICATION
EXAMPLE 1
Inhibition of Lymphocyte Recruitment to Colon Induction of Colitis
in Mice
[0022] BALB/c mice were given access to a 5% solution of dextran
sodium sulfate (DSS) in their drinking water for a period of 10
days, as previously described (Lab. Invest. 69:238-249, 1993).
During this time period, the mice developed clinical symptoms of
colitis including softening of stools and bloody diarrhea.
Multifocal epithelial injury and ulceration, similar to ulcerative
colitis in humans, was evident on histologic examination of colonic
mucosa from affected mice. Moreover, affected mice lost 20-30% of
their initial body weight by day 10.
Antibody blockade of .beta.7 and MAdCAM Interactions
[0023] To determine the efficacy of .beta.7-specific antibodies in
blocking the recruitment of lymphocytes to the colon, BALB/c mice
were given daily intraperitoneal (i.p.) injections of 100 .mu.g of
monoclonal antibodies against .beta.7, consisting of either FIB21
or FIB30 in saline, as previously characterized and described
(Berlin, C., et al., Cell 74:185-195, 1993; Michie, S. A., et al,
Am. J. Pathol. 143:1688-1698, 1993; Hamann, A., et al., J. Immunol.
152:3282-3293, 1994) or an isotype-matched control rat monoclonal
antibody at the same dose (Andrew et al., supra) over the 10 day
course of DSS treatment.
Methods of Evaluation
[0024] Two methods were used to evaluate efficacy of the antibody
therapy to inhibit leukocyte infiltration and mucosal injury in the
colitic mouse. In the first method, treatment was judged
histologically by two blinded observers using a scoring system for
the evaluation of epithelial injury and degree of leukocyte
cellular infiltration (Table 1). For this assessment, colon tissue
was first fixed in 10% neutral buffered formalin, dehydrated,
embedded in paraffin, sectioned, and the sections were stained with
hematoxylin and eosin prior to examination.
1TABLE PATHOLOGY EVALUATION Grade Definition INFLAMMATION Normal
(0) Absence of clusters of polymorphonoclear leukocytes (PMNs) or
mononuclear cells in the lamina propria; absence of intraepithelial
PMNs Mild (1) Focal aggregates of PMNs and/or mononuclear cells in
the lamina propria (equivocal or slight) or presence of isolated
intraepithelial PMNs in 3 or fewer crypts per cross-section
Moderate (2) Focal aggregates of PMNs and/or mononuclear cells in
the lamina propria (multi-focal or diffuse 2-5X) or intraepithelial
PMNs in more than 3 crypts per cross-section Severe (3) Diffuse
infiltration of PMNs or mononuclear cells in the lamina propria
(diffuse > 5X) or crypt abscesses STRUCTURAL OR EPITHELIAL
ALTERATIONS Normal (0) Tight crypts, no erosion, columnar
epithelial cells Mild (1) Epithelial immaturity; equivocal
irregularity of epithelial surface Moderate (2) At least two foci
of crypt branching or loss of crypts (<50%); loss of surface
epithelium Severe (3) Diffuse or multifocal branching or loss of
crypts (>50%); fibrosis; complete loss of epithelium (focal)
Additional histologic assessment was performed using
immunohistochemistry for the detection and semiquantification of
lymphocytes expressing .beta.7 integrins and mucosal venules
expressing MAdCAM. As previously described (Ringler, D.J., et al.,
Am. J. Pathol. 134:373-383, 1989), colon tissue was first
snap-frozen in OCT compound, sectioned while frozen, and the
sections were subsequently fixed in acetone for 10 min at 4.degree.
C. After washing in phosphate buffered saline (PBS), # nonspecific
antibody binding sites were blocked with 10% normal rabbit serum
diluted in PBS for 10 min, followed in sequence with washes by
FIB21 antibody at 20 .mu.g/ml in PBS for 30 min at room temperature
(RT), biotinylated rabbit anti-rat polyclonal antibody,
avidinperoxidase complexes, and finally the chromogen,
diaminobenzidine and hydrogen peroxide diluted in Tris buffer.
[0025] In the second method, recruitment of lymphocytes to the
colon was quantitatively assessed using radiolabeled mesenteric
lymph node lymphocytes from syngeneic donor mice. The experimental
design of the animal experiments was similar to that described
above except that BALB/c mice were placed on 5% DSS for 9 days
(instead of 10) and on day 8, mice were given i.p. injections of
100 .mu.g of FIB21 (anti-.beta.7), MECA-367 (anti-MAdCAM), a
mixture of both, or an isotype-matched control monoclonal antibody
in saline. On day 9, mesenteric lymph node cells were isolated from
donor syngeneic BALB/c mice, labeled with .sup.51Cr, and
5.0.times.10.sup.6 cells/mouse were incubated for 30 minutes at
37.degree. C. with 500 .mu.g control antibody, 250 .mu.g of
MECA-367, 500 .mu.g FIB21, or both (total amount is 750 .mu.g) in
saline. The labeled cells and antibody were then injected
intravenously (i.v.) into the DSS-treated recipient mice.
Full-length colons were harvested from all experimental animals 1
hour after injection, and .gamma.-irradiation was measured using a
.gamma.-counter.
Data Analysis
[0026] Differences between mean scores obtained for each group of
animals were assessed for statistical significance using a paired
Student's t-test. Differences between means were considered
significant when P<0.05.
Results
[0027] Histologically, inflammation and epithelial injury to the
mucosa were most severe in the descending colon, rectum and cecum.
Analysis of frozen tissue sections of colon by immunohistochemistry
revealed that the most significant recruitment of .beta.7.sup.+
lymphocytes was to the right colon. In addition, the level of
expression of the mucosal vascular addressin, MAdCAM-1, was found
to be expressed only at low levels in vessels in the intestinal
mucosa early in DSS treatment (3 days), but increased dramatically
after 9 days of DSS treatment, supporting the conclusion that
.beta.7 and MAdCAM-1 interactions are relevant to the inflammatory
process in the colonic mucosa during DSS-induced colitis.
[0028] Histologic evaluation of mice exposed to a 10-day course of
DSS and daily therapy using .beta.7-specific antibodies
demonstrated that substantial reductions of leukocyte recruitment
(P<0.01 for FIB30 and P<0.001 for FIB21) and epithelial
injury (P<0.05) occurred in right (ascending) colon compared to
animals receiving a control antibody at the same dose (FIGS. 1a and
1b). Furthermore, analysis using immunohistochemistry of frozen
sections from these animals suggested that the number of
.beta.7.sup.+ cells recruited to the right colon, but not other
sections of colon, during DSS treatment was reduced.
[0029] Lymphocyte recruitment to inflamed colon was then
quantitatively assessed using radiolabeled mesenteric lymphocytes
taken from syngeneic donors. One hour after injection of these
cells in DSS-treated recipients, there was a trend towards a
reduction in the number of .sup.51Cr-labeled cells recruited to
colon in mice that were treated with either .beta.7-specific
antibodies or the MAdCAM-specific antibodies, but not in mice
treated with the isotope-matched control antibodies (FIG. 2).
EXAMPLE 2
Resolution of Villus Alterations in the Common Marmoset (Callithrix
jacchus) with Malabsorptive Enteritis
Description of Model
[0030] Common marmosets (Callithrix jacchus) are a new world
nonhuman primate that, under captive conditions at the New England
Regional Primate Research Center (NERPRC), develop a
steroid-nonresponsive, spontaneous malabsorption syndrome
characterized by weight loss, diarrhea, and small intestinal
mucosal changes consistent with loss of absorptive capacity. These
histologic changes include small intestinal villus atrophy and
fusion, and a mononuclear leukocyte infiltrate within the lamina
propria similar to Celiac disease (nontropical sprue) in humans.
Retrospective analysis from the pathology archive files at NERPRC
demonstrated that up to 80% of common marmosets have, to various
degrees, malabsorptive enteritis at the time of postmortem
examination.
Antibody Therapy Protocol
[0031] Adult common marmosets were selected for study from the
colony-at-large at NERPRC. Base-line studies on all animals
included physical examination, complete blood count (CBC), blood
chemistry profile, serum B12, c-reactive protein, and
full-thickness jejunal biopsy by laparotomy. Following recovery
from abdominal surgery, the animals were treated for 14 days with 2
mg/kg/day of ACT-1 monoclonal antibody, a blocking monoclonal
antibody against a conformational epitope of .alpha.4.beta.7
(Schweighoffer, T., et al., J. Immunol. 151:717-729, 1993).
Previous studies indicated that this antibody cross-reacted to
Callithrix .alpha.4.beta.7. All assessments that were performed
prior to antibody therapy were repeated between the 10th and 14th
day of antibody therapy.
Analysis of Jejunal Biopsies
[0032] Full-thickness jejunal biopsies from each marmoset were
evaluated histologically by two independent pathologists, and
villus architecture was scored according to the following grading
criteria:
2 Villus atrophy 0 normal mucosal thickness and villus height 1
mild atrophy; slight shortening of villi; height approximately 75%
of normal 2 moderate atrophy; villi approximately 33-50% normal
height 3 severe atrophy; short (<33% normal) or no observable
villi Villus fusion 0 normal; no fusion 1 1-2 villi in specimen
fused 2 Between 1-2 and 50% of villi in specimen fused 3 >50%
villi in specimen fused
Data Analysis
[0033] Differences between mean scores obtained for each group of
animals were assessed for statistical significance using a paired
Student's t-test. Differences between means were considered
significant when P<0.05.
Results
[0034] The mean scores for villus fusion and atrophy before and
after antibody therapy with the ACT-1 monoclonal antibody are shown
in FIGS. 3 and 4, respectively. As demonstrated, there was almost
complete resolution of villus atrophy (P<0.01) and a trend for
improvement of villus fusion after a two-week course of therapy
with the ACT-1 antibody. The effect was not secondary to
nonspecific effects of exposure to foreign immunoglobulin since
other animals treated with various monoclonal antibodies directed
against epitopes other than that recognized by ACT-1 were
ineffective in reducing villus fusion and atrophy scores.
EXAMPLE 3
Resolution of Colitis in the Cotton Top Tamarin
Description of Model
[0035] The cotton-top tamarin (Saguinus oedipus) is a New World
nonhuman primate which develops a spontaneous colitis similar to
ulcerative colitis in man.
[0036] ACT-1 was known to cross-react in the tamarin because of
immunohistologic staining with ACT-1 antibody of colitic mucosa
from affected animals. These initial pilot studies demonstrated
that from 40-80% of mononuclear cells within the lamina propria of
colon from affected animals were .alpha.4.beta.7+ similar to human
colitic mucosa.
Methods
[0037] Colitic animals were chosen from the colony-at-large based
upon gross observation of diarrhea and weight loss. All candidate
animals were then subjected to colon biopsy to confirm the presence
of colitis, as defined as a histologic inflammatory activity score
of 2 or 3. The scoring system used was originally described in
Madara, J. L. et al., Gastroenterology 88:13-19 (1985). Briefly,
inflammatory activity scores were based upon the relative numbers
of neutrophils within the lamina propria, crypt lumena, crypt
epithelium, and surface epithelium. All biopsy samples were scored
and categorized into four groups, with 0 representing normal mucosa
and 3 representing the most severe and inflamed mucosa. Scores of 0
and 1 do not represent symptomatic colitis, while scores of 2 to 3
represent mild to severe colitic activity. Within 5 days of
confirmation of colitis, the animals began immunotherapy with ACT-1
monoclonal antibody.
[0038] Four colitic animals received ACT-1 monoclonal antibody at a
dose of 2 mg/kg/day intravenously (I.V.) the first day followed by
intramuscularly (I.M.) injections for 7 consecutive days
thereafter. The dosing regime was the same as that used in the
common marmoset study above.
[0039] Colon biopsies were again obtained at the time of the first
antibody infusion (Day 0) and on days 5, 10 and 20. The biopsies
were evaluated by an independent pathologist. Additional colon
biopsies were frozen for immunohistology. Animal caretakers
evaluated stool consistency on a daily basis by categorizing stool
as diarrhea, semi-solid, or solid. Animals were weighed every other
day, while blood was drawn at the same intervals for flow
cytometry, hematology, and storage of serum or plasma for further
analyses, such as antibody concentration, anti-mouse IgG titer,
clinical chemistry, or acute phase proteins.
Results/Progress
[0040] All four animals maintained either a grade 2 or 3 colitic
inflammatory activity in both the pre-treatment and Day 0 biopsy
samples, which for 3 animals was separated by 5 days. In addition,
changes within the mucosal architecture of all four animals
demonstrated that these four animals had colitis of a long-lasting
nature. Therefore, all animals appeared to have a chronic disease
course.
[0041] With respect to stool consistency, diarrhea resolved in all
four animals by day 8 of ACT-1 immunotherapy (FIG. 4). All animals
maintained solid stools for approximately 1 week after termination
of antibody injections (FIG. 4). One animal (Sgo 63-93) has had
solid stool from Day 4 until the end of the protocol at Day 20
(FIG. 4). Two animals (Sgo 129-91 and Sgo 17-85) had slight
relapses to semi-solid stools after Day 14 in the study (FIG. 4).
The fourth animal (Sgo 326-84) showed a persistent
improvement/resolution of diarrhea from Day 6 to Day 20.
[0042] With respect to histologic changes, all four animals have
shown improvement in inflammatory activity during or after ACT-1
immunotherapy. The colitis in two animals (Sgo 129-91 and Sgo
17-85) completely resolved by Day 10 (FIG. 6). Another animal (Sgo
63-93) did not show complete abrogation of colitis activity until
Day 20 (FIG. 6), while mucosal biopsy scores from the fourth animal
(Sgo 326-84) showed improvement during the entire study period
(FIG. 6; two biopsies on day 20 in Sgo 326-84 were scored as 0 and
1). Furthermore, animal 326-84 gained 20% of its original body
weight during the study period.
[0043] To detect antibody administered in vivo, flow cytometry and
immunohistology were performed. Flow cytometry without a primary
antibody showed excellent labeling to peripheral blood lymphocytes
in animals at all time points after antibody administration.
Immunohistology on colon biopsies using no primary antibody in the
sequence from three animals on samples up to and including Day 10
showed excellent labeling of lymphocytes within the lamina propria
on the samples from Days 5 and 10 but not, as expected, from Day 0
prior to antibody infusion. Collectively, these results showed that
ACT-1 antibody localized to the target site, namely lymphocytes
within the peripheral blood and specifically to the extravascular
compartment within colitic mucosa.
Summary
[0044] By histologic criteria and stool consistency, ACT-1 was
efficacious in improving colitis in the cotton top tamarin.
[0045] There appeared to be a good correlation between histologic
inflammatory activity scores and stool consistency. Noteworthy is
the observation that stool consistency generally improved in 1-2
days in animals receiving ACT-1 antibody.
Equivalents
[0046] Those skilled in the art will know, or be able to ascertain,
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
all other equivalents are intended to be encompassed by the
following claims.
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