U.S. patent application number 11/634822 was filed with the patent office on 2007-11-01 for treatment for inflammatory bowel disease.
This patent application is currently assigned to Biogen Idec MA Inc., a Massachusetts corporation. Invention is credited to Linda C. Burkly, Roy R. Lobb.
Application Number | 20070253902 11/634822 |
Document ID | / |
Family ID | 27403472 |
Filed Date | 2007-11-01 |
United States Patent
Application |
20070253902 |
Kind Code |
A1 |
Lobb; Roy R. ; et
al. |
November 1, 2007 |
Treatment for inflammatory bowel disease
Abstract
A method for the treatment of inflammatory bowel disease (IBD)
is disclosed. The method comprises administration of an antibody,
polypeptide or other molecule recognizing VLA-4, a surface molecule
expressed on most types of white blood cells and involved in
leukocyte adhesion to endothelium and other tissus in the gut.
Inventors: |
Lobb; Roy R.; (Westwood,
MA) ; Burkly; Linda C.; (West Newton, MA) |
Correspondence
Address: |
FISH & RICHARDSON
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Biogen Idec MA Inc., a
Massachusetts corporation
|
Family ID: |
27403472 |
Appl. No.: |
11/634822 |
Filed: |
December 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10252978 |
Sep 23, 2002 |
7176184 |
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11634822 |
Dec 6, 2006 |
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09157452 |
Sep 21, 1998 |
6482409 |
|
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10252978 |
Sep 23, 2002 |
|
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08950660 |
Oct 15, 1997 |
5932214 |
|
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09157452 |
Sep 21, 1998 |
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08456124 |
May 31, 1995 |
|
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08950660 |
Oct 15, 1997 |
|
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08373857 |
Jan 18, 1995 |
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08456124 |
May 31, 1995 |
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08284603 |
Aug 11, 1994 |
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08373857 |
Jan 18, 1995 |
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PCT/US93/00924 |
Feb 2, 1993 |
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08373857 |
Jan 18, 1995 |
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07835139 |
Feb 12, 1992 |
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PCT/US93/00924 |
Feb 2, 1993 |
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Current U.S.
Class: |
424/1.69 ;
424/134.1; 514/20.9 |
Current CPC
Class: |
Y10S 514/885 20130101;
A61P 29/00 20180101; A61K 51/1027 20130101; C07K 14/70542 20130101;
A61K 47/6829 20170801; C07K 16/2842 20130101; A61K 47/6827
20170801; A61P 1/00 20180101; A61K 47/6849 20170801; A61K 47/6825
20170801 |
Class at
Publication: |
424/001.69 ;
424/134.1; 514/008 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 39/00 20060101 A61K039/00; A61K 51/00 20060101
A61K051/00; A61P 1/00 20060101 A61P001/00 |
Claims
1.-25. (canceled)
26. A method for the treatment of inflammatory bowel disease,
comprising administering to a mammal suffering from inflammatory
bowel disease a composition comprising a soluble fibronectin
polypeptide comprising an alternatively spliced non-type III
connecting segment or a fragment thereof.
27. The method according to claim 26, wherein the mammal is a
human.
28. The method according to claim 26, wherein the mammal has
ulcerative colitis.
29. The method according to claim 26, wherein the mammal has
Crohn's Disease.
30. The method according to claim 26, wherein the composition is
administered during an acute flareup of the inflammatory bowel
disease.
31. The method according to claim 26, wherein the fibronectin
polypeptide or fragment thereof is a component of a chimeric
molecule.
32. The method according to claim 26, wherein the composition is
administered intravenously.
33. The method according to claim 31, wherein the chimeric molecule
further comprises a toxin moiety.
34. The method according to claim 33, wherein the toxin moiety is a
cytotoxic peptide selected from the group consisting of Diphtheria
toxin A, Pseudomonas Exotoxin, Ricin A, Abrin A, Schigella toxin,
or Gelonin.
35. The method according to claim 33, wherein the toxin moiety is a
radionucleotide or a chemotherapeutic agent.
36. The method according to claim 31, wherein the chimeric molecule
further comprises an additional peptide that increases solubility
or in vivo life time of the soluble fibronectin or fragment
thereof.
37. The method according to claim 36, wherein the additional
peptide is a heavy chain constant region of human IgG1.
38. The method according to claim 36, wherein the additional
peptide comprises at least one of the CH.sub.2 or CH.sub.3 regions
of the heavy chain of human IgG1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 10/252,978, filed on Sep. 23, 2002,
which is a continuation application of U.S. patent application Ser.
No. 09/157,452, filed on Sep. 21, 1998, now U.S. Pat. No.
6,482,409, which is a continuation application of U.S. patent
application Ser. No. 08/950,660, filed on Oct. 15, 1997, now U.S.
Pat. No. 5,932,214, which is a file wrapper continuation
application of U.S. patent application Ser. No. 08/456,124, filed
on May 31, 1995, now abandoned, which is a continuation-in-part
application U.S. patent application Ser. No. 08/373,857, filed on
Jan. 18, 1995, now abandoned, which is a continuation-in-part
application of U.S. patent application Ser. No. 08/284,603, filed
on Aug. 11, 1994, now abandoned, and of International Patent
Application No. PCT/US93/00924, filed on Feb. 2, 1993, which is a
continuation-in-part application of U.S. patent application Ser.
No. 07/835,139, filed Feb. 12, 1992, now abandoned. The contents of
the prior applications are hereby incorporated by reference in
their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to a treatment for
inflammatory bowel disease (IBD). More particularly, this invention
relates to the use of antibodies recognizing the integrin VLA-4
(very late antigen-4) in the treatment of IBD.
BACKGROUND OF THE INVENTION
[0003] Inflammatory bowel disease, or IBD, is a collective term
encompassing ulcerative colitis and Crohn's disease (ileitis),
which are chronic inflammatory disorders of the gastrointestinal
tract. Ulcerative colitis is confined to the large intestine
(colon) and rectum, and involves only the inner lining of the
intestinal wall. Crohn's disease may affect any section of the
gastrointestinal tract (i.e., mouth, esophagus, stomach, small
intestine, large intestine, rectum and anus) and may involve all
layers of the intestinal wall. Both diseases are characterized by
abdominal pain and cramping, diarrhea, rectal bleeding and fever.
The symptoms of these diseases are usually progressive, and
sufferers typically experience periods of remission followed by
severe flareups.
[0004] IBD affects an estimated two million people in the United
States alone. Although IBD is not considered a fatal illness,
prolonged disease can lead to severe malnutrition affecting growth
or to the formation of abscesses or intestinal scar tissue, leading
in turn to infection or bowel obstruction.
[0005] IBD has no cure, and the exact causes of IBD are not yet
understood. Conventional treatments for IBD have involved
anti-inflammatory drugs, immunosuppressive drugs and surgery.
Sulfasalazine and related drugs having the bioactive
5-amino-salicylic acid (5-ASA) moiety are widely used to control
moderate IBD symptoms and to maintain remission. Severe
inflammation is often treated with powerful corticosteroids and
sometimes ACTH or with immunosuppressants such as 6-mercaptopurine
and azathioprine. The most common surgical treatments for severe
chronic IBD are intestinal resections and, ultimately, colectomy,
which is a complete cure only for ulcerative colitis.
[0006] Severe side effects are associated with the drugs commonly
prescribed for IBD, including nausea, dizziness, changes in blood
chemistry (including anemia and leukopenia), skin rashes and drug
dependence; and the surgical treatments are radical procedures that
often profoundly alter the everyday life of the patient.
Accordingly, there is a great need for treatments for IBD that are
effective yet less severe in their side effects and are less
invasive of the IBD sufferer's body and quality of life.
[0007] The search for the causes of IBD and more effective
treatments has led several investigators to study diseased and
normal tissue on a cellular level. This has led to observations of
variations in the normal content of intestinal mucin (Podolsky,
1988 [1]) and to the observation of colonic glycoproteins that
emerge only in diseased tissue (Podolsky and Fournier, 1988a [2],
1988b [3]). Researchers have observed that the cell adhesion
molecule ICAM-1 is expressed at elevated levels in IBD tissue
(Malizia et al., 1991 [4]). This molecule is thought to mediate
leukocyte recruitment to sites of inflammation through adhesion to
leukocyte surface ligands, i.e., LFA-1 (CD11a/CD18 complex) on all
leukocytes and Mac-1 (CD11b/CD18) on phagocytes. (See, e.g.,
Springer, 1990 [5].) Because flareups of IBD are often accompanied
by increased concentrations of neutrophils and lymphocytes in the
intestinal submucosa, blocking of interactions between endothelial
cell receptors (such as ICAM-1) and their leukocyte ligands (such
as LFA-1, Mac-1) has been proposed as a treatment for IBD.
[0008] Another cell adhesion molecule, VCAM-1 (vascular cell
adhesion molecule-1) is expressed on inflamed endothelium and has
been shown to recognize the .alpha..sub.4.beta..sub.1 integrin,
VLA-4, expressed on the surface of all leukocytes except
neutrophils (Springer, 1990 [5]). VCAM-1 also has been found to be
expressed constitutively in noninflamed tissue, including Peyer's
patch follicular dendritic cells (Freedman et al., 1990 [6]; Rice
et al., 1991 [7]). Additionally, besides mediating cell adhesion
events, VCAM-1 also has recently been determined to play a
costimulatory role, through VLA-4, in T cell activation (Burkly et
al., 1991 [8]; Damle and Arrufo, 1991 [9]; van Seventer et al.,
1991 [10]). Accordingly, further study of VCAM-1 has been taken up
to investigate whether it plays a role as a regulator of the immune
response as well as a mediator of adhesion in vivo.
[0009] It has now been surprisingly discovered that administering
anti-VLA-4 antibody significantly reduces acute inflammation in a
primate model for IBD. Cotton top tamarins suffering from a
spontaneous intestinal inflammation comparable to ulcerative
colitis in humans that were treated with an anti-VLA-4 antibody
(HP1/2) showed significant reduction in inflammation of biopsied
intestinal tissue.
SUMMARY OF THE INVENTION
[0010] Accordingly, the present invention provides novel methods
for the treatment of IBD and further provides new pharmaceutical
compositions useful in the treatment of IBD. In particular, the
present invention provides a method comprising the step of
administering to an IBD sufferer a VLA-4 blocking agent, e.g., an
anti-VLA-4 antibody, such as antibody HP1/2 Also contemplated is
the use of analogous antibodies, antibody fragments, soluble
proteins and small molecules that mimic the action of anti-VLA-4
antibodies in the treatment of IBD.
DETAILED DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic depicting structure of VCAM 2D-IgG
fusion protein described in Example V. VCAM 2D-IgG is a soluble
form of the ligand for VLA4 (VCAM1) and consists of the two
N-terminal domains of VCAM 1 fused to the human IgG1 heavy chain
constant region sequences (Hinges, C.sub.H2 and C.sub.H3).
[0012] FIG. 2 is graph depicting VCAM-IgG inhibitory dose response
after single intraperitoneal doses in mouse contact
hypersensitivity. VCAM-IgG tested over a dose range of 1.25 to 20
mg/kg (25-400 .mu.g/mouse) maximally inhibited the mouse ear
swelling response about 4/5 as well as the PS/2 antibody. Maximal
inhibition occurred at the 10 mg/kg dose. The ED50 was about 2.5
mg/kg, i.p. (50 .mu.g/mouse).
[0013] FIG. 3 is a graph depicting inhibition of the mouse ear
swelling response by VCAM-IgG in mouse contact hypersensitivity. In
a confirmation study, VCAM-IgG tested at 20 mg/kg i.p. (400
.mu.g/mouse) maximally inhibited the mouse ear swelling response as
effectively as the PS/2 8 mg/kg i.v. antibody dose.
[0014] FIG. 4 is a graph depicting the effect of VCAM 2D-IgG fusion
protein and controls on prevention of diabetes after adoptive
transfer of spleen cells; the frequency of recipients which became
diabetic and day of disease onset are shown for transfer of
2.times.10.sup.7 splenocytes from diabetic (D) NOD donors with an
irrelevant rat LFA-3Ig fusion protein treatment (closed squares),
and with VCAM 2D-IgG treatment (open circles) or of recipients
which received PBS alone without cells transferred (closed
triangles); the splenocytes were transferred with VCAM 2D-IgG or
rat LFA-3Ig, and then VCAM 2D-IgG or rat LFA-3Ig was injected every
other day through day 17 post-transfer (n=5 for all groups).
[0015] FIG. 5 is a graph depicting the effect of VCAM-Ig (30 mg,
aerosol given 30 min before antigen challenge) on airway
hyperresponsiveness in dual responder sheep. This dose resulted in
significant but partial inhibition of LPR, but no effect on
AHR.
[0016] FIG. 6 is a graph depicting the effect of VCAM-Ig (60 mg,
aerosol given 30 min before antigen challenge) on airway
hyperresponsiveness in dual responder sheep. This dose resulted in
significant but partial inhibition of LPR, and inhibition of
AHR.
[0017] FIG. 7 is a graph depicting the effect of VCAM-Ig (30 mgs,
aerosol given 30 min. before antigen challenge and 8 h. after
challenge) on airway hyperresponsiveness in dual responder sheep.
This dose resulted in complete inhibition of LPR, but no inhibition
of AHR.
[0018] FIG. 8 is a graph depicting the effect of VCAM-Ig (15 mgs,
aerosol given 2, 8 and 24 h. after antigen challenge) on airway
hyperresponsiveness in dual responder sheep. This dose resulted in
a significant but partial inhibition of LPR, and inhibition of
AHR.
[0019] FIG. 9 is a graph depicting the effect of VCAM-Ig (30 mgs,
aerosol given 2, and 24 h. after antigen challenge) on airway
hyperresponsiveness in dual responder sheep. This optimal dose
resulted in complete inhibition of both LPR and AHR.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The technology for producing monoclonal antibodies is well
known. Briefly, an immortal cell line (typically myeloma cells) is
fused to lymphocytes (typically splenocytes) from a mammal
immunized with whole cells expressing a given antigen, e.g., VLA-4,
and the culture supernatants of the resulting hybridoma cells are
screened for antibodies against the antigen. (See, generally,
Kohler and Milstein, 1975 [11].)
[0021] Immunization may be accomplished using standard procedures.
The unit dose and immunization regimen depend on the species of
mammal immunized, its immune status, the body weight of the mammal,
etc. Typically, the immunized mammals are bled and the serum from
each blood sample is assayed for particular antibodies using
appropriate screening assays. For example, anti-VLA-4 antibodies
may be identified by immunoprecipitation of .sup.125I-labeled cell
lysates from VLA-4-expressing cells. (See, Sanchez-Madrid et al.,
1986 [13] and Hemler et al., 1987 [14].) Anti-VLA-4 antibodies may
also be identified by flow cytometry, e.g., by measuring
fluorescent staining of Ramos cells incubated with an antibody
believed to recognize VLA-4 (see, Elices et al., 1990 [15]). The
lymphocytes used in the production of hybridoma cells typically are
isolated from immunized mammals whose sera have already tested
positive for the presence of anti-VLA-4 antibodies using such
screening assays.
[0022] Typically, the immortal cell line (e.g., a myeloma cell
line) is derived from the same mammalian species as the
lymphocytes. Preferred immortal cell lines are mouse myeloma cell
lines that are sensitive to culture medium containing hypoxanthine,
aminopterin and thymidine ("HAT medium"). HAT-sensitive mouse
myeloma cells may be fused to mouse splenocytes, e.g., using 1500
molecular weight polyethylene glycol ("PEG 1500"). Hybridoma cells
resulting from the fusion are then selected using HAT medium, which
kills unfused and unproductively fused myeloma cells (unfused
splenocytes die after several days because they are not
transformed). Hybridomas producing a desired antibody are detected
by screening the hybridoma culture supernatants. For example,
hybridomas prepared to produce anti-VLA-4 antibodies may be
screened by testing the hybridoma culture supernatant for secreted
antibodies having the ability to bind to a recombinant
.alpha..sub.4-subunit-expressing cell line, such as transfected
K-562 cells (see, Elices et al., [15]).
[0023] To produce anti VLA-4-antibodies, hybridoma cells that test
positive in such screening assays may be cultured in a nutrient
medium under conditions and for a time sufficient to allow the
hybridoma cells to secrete the monoclonal antibodies into the
culture medium. Tissue culture techniques and culture media
suitable for hybridoma cells are well known. The conditioned
hybridoma culture supernatant may be collected and the anti-VLA-4
antibodies optionally further purified by well-known methods.
[0024] Alternatively, the desired antibody may be produced by
injecting the hybridoma cells into the peritoneal cavity of a mouse
primed with 2,6,10,14-tetramethylpentadecane (PRISTANE; Sigma
Chemical Co., St. Louis Mo.). The hybridoma cells proliferate in
the peritoneal cavity, secreting the antibody, which accumulates as
ascites fluid. The antibody may be harvested by withdrawing the
ascites fluid from the peritoneal cavity with a syringe.
[0025] Several anti-VLA-4 monoclonal antibodies have been
previously described (see, e.g., Sanchez-Madrid et al., 1986 [12];
Hemler et al. (1987) [13]; Pulido et al. (1991) [14]). For the
experiments herein, an anti-VLA-4 monoclonal antibody designated
HP1/2 (obtained from Biogen, Inc., Cambridge, Mass.) was used. The
variable regions of the heavy and light chains of the anti-VLA-4
antibody HP1/2 have been cloned, sequenced and expressed in
combination with constant regions of human immunoglobulin heavy and
light chains. Such a chimeric HP1/2 antibody is similar in
specificity and potency to the murine HP1/2 antibody, and may be
useful in methods of treatment according to the present invention.
Similarly, humanized recombinant anti-VLA-4 antibodies may be
useful in these methods. The HP1/2 V.sub.H DNA sequence and its
translated amino acid sequences are set forth in SEQ ID NO: 1 and
SEQ ID NO: 2, respectively. The HP1/2 V.sub.K DNA sequence and its
translated amino acid sequence are set forth in SEQ ID NO: 3 and
SEQ ID NO: 4, respectively.
[0026] Monoclonal antibodies such as HP1/2 and other anti-VLA-4
antibodies (e.g., Mab HP2/1, HP2/4, L25, P4C2) capable of
recognizing the .alpha. chain of VLA-4 will be useful in the
present invention. It is most preferred that the antibodies will
recognize the B1 or B2 epitopes of the VLA-.alpha..sub.4 chain
(see, Pulido et al. (1991) [15]). While not wishing to be bound by
one scientific theory, anti-VLA-4 antibodies used according to the
method of the present invention may specifically inhibit, at least
for an initial period, the migration of VLA-4-expressing leukocytes
to inflamed sections of the gut. Or, the release of inflammatory
mediators and cytokines by leukocytes already recruited to IBD
tissue may be blocked by anti-VLA-4 antibodies that prevent some
form of VCAM-1-mediated signal transduction, such as the T cell
co-activation observed previously (e.g., Burkly et al. 1991 [8]).
Monoclonal antibody HP1/2 has been shown to block leukocyte
adhesion to VCAM-1-expressing cells but not to promote
VLA-4-mediated T cell activation.
[0027] The method of the present invention comprises administering
to a mammal suffering from inflammatory bowel disease a composition
comprising a VLA-4 blocking agent, e.g., an anti-VLA-4 antibody.
The examples below set forth the results observed in cotton top
tamarins. The physiological and histochemical similarities between
a spontaneous chronic diffuse colitis observed in the cotton top
tamarin (CTT) and IBD humans has been documented (see, e.g.,
Podolsky et al., 1985a [16], Podolsky et al., 1985b [17]). Prior
studies have also demonstrated parallel responses in CTTs to
therapeutic compounds used in the management of the human IBD (see,
e.g., Madara et al., 1985 [18]). Accordingly, the results reported
herein will be relevant and applicable to, and the method claimed
will be useful in any mammal, including humans, suffering from
IBD.
[0028] The VLA-4 blocking agent, e.g., an anti-VLA-4 antibody,
administered in accordance with the present invention may be
administered prophylactically to a chronic IBD sufferer, to bring
about or maintain remission of the disease; however, preferably the
method of the present invention is used to treat acute flareups of
the disease.
[0029] The VLA-4 blocking agent, e.g., an anti-VLA-4 antibody, can
be administered in the form of a composition, e.g., a composition
comprising an anti-VLA-4 antibody and a pharmaceutically acceptable
carrier. Preferably, the composition will be in a form suitable for
intravenous injection. For acute flareups of ulcerative colitis or
Crohn's disease, dosages of antibodies from 0.05 mg/kg-patient/day
to 5.0 mg/kg-patient/day (preferably from 0.5 mg/kg-patient/day to
2.0 mg/kg-patient/day) may be used, although higher or lower
dosages may be indicated with consideration to the age,
sensitivity, tolerance, and other characteristics of the patient,
the acuteness of the flareup, the history and course of the
disease, plasma level and half-life of the antibody employed and
its affinity for its recognition site, and other similar factors
routinely considered by an attending physician. For maintenance of
remission from active disease, dosages from 0.05 mg/kg-patient/day
to 5.0 mg/kg-patient/day (preferably from 0.5 mg/kg-patient/day to
2.0 mg/kg-patient/day) may be used, although higher or lower
dosages may be indicated and employed with advantageous effects
considering the age, sensitivity, tolerance, and other
characteristics of the patient, the pattern of flareups, the
history and course of the disease, the plasma level and half-life
of the agent, e.g., an antibody, employed and its affinity for its
recognition site, and other similar factors routinely considered by
an attending physician. Dosages may be adjusted, for example, to
provide a particular plasma level of an agent, e.g., an antibody,
e.g., in the range of 5-30 .mu.g/ml, more preferably 10- 15
.mu.g/ml, for murine antibodies, and to maintain that level, e.g.,
for a period of time (e.g., 1 week) or until clinical results are
achieved (e.g., flareup subsides). Chimeric and humanized
antibodies, which would be expected to be cleared more slowly, will
require lower dosages to maintain an effective plasma level. Also,
antibodies or fragments having high affinity for VLA-4 will need to
be administered less frequently or in lower doses than antibodies
or antibody fragments of lesser affinity.
[0030] Suitable pharmaceutical carriers include, e.g., sterile
saline, physiological buffer solutions and the like. The
pharmaceutical compositions may additionally be formulated to
control the release of the active ingredients or prolong their
presence in the patient's system. Numerous suitable drug delivery
systems are known for this purpose and include, e.g., hydrogels,
hydroxmethylcellulose, microcapsules, liposomes, microemulsions,
microspheres, and the like. Phosphate buffered saline (PBS) is a
preferred carrier for injectible compositions.
[0031] It will also be recognized that for the purposes of the
present invention, antibodies capable of binding to the
.alpha..sub.4 subunit of VLA-4 should be employed. It is preferred
that monoclonal antibodies be used.
[0032] In addition to naturally produced antibodies, suitable
recombinant antibodies capable of binding to VLA-4 may
alternatively be used. Such recombinant antibodies include
antibodies produced via recombinant DNA techniques, e.g., by
transforming a host cell with a suitable expression vector
containing DNA encoding the light and heavy immunoglobulin chains
of the desired antibody, and recombinant chimeric antibodies,
wherein some or all of the hinge and constant regions of the heavy
and/or the light chain of the anti-VLA-4 antibody have been
substituted with corresponding regions of an immunoglobulin light
or heavy chain of a different species (i.e., preferably the same
species as the IBD sufferer being treated, to minimize immune
response to the administered antibody). (See, e.g., Jones et al.,
1986 [19], Ward et al., 1989 [20], and U.S. Pat. No. 4,816,397
(Boss et al.) [21], all incorporated herein by reference.)
Recombinant antibodies specifically contemplated herein include
CDR-grafted antibodies or "humanized" antibodies, wherein the
hypervariable regions of, e.g., murine antibodies are grafted onto
framework regions of, e.g., a human antibody. (See, e.g., Riechmann
et al., 1988 [22]; Man Sung Co et al., 1991 [23]; Brown, Jr., 1991
[24].)
[0033] Furthermore, VLA-4-binding fragments of anti-VLA-4
antibodies, such as Fab, Fab', F(ab').sub.2, and F(v) fragments;
heavy chain monomers or dimers; light chain monomers or dimers; and
dimers consisting of one heavy chain and one light chain are also
contemplated herein. Such antibody fragments may be produced by
chemical methods, e.g., by cleaving an intact antibody with a
protease, such as pepsin or papain, or via recombinant DNA
techniques, e.g., by using host cells transformed with truncated
heavy and/or light chain genes. Heavy and light chain monomers may
similarly be produced by treating an intact antibody with a
reducing agent such as dithiothreitol or .beta.-mercaptoethanol or
by using host cells transformed with DNA encoding either the
desired heavy chain or light chain or both.
[0034] As an alternative to hybridoma technology, antibody
fragments having the desired anti-VLA-4 specificities may be
isolated by phage cloning methods. (See, e.g., Clackson et al.,
1991 [25].)
[0035] Also, from the discussion herein it will be apparent that
other VLA-4 blocking agents can be used in the methods described
herein. For the purposes of the invention a VLA-4 blocking agent
refers to an agent, e.g., a polypeptide or other molecule, which
can inhibit or block VLA-4-mediated binding or which can otherwise
modulate VLA-4 function, e.g., by inhibiting or blocking
VLA-4-ligand mediated VLA-4 signal transduction and which is
effective in the treatment of IBD, preferably in the same manner as
are anti-VLA-4 antibodies.
[0036] A VLA-4 blocking agent is a molecule which has one or more
of the following properties: (1) it coats, or binds to, a VLA-4
antigen on the surface of a VLA-4 bearing cell with sufficient
specificity to inhibit a VLA-4-ligand/VLA-4 interaction, e.g., the
VLA-4/VCAM-1interaction; (2) it coats, or binds to, a VLA-4 antigen
on the surface of a VLA-4 bearing cell with sufficient specificity
to modify, and preferably to inhibit, transduction of a
VLA-4-mediated signal, e.g., VLA-4/VCAM-1-mediated signaling; (3)
it coats, or binds to, a VLA-4-ligand, e.g., VCAM-1 or fibronectin,
with sufficient specificity to inhibit the VLA-4/VLA-4-ligand
interaction; (4) it coats, or binds to, a VLA-4-ligand, e.g.,
VCAM-1 or fibronectin, with sufficient specificity to modify, and
preferably to inhibit, transduction of VLA-4-ligand mediated VLA-4
signaling, e.g., VCAM-1-mediated VLA-4 signaling. In preferred
embodiments the VLA-4 blocking agent has one or both of properties
1 and 2. In other preferred embodiments the VLA-4 blocking agent
has one or both of properties 3 and 4.
[0037] For purposes of the invention, any agent capable of binding
to VLA-4 antigens on the surface of VLA-4 bearing cells and which
effectively blocks or coats VLA-4 antigens, is considered to be an
equivalent of the monoclonal antibody used in the examples
herein.
[0038] As discussed herein, the blocking agents used in methods of
the invention are not limited to antibodies or antibody
derivatives, but may be other molecules, e.g., soluble forms of
other proteins which bind VLA-4, e.g., the natural binding proteins
for VLA-4. These binding agents include soluble VCAM-1 or VCAM-1
peptides, VCAM-1 fusion proteins, bifunctional VCAM-1/Ig fusion
proteins, fibronectin, fibronectin having an alternatively spliced
non-type III connecting segment, and fibronectin peptides
containing the amino acid sequence EILDV (SEQ ID NO: 17) or a
similar conservatively substituted amino acid sequence. These
binding agents can act by competing with the cell-surface binding
protein for VLA-4 or by otherwise altering VLA-4 function. For
example, a soluble form of VCAM-1 (see, e.g., Osborn et al. 1989
[26]) or a fragment thereof may be administered to bind to VLA-4,
and preferably compete for a VLA-4 binding site, thereby leading to
effects similar to the administration of anti-VLA-4 antibodies.
Soluble VCAM-1 fusion proteins can be used in the methods described
herein. For example, VCAM-1, or a fragment thereof which is capable
of binding to VLA-4 antigen on the surface of VLA-4 bearing cells,
e.g., a fragment containing the two N-terminal domains of VCAM-1,
can be fused to a second peptide, e.g., a peptide which increases
the solubility or the in vivo life time of the VCAM-1 moiety. The
second peptide can be a fragment of a soluble peptide, preferably a
human peptide, more preferably a plasma protein, or a member of the
immunoglobulin super family. In particularly preferred embodiments
the second peptide is IgG or a portion or fragment thereof, e.g.,
the human IgG1 heavy chain constant region. A particularly
preferred fusion protein is the VCAM 2D-IgG fusion.
[0039] Included in the invention as VLA-4 blocking agents are (at
least) peptides (preferably peptides of less than 5 or 10 amino
acid resides in length), peptide mimetics, carbohydrates, and small
molecules, such as oligosaccharides, capable of blocking VLA-4 in
any of the ways described herein, e.g., by binding VLA-4 antigens
on the surface of VLA-4-bearing cells or by binding to
VLA-4-ligands. Small molecules such as oligosaccharides that mimic
the binding domain of a VLA-4 ligand and fit the receptor domain of
VLA-4 may also be employed. (See, J. J. Devlin et al., 1990 [24],
J. K. Scott and G. P. Smith, 1990 [25], and U.S. Pat. No. 4,833,092
(Geysen) [26], all incorporated herein by reference.) Examples of
small molecules useful in the invention can be found in Adams et
al. U.S. Ser. No. 08/376,372, filed Jan. 23, 1995, hereby
incorporated by reference.
[0040] In preferred embodiments more than one VLA-4 blocking agent
is administered to a patient, e.g., a VLA-4 blocking agent which
binds to VLA-4 can be combined with a VLA-4 blocking agent which
binds to VCAM-1.
[0041] The use of such VLA-4 blocking agents, e.g., VLA-4-binding
polypeptides or molecules that effectively decrease inflammation in
IBD tissue in treated subjects is contemplated herein as an
alternative method for treatment of IBD.
[0042] It is also contemplated that anti-VLA-4 antibodies may be
used in combination with other antibodies having a therapeutic
effect on IBD. For instance, to the extent that the beneficial
effects reported herein are due to the inhibition of leukocyte
recruitment to endothelium, combinations of anti-VLA-4 antibodies
with other antibodies that interfere with the adhesion between
leukocyte antigens and endothelial cell receptor molecules may be
advantageous. For example, in addition to the use of anti-VLA-4
antibodies in accordance with this invention, the use of
anti-ELAM-1 antibodies, anti-VCAM-1 antibodies, anti-ICAM-1
antibodies, anti-CDX antibodies, anti-CD18 antibodies, and/or
anti-LFA-1 antibodies may be advantageous.
[0043] When formulated in the appropriate vehicle, the
pharmaceutical compositions contemplated herein may be administered
by any suitable means such as orally, intraesophageally or
intranasally, as well as subcutaneously, intramuscularly,
intravenously, intra-arterially, or parenterally. Ordinarily
intravenous (i.v.) or parenteral administration will be preferred
to treat flareup conditions; oral administration in a timed release
vehicle will be preferred to maintain remission.
[0044] Improvement for IBD patients as a result of the methods of
this invention can be evaluated by any of a number of methods known
to practitioners in this art. For example, improvement in observed
symptomology such as the Truelove-Witts criteria (see, e.g.,
Lichtiger, et al., 1990 [30]) may be used, or specimens of colon
tissue may be biopsied and characterized histologically (see, e.g.,
Madara et al., 1985 [18]).
[0045] In another aspect the invention features a chimeric molecule
which includes: (1) a VLA-4 targeting moiety, e.g., a VCAM-1 moiety
capable of binding to VLA-4 antigen on the surface of VLA-4 bearing
cells; (2) optionally, a second peptide, e.g., one which increases
solubility or in vivo life time of the VLA-4 targeting moiety,
e.g., a member of the immunoglobulin super family or fragment or
portion thereof, e.g., a portion or a fragment of IgG, e.g., the
human IgG1 heavy chain constant region, e.g., C.sub.H2 and C.sub.H3
hinge regions; and (3) a toxin moiety. The VLA-4 targeting moiety
can be any naturally occurring VLA-4 ligand or fragment thereof,
e.g., a VCAM-1 peptide, fibronectin, fibronectin having an
alternatively spliced non-type III connecting segment, and
fibronectin peptides containing the amino acid sequence EILDV (SEQ
ID NO: 17) or a similar conservatively substituted amino acid
sequence. A preferred targeting moiety is a soluble VCAM-1
fragment, e.g., the N-terminal domains 1 and 2 of the VCAM-1
molecule. The toxin moiety can be any agent which kills or
inactivates a cell when the toxin is targeted to the cell by the
VLA-4 targeting moiety. Toxin moieties include: cytotoxic peptide
moieties, e.g., Diphtheria toxin A, Pseudomonas Exotoxin, Ricin A,
Abrin A, Schigella toxin, or Gelonin; radionucleotides; and
chemotherapeutic agents.
[0046] The chimeric molecule can be used to treat a subject, e.g.,
a human, at risk for a disorder, e.g., IBD, characterized by the
presence of cells bearing VLA-4, and preferably activated
VLA-4.
[0047] The methods and compositions of the present invention will
be further illuminated by reference to the following examples,
which are presented by way of illustration and not of
limitation.
EXAMPLE I
VCAM1 Expression in the Colon
[0048] Experiments were performed to determine whether active IBD
involved the expression of endothelial cell surface proteins
involved in leukocyte adhesion. Expression of VCAM-1 in colon
tissue of IBD sufferers and normal or uninvolved colon tissue
controls was evaluated. Human colonoscopic biopsy tissue samples
were obtained, with informed consent, and prepared as frozen
sections by mounting in OCT compound (TissueTek) and quick freezing
in isopentane/liquid nitrogen. The human colon samples were from
normal colon, active ulcerative colitis colon (UC-active), inactive
ulcerative colitis colon (UC-inactive), uninvolved ulcerative
colitis colon (UC-uninvolved), active Crohn's Disease colon
(CD-active), and uninvolved Crohn's Disease colon
(CD-uninvolved).
[0049] Frozen sections (.about.4.mu.) were placed on gelatin-coated
slides (1% gelatin, heated at 60.degree. C. for 1-2 min., air
dried, 1% formaldehyde at room temp., air dried), air dried 30
minutes, fixed in acetone for ten minutes at 4.degree. C., washed
three times in PBS and treated with 0.3% H.sub.20.sub.2 in methanol
(30 min., room temp.). The slides were then washed with PBS for 30
minutes, incubated with dilute normal human serum (1:100), and
incubated with anti-VCAM-1 antibody 4B9 (1:100; obtained as a gift
from Dr. John Harlan) for 60 minutes at room temperature. Control
slides were incubated with an anti-bovine serum albumin (anti-BSA)
antibody (Sigma Chemical Co., St. Louis Mo.). The samples were then
washed with PBS for 10 minutes and incubated with a secondary
biotinylated rabbit anti-mouse immunoglobulin (Dako Corp., Santa
Barbara, Calif.) for 60 minutes at room temperature, then
visualized using avidin-linked peroxidase (VECTASTAIN, Vector Labs,
Burlingame Calif.).
[0050] The results of these tests are set forth in the following
TABLE I: TABLE-US-00001 TABLE I Endothelial Cell Staining In Human
Tissue VCAM-1 Expression Tissue (n) n (%) Normal (11) 6 (54.4) UC
active (23) 14 (60.9) UC inactive (8) 5 (62.5) UC uninvolved (10) 4
(40.0) CD active (9) 5 (55.5) CD uninvolved (12) 7 (58.3)
[0051] These data confirm the observations such as those reported
by Freedman et al. [6] and Rice et al. [7] that VCAM-1 is expressed
in both IBD-involved colon tissue and in normal colon tissue. In
both CD and UC tissues, VCAM-1 was observed by immunocytochemistry
in about 60% of samples.
EXAMPLE II
Anti-VLA-4 Antibody Recognition of CTT White Blood Cells
[0052] An anti-VLA-4 monoclonal antibody (HP1/2, obtained from
Biogen, Inc., Cambridge Mass.) was tested to confirm that it
recognized an epitope on CTT leukocytes.
[0053] Blood samples (3 ml) from CTTs were heparinized and the CTT
peripheral blood mononuclear leukocytes (PBLs) were isolated using
a Ficoll-Hypaque gradient (Pharmacia) according to the
manufacturer's instructions for isolation of human PBLs. CTT PBLs
were examined for their ability to bind to the murine anti-human
VLA-4 monoclonal antibodies HP1/2 and HP2/1 by FACS analysis using
a Becton Dickenson FACStar and standard techniques (see, e.g., Lobb
et al., 1991a [31]). Both monoclonal antibodies bound to CTT PBLs,
indicating that both human and CTT VLA4 have similar epitopes
recognized by these two antibodies.
[0054] CTT PBLs were also observed to adhere to microtiter plates
coated with immobilized recombinant soluble human VCAM-1 (Biogen,
Inc.), which binding was blocked by HP1/2 and HP2/1. These results
show that CTT PBLs bind to VCAM-1 in a VLA-4-dependent manner, and
that HP1/2 and HP2/1 block the interaction of CTT VLA-4 with human
VCAM-1. (Cf. Lobb et al., 1991b [32].)
EXAMPLE III
Cotton Top Tamarin Trials
[0055] A stock solution in sterile saline of the Anti-VLA-4
antibody, HP1/2 (IgG1), and a placebo control (saline only), were
prepared for administration to ten cotton top tamarins (CTTs)
exhibiting symptoms of spontaneous colitis (i.e., diarrhea, etc.;
see, Madara et al. [18]). Five CTTs received HP1/2 and five
received placebo, by intravenous injection. The CTTs receiving
HP1/2 were injected with 1 mg HP1/2 per day (i.e., about 2
mg/kg/day, based on approximate half-kilogram weight of a CTT) for
eight days (on Days 0, 1, 2, 3, 4, 5, 6, and 7 of the trial). Colon
tissue samples obtained from the animals were biopsied every other
day (on Days 0, 2, 4, 6, 8, and 10 of the trial).
[0056] Data from the biopsies were used to determine an acute
inflammation index for each animal, giving a semi-quantitative
analysis of the course of the colitis. (See, Madara et al. [18].)
The inflammation indices before the trial began (Day 0) and at the
end of the trial at Day 10 are set forth in Table II, below:
("Treated CTTs" received antibody HP1/2; "Control CTTs" received
placebo) TABLE-US-00002 TABLE II Day O Day 10 AII* AII Treated CTTs
1 2 0 2 1 0 3 1 0 4 2 0 5 2 1 MEAN 1.6 0.2 Control CTTs C1 2 0 C2 2
1 C3 1 1 C4 2 2 C5 2 2 MEAN 1.8 1.2 *AII = acute inflammation
index
[0057] These results show that treatment with anti-VLA4 antibody
resulted in a significant (p<0.01) decrease in acute
inflammation index.
EXAMPLE IV
[0058] The trial described in Example III was repeated using 14
CTTs, 7 receiving HP1/2 and 7 receiving placebo. The change in
acute inflammation index from Day 0 to Day 10 is set forth in Table
III: TABLE-US-00003 TABLE III Day 0 Day 10 AII AII Treated CTTs 6 2
0 7 2 0 8 2 0 9 2 0 10 2 0 11 2 1 12 2 2 MEAN 2.0 0.43 Control CTTs
C6 2 2 C7 2 2 C8 1 1 C9 2 1 C10 2 1 C11 2 0 C12 1 0 MEAN 1.71
1.00
[0059] The foregoing results show a significant decrease in acute
inflammation in the CTTs receiving HP1/2.
EXAMPLE V
[0060] VCAM 2D-IgG is a soluble form of the ligand for VLA4 (VCAM1)
which consists of the two N-terminal domains of VCAM1 fused to the
human IgG1 heavy chain constant region sequences (Hinges, C.sub.H2
and C.sub.H3). The VCAM 2D-IgG DNA sequence and its translated
amino acid sequence are shown in SEQ ID NO: 5 and SEQ ID NO:12,
respectively. In other systems, administration of this fusion
peptide has been shown to have effects which are similar to the
administration of anti-VLA-4 monoclonal antibody. FIG. 1
illustrates the fusion protein structure. The fusion protein was
constructed by recombinant techniques as described below.
Isolation of cDNA of Human IgG1 Heavy Chain
Region and Construction of Plasmid pSAB144
[0061] In order to isolate a cDNA copy of the human IgG1 heavy
chain region, RNA was prepared from COS7 cells which has been
transiently transfected by the plasmid VCAM1-IgG1(also known as
pSAB133). Construction of plasmid VCAM1-IgG1 is described in PCT
patent application WO 90/13300. The RNA was reverse transcribed to
generate cDNA using reverse transcriptase and random hexamers as
the primers. After 30 min. at 42.degree. C., the reverse
transcriptase reaction was terminated by incubation of the reaction
at 95.degree. C. for 5 min. The cDNA was then amplified by PCR
(Polymerase Chain Reaction, see, e.g., Sambrook et al., Molecular
Cloning, Vol. 3, pp. 14.1-14.35 (Cold Spring Harbor; 1989) [34])
using the following kinased primers: 370-31 (SEQ ID NO: 6 and SEQ
ID NO:13): TABLE-US-00004 5'TCGTC GAC AAA ACT CAC ACA TGC C Asp Lys
Thr His Thr Cys
[0062] which contains a SalI site, and 370-32 (SEQ ID NO: 7):
TABLE-US-00005 5' GTAAATGAGT GCGGCGGCCG CCAA,
which encodes the carboxy terminal lysine of the IgG1 heavy chain
constant region, followed by a NotI site.
[0063] The PCR amplified cDNA was purified by agarose gel
electrophoresis and glass bead elution for cloning in plasmid
pNN03. Plasmid pNN03 was constructed by removing the synthetic
polylinker sequence from the commercially available plasmid pUC8
(Pharmacia, Piscataway, N.J.) by restriction endonuclease digestion
and replacing the synthetic polylinker sequence with the following
novel synthetic sequence (SEQ ID NO: 8): TABLE-US-00006 GCGGCCGCGG
TCCAACCACC AATCTCAAAG CTTGGTACCC GGGAATTCAG ATCTGCAGCA TGCTCGAGCT
CTAGATATCG ATTCCATGGA TCCTCACATC CCAATCCGCG GCCGC.
[0064] The purified PCR amplified cDNA fragment was ligated to
pNN03 which had been cleaved with EcoRV, dephosphorylated, and
purified by low melt agarose gel electrophoresis. The ligation
reaction was used to transform E. coli JA221 and the resulting
colonies were screened for a plasmid containing an insert of
approximately 700 bp. The identity of the correct insert was
confirmed by DNA sequence analysis, and the plasmid was designated
pSAB144.
Construction of Plasmid pSAB142
[0065] The plasmid pSAB142 was constructed as follows. cDNA
prepared from COS cells transfected with pSAB133 (as described in
the previous section) was subjected to PCR amplification using
oligonucleotides 370-01 and 370-29. Oligonucleotide 370-01 includes
a NotI site and the nucleotides corresponding to amino acids 1
through 7 of the VCAM-1 signal sequence (SEQ ID NO:9 and SEQ ID
NO:14): TABLE-US-00007 5' GAGCTCGAGG CGGCCGCACC ATG CCT GGG AAG ATG
GTC Met Pro Gly Lys Met Val GTG Val
[0066] Oligonucleotide 370-29 corresponds to the VCAM-1 amino acids
214-219 and includes a SalI site (SEQ ID NO: 10): TABLE-US-00008
5'AA GTC GAC TTG CAA TTC TTT TAC
The amplified DNA fragment was ligated to the vector fragment of
pNN03, cleaved by EcoRV. Construction of pSAB 132
[0067] pJOD-S (Barsoum, J., DNA and Cell Biol., 9, pp.293-300
(1990) [35]) was modified to insert a unique NotI site downstream
from the adenovirus major late promoter so that NotI fragments
could be inserted into the expression vector. pJOD-S was linearized
by NotI cleavage of the plasmid DNA. The protruding 5' termini were
blunt-ended using Mung Bean nuclease, and the linearized DNA
fragment was purified by low melting temperature agarose gel
electrophoresis. The DNA fragment was religated using T4 DNA
ligase. The ligated molecules were then transformed into E. coli
JA221. Colonies were screened for the absence of a NotI site. The
resulting vector was designated pJOD-S delta Notl. pJOD-8 delta
Notl was linearized using SalI and the 5' termini were
dephosphorylated using calf alkaline phosphatase. The linearized
DNA fragment was purified by low melting temperature agarose gel
electrophoresis and ligated in the presence of phosphorylated
oligonucleotide ACE175, which has the following sequence
TABLE-US-00009 (SEQ ID NO: 11): TCGACGCGGC CGCG
[0068] The ligation mixture was transformed into E. coli JA221, and
colonies were screened for the presence of a plasmid having a NotI
site. The desired plasmid was named pMDR901.
[0069] In order to delete the two SV40 enhancer repeats in the Sv40
promoter which controls transcription of the DHFR cDNA, pMDR901 and
pJOD.DELTA.e-tPA (Barsoum, DNA and Cell Biol., 9, pp. 293-300
(1990) [35]), both were cleaved with AatII and DraIII. The 2578 bp
AatII-DraIII fragment from pMDR901 and the 5424 bp AatII-DraIII
fragment from pJOD.DELTA.e-tPA were isolated by low melting
temperature agarose gel electrophoresis and ligated together.
Following transformation into E. coli JA221, the resulting plasmid,
pMDR902, was isolated. pSAB132 was then formed by eliminating the
EcoRI-NotI fragment of pMDR902 containing the adenovirus major late
promoter and replacing it with an 839 bp EcoRI-NotI fragment from
plasmid pCMV-B (Clontech, Palo Alto, Calif.) containing the human
cytomegalovirus immediate early promoter and enhancer.
Construction of pSAB146
[0070] pSAB144 was cleaved with SalI and NotI, and the 693 bp
fragment isolated. pSAB142 was cleaved with NotI and SalI and the
664 bp fragment was isolated. The two fragments were ligated to
pSAB132 which had been cleaved with NotI, and the 5' termini
dephosphorylated by calf alkaline phosphatase. The resulting
plasmid, pSAB146, contained the DNA sequence encoding the VCAM-1
signal sequence, the amino terminal 219 amino acids of mature
VCAM-1, ten amino acids of the hinge region of IgG1 and the CH2 and
CH3 constant domains of IgG1.
Production of VCAM 2D-IgG from a Stably Transformed CHO Cell
Line
[0071] A recombinant VCAM 2D-IgG expression vector was constructed
as described below and transfected into CHO cells to produce a cell
line continuously secreting VCAM 2D-IgG.
[0072] The 1.357 kb NotI fragment containing the VCAM 2D-IgG coding
sequence of pSAB146 was purified by agarose gel electrophoresis.
This fragment was ligated into the NotI cloning site of the
expression vector pMDR901, which uses the adenovirus 2 major late
promoter for heterologous gene expression and the selectable,
amplifiable dihydrofolate reductase (dhfr) marker. The ligated DNA
was used to transform E. coli DH5. Colonies containing the plasmid
with the desired, correctly oriented insert were identified by the
presence of 5853 and 3734 bp fragments upon digestion with HindIII;
and 4301, 2555, 2293, and 438 bp fragments upon digestion with
BglII. The resultant recombinant VCAM 2D-IgG expression vector was
designated pEAG100. The identity of the correct insert was
confirmed by DNA sequence analysis.
[0073] The recombinant expression plasmid pEAG100 was
electroporated into dhfr-deficient CHO cells according to the
published protocol of J. Barsoum (DNA Cell Biol 9: 293-300, 1990
[35]), with the following changes: 200 .mu.g of PvuI-linearized
pEAG100 plasmid and 200 .mu.g of sonicated salmon sperm DNA were
used in the electroporation protocol. In addition, cells were
selected in alpha-complete medium supplemented with 200 nM
methotrexate.
[0074] To determine expression levels of secreted VCAM 2D-IgG,
clones were transferred to a flat bottom 96 well microtiter plate,
grown to confluency and assayed by ELISA as described below.
[0075] Wells of Immulon 2 plates (Dynatech, Chantilly, Va.) were
each coated with anti-VCAM MAb 4B9 (isolated and purified on
Protein A Sepharose as described by Carlos et al, 1990 [56]) with
100 .mu.l of anti-VCAM 4B9 MAb diluted to 10 .mu.g/ml in 0.05 M
sodium carbonate/bicarbonate buffer, pH 9.6, covered with Parafilm,
and incubated overnight at 4.degree. C. The next day, the plate
contents were dumped out and blocked with 200 .mu.l/well of a block
buffer (5% fetal calf serum in 1.times.PBS), which had been
filtered through a 2 filter. The buffer was removed after a 1 hour
incubation at room temperature and the plates were washed twice
with a solution of 0.05% Tween-20 in 1.times.PBS. Conditioned
medium was added at various dilutions. As a positive control, an
anti-mouse Ig was also included. Block buffer and LFA-3TIP
constituted as negative controls. The samples and controls were
incubated at room temperature for 2 hours.
[0076] The plates were then washed twice with a solution of 0.05%
Tween-20 in 1.times.PBS. Each well, except for the positive control
well, was then filled with 50 .mu.l of a 1:2000 dilution of
HRP-Donkey anti-human IgG (H+L) (Jackson Immune Research
Laboratories, Inc.; West Grove, Pa.) in block buffer. The positive
control well was filled with 50 .mu.l of a 1:2000 dilution of
HRP-Goat anti-mouse IgG (H+L)(Jackson Immune Research Laboratories,
Inc.; West Grove, Pa.) in block buffer. The plates were then
incubated for 1 hour at room temperature.
[0077] The HRP conjugated Ab solutions were removed, and the wells
were washed twice with 0.05% Tween-20 in 1.times.PBS. Then, 100
.mu.l of HRP-substrate buffer was added to each well at room
temperature. HRP-substrate buffer was prepared as follows: 0.5 ml
of 42 mM 3,3', 5,5'-tetramethylbenzidine (TMB), (ICN
Immunobiologicals, Lisle, S.C., Catalogue No. 980501) in DMSO
(Aldrich) was slowly added to 50 ml of substrate buffer (0.1 M
sodium acetate/citric acid, pH 4.9); followed by addition of 7.5
.mu.l of 30% hydrogen peroxide (Sigma, Catalogue No. H-1009).
[0078] The development of a blue color in each well was monitored
at 650 nm on a microtiter plate reader. After 7-10 minutes, the
development was stopped by the addition of 100 .mu.l of 2N Sulfuric
acid. The resulting yellow color was read at 450 nm on a microtiter
plate reader. A negative control well was used to blank the
machine.
Purification of VCAM 2D-IgG
[0079] CHO cells expressing VCAM 2D-IgG were grown in roller
bottles on collagen beads. Conditioned medium (5 Liters) was
concentrated to 500 ml using an Amicon S1Y10 spiral ultrafiltration
cartridge (Amicon, Danvers, Mass.). The concentrate was diluted
with 1 liter of Pierce Protein A binding buffer (Pierce, Rockford,
Ill.) and gravity loaded onto a 10 ml Protein A column (Sepharose 4
Fast Flow, Pharmacia, Piscataway, N.J.). The column was washed 9
times with 10 ml of Protein A binding buffer and then 7 times with
10 ml of PBS. VCAM 2D-IgG was eluted with twelve-5 ml steps
containing 25 mM H.sub.3PO.sub.4 pH 2.8, 100 mM NaCl. The eluted
samples were neutralized by adding 0.5 M Na.sub.2HPO.sub.4 pH 8.6
to 25 mM. Fractions were analyzed for absorbance at 280 nm and by
SDS-PAGE. The three peaks fractions of highest purity were pooled,
filtered, aliquoted and stored at -70.degree. C. By SDS-PAGE, the
product was greater than 95% pure. The material contained less than
1 endotoxin unit per mg of protein. In some instances, it was
necessary to further purify the Protein A eluate product on
Q-Sepharose FF (Pharmacia). The protein A eluate was diluted with 3
volumes of 25 mM Tris HCl pH 8.0 and loaded onto a Q-Sepharose FF
column at 10 mg VCAM 2D-IgG per ml of resin. The VCAM 2D-IgG was
then eluted from the Q-Sepharose with PBS.
Evaluation of VCAM 2D-IgG
[0080] The following examples (Example VI-VIII) have been included
to provide evidence that the VCAM-Ig parallels the efficacy of
anti-.alpha.4 monoclonal antibodies in vivo in three out of three
models studied below. Two murine models, contact hypersensitivity
model and NOD diabetic mouse model have been tested with the
VCAM-Ig fusion protein, as well as the sheep asthma model. The
sheep asthma model is quite distinct from the mouse models in that:
1) it is a different species; 2) a different organ (lung, versus
skin or pancreas); 3) the leukocytes targeted are unknown (i.e. not
clearly T cell dependent classical immune reaction); 4) aerosol
versus i.v./i.p. administration; 5) mAbs used are different
(anti-murine-.alpha.4 mAbs PS/2 and R1/2 in mice versus
anti-human-.alpha.4 mAb HP1/2 in sheep). Therefore, using the
correlation between VCAM-Ig and the anti-.alpha.4 mAbs provided by
the examples below, strong evidence has been generated that the
VCAM-Ig fusion protein would be effective in the treatment of IBD
because the anti-human-.alpha.4 mAb HP1/2 has been proven effective
in the cotton top tamarin trials described above.
EXAMPLE VI
The Effect of VCAM-Ig on the Murine Contact Hypersensitivity
Response
[0081] The experiments described below can be performed essentially
as follows.
[0082] BALB/c mice (purchased from Taconic Farms, Germantown, N.Y.)
were sensitized to 2,4-Dinitrofluorobenzene (DNFB)(Aldrich Chemical
Co., Wis.) by the application of 25 .mu.g of 0.5% DNFB in an
acetone/olive oil vehicle (4:1 v/v) on day 0 and again on day 1 to
the shaved abdominal skin (see, Current Protocols in Immunology,
John Wiley and Sons, NY 1001, Section 4.2). Mice were sensitized to
oxazalone by the application of 150 .mu.l of 3.0% oxazalone in 100%
ethanol to the shaved abdominal skin on day 0 (see, Current
Protocols in Immunology, John Wiley and Sons, NY 1001, Section
4.2). For the day 0 sensitization procedure the mice were lightly
anesthetized with avertin (0.010-0.015 ml/h body weight of 2.5%
avertin stock, intraperitoneal). Immediately prior to challenge,
baseline ear thickness measurements were recorded in units of
10.sup.-4 inches, for both ears of all of the mice, using a
Mitutoyo engineer's micrometer (see, Current Protocols in
Immunology, John Wiley and Sons, NY 1001, Section 4.2). All
measurements were done in quadruplicate and summarized as mean
thickness.+-.SEM. The DNFB response was elicited on day 5 by
skin-painting the dorsal surface of the left ear with 10 .mu.l of
0.2% DNFB in 4:1 acetone/olive oil. The dorsal surface of the right
(control) ear was painted with 10 .mu.l of vehicle only and was a
specificity control for the reaction. The oxazalone response was
elicited on day 6 by painting the dorsal surface of the left ear
with 20 .mu.l of 1% oxazalone in ethanol. The right ear was painted
with 20 .mu.l of ethanol. The CHS response was assessed by
obtaining 24-h ear thickness measurements. The average change in
left ear thickness (.DELTA.T) for each group was determined by
substituting baseline ear thickness from 24-h ear thickness. The
immunomodulatory effect of the antibody treatment was evaluated by
calculating the percentage inhibition as follows: %
Inhibition=[1-.DELTA.T treatment/.DELTA.T control].times.100%.
[0083] VCAM-IgG was administered to mice in varying intraperitoneal
doses, 4-6 hours prior to challenge. VCAM-IgG was administered in
pyrogen-free, azide-free PBS. As shown in FIG. 2, VCAM-IgG tested
over a dose range of 1.25 to 20 mg/kg (25-400 .mu.g/mouse)
maximally inhibited the mouse ear swelling response about 4/5 as
well as the PS/2 antibody, which is a IgG2b rat anti-mouse
.alpha.-4 antibody (anti-VLA4 antibody). Maximal inhibition was
observed at the 10 mg/kg dose. The ED50 was about 2.5 mg/kg, i.p.
(50 .mu.g/mouse).
[0084] In a confirmation study (FIG. 3), VCAM-IgG tested at 20
mg/kg i.p. (400 .mu.g/mouse) maximally inhibited the mouse ear
swelling response as effectively as the PS/2 8 mg/kg i.v. antibody
dose.
[0085] In summary, VCAM-IgG at optimal dose can block the skin CHR
response by about 40-50%, just as well as does an optimal dose of
the PS/2 antibody.
EXAMPLE VII
The Effect of VCAM-Ig Treatment on Adoptive Transfer of
Diabetes
[0086] For the adoptive transfer of diabetes experiments, NOD mice
were obtained from Taconic Farms (Germantown, N.Y.) or from the
Joslin Diabetes Center (Boston, Mass.). Spontaneously diabetic (D)
females of recent onset (13-20 weeks of age) were used as spleen
cell donors and 8 week old nondiabetic (Y) females served as
recipients. Spleen cells from 4 week old nondiabetic (Y) female
donors which fail to transfer disease were used as a negative
control.
[0087] Recipient mice were placed on acidified water (1:8400
dilution of concentrated HCl in water) one week prior to sublethal
irradiation (775 rad) performed in split doses (300 rad, 300 rad,
and 175 rad) on each of three days (day -2, -1, and the day of
transfer), in order to minimize any incidence of intestinal
infection subsequent to high dose irradiation (Gamma Cell 1000
Cesium.sup.137 source, Nordion International, Inc., Ontario,
Canada). Spleens were harvested from diabetic donors or from
nondiabetic controls, cell suspensions made and red cells lysed
with Hemolytic Geys solution. Spleen cells were injected
intravenously (2-3.times.10.sup.7 in 0.2 ml PBS) and were
pretreated with either 100 .mu.g VCAM 2D-IgG or 100 .mu.g of
irrelevant LFA-3Ig fusion protein control. Another group received
PBS alone without cells transferred. The fusion protein LFA-3Ig
(LFA-3TIP) was isolated and purified as described in PCT US92/02050
and Miller et al., 1993 [36]. The VCAM 2D-IgG fusion protein or
irrelevant LFA-3Ig protein was administered at a dose of 100
.mu.g/0.2 ml intraperitoneally twice weekly through day 17. This
concentration was sufficient to provide a serum level of fusion
protein sufficient to saturate VLA4-positive cells, the serum
levels determined by ELISA as described above. Diabetes onset was
monitored as described above.
[0088] The results of the evaluation are shown in FIG. 4. As shown
in this Figure, VCAM 2D-IgG fusion protein significantly inhibits
the onset of diabetes in recipients of cells from diabetic donor
mice (D/VCAM-Ig, open circles) with 60% incidence by day 30
post-transfer, as compared to the mice which received cells from
diabetic donor (data not shown) and LFA-3Ig irrelevant control Ig
fusion protein (D/LFA-3 Ig) which had already achieved 60%
incidence by day 15 post-transfer. Mice which received no cells
(PBS only) did not develop disease. There were n=5 mice per
experimental group.
EXAMPLE VIII
The Effect of VCAM-Ig Treatment on the Sheep Model of Airways
Hvper-Responsiveness
[0089] Experiments were performed essentially as described by
Abraham et al. [33]. Briefly, allergic sheep having natural
allergic cutaneous reaction to 1:1000 or 1:10,000 dilutions of
Ascaris suum extract (Greer Diagnostics, Lenoir N.C.) were tested,
and sheep demonstrating both early and late phase airway response
("dual responders") to inhalation challenge with Ascaris suum
antigen were selected. To measure breathing mechanics and physical
changes in the airways, the sheep were restrained in a prone
position with heads immobilized. A balloon catheter was advanced
through one nostril under topical anesthesia with 2% lidocaine
solution to the lower esophagus, and a cuffed endotracheal tube was
advanced through the other nostril (using a flexible fiberoptic
bronchoscope as a guide) for the measurement of airway mechanics
and during aerosol challenges. Pleural pressure was estimated with
the esophageal balloon catheter (filled with 1 ml of air)
positioned 5-10 cm from the gastroesophageal junction. In this
position, end expiratory pleural pressure ranged between -2 and -5
cm H.sub.2O. Once the balloon was placed, it was secured so that it
remained in position for the duration of the experiment. Lateral
pressure in the trachea was measured with a sidehole catheter,
(inner diam. 2.5 mm) advanced through and positioned distal to the
tip of the endotracheal tube. Transpulmonary pressure (the
difference between tracheal and pleural pressure) was measured with
a differential pressure transducer catheter system (MP45, Validyne,
Northridge, Calif.). The pressure transducer catheter system showed
no phase shift between pressure and flow to a frequency of 9
H.sub.Z. Pulmonary resistance (R.sub.L) was measured by connecting
the proximal end of the endotracheal tube to a Fleich
pneumotachograph (Dyna Sciences, Blue Bell Pa.). Signals indicating
flow and transpulmonary pressure were recorded on an oscilloscope
recorder (Model DR-12; Electronics for Medicine, White Plains,
N.Y.) linked to a computer for automatic calculation of pulmonary
resistance (R.sub.L) from transpulmonary pressure, respiratory
volume (obtained by digital integration) and flow by the mid-volume
technique, analyzed from 5-10 breaths. Thoracic gas volume
(V.sub.tg) was measured immediately after determination of R.sub.L
in a constant volume body plethysmograph. Specific lung resistance
(SR.sub.L) was calculated from these values
(SR.sub.L=V.sub.tg.times.R.sub.L).
[0090] Airway responsiveness was determined by performing dose
response curves to inhaled carbachol. The dose response curves were
plotted using measurements of SR.sub.L taken immediately after
inhalation of buffer (PBS) alone and after each consecutive
administration of 10 breaths of increasing concentrations of
carbachol in PBS. The concentrations of carbachol were 0.25%, 0.5%,
1.0%, 2.0% and 4.0% wt/vol in PBS. The provocation test was
discontinued when SR.sub.L increased over 400% from the post-PBS
value or after the highest carbachol concentration had been
administered. Airway responsiveness was determined by calculating
from the dose response curves the cumulative carbachol dose in
breath units (BU) that increased specific lung resistance 400% over
the post buffer value (PD.sub.400%). One breath unit was defined as
one breath of a 1% wt/vol carbachol solution. Thus, the greater the
suppression of airway hyper-responsiveness, the greater the number
of breath units would be required before observing the same
bronchoconstriction as seen in the controls.
[0091] Each sheep was subjected to a trial as a control in which a
placebo (PBS without additive) was used as a pretreatment 30
minutes before allergen challenge with Ascaris suum antigen (Greer
Diagnostics, Lenoir, N.C.). The antigen solution was delivered as
an aerosol using a disposable medical nebulizer (RAINDROP.RTM.,
Puritan Bennett, Lenexa, Kans.) that provided an aerosol with a
mass median aerodynamic diameter of 3.2 .mu.M (geometric SD 1.9) as
determined by an Andersen cascade impactor. The Ascaris suum
extract was diluted in PBS to a concentration of 82,000 Protein
Nitrogen Units(PNU)/ml. The output of the nebulizer was directed
into a plastic T-tube, one end of which was connected to the
inspiratory port of a Harvard respirator. A dosimeter connected to
the nebulizer consisting of a solenoid valve and a 20 psi
compressed air source and the solenoid valve was activated at the
beginning of the inspiratory cycle of the Harvard respirator for
one second. The aerosol delivered at a tidal volume of 500 ml and a
rate of 20 breaths per min. for 20 min. Each sheep was challenged
with an equivalent dose of antigen (400 breaths) in the control and
VCAM1-IgG1 trials. Carbachol aerosols for the dose response curves
were also generated by nebulizer as described above.
[0092] An airway challenge trial using five pairs of responder
allergic sheep was conducted in order to investigate the efficacy
of VCAM1-IgG1 (VCAM-Ig) fusion protein in the sheep model of
airways hyper-responsiveness. The efficacy of the aerosol delivery
of the VCAM-Ig was investigated. VCAM-Ig was delivered via
nebulizer in the form of an aerosol.
[0093] In order to optimize therapeutic efficacy, aerosolized
VCAM-Ig was first administered at different dosing regimens. These
experiments are summarized in FIGS. 5-9. In all the experiments,
the control sheep received placebo. In the first experiment, two
animals were given 30 mg VCAM-Ig (1 mg/kg) at 30 minutes prior to
antigen challenge, which is the standard time used for other
therapeutic agents. Under these conditions significant but partial
inhibition of the late phase response (LPR) but no effect on
airways hyper-responsiveness (AHR) was observed (FIG. 5). This
result was not surprising as VCAM-Ig was found previously to be a
little less potent than mAb HP1/2. In the second experiment,
therefore, the dose of VCAM-Ig was increased to 60 mg. This dose
resulted in the partial blockage of LPR as in the previous
experiment, but now AHR was blocked too (FIG. 6). However, due to
the serious problems which resulted from attempting to aerosolize
such a large volume, in subsequent experiments dosages were
administered at different time intervals. In the third experiment,
30 mg of VCAM-Ig were administered at 30 minutes prior to and 8
hours after antigen challenge. Here the LPR was blocked completely
but no blockage of the AHR was observed (FIG. 7). With respect to
the LPR this represented a single dose 30 minutes prior to antigen
challenge (equivalent to experiment 1) because the second dose at 8
hours was given after the LPR was largely over. In the fourth
experiment, 15 mg of VCAM-Ig were administered at 2, 8 and 24
hours. Here partial blockage of the LPR and blockage of the AHR was
observed (FIG. 8). In the final experiment, 30 mg of VCAM-Ig were
administered at 2 and 24 hours and resulted in complete blockage of
both the LPR and AHR (FIG. 9). This optimal dosage was tested on
four animals with the same result.
[0094] In summary, ten animals have all shown partial or complete
inhibition of the LPR versus a placebo control, and complete
inhibition of both the LPR and AHR can be achieved under optimal
conditions (30 mg of VCAM-Ig administered at 2 and 24 hours after
antigen challenge).
[0095] The foregoing examples are intended as an illustration of
the method of the present invention and are not presented as a
limitation of the invention as claimed hereinafter. From the
foregoing disclosure, numerous modifications and additional
embodiments of the invention will be apparent to those experienced
in this art. For example, actual dosage used, the type of antibody,
antibody fragment or analog used, mode of administration, exact
composition, time and manner of administration of the treatment,
and many other features all may be varied without departing from
the above description. All such modifications and additional
embodiments are within the contemplation of this application and
within the scope of the appended claims.
Cited Publications
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Colitis: Potential Meaning in Heterogeneity, " Inflammatory Bowel
Diseases: Basic Research and Clinical Implications, Falk Symposium,
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"Alterations in Mucosal Content of Colonic Glycoconjugates in
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D. Fournier, "Emergence of Antigenic Glycoprotein Structures in
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"Vascular and Nonvascular Expression of INCAM-110, Amer. J.
Pathology, 138(2), pp. 385-93 (1991). [0103] [8] L. Burkly, et al.,
"Signaling by Vascular Cell Adhesion Molecule-1 (VCAM-1) Through
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Aruffo, "Vascular Cell Adhesion Molecule 1 Induces T-cell Antigen
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Natl. Acad. Sci. USA, 88, pp. 6403-7 (1991). [0105] [10] G. van
Seventer et al., "Analysis of T Cell Stimulation by Superantigen
Plus Major Histocompatibility Complex Class II Molecules or by CD3
Monoclonal Antibody: Costimulation by Purified Adhesion Ligands
VCAM-1, ICAM-1, but Not ELAM-1," J. Exp. Medicine, 174, pp. 901-13
(1991). [0106] [11] Kohler and Milstein, "Continuous Cultures of
Fused Cells Secreting Antibody of Predefined Specificity," Nature,
256, pp. 495-7 (1975). [0107] [12] F. Sanchez-Madrid et al.,
"VLA-3: A novel polypeptide association within the VLA molecular
complex: cell distribution and biochemical characterization," Eur.
J. Immunol., 16, pp. 1343-9 (1986). [0108] [13] M. E. Hemler et
al., "Characterization of the Cell Surface Heterodimer VLA-4 and
Related Peptides," J. Biol. Chem., 262(24), pp. 11478-85 (1987).
[0109] [14] M. Elices et al., "VCAM-1 on Activated Endothelium
Interacts with the Leukocyte Integrin VLA-4 at a Site Distinct from
the VLA-4/Fibronectin Binding Site," Cell, 60, pp. 577-84 (1990).
[0110] [15] R. Pulido et al., "Functional Evidence for Three
Distinct and Independently Inhibitable Adhesion Activities Mediated
by the Human Integrin VLA-4," J. Biol. Chem., 266(16), pp. 10241-5
(1991). [0111] [16] D. Podolsky, et al., "Colonic Mucin Composition
in Primates Selective Alterations Associated with Spontaneous
Colitis in the Cotton-top Tamarin," Gastroenterology, 88, pp. 20-5
(1985). [0112] [17] D.-Podolsky et al., "Spontaneous Colitis In
Cotton-Top Tamarins: Histologic, Clinical and Biochemical Features
of an Animal Model of Chronic Colitis," Digestive Diseases and
Sciences, 30(4), Abstract, p. 396 (1985). [0113] [18] J. Madara et
al., "Characterization of Spontaneous Colitis in Cotton-Top Tamarin
(Saguinus oedipus) and Its Response to Sulfasalazine,"
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al., "Replacing the Complementarity-Determining Regions in a Human
Antibody with Those From a Mouse," Nature, 321, pp. 522-25 (1986).
[0115] [20] E. Ward et al., "Binding Activities of a Repertoire of
Single Immunoglobulin Variable Domains Secreted From Escherichia
coli," Nature, 341, pp. 544-6 (1989). [0116] [21] U.S. Pat. No.
4,816,397, Boss et al., "Multichain Polypeptides or Proteins And
Processes For Their Production", issued Mar. 28, 1989. [0117] [22]
L. Riechmann et al., "Reshaping Human Antibodies for Therapy,"
Nature, 332, pp. 323-7 (1988). [0118] [23] Man Sun Co et al.,
"Humanized Antibodies for Antiviral Therapy," Proc. Natl. Acad.
Sci. USA, 88, pp. 2869-73 (1990). [0119] [24] P. Brown, Jr. et al.,
"Anti-Tac-H, a Humanized Antibody to the Interleukin 2 Receptor,
Prolongs Primate Cardiac Allograft Survival," Proc. Natl. Acad.
Sci. USA, 88, pp. 2663-7 (1990). [0120] [25] T. Clackson et al.,
"Making Antibody Fragments Using Phage Display Libraries," Nature,
352, pp. 624-28 (1991). [0121] [26] L. Osborn et al., "Direct
Expression Cloning of Vascular Cell Adhesion Molecule 1, a
Cytokine-induced Endothelial Protein That Binds to Lymphocytes,"
Cell, 59, pp. 1203-11 (1989). [0122] [27] J. Devlin et al., "Random
Peptide Libraries: A Source of Specific Protein Binding Molecules,"
Science, 249, pp. 400-406 (1990). [0123] [28] J. Scott and G.
Smith, "Searching for Peptide Ligands with an Epitope Library,"
Science, 249, pp. 386-90 (1990). [0124] [29] U.S. Pat. No.
4,833,092, Geysen, "Method For Determining Mimotopes", issued May
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Report: Cyclosporin in Treatment of Severe Active Ulcerative
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al., "Expression and Functional Characterization of a Soluble Form
of Endothelial-Leukocyte Adhesion Molecule 1," J. Immunol., 147(1),
pp. 124-29 (1991). [0127] [32] R. Lobb et al., "Expression and
Functional Characterization of a Soluble Form of Vascular Cell
Adhesion Molecule 1," Biochem. Biophys. Res. Commun., 178(3), pp.
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Markers of Inflammation in the Airways of Allergic Sheep with and
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138, 1565-1571 (1988) [0129] [34] Sambrook et al., "Polymerase
Chain Reaction", Molecular Cloning, 3, 370-31 (1989) [0130] [35] J.
Barsoum, DNA and Cell Biol., 9, 293-300 (1990) [0131] [36] Miller
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[0132] The foregoing documents are incorporated herein by
reference.
Sequence CWU 1
1
17 1 363 DNA Mus musculus CDS (1)...(363) misc_feature (1) /note=
"pBAG159 insert HP1/2 heavy chain variable regions;amino acid 1 is
Glu (E) but Gln (Q) may be substituted as shown in SEQ ID NO15 and
16" 1 gar gtc aaa ctg cag cag tct ggg gca gag ctt gtg aag cca ggg
gcc 48 Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly
Ala 1 5 10 15 tca gtc aag ttg tcc tgc aca gct tct ggc ttc aac att
aaa gac acc 96 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile
Lys Asp Thr 20 25 30 tat atg cac tgg gtg aag cag agg cct gaa cag
ggc ctg gag tgg att 144 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln
Gly Leu Glu Trp Ile 35 40 45 gga agg att gat cct gcg agt ggc gat
act aaa tat gac ccg aag ttc 192 Gly Arg Ile Asp Pro Ala Ser Gly Asp
Thr Lys Tyr Asp Pro Lys Phe 50 55 60 cag gtc aag gcc act att aca
gcg gac acg tcc tcc aac aca gcc tgg 240 Gln Val Lys Ala Thr Ile Thr
Ala Asp Thr Ser Ser Asn Thr Ala Trp 65 70 75 80 ctg cag ctc agc agc
ctg aca tct gag gac act gcc gtc tac tac tgt 288 Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca gac gga
atg tgg gta tca acg gga tat gct ctg gac ttc tgg ggc 336 Ala Asp Gly
Met Trp Val Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly 100 105 110 caa
ggg acc acg gtc acc gtc tcc tca 363 Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 2 121 PRT Mus musculus 2 Glu Val Lys Leu Gln Gln Ser
Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser
Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His
Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Arg Ile Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp Pro Lys Phe 50 55
60 Gln Val Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Trp
65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Asp Gly Met Trp Val Ser Thr Gly Tyr Ala Leu
Asp Phe Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 3 318 DNA Mus musculus CDS (1)...(318) misc_feature (1) /note=
"pBAG172 insert HP1/2 light chain variable region" 3 agt att gtg
atg acc cag act ccc aaa ttc ctg ctt gtt tca gca gga 48 Ser Ile Val
Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 gac
agg gtt acc ata acc tgc aag gcc agt cag agt gtg act aat gat 96 Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25
30 gta gct tgg tac caa cag aag cca ggg cag tct cct aaa ctg ctg ata
144 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45 tat tat gca tcc aat cgc tac act gga gtc cct gat cgc ttc
act ggc 192 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60 agt gga tat ggg acg gat ttc act ttc acc atc agc
act gtg cag gct 240 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser
Thr Val Gln Ala 65 70 75 80 gaa gac ctg gca gtt tat ttc tgt cag cag
gat tat agc tct ccg tac 288 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln
Asp Tyr Ser Ser Pro Tyr 85 90 95 acg ttc gga ggg ggg acc aag ctg
gag atc 318 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 4 106
PRT Mus musculus 4 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu
Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg
Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly
Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp
Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 5 1347 DNA Homo
sapiens CDS (1)...(1338) VCAM-1 gene sequence (1)...(219) This
portion of the sequence corresponds, in part, to Exons I, II and
III nucleotide sequence of Cybulsky et al. Proc. Nat'l. Acad. Sci.
USA 88 7861 (1991). Hinge regions (220)...(229) This portion of the
sequence corresponds, in part, to Fig. 12A in PCT/US92/02050 and
represents the hinge region of Human IgG1 heavy chain constant
region. Heavy chain constant region 2 (230)...(338) This portion of
the sequence corresponds, in part, to Fig. 12A in PCT/US92/02050
and represents the heavy chain constant region 2 of Human IgG1
heavy chain constant region. Heavy chain constant region 3
(339)...(446) This portion of the sequence corresponds, in part, to
Fig. 12A in PCT/US92/02050 and represents the heavy chain constant
region 3 of Human IgG1 heavy chain constant region. 5 atg cct ggg
aag atg gtc gtg atc ctt gga gcc tca aat ata ctt tgg 48 Met Pro Gly
Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp 1 5 10 15 ata
atg ttt gca gct tct caa gct ttt aaa atc gag acc acc cca gaa 96 Ile
Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu 20 25
30 tct aga tat ctt gct cag att ggt gac tcc gtc tca ttg act tgc agc
144 Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser
35 40 45 acc aca ggc tgt gag tcc cca ttt ttc tct tgg aga acc cag
ata gat 192 Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln
Ile Asp 50 55 60 agt cca ctg aat ggg aag gtg acg aat gag ggg acc
aca tct acg ctg 240 Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr
Thr Ser Thr Leu 65 70 75 80 aca atg aat cct gtt agt ttt ggg aac gaa
cac tct tac ctg tgc aca 288 Thr Met Asn Pro Val Ser Phe Gly Asn Glu
His Ser Tyr Leu Cys Thr 85 90 95 gca act tgt gaa tct agg aaa ttg
gaa aaa gga atc cag gtg gag atc 336 Ala Thr Cys Glu Ser Arg Lys Leu
Glu Lys Gly Ile Gln Val Glu Ile 100 105 110 tac tct ttt cct aag gat
cca gag att cat ttg agt ggc cct ctg gag 384 Tyr Ser Phe Pro Lys Asp
Pro Glu Ile His Leu Ser Gly Pro Leu Glu 115 120 125 gct ggg aag ccg
atc aca gtc aag tgt tca gtt gct gat gta tac cca 432 Ala Gly Lys Pro
Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro 130 135 140 ttt gac
agg ctg gag ata gac tta ctg aaa gga gat cat ctc atg aag 480 Phe Asp
Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys 145 150 155
160 agt cag gaa ttt ctg gag gat gca gac agg aag tcc ctg gaa acc aag
528 Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
165 170 175 agt ttg gaa gta acc ttt act cct gtc att gag gat att gga
aaa gtt 576 Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly
Lys Val 180 185 190 ctt gtt tgc cga gct aaa tta cac att gat gaa atg
gat tct gtg ccc 624 Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met
Asp Ser Val Pro 195 200 205 aca gta agg cag gct gta aaa gaa ttg caa
gtc gac aaa act cac aca 672 Thr Val Arg Gln Ala Val Lys Glu Leu Gln
Val Asp Lys Thr His Thr 210 215 220 tgc cca ccg tgc cca gca cct gaa
ctc ctg ggg gga ccg tca gtc ttc 720 Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 ctc ttc ccc cca aaa
ccc aag gac acc ctc atg atc tcc cgg acc cct 768 Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 gag gtc aca
tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 816 Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 aag
ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 864 Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285 aag ccg cgg gag gag cag tac aac agc acg tac cgg gtg gtc agc gtc
912 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300 ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac
aag tgc 960 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys 305 310 315 320 aag gtc tcc aac aaa gcc ctc cca gcc ccc atc
gag aaa acc atc tcc 1008 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser 325 330 335 aaa gcc aaa ggg cag ccc cga gaa
cca cag gtg tac acc ctg ccc cca 1056 Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 tcc cgg gat gag ctg
acc aag aac cag gtc agc ctg acc tgc ctg gtc 1104 Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 aaa ggc
ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 1152 Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380 cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac
1200 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp 385 390 395 400 ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac
aag agc agg tgg 1248 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp 405 410 415 cag cag ggg aac gtc ttc tca tgc tcc
gtg atg cat gag gct ctg cac 1296 Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His 420 425 430 aac cac tac acg cag aag
agc ctc tcc ctg tct ccg ggt aaa 1338 Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 445 tgagtgcgg 1347 6 24 DNA
Homo sapiens CDS (6)...(23) 6 tcgtc gac aaa act cac aca tgc c 24
Asp Lys Thr His Thr Cys 1 5 7 24 DNA Artificial Sequence primer for
PCR 7 gtaaatgagt gcggcggccg ccaa 24 8 115 DNA Artificial Sequence
synthetic polylinker sequence 8 gcggccgcgg tccaaccacc aatctcaaag
cttggtaccc gggaattcag atctgcagca 60 tgctcgagct ctagatatcg
attccatgga tcctcacatc ccaatccgcg gccgc 115 9 41 DNA Homo sapiens
CDS (21)...(41) 9 gagctcgagg cggccgcacc atg cct ggg aag atg gtc gtg
41 Met Pro Gly Lys Met Val Val 1 5 10 23 DNA Homo sapiens 10
aagtcgactt gcaattcttt tac 23 11 14 DNA Artificial Sequence
phosphorylated oligonucleotide 11 tcgacgcggc cgcg 14 12 446 PRT
Homo sapiens 12 Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn
Ile Leu Trp 1 5 10 15 Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile
Glu Thr Thr Pro Glu 20 25 30 Ser Arg Tyr Leu Ala Gln Ile Gly Asp
Ser Val Ser Leu Thr Cys Ser 35 40 45 Thr Thr Gly Cys Glu Ser Pro
Phe Phe Ser Trp Arg Thr Gln Ile Asp 50 55 60 Ser Pro Leu Asn Gly
Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu 65 70 75 80 Thr Met Asn
Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr 85 90 95 Ala
Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile 100 105
110 Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu
115 120 125 Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val
Tyr Pro 130 135 140 Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp
His Leu Met Lys 145 150 155 160 Ser Gln Glu Phe Leu Glu Asp Ala Asp
Arg Lys Ser Leu Glu Thr Lys 165 170 175 Ser Leu Glu Val Thr Phe Thr
Pro Val Ile Glu Asp Ile Gly Lys Val 180 185 190 Leu Val Cys Arg Ala
Lys Leu His Ile Asp Glu Met Asp Ser Val Pro 195 200 205 Thr Val Arg
Gln Ala Val Lys Glu Leu Gln Val Asp Lys Thr His Thr 210 215 220 Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230
235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355
360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 13 6 PRT Artificial
Sequence synthetically generated peptide 13 Asp Lys Thr His Thr Cys
1 5 14 7 PRT Homo sapiens 14 Met Pro Gly Lys Met Val Val 1 5 15 363
DNA Mus musculus CDS (1)...(363) misc_feature (1) /note= "pBAG159
insert HP1/2 heavy chain variable regions 15 car gtc aaa ctg cag
cag tct ggg gca gag ctt gtg aag cca ggg gcc 48 Gln Val Lys Leu Gln
Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 tca gtc aag
ttg tcc tgc aca gct tct ggc ttc aac att aaa gac acc 96 Ser Val Lys
Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 tat
atg cac tgg gtg aag cag agg cct gaa cag ggc ctg gag tgg att 144 Tyr
Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40
45 gga agg att gat cct gcg agt ggc gat act aaa tat gac ccg aag ttc
192 Gly Arg Ile Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp Pro Lys Phe
50 55 60 cag gtc aag gcc act att aca gcg gac acg tcc tcc aac aca
gcc tgg 240 Gln Val Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr
Ala Trp 65 70 75 80 ctg cag ctc agc agc ctg aca tct gag gac act gcc
gtc tac tac tgt 288 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 gca gac gga atg tgg gta tca acg gga tat
gct ctg gac ttc tgg ggc 336 Ala Asp Gly Met Trp Val Ser Thr Gly Tyr
Ala Leu Asp Phe Trp Gly 100 105 110 caa ggg acc acg gtc acc gtc tcc
tca 363 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 16 121 PRT Mus
musculus 16 Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn
Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Ser Gly
Asp Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Val Lys Ala Thr Ile
Thr Ala Asp Thr Ser Ser Asn Thr Ala Trp 65 70 75 80 Leu Gln Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asp
Gly Met Trp Val Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly 100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 17 5 PRT Artificial Sequence Exemplary motif 17 Glu Ile Leu Asp
Val 1 5
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