U.S. patent application number 11/192449 was filed with the patent office on 2005-12-22 for method for the treatment of fibrosis.
This patent application is currently assigned to BIOGEN IDEC MA INC.. Invention is credited to Gotwals, Philip J., Kotelianski, Victor.
Application Number | 20050281818 11/192449 |
Document ID | / |
Family ID | 26828876 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050281818 |
Kind Code |
A1 |
Gotwals, Philip J. ; et
al. |
December 22, 2005 |
Method for the treatment of fibrosis
Abstract
Disclosed is a method of treating fibrosis in a human or animal
subject. The method comprises administering to the subject an
effective amount of an antibody to an integrin or fragment
thereof.
Inventors: |
Gotwals, Philip J.;
(Needham, MA) ; Kotelianski, Victor; (Boston,
MA) |
Correspondence
Address: |
Kevin J. McGough
Coleman Sudol Sapone, P.C.
714 Colorado Avenue
Bridgeport
CT
06605-1601
US
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
26828876 |
Appl. No.: |
11/192449 |
Filed: |
July 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11192449 |
Jul 28, 2005 |
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10625260 |
Jul 22, 2003 |
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10625260 |
Jul 22, 2003 |
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10061658 |
Feb 1, 2002 |
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6652856 |
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10061658 |
Feb 1, 2002 |
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09557092 |
Apr 21, 2000 |
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60130847 |
Apr 22, 1999 |
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60137214 |
Jun 1, 1999 |
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Current U.S.
Class: |
424/144.1 |
Current CPC
Class: |
A61P 17/02 20180101;
A61P 19/04 20180101; A61K 2039/505 20130101; A61P 11/00 20180101;
A61P 1/00 20180101; C07K 16/2839 20130101; A61P 13/12 20180101;
A61P 21/00 20180101; A61P 19/02 20180101; A61P 25/00 20180101; A61P
29/00 20180101; A61P 43/00 20180101; A61P 9/00 20180101; A61P 13/00
20180101; A61P 17/00 20180101; A61P 11/02 20180101; A61P 1/16
20180101; A61P 37/00 20180101; A61P 27/02 20180101; C07K 16/2842
20130101 |
Class at
Publication: |
424/144.1 |
International
Class: |
A61K 039/395 |
Claims
1. A method for treating a subject suffering from a fibrotic
condition, comprising administering to the subject a pharmaceutical
composition, the pharmaceutical composition comprising an antibody
molecule that antagonizes an interaction of .alpha.1.beta.1 with
its ligand.
2. A method according to claim 1, wherein the antibody is selected
from the group consisting a human antibody or a fragment of a human
antibody, a chimeric antibody or fragment of a chimeric antibody,
or a humanized antibody and fragments thereof or a fragment of a
humanized antibody.
3. A method of claim 1, wherein the antibody is a monoclonal
antibody.
4. A method of claim 1, wherein the antibody or antibody fragment
is administered to the subject (a) around once or twice
approximately every seven days (b) in an amount of between about
0.3 mg/kg/day to about 5 mg/kg/day.
5. A method of claim 1, wherein the subject is a human.
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. A method of claim 1, wherein the composition is administered
parenterally.
13. (canceled)
14. (canceled)
15. A method for treating a subject suffering from a fibrotic
condition, comprising administering to the subject an antibody or
fragment of an antibody selected from the group consisting of AQC2
(ATCC PTA-3580); AJH10(ATCC PTA-3580); 1B3.1 (ATCC No. HB10336); or
TS2/7.1 (ATCC No. HB-245).
16. A method of claim 1, wherein the subject suffers from fibrosis
of an internal organ.
17. A method of claim 16, wherein the internal organ is the liver,
kidney, heart blood vessels, or gastrointestinal tract.
18. A method of claim 1, wherein the subject suffers from
myelofibrosis, liver cirrhosis, mesangial proliferative
glomerulonephritis, crescentic glomerulonephritis, diabetic
nephropathy, renal interstitial fibrosis, or HIV associated
nephropathy.
19. A method of claim 1, wherein the fibrosis is dermal
fibrosis.
20. A method of claim 19, wherein the subject suffers from
scleroderma, morphea, keloids, hypertrophic scars, familial
cutaneous collagenoma, or connective tissue nevi of the collagen
type.
21. A method of claim 1, wherein the fibrosis is fibrosis of the
eye.
22. A method claim 21, wherein the subject suffers from diabetic
retinopathy, postsurgical scarring, or proliferative
vitreoretinopathy
Description
RELATED APPLICATIONS
[0001] This application is a continuation of prior U.S. provisional
application Ser. No. 60/130,847 filed on Apr. 22, 1999 as a
continuation in part of prior U.S. provisional application Ser. No.
60/137,214 filed on Jun. 1, 1999.
FIELD OF THE INVENTION
[0002] This invention relates to methods for treating fibrosis in
subjects in need of such treatment.
BACKGROUND OF THE INVENTION
[0003] Collagen is a fibril-forming protein which is essential for
maintaining the integrity of the extracellular matrix found in
connective tissues. The production of collagen is a highly
regulated process, and its disturbance may lead to the development
of tissue fibrosis. While the formation of fibrous tissue is part
of the normal beneficial process of healing after injury, in some
circumstances there is an abnormal accumulation of fibrous
materials such that it may ultimately lead to organ failure (Border
et al. (1994) New Engl. J. Med. 331:1286-1292). Injury to any organ
leads to a stereotypical physiological response: platelet-induced
hemostasis, followed by an influx of inflammatory cells and
activated fibroblasts. Cytokines derived from these cell types
drive the formation of new extracellular matrix and blood vessels
(granulation tissue). The generation of granulation tissue is a
carefully orchestrated program in which the expression of protease
inhibitors and extracellular matrix proteins is upregulated, and
the expression of proteases is reduced, leading to the accumulation
of extracellular matrix.
[0004] Central to the development of fibrotic conditions, whether
induced or spontaneous, is stimulation of fibroblast activity. The
influx of inflammatory cells and activated fibroblasts into the
injured organ depends on the ability of these cell types to
interact with the interstitial matrix comprised primarily of
collagens. The major cell surface collagen receptors are the
.alpha.1.beta.1 (VLA-1) and .alpha.2.beta.1 (VLA-2) integrins. Both
integrins have been implicated in cell adhesion and migration on
collagen (Keely et al. (1995) J. Cell Sci. 108:595-607 and Gotwals
et al. (1996) J. Clin. Invest. 97: 2469-2477); in promoting
contraction of collagen matrices (Gotwals et al. (1996) J. Clin.
Invest. 97: 2469-2477 and Schiro, (1991) Cell 67:403-410), and in
regulating the expression of genes involved in the remodeling of
the extracellular matrix (Riikonen et al. (1995) J. Biol. Chem.
270:1-5 and Langholz et al. (1995) J. Cell Biol. 131: 1903-1915).
For example, when fibroblasts contact a collagen matrix, signaling
through the .alpha.1.beta.1 integrin down-regulates collagen I
expression, while signaling through .alpha.2.beta.1 up-regulates
the expression of matrix metalloproteases (Langholz et al. (1995)
J. Cell Biol. 131: 1903-1915).
[0005] Many of the diseases associated with the proliferation of
fibrous tissue are both chronic and often debilitating, including
for example, skin diseases such as scleroderma. Some, including
pulmonary fibrosis, can be fatal due in part to the fact that the
currently available treatments for this disease have significant
side effects and are generally not efficacious in slowing or
halting the progression of fibrosis [Nagler et al. 1996, Am. J.
Respir. Crit. Care Med., 154:1082-86].
[0006] There is, accordingly, a continuing need for new
anti-fibrotic agents.
[0007] In contrast to the trends in research in the field of
anti-fibrotic therapy which has focused on upstream cytokine
mediators of fibrosis, such as TGF-B, we propose the use of
antibody molecules comprising antigen binding regions derived from
the heavy or light chain variable regions of an anti-VLA antibody,
for use in anti-fibrotic treatment and specifically for treatment
of pulmonary fibrosis.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method of treating fibrosis
in a subject. Specifically, the invention provides a method for
treating fibrosis, comprising administering to a patient a
pharmaceutical composition comprising an effective amount of an
antibody molecule comprising antigen binding regions derived from
the light and heavy chain variable regions of an anti-VLA antibody.
In a preferred embodiment, the anti-VLA antibody is selected from
the group consisting of anti-VLA-1, -2, -3, -4, -5, -6. In a most
preferred embodiment, the invention provides a method for treating
pulmonary fibrosis, comprising administering to a patient a
pharmaceutical composition, the pharmaceutical composition
comprising an effective amount of an antibody molecule comprising
antigen binding regions derived from the light and heavy chain
variable regions of an anti-VLA-1 and anti-VLA-2 antibody.
[0009] The anti-VLA antibody can be selected from the group
consisting of a human antibody, a chimeric antibody, a humanized
antibody and fragments thereof. The anti-VLA antibody can be a
monoclonal or polyclonal antibody.
[0010] The invention further provides a method for treating
fibrosis in a subject that is a human or animal subject.
[0011] All of the cited literature in the preceding section, as
well as the cited literature included in the following disclosure,
are hereby incorporated by reference.
DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. The .alpha.1-I domain binds collagen. A. Increasing
concentrations of the human .alpha.1-I domain were bound to plates
previously coated with 1 .mu.g/ml collagen I (squares) or collagen
IV (circles). Values shown have been corrected for background
binding to BSA. B. 2 .mu.g/ml human .alpha.1-I domain was mixed
with increasing concentration of an anti-human .alpha.1.beta.1
integrin antibody 5E8D9 (squares) or an anti-human
.alpha.2.beta.1-integrin antibody A2IIE10 (circles), and then bound
to plates previously coated with 1 .mu.g/ml collagen IV. C. Plates
were coated with 1 .mu.g/ml collagen IV or 3% BSA. .alpha.1-I
domain (2 .mu.g/ml) was subsequenctly bound to coated plates plates
in the presence of 1 mM Mn.sup.2+, 1 mM Mg.sup.2+, or 5 mM EDTA.
Data shown is representative of three independent experiments.
[0013] FIG. 2. Identification of a blocking mAb to the .alpha.1-I
domain. A. Increasing concentration of mAbs AEF3 (triangles) or
AJH10 (ATCC NO. ______) (circles) were bound to plates coated with
30 .mu.g/ml .alpha.1-I domain. B. The .alpha.1-I domain was treated
with increasing concentrations of mAb AJH10 (ATCC NO. ______)
(diamonds) or mAb BGC5 (squares) and bound collagen IV (2 .mu.g/ml)
coated plates. C. K562-.alpha.1 cell were treated with increasing
concentration of mAbs AEF3(triangles) or AJH10 (ATCC NO. ______)
(circles) and bound to collagen IV (5 .mu.g/ml) coated plates.
45-50% of cells added to each well adhered to collagen IV. Data
shown is representative of three independent experiments.
[0014] FIG. 3. Species Cross-reactivity of the blocking mAbs. A.
Detergents lysates from (1) sheep vascular smooth muscle, (2) human
leukemia K562-.alpha.1 cells or (3) purified R.DELTA.H GST-I
domain; (4) Rat GST-.alpha.1 I domain; and (5) human GST-.alpha.1 I
domain were separated by 10-20% SDS-PAGE under non-reducing
conditions, and immunoblotted with function-blocking mAb AJH10
(ATCC NO. ______). Molecular weight markers are shown on the left;
non-reduced .alpha.1.beta.1 integrin migrates at .about.180 kDa;
GST-I domain migrates at .about.45 kDa. B. Rabbit vascular smooth
muscle cells were incubated with either mAb AJH10 (ATCC NO.
______)(bottom) or murine IgG control (top) and analyzed by
fluorescence activated cell sorter (FACS).
[0015] FIG. 4. Location of the Epitope for the anti-.alpha.1 I
domain Blocking mAbs. A. Amino acid sequence of the rat (top) and
human (below) .alpha.1-I domain. The residues that comprise the
MIDAS (metal ion dependent adhesion site) motif are shown in bold.
The human amino acids that replaced the corresponding rat residues
(R.DELTA.H) are shown below the rat sequence in the boxed region.
For clarity, residue numbering in the text refers to this figure.
B. Increasing concentrations of mAb AJH10 (ATCC NO. ______) were
bound to plates coated with 30 .mu.g/ml human (circles), rat
(triangles) or R.DELTA.H (squares) .alpha.1-I domain. Data shown is
representative of three experiments.
[0016] FIG. 5. Amino acid sequence of the human .alpha.1-I
domain.
[0017] FIG. 6. Cation Stabilizes the Expression of the Epitope. A.
0.5 .mu.g of blocking mAb AJH10 (ATCC NO. ______) or non-blocking
mAb AEF3 in the presence of 5 mM EDTA (open) or 1 mM MnCl.sub.2
(solid) were bound to plates previously coated with 1 .mu.g/ml
affinity purified, human .alpha.1.beta.1 integrin. B. 5 .mu.g/ml
AJH10 (ATCC NO. ______) or AEF3 were incubated with K562-.alpha.1
cells in the presence of 2 mM MnCl.sub.2 (solid), or following a
wash with 5 mM EDTA (open). Bound antibody was measured by FACS and
is reported as the mean fluorescence intensity (MFI).
[0018] FIG. 7. Denaturation of the .alpha.1-I domain by Urea. 0.6
.mu.M rat .alpha.1 I domain, in the presence of no cation
(squares), or 1 mM MnCl.sub.2 (circles) and increasing
concentrations of urea were analyzed at 25.degree. C. using an
excitation wavelength of 280 nm. Fluorescence data from the
emission spectra at 350 nm are plotted as a function of urea
concentration and standardized using the change in fluorescence for
each of the test conditions as a measure of the total fraction
unfolded.
[0019] FIG. 8. Circular dichroism spectra of thermally denatured
.alpha.1-I domain. Temperature dependent, circular dichroism
measurements at fixed wavelength (222 nM) were performed using 55
.mu.M .alpha.1-I domain in the absence (solid line), or presence of
2 mM Mg.sup.2+ (dot-dash line), or 2 mM Mn.sup.2+ (dotted line).
Data are expressed as (A) continuous temperature dependence of
molar ellipticity per residue, and (B) first derivative curves
after smoothing the corresponding data curves shown in panel A.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present application is directed to the discovery that
antibodies to integrins and fragments thereof can be used for the
treatment of fibrosis.
[0021] The methods of the present invention contemplate the use of
antibodies to integrins where the integrins contemplated include
molecules which comprise a .beta. chain, including but not limited
to .beta.1, .beta.2, .beta.3, .beta.4, .beta.5, .beta.6, .beta.7,
.beta.8, non-covalently bound to an .alpha. chain, including but
not limited to .alpha.1, .alpha.2, .alpha.3, .alpha.4, .alpha.5,
.alpha.6, .alpha.7, .alpha.8, .alpha.9, .alpha.10, .alpha.V,
.alpha.L, .alpha.M, .alpha.X, .alpha.D, .alpha.E, .alpha.IIb.
Examples of the various integrins contemplated for use in the
invention include, but are not limited to:
[0022] .alpha.1.beta.1, .alpha.2.beta.1, .alpha.3.beta.1,
.alpha.4.beta.1, .alpha.5.beta.1, .alpha.6.beta.1, .alpha.7.beta.1,
.alpha.8.beta.1, .alpha.9.beta.1, .alpha.10.beta.1,
.alpha.V.beta.1, .alpha.L.beta.1, .alpha.M.beta.1, .alpha.X.beta.1,
.alpha.D.beta.1, .alpha.IIb.beta.1, .alpha.E.beta.1;
[0023] .alpha.1.beta.2, .alpha.2.beta.2, .alpha.3.beta.2,
.alpha.4.beta.2, .alpha.5.beta.2, .alpha.6.beta.2, .alpha.7.beta.2,
.alpha.8.beta.2, .alpha.9.beta.2, .alpha.10.beta.2,
.alpha.V.beta.2, .alpha.L.beta.2, .alpha.M.beta.2, .alpha.X.beta.2,
.alpha.D.beta.2, .alpha.IIb.beta.2, .alpha.E.beta.2;
[0024] .alpha.1.beta.3, .alpha.2.beta.3, .alpha.3.beta.3,
.alpha.4.beta.3, .alpha.5.beta.3, .alpha.6.beta.3, .alpha.7.beta.3,
.alpha.8.beta.3, .alpha.9.beta.3, .alpha.10.beta.3,
.alpha.V.beta.3, .alpha.L.beta.3, .alpha.M.beta.3,
.alpha.X,.beta.3, .alpha.D.beta.3, .alpha.IIb.beta.3,
.alpha.E.beta.3;
[0025] .alpha.1.beta.4, .alpha.2.beta.4, .alpha.3.beta.4,
.alpha.4.beta.4, .alpha.5.beta.4, .alpha.6.beta.4, .alpha.7.beta.4,
.alpha.8.beta.4, .alpha.9.beta.4, .alpha.10.beta.4,
.alpha.V.beta.4, .alpha.L.beta.4, .alpha.M.beta.4, .alpha.X.beta.4
.alpha.D.beta.4, .alpha.IIb.beta.4, .alpha.E.beta.4;
[0026] .alpha.1.beta.5, .alpha.2.beta.5, .alpha.3.beta.5,
.alpha.4.beta.5, .alpha.5.beta.5, .alpha.6.beta.5, .alpha.7.beta.5,
.alpha.8.beta.5, .alpha.9.beta.5, .alpha.10.beta.5,
.alpha.V.beta.5, .alpha.L.beta.5, .alpha.M.beta.5, .alpha.X.beta.5,
.alpha.D.beta.5, .alpha.IIb.beta.5, .alpha.E.beta.5;
[0027] .alpha.1.beta.6, .alpha.2.beta.6, .alpha.3.beta.6,
.alpha.4.beta.6, .alpha.5.beta.6, .alpha.6.beta.6, .alpha.7.beta.6,
.alpha.8.beta.6, .alpha.9.beta.6, .alpha.10.beta.6,
.alpha.V.beta.6, .alpha.L.beta.6, .alpha.M.beta.6, .alpha.X.beta.6,
.alpha.D.beta.6, .alpha.IIb.beta.6, .alpha.E.beta.6;
[0028] .alpha.1.beta.7, .alpha.2.beta.7, .alpha.3.beta.7,
.alpha.4.beta.7, .alpha.5.beta.7, .alpha.6.beta.7, .alpha.7.beta.7,
.alpha.8.beta.7, .alpha.9.beta.7, .alpha.10.beta.7,
.alpha.V.beta.7, .alpha.L.beta.7, .alpha.M.beta.7, .alpha.X.beta.7,
.alpha.D.beta.7, .alpha.IIb.beta.7, .alpha.E.beta.7;
[0029] .alpha.1.beta.8, .alpha.2.beta.8, .alpha.3.beta.8,
.alpha.4.beta.8, .alpha.5.beta.8, .alpha.6.beta.8, .alpha.7.beta.8,
.alpha.8.beta.8, .alpha.9.beta.8, .alpha.10.beta.8,
.alpha.V.beta.8, .alpha.L.beta.8, .alpha.M.beta.8, .alpha.X.beta.8,
.alpha.D.beta.8, .alpha.IIb.beta.8, .alpha.E.beta.8;
[0030] The methods of the present invention also contemplate the
use of antibodies to integrin fragments including for example
antibodies to a .beta. chain alone, including but not limited to
.beta.1, .beta.2, .beta.3, .beta.4, .beta.5, .beta.6, .beta.7,
.beta.8, as well as an a chain alone, including but not limited to
.alpha.1, .alpha.2, .alpha.3, .alpha.2, .alpha.5, .alpha.6,
.alpha.7, .alpha.7, .alpha.8, .alpha.9, .alpha.10, .alpha.V,
.alpha.L, .alpha.M, .alpha.X, .alpha.D, .alpha.E, .alpha.IIb. In
addition, the methods of the present invention further contemplate
the use of antibodies to integrin fragments including for example
antibodies to the I domain of the .alpha. chain, including but not
limited to the I domain from .alpha.1.beta.1 (Briesewitz et al.,
1993 J. Biol. Chem. 268:2989); .alpha.2.beta.1 (Takada and Hemler,
1989 J Cell Biol 109:397), .alpha.L.beta.2 (Larson et al., 1989 J
Cell Biol 108:703), .alpha.M.beta.2 (Corbi et al., 1988 J Biol Chem
263:12403), .alpha.X.beta.2 (Corbi et al., 1987 EMBO J 6:4023),
.alpha.D.beta.2 (Grayson et al., 1988 J Exp Med 188:2187),
.alpha.E.beta.7 (Shaw et al., 1994 J Biol Chem 269:6016). In a
preferred embodiment, the alpha1-I domain antigenic determinant
comprises an amino acid sequence of at least 6 contiguous amino
acids, wherein the contiguous sequence is found within the sequence
of FIG. 5. Moreover, in a preferred embodiment, the contiguous
sequence is Val-Gln-Arg-Gly-Gly-Arg.
[0031] In a preferred embodiment the invention contemplates the use
of antibodies to VLA-1, -2, -3, -4, -5, -6, in which each of the
molecules comprise a .beta.1 chain non covalently bound to a
.alpha. chain, (.alpha.1, .alpha.2, .alpha.3, .alpha.4, .alpha.5,
.alpha.6), respectively. In a most preferred embodiment, the
invention contemplates using anti-VLA-1 and anti-VLA-2 antibodies
for the treatment of pulmonary fibrosis.
[0032] Methods for producing integrins for use in the present
invention are known to those of skill in the art (see for e.g.
Springer et al. 1990, Nature 346:425-434).
[0033] Embodiments of the present invention include polyclonal and
monoclonal antibodies to integrins and fragments thereof. Preferred
embodiments of the present invention include a monoclonal antibody,
including for example, an anti-VLA antibody homolog. Preferred
antibodies and homologs for treatment, in particular for human
treatment, include human antibody homologs, humanized antibody
homologs, chimeric antibody homologs, Fab, Fab', F(ab')2 and F(v)
antibody fragments, and monomers or dimers of antibody heavy or
light chains or mixtures thereof. Thus, monoclonal antibodies
against an integrin molecule or fragment thereof are the preferred
binding agent in the method of the invention.
[0034] As used herein, the term "antibody homolog" includes intact
antibodies consisting of immunoglobulin light and heavy chains
linked via disulfide bonds. The term "antibody homolog" is also
intended to encompass a protein comprising one or more polypeptides
selected from immunoglobulin light chains, immunoglobulin heavy
chains and antigen-binding fragments thereof which are capable of
binding to one or more antigens (i.e., VLA-1, VLA-2, VLA-3, VLA-4,
VLA-5, VLA-6). The component polypeptides of an antibody homolog
composed of more than one polypeptide may optionally be
disulfide-bound or otherwise covalently crosslinked.
[0035] Accordingly, therefore, "antibody homologs" include intact
immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as
subtypes thereof), wherein the light chains of the immunoglobulin
may be of types kappa or lambda. "Antibody homologs" also include
portions of intact antibodies that retain antigen-binding
specificity, for example, Fab fragments, Fab' fragments, F(ab')2
fragments, F(v) fragments, heavy chain monomers or dimers, light
chain monomers or dimers, dimers consisting of one heavy and one
light chain, and the like. Thus, antigen-binding fragments, as well
as full-length dimeric or trimeric polypeptides derived from the
above-described antibodies are themselves useful.
[0036] As used herein, a "humanized antibody homolog" is an
antibody homolog, produced by recombinant DNA technology, in which
some or all of the amino acids of a human immunoglobulin light or
heavy chain that are not required for antigen binding have been
substituted for the corresponding amino acids from a nonhuman
mammalian immunoglobulin light or heavy chain.
[0037] As used herein, a "chimeric antibody homolog" is an antibody
homolog, produced by recombinant DNA technology, in which all or
part of the hinge and constant regions of an immunoglobulin light
chain, heavy chain, or both, have been substituted for the
corresponding regions from another immunoglobulin light chain or
heavy chain. In another aspect the invention features a variant of
a chimeric molecule which includes: (1) a VLA targeting moiety; (2)
optionally, a second peptide, e.g., one which increases solubility
or in vivo life time of the VLA 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 IgGl heavy
chain constant region, e.g., CH2 and CH3 hinge regions; and a toxin
moiety. The chimeric molecule can be used to treat a subject, e.g.,
a human, at risk for disorder related to proliferation of
epithelial cells such as hair follicles and the like.
[0038] As used herein, a "human antibody homolog" is an antibody
homolog produced by recombinant DNA technology, in which all of the
amino acids of an immunoglobulin light or heavy chain that are
derived from a human source.
[0039] A "a subject with a fibrotic condition" refers to, but is
not limited to, subjects afflicted with fibrosis of an internal
organ, subjects afflicted with a dermal fibrosing disorder, and
subjects afflicted with fibrotic conditions of the eye.
[0040] Fibrosis of internal organs (e.g., liver, lung, kidney,
heart blood vessels, gastrointestinal tract), occurs in disorders
such as pulmonary fibrosis, myelofibrosis, liver cirrhosis,
mesangial proliferative glomerulonephritis, crescentic
glomerulonephritis, diabetic nephropathy, renal interstitial
fibrosis, renal fibrosis in patients receiving cyclosporin, and HIV
associated nephropathy. In a preferred embodiment, the invention
contemplates using the anti-VLA antibodies for the treatment of
pulmonary fibrosis. In a most preferred embodiment, the invention
contemplates using anti-VLA-1 and anti-VLA-2 antibodies for the
treatment of pulmonary fibrosis.
[0041] Dermal fibrosing disorders include, but are not limited to,
scleroderma, morphea, keloids, hypertrophic scars, familial
cutaneous collagenoma, and connective tissue nevi of the collagen
type.
[0042] Fibrotic conditions of the eye include conditions such as
diabetic retinopathy, postsurgical scarring (for example, after
glaucoma filtering surgery and after cross-eye surgery), and
proliferative vitreoretinopathy.
[0043] Additional fibrotic conditions which may be treated by the
methods of the present invention include: rheumatoid arthritis,
diseases associated with prolonged joint pain and deteriorated
joints; progressive systemic sclerosis, polymyositis,
dermatomyositis, eosinophilic fascitis, morphea, Raynaud's
syndrome, and nasal polyposis.
[0044] In addition, fibrotic conditions which may be treated the
methods of present invention also include inhibiting overproduction
of scarring in patients who are known to form keloids or
hypertrophic scars, inhibiting or preventing scarring or
overproduction of scarring during healing of various types of
wounds including surgical incisions, surgical abdominal wounds and
traumatic lacerations, preventing or inhibiting scarring and
reclosing of arteries following coronary angioplasty, preventing or
inhibiting excess scar or fibrous tissue formation associated with
cardiac fibrosis after infarction and in hypersensitive
vasculopathy.
[0045] An "effective amount" (when used in the toleragenic context)
is an amount sufficient to effect beneficial or desired clinical
results. An effective amount can be administered in one or more
administrations. In terms of treatment, an "effective amount" of an
antibody for use in the present invention, including for example an
anti-VLA antibody, is an amount sufficient to palliate, ameliorate,
stabilize, reverse, slow or delay progression of a fibrotic
condition in accordance with clinically acceptable standards for
disorders to be treated or for cosmetic purposes. Detection and
measurement of indicators of efficacy may be measured by a number
of available diagnostic tools, including, for example, by physical
examination including blood tests, pulmonary function tests, and
chest X-rays; CT scan; bronchoscopy; bronchoalveolar lavage; lung
biopsy and CT scan.
[0046] Methods of Making Antibodies
[0047] The technology for producing monoclonal antibodies,
including for example, anti-integrin monoclonal antibodies is well
known. See for example, Mendrick et al. 1995, Lab. Invest.
72:367-375 (mAbs to murine anti-.alpha.1.beta.1 and
anti-.alpha.2.beta.1); Sonnenberg et al. 1987 J. Biol. Chem.
262:10376-10383 (mAbs to murine anti-.alpha.6.beta.1); Yao et al.
1996, J Cell Sci 1996 109:3139-50 (mAbs to murine
anti-.alpha.7.beta.1); Hemler et al. 1984, J Immunol 132:3011-8
(mAbs to human .alpha.1.beta.1); Pischel et al. 1987 J Immunol
138:226-33 (mAbs to human .alpha.2.beta.1); Wayner et al. 1988, J
Cell Biol 107:1881-91 (mAbs to human .alpha.3.beta.1); Hemler et
al. 1987 J Biol Chem 262:11478-85 (mAbs to human .alpha.4.beta.1);
Wayner et al. 1988 J. Cell Biol 107:1881-91 (mAbs to human
.alpha.5.beta.1); Sonnenberg et al. 1987, J. Biol. Chem.
262:10376-10383 (mAbs to human .alpha.6.beta.1); A Wang et al. 1996
Am. J. Respir. Cell Mol Biol. 15:664-672 (mAbs to human
.alpha.9.beta.1); Davies et al. 1989 J Cell Biol 109:1817-26 (mAbs
to human .alpha.V.beta.1); Sanchez-Madrid et al. 1982, Proc Natl
Acad Sci USA 79:7489-93 (mAbs to human .alpha.L.beta.2); Diamond et
al. 1993, J Cell Biol 120:1031-43 (mAbs to human .alpha.M.beta.2);
Stacker et al. 1991 J Immunol 146:648-55 (mAbs to human
.alpha.X.beta.2); Van der Vieren et al 1995 Immunity 3:683-90 (mAbs
to human .alpha.D.beta.2); Bennett et al. 1983 Proc Natl Acad Sci
USA 80:2417-21 (mAbs to human .alpha.IIb.beta.2); Hessle et al.
1984, Differentiation 26:49-54 (mAbs to human .alpha.6.beta.4);
Weinacker et al. 1994 J Biol Chem 269:6940-8 (mAbs to human
.alpha.V.beta.5); Weinacker et al. 1994 J Biol Chem 269:6940-8
(mAbs to human .alpha.V.beta.6); Cerf-Bensussan et al 1992 Eur J
Immunol 22:273-7 (mAbs to human .alpha.E.beta.7); Nishimura et al.
1994 J Biol Chem 269:28708-15 (mAbs to human .alpha.V.beta.8);
Bossy et al. 1991 EMBO J 10:2375-85 (polyclonal antisera to human
.alpha.8.beta.1); Camper et al. 1998 J. Biol. Chem. 273:20383-20389
(polyclonal antisera to human .alpha.10.beta.1).
[0048] In general, an 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,
arninopterin and thymidine ("HAT medium"). Typically, HAT-sensitive
mouse myeloma cells are fused to mouse splenocytes 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 ftised 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 antibodies may be screened
by testing the hybridoma culture supernatant for secreted
antibodies having the ability to bind to a recombinant
VLA-expressing cell line.
[0049] To produce anti-VLA antibody homologs that are intact
immunoglobulins, hybridoma cells that tested positive in such
screening assays were 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 antibodies optionally further
purified by well-known methods.
[0050] Alternatively, the desired antibody may be produced by
injecting the hybridoma cells into the peritoneal cavity of an
unimmunized mouse. 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.
[0051] Fully human monoclonal antibody homologs against VLA are
another preferred binding agent which may block antigens in the
method of the invention. In their intact form these may be prepared
using in vitro-primed human splenocytes, as described by Boerner et
al., 1991, J. Inmmunol. 147:86-95, "Production of Antigen-specific
Human Monoclonal Antibodies from In Vitro-Primed Human
Splenocytes".
[0052] Alternatively, they may be prepared by repertoire cloning as
described by Persson et al., 1991, Proc. Nat. Acad. Sci. USA 88:
2432-2436, "Generation of diverse high-affinity human monoclonal
antibodies by repertoire cloning" and Huang and Stollar, 1991, J.
Immunol. Methods 141: 227-236, "Construction of representative
immunoglobulin variable region CDNA libraries from human peripheral
blood lymphocytes without in vitro stimulation". U.S. Pat. No.
5,798,230 (Aug. 25, 1998, "Process for the preparation of human
monoclonal antibodies and their use") describes preparation of
human monoclonal antibodies from human B cells. According to this
process, human antibody-producing B cells are immortalized by
infection with an Epstein-Barr virus, or a derivative thereof, that
expresses Epstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2
function, which is required for immortalization, is subsequently
shut off, which results in an increase in antibody production.
[0053] In yet another method for producing fully human antibodies,
U.S. Pat. No. 5,789,650 (Aug. 4, 1998, "Transgenic non-human
animals for producing heterologous antibodies") describes
transgenic non-human animals capable of producing heterologous
antibodies and transgenic non-human animals having inactivated
endogenous immunoglobulin genes. Endogenous immunoglobulin genes
are suppressed by antisense polynucleotides and/or by antiserum
directed against endogenous immunoglobulins. Heterologous
antibodies are encoded by immunoglobulin genes not normally found
in the genome of that species of non-human animal. One or more
transgenes containing sequences of unrearranged heterologous human
immunoglobulin heavy chains are introduced into a non-human animal
thereby forming a transgenic animal capable of functionally
rearranging transgenic immunoglobulin sequences and producing a
repertoire of antibodies of various isotypes encoded by human
immunoglobulin genes. Such heterologous human antibodies are
produced in B-cells which are thereafter immortalized, e.g., by
fusing with an immortalizing cell line such as a myeloma or by
manipulating such B-cells by other techniques to perpetuate a cell
line capable of producing a monoclonal heterologous, fully human
antibody homolog.
[0054] Yet another preferred binding agent which may block VLA
antigens in the method of the invention is a humanized antibody
homolog having the capability of binding to a VLA protein.
Following the early methods for the preparation of chimeric
antibodies, a new approach was described in EP 0239400 (Winter et
al.) whereby antibodies are altered by substitution of their
complementarity determining regions (CDRs) for one species with
those from another. This process may be used, for example, to
substitute the CDRs from human heavy and light chain Ig variable
region domains with alternative CDRs from murine variable region
domains. These altered Ig variable regions may subsequently be
combined with human Ig constant regions to created antibodies which
are totally human in composition except for the substituted murine
CDRs. Such CDR-substituted antibodies would be predicted to be less
likely to elicit an immune response in humans compared to chimeric
antibodies because the CDR-substituted antibodies contain
considerably less non-human components. The process for humanizing
monoclonal antibodies via CDR "grafting" has been termed
"reshaping". (Riechmann et al., 1988 Nature 332: 323-327,
"Reshaping human antibodies for therapy"; Verhoeyen et al., 1988,
Science 239: 1534-1536, "Reshaping of human antibodies using
CDR-grafting in Monoclonal Antibodies".
[0055] Typically, complementarity determining regions (CDRs) of a
murine antibody are transplanted onto the corresponding regions in
a human antibody, since it is the CDRs (three in antibody heavy
chains, three in light chains) that are the regions of the mouse
antibody which bind to a specific antigen. Transplantation of CDRs
is achieved by genetic engineering whereby CDR DNA sequences are
determined by cloning of murine heavy and light chain variable (V)
region gene segments, and are then transferred to corresponding
human V regions by site directed mutagenesis. In the final stage of
the process, human constant region gene segments of the desired
isotype (usually gamma I for CH and kappa for CL) are added and the
humanized heavy and light chain genes are co-expressed in mammalian
cells to produce soluble humanized antibody.
[0056] The transfer of these CDRs to a human antibody confers on
this antibody the antigen binding properties of the original murine
antibody. The six CDRs in the murine antibody are mounted
structurally on a V region "framework" region. The reason that
CDR-grafting is successful is that framework regions between mouse
and human antibodies may have very similar 3-D structures with
similar points of attachment for CDRS, such that CDRs can be
interchanged. Such humanized antibody homologs may be prepared, as
exemplified in Jones et al., 1986 Nature 321: 522-525, "Replacing
the complementarity-determining regions in a human antibody with
those from a mouse"; Riechmann, 1988, Nature 332:323-327,
"Reshaping human antibodies for therapy"; Queen et al., 1989, Proc.
Nat. Acad. Sci. USA 86:10029, "A humanized antibody that binds to
the interleukin 2 receptor" and Orlandi et al., 1989, Proc. Natl.
Acad. Sci. USA 86:3833 "Cloning Immunoglobulin variable domains for
expression by the polymerase chain reaction".
[0057] Nonetheless, certain amino acids within framework regions
are thought to interact with CDRs and to influence overall antigen
binding affinity. The direct transfer of CDRs from a murine
antibody to produce a humanized antibody without any modifications
of the human V region frameworks often results in a partial or
complete loss of binding affinity. In a number of cases, it appears
to be critical to alter residues in the framework regions of the
acceptor antibody in order to obtain binding activity.
[0058] Queen et al., 1989, Proc. Nat. Acad. Sci. USA 86:
10029-10033, "A humanized antibody that binds to the interleukin 2
receptor" and WO 90/07861 (Protein Design Labs Inc.) have described
the preparation of a humanized antibody that contains modified
residues in the framework regions of the acceptor antibody by
combining the CDRs of a murine mAb (anti-Tac) with human
immunoglobulin framework and constant regions. They have
demonstrated one solution to the problem of the loss of binding
affinity that often results from direct CDR transfer without any
modifications of the human V region framework residues; their
solution involves two key steps. First, the human V framework
regions are chosen by computer analysts for optimal protein
sequence homology to the V region framework of the original murine
antibody, in this case, the anti-Tac MAb. In the second step, the
tertiary structure of the murine V region is modelled by computer
in order to visualize framework amino acid residues which are
likely to interact with the murine CDRs and these murine amino acid
residues are then superimposed on the homologous human framework.
Their approach of employing homologous human frameworks with
putative murine contact residues resulted in humanized antibodies
with similar binding affinities to the original murine antibody
with respect to antibodies specific for the interleukin 2 receptor
(Queen et al., 1989 [supra]) and also for antibodies specific for
herpes simplex virus (HSV) (Co. et al., 1991, Proc. Nat. Acad. Sci.
USA 88: 2869-2873, "Humanised antibodies for antiviral
therapy".
[0059] According to the above described two step approach in WO
90/07861, Queen et al. outlined several criteria for designing
humanized immunoglobulins. The first criterion is to use as the
human acceptor the framework from a particular human immunoglobulin
that is usually homologous to the non-human donor immunoglobulin to
be humanized, or to use a consensus framework from many human
antibodies. The second criterion is to use the donor amino acid
rather than the acceptor if the human acceptor residue is unusual
and the donor residue is typical for human sequences at a specific
residue of the framework. The third criterion is to use the donor
framework amino acid residue rather than the acceptor at positions
immediately adjacent to the CDRS
[0060] One may use a different approach (see Tempest, 1991,
Biotechnology 9: 266-271, "Reshaping a human monoclonal antibody to
inhibit human respiratory syncytial virus infection in vivo") and
utilize, as standard, the V region frameworks derived from NEWM and
REI heavy and light chains respectively for CDR-grafting without
radical introduction of mouse residues. An advantage of using the
Tempest et al., 1991 approach to construct NEWM and REI based
humanized antibodies is that the 3dimensional structures of NEWM
and REI variable regions are known from x-ray crystallography and
thus specific interactions between CDRs and V region framework
residues can be modeled.
[0061] Regardless of the approach taken, the examples of the
initial humanized antibody homologs prepared to date have shown
that it is not a straightforward process. However, even
acknowledging that such framework changes may be necessary, it is
not possible to predict, on the basis of the available prior art,
which, if any, framework residues will need to be altered to obtain
functional humanized antibodies of the desired specificity. Results
thus far indicate that changes necessary to preserve specificity
and/or affinity are for the most part unique to a given antibody
and cannot be predicted based on the humanization of a different
antibody.
[0062] Subjects
[0063] The subject treatments are effective on both human and
animal subjects afflicted with these conditions. Animal subjects to
which the invention is applicable extend to both domestic animals
and livestock, raised either as pets or for commercial purposes.
Examples are dogs, cats, cattle, horses, sheep, hogs and goats.
[0064] Pharmaceutical Preparations
[0065] In the methods of the invention the anti-VLA antibodies may
be administered parenterally. The term "parenteral" as used herein
includes subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion
techniques.
[0066] The antibody homologs are preferably administered as a
sterile pharmaceutical composition containing a pharmaceutically
acceptable carrier, which may be any of the numerous well known
carriers, such as water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol, and the like, or combinations thereof.
The compounds of the present invention may be used in the form of
pharmaceutically acceptable salts derived from inorganic or organic
acids and bases. Included among such acid salts are the following:
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate
and undecanoate. Base salts include anmmonium salts, alkali metal
salts, such as sodium and potassium salts, alkaline earth metal
salts, such as calcium and magnesium salts, salts with organic
bases, such as dicyclohexylamine salts, N-methyl-D-glucamine,
tris(hydroxymethyl)methylamine and salts with amino acids such as
arginine, lysine, and so forth. Also, the basic nitrogen-containing
groups can be quaternized with such agents as lower alkyl halides,
such as methyl, ethyl, propyl, and butyl chloride, bromides and
iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and
diamyl sulfates, long chain halides such as decyl, lauryl, myristyl
and stearyl chlorides, bromides and iodides, aralkyl halides, such
as benzyl and phenethyl bromides and others. Water or oil-soluble
or dispersible products are thereby obtained.
[0067] The pharmaceutical compositions of this invention comprise
any of the compounds of the present invention, or pharmaceutically
acceptable derivatives thereof, together with any pharmaceutically
acceptable carrier. The term "carrier" as used herein includes
acceptable adjuvants and vehicles. Pharmaceutically acceptable
carriers that may be used in the pharmaceutical compositions of
this invention include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat.
[0068] According to this invention, the pharmaceutical compositions
may be in the form of a sterile injectable preparation, for example
a sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as do natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions.
[0069] The pharmaceutical compositions of this invention may be
given orally. If given orally, they can be administered in any
orally acceptable dosage form including, but not limited to,
capsules, tablets, aqueous suspensions or solutions. In the case of
tablets for oral use, carriers which are commonly used include
lactose and corn starch. Lubricating agents, such as magnesium
stearate, are also typically added. For oral administration in a
capsule form, useful diluents include lactose and dried corn
starch. When aqueous suspensions are required for oral use, the
active ingredient is combined with emulsifying and suspending
agents. If desired, certain sweetening, flavoring or coloring
agents may also be added.
[0070] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions can be formulated in
a suitable lotion or cream containing the active components
suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Topically-transdermal patches may also be used. For topical
applications, the pharmaceutical compositions may be formulated in
a suitable ointment containing the active component suspended or
dissolved in one or more carriers. Carriers for topical
administration of the compounds of this invention include, but are
not limited to, mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene, polyoxypropylene compound,
emulsifying wax and water. Alternatively, the pharmaceutical
compositions can be formulated in a suitable lotion or cream
containing the active components suspended or dissolved in one or
more pharmaceutically acceptable carriers. Suitable carriers
include, but are not limited to, mineral oil, sorbitan
monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol and water.
[0071] In addition to the direct topical application of the
preparations they can be topically administered by other methods,
for example, encapsulated in a temperature and/or pressure
sensitive matrix or in film or solid carrier which is soluble in
body fluids and the like for subsequent release, preferably
sustained-release of the active component. As appropriate
compositions for topical application there may be cited all
compositions usually employed for topically administering
therapeutics, e.g., creams, genies, dressings, shampoos, tinctures,
pastes, ointments, salves, powders, liquid or semiliquid
formulation and the like. Application of said compositions may be
by aerosol e.g. with a propellent such as nitrogen carbon dioxide,
a freon, or without a propellent such as a pump spray, drops,
lotions, or a semisolid such as a thickened composition which can
be applied by a swab. In particular compositions, semisolid
compositions such as salves, creams, pastes, genies, ointments and
the like will conveniently be used.
[0072] Particular compositions for use in the method of the present
invention are those wherein the anti-VLA antibody is formulated in
vesicles such as liposome-containing compositions. Liposomes are
vesicles formed by amphiphatic molecules such as polar lipids, for
example, phosphatidyl cholines, ethanolamines and serines,
sphingomyelins, cardiolipins, plasmalogens, phosphatidic acids and
cerebiosides. Liposomes are formed when suitable amphiphathic
molecules are allowed to swell in water or aqueous solutions to
form liquid crystals usually of multilayer structure comprised of
many bilayers separated from each other by aqueous material (also
referred to as coarse liposomes). Another type of liposome known to
be consisting of a single bilayer encapsulating aqueous material is
referred to as a unilamellar vesicle. If watersoluble materials are
included in the aqueous phase during the swelling of the lipids
they become entrapped in the aqueous layer between the lipid
bilayers.
[0073] A particularly convenient method for preparing liposome
formulated forms of anti-VLA antibodies is the method described in
EP-A-253,619, incorporated herein by reference. In this method,
single bilayered liposomes containing encapsulated active
ingredients are prepared by dissolving the lipid component in an
organic medium, injecting the organic solution of the lipid
component under pressure into an aqueous component while
simultaneously mixing the organic and aqueous components with a
high speed homogenizer or mixing means, whereupon the liposomes are
formed spontaneously. The single bilayered liposomes containing the
encapsulated active ingredient can be employed directly or they can
be employed in a suitable pharmaceutically acceptable carrier for
topical administration. The viscosity of the liposomes can be
increased by the addition of one or more suitable thickening agents
such as, for example xanthan gum, hydroxypropyl cellulose,
hydroxypropyl methylcellulose and mixtures thereof. The aqueous
component may consist of water alone or it may contain
electrolytes, buffered systems and other ingredients, such as, for
example, preservatives. Suitable electrolytes which can be employed
include metal salts such as alkali metal and alkaline earth metal
salts. The preferred metal salts are calcium chloride, sodium
chloride and potassium chloride. The concentration of the
electrolyte may vary from zero to 260 mM, preferably from 5 mM to
160 mM. The aqueous component is placed in a suitable vessel which
can be adapted to effect homogenization by effecting great
turbulence during the injection of the organic component.
Homogenization of the two components can be accomplished within the
vessel, or, alternatively, the aqueous and organic components may
be injected separately into a mixing means which is located outside
the vessel. In the latter case, the liposomes are formed in the
mixing means and then transferred to another vessel for collection
purpose.
[0074] The organic component consists of a suitable non-toxic,
pharmaceutically acceptable solvent such as, for example ethanol,
glycerol, propylene glycol and polyethylene glycol, and a suitable
phospholipid which is soluble in the solvent. Suitable
phospholipids which can be employed include lecithin,
phosphatidylcholine, phosphatydylserine, phosphatidylethanol-amine,
phosphatidylinositol, lysophosphatidylcholine and phospha-tidyl
glycerol, for example. Other lipophilic additives may be employed
in order to selectively modify the characteristics of the
liposomes. Examples of such other additives include stearylamine,
phosphatidic acid, tocopherol, cholesterol and lanolin
extracts.
[0075] In addition, other ingredients which can prevent oxidation
of the phospholipids may be added to the organic component.
Examples of such other ingredients include tocopherol, butylated
hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate and
ascorbyl oleate. Preservatives such a benzoic acid, methyl paraben
and propyl paraben may also be added.
[0076] Apart from the above-described compositions, use may be made
of covers, e.g. plasters, bandages, dressings, gauze pads and the
like, containing an appropriate amount of an anti-VLA antibody
therapeutic. In some cases use may be made of plasters, bandages,
dressings, gauze pads and the like which have been impregnated with
a topical formulation containing the therapeutic formulation.
[0077] The pharmaceutical compositions of this invention may also
be administered by nasal aerosol or inhalation through the use of a
nebulizer, a dry powder inhaler or a metered dose inhaler. Such
compositions are prepared according to techniques well-known in the
art of pharmaceutical formulation and may be prepared as solutions
in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, and/or other conventional solubilizing or dispersing
agents.
[0078] According to another embodiment compositions containing a
compound of this invention may also comprise an additional agent
selected from the group consisting of corticosteroids,
antiinflammatories, immunosuppressants, antimetabolites, and
immunomodulators. Specific compounds within each of these classes
may be selected from any of those listed under the appropriate
group headings in "Comprehensive Medicinal Chemistry", Pergamon
Press, Oxford, England, pp. 970-986 (1990), the disclosure of which
is herein incorporated by reference. Also included within this
group are compounds such as theophylline, sulfasalazine and
aminosalicylates (antiinflammatories); cyclosporin, FK-506, and
rapamycin (immunosuppressants); cyclophosphamide and methotrexate
(antimetabolites); steroids (inhaled, oral or topical) and
interferons (immunomodulators).
[0079] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated, and the particular mode of
administration. It should be understood, however, that a specific
dosage and treatment regimen for any particular patient will depend
upon a variety of factors, including the activity of the specific
compound employed, the age, body weight, general health, sex, diet,
time of administration, rate of excretion, drug combination, and
the judgment of the treating physician and the severity of the
particular disease being treated. The amount of active ingredient
may also depend upon the therapeutic or prophylactic agent, if any,
with which the ingredient is co-administered.
[0080] The dosage and dose rate of the compounds of this invention
effective to produce the desired effects will depend on a variety
of factors, such as the nature of the inhibitor, the size of the
patient, the goal of the treatment, the nature of the pathology to
be treated, the specific pharmaceutical composition used, and the
judgment of the treating physician. Dosage levels of between about
0.001 and about 100 mg/kg body weight per day, preferably between
about 0.1 and about 50 mg/kg body weight per day of the active
ingredient compound are useful. Most preferably, the antibody
homlogs will be administered at a dose ranging between about 0.1
mg/kg body weight/day and about 20 mg/kg body weight/day,
preferably ranging between about 0.1 mg/kg body weight/day and
about 10 mg/kg body weight/day and at intervals of every 1-14 days.
Preferably, an antibody composition is administered in an amount
effective to provide a plasma level of antibody of at least 1
.mu.g/ml.
[0081] Persons having ordinary skill in the art can readily test if
an antagonist of the invention is having it intended effect. For
instance, cells contained in a sample of the individual's
epithelium are probed for the presence of the agent in vitro (or ex
vivo) using a second reagent to detect the administered agent. For
example, this may be a fluorochrome labelled antibody specific for
the administered agent which is then measured by standard FACS
(fluorescence activated cell sorter) analysis. Alternatively,
presence of the administered agent is detected in vitro (or ex
vivo) by the inability or decreased ability of the individual's
cells to bind the same agent which has been itself labelled (e.g.,
by a fluorochrome). The preferred dosage should produce detectable
coating of the vast majority of hedgehog-positive cells.
Preferably, coating is sustained in the case of an antibody homolog
for a 1-14 day period.
[0082] Practice of the present invention will employ, unless
indicated otherwise, conventional techniques of cell biology, cell
culture, molecular biology, microbiology, recombinant DNA, protein
chemistry, and immunology, which are within the skill of the art.
Such techniques are described in the literature. See, for example,
Molecular Cloning: A Laboratory Manual, 2nd edition. (Sambrook,
Fritsch and Maniatis, eds.), Cold Spring Harbor Laboratory Press,
1989; DNA Cloning, Volumes I and II (D. N. Glover, ed), 1985;
Oligonucleotide Synthesis, (M. J. Gait, ed.), 1984; U.S. Pat. No.
4,683,195 (Mullis et al.,); Nucleic Acid Hybridization (B. D. Hames
and S. J. Higgins, eds.), 1984; Transcription and Translation (B.
D. Hames and S. J. Higgins, eds.), 1984; Culture of Animal Cells
(R. I. Freshney, ed). Alan R. Liss, Inc., 1987; Immobilized Cells
and Enzymes, IRL Press, 1986; A Practical Guide to Molecular
Cloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and
155 (Wu et al., eds), Academic Press, New York; Gene Transfer
Vectors for Mammalian Cells (J. H. Miller and M. P. Calos, eds.),
1987, Cold Spring Harbor Laboratory; Immunoclemical Methods in Cell
and Molecular Biology (Mayer and Walker, eds.), Academic Press,
London, 1987; Handbook of Experiment Immunology, Volumes I-IV (D.
M. Weir and C. C. Blackwell, eds.), 1986; Manipulating the Mouse
Embryo, Cold Spring Harbor Laboratory Press, 1986.
[0083] The following Examples are provided to illustrate the
present invention, and should not be construed as limiting
thereof.
EXAMPLES
Example 1
[0084] Treatment of Animals
[0085] Male C57/BL6 mice weighing 28-30 g, were housed in plastic
cages in groups of 4 in facilities approved by the American
Association for Accreditation of Laboratory Animal Care. The
animals were allowed to acclimate for one week to laboratory
conditions prior to starting the experiments. They had access to
Rodent Laboratory Chow 5001 (Purina Mills, Inc., St. Louis, Mo.)
and water ad libitum and housed in a room which gets filtered air
and has 12 hr light /12 hr dark cycle. Mice were assigned into the
following groups:
1 GROUP TREATMENT A Saline + Phosphate buffered saline B Saline +
Ha4/8 control IgG C Bleomycin + Ha4/8 control IgG D Bleomycin +
Ha31/8 (hamster anti-.alpha.1 .beta.1 rat integrin antibody) E
Bleomycin + Ha1/29 (hamster anti-.alpha.2 .beta.1 rat integrin
antibody)
[0086] Bleomycin sulfate was dissolved in pyrogen free sterile
isotonic saline just before intratraceheal (IT) instillation. Under
methoxyflurane anaesthesia mice in appropriate groups received by
intratracheal administration either 100 .mu.l of sterile isotonic
solution or 0.08 units of bleomycin solution in 100 .mu.l.
Antibodies (4 mg/kg) were administered by intraperitoneal injection
to mice in appropriate groups three times a week for 21 days post
installation. Thereafter, the animals in each group were killed by
an overdose of sodium pentobarbital (100-125 mg/kg ip) and their
lungs processed for bronchoalveolar lavage, biochemical and
histopathological studies.
[0087] Monoclonal Antibodies
[0088] Ha31/8 (hamster anti-.alpha.1.beta.1 integrin IgG); Hal/29
(hamster anti-rat .alpha.2.beta.1 integrin IgG); and Ha4/8 (hamster
anti-rat control IgG) are described in Mendrick and Kelly, Lab.
Invest. 69:690-702 (1993); Mendrick et al, Lab. Invest. 72:367-375
(1995), and commercially available (Pharmingen, San Diego,
Calif.).
[0089] Determination of Total Cell Number and Protein Levels in
Broncoalveolar Lavage
[0090] After cannulation of the trachea the lungs were lavaged with
5 ml of isotonic saline, given in five aliquots of 1 ml. The saline
was administered with a syringe through the cannula, the chest wall
was gently massaged, and the fluid was withdrawn. The fluid was
centrifuged at 1500 g for 20 minutes at 4 degree C., and resusended
in isotonic saline solultion. The protein content for the
supernatant from broncoalveolar lavage specimens was detemined by a
method of Lowry et al., J Biol. Chem. 1193: 265-275 (1951), with
bovine serume albumin as a standard. Total leukocyte count of cells
in suspension was determined in a Coulter Counter (Coulter
Electronics, Hialeah, Fla.).
[0091] Determination of Hydoxyproline
[0092] The lungs of animals used for biochemical studies were
perfused in situ via the right ventricle with ice-cold isotonic
saline to wash out blood from the pulmonary vasculature through an
opening in the left auricle. The lung lobes were quickly dissected
free of non-parenchymal tissue, dropped in liquid nitrogen for
quick freezing and then stored at -80C. The frozen lungs were later
thawed and homogenized in 0.1 M KCl, 0.02 M Tris buffer (pH 7.6)
with a Polytron homogenizer. Hydroxyproline content of the lung
homogenate as a measure of collagen content was quantitated by the
techniques of Woessner, Arch. Biochem. Biophys. 93: 440-447
(1961).
[0093] Results
[0094] In this study, we tested the hypothesis that neutralizing
antibody for integrin .alpha.1.beta.1 (anti.alpha.1.beta.1) or
integrin .alpha.2.beta.1 (anti.alpha.2.beta.1) may reduce bleomycin
(BL)-induced lung fibrosis in vivo. Male C57/BL6 mice were
intratraceally (IT) injected saline (SA) or BL at 0.08 U in 0.1 ml
followed by intraperitoneal (IP) injection of the antibody (100
.mu.g in 0.2 ml) three times a week. Twenty-one days after the IT
instillation, mice were killed for bronchoalveolar lavage (BAL),
biochemical and histopathological analysis.
[0095] Results:
2 Hydroxy- Total BAL BAL Group Treatment proline cells protein (n)
(IT + IP) (.mu.g/lung) (.times.10.sup.-3/lung) (.mu.g/lung) A(12)
SA + PBS 231 .+-. 15 2.2 .+-. 0.14 171 .+-. 21 B(10) SA + IgG 225
.+-. 21 2.3 .+-. 0.24 167 .+-. 24 C(9) BL + IgG 371 .+-. 42* 6.7
.+-. 0.99* 1079 .+-. 292* D(11) BL + anti.alpha.1.beta.1 230 .+-.
20 6.5 .+-. 1.17* 1238 .+-. 244* E(10) BL + anti.alpha.2.beta.1 221
.+-. 14 4.2 .+-. 0.72 945 .+-. 184* *Significantly higher than
other group
[0096] Lung histopathology showed fibrotic lesions in C group of
mice, but lungs from D and E groups indicated somewhat reduced
fibrosis compared to C group. Our data demonstrated that treatment
with anti.alpha.1.beta.1 or anti.alpha.2.beta.1 antibody reduced
BL-induced lung collagen accumulation in mice. However, treatment
with either antibody did not affect BL-induced increases in the BAL
cell number and protein level, except for anti.alpha.2.beta.1 which
reduced the total BAL cells. It is concluded that integrins
.alpha.1.beta.1 and .alpha.1.beta.1 play important roles in
BL-induced pulmonary fibrosis and the use of anti.alpha.1.beta.1 or
anti.alpha.2.beta.1 antibody has great antifibrotic potential in
vivo.
Example 2
[0097] Histopathological Study
[0098] After lung lavage, the thoracic cavity was opened and the
heart and lungs were removed en bloc. The lungs were instilled with
a 1% glutaraldehyde-parafornaldehyde fixative in 0.12M cacodylate
buffer at 400 m Osm at 30 cm H.sub.2O presure. The lungs are fixed
via this pressure for about 2 hours and then stored in fixative
with the tracheas occluded. Before embedding, the lung was isolated
from the heart and all non-pulmonary tissue by blunt dissection and
removed. Blocks of tissue were cut from at least two sagittal slabs
(2-3 mm thick) from the right cranial, right caudal, and left lung
lobes of each lung. Each block was cut with about a 1 cm.sup.2
face. The blocks were dehydrated in a graded series of ethanol and
embedded in paraffin. Sections (5 .mu.m thick) were cut from the
paraffin blocks and stained with haematoxylin and eosin for
histological evaluations.
[0099] Data Analysis and Interpretation
[0100] The data are analyzed in terms of average values with their
standard deviations and standard errors of means. Student's t-test,
chi-square distributions, correlation coefficient, analysis of
variance (ANOVA) and multiple comparison test will be applied to
judge the significance of differences between the control and
treatment groups using a computer based statistical package
(SAS/STAT Guide, 6th Ed. Cary, N.C. pp. 183-260 (1985)).
[0101] Histopathological Examination of Lungs
[0102] Histopathological examination of lungs was carried out on
mice sacrificed at 21 days after intratracheal instillation of
saline or bleomycin. The mice treated with saline and control IgG
(Group B) had no visible lesions and displayed interalveolar septa
with a normal thin appearance. In contrast, mice treated with
bleomycin and control IgG (Group C) had lesions varying from
multifocal locations in proximal acini to a diffuse distribution
that occasionally involved the pleura. In diffuse lesions, alveolar
spaces were often obliterated by organized connective tissue and
fibrotic lesions. In the multifocal lesion interalveolar septa were
thickened and lined by hypertrophied and hyperplastic cuboidal
epithelial cells and abundant airway inflammatory cells. The lungs
of mice treated with bleomycin and either anti-alpha1 (Group D) and
alpha2 (Group E) integrin antibodies appeared more like those in
Group B. Group D and E animals exhibited only a limited number of
fibrotic lesions, with mild multifocal septal thickening and small
aggregates of mononuclear cells.
Example 3
[0103] Cloning and mutagenesis of the .alpha.1-I domain. Human and
rat .alpha.1.beta.1 integrin I domain sequences were amplified from
full length cDNAs (Kern, et al. (1994) J. Biol. Chem. 269,
22811-22816; Ignatius et al. (1990) J. Cell Biol. 111, 709-720) by
the polymerase chain reaction (PCR) (PCR CORE Kit; Boehringer
Mannheim, GmbH Germany), using either human specific
(5'-CAGGATCCGTCAGCCCCACATTTCAA-3' [forward];
5'-TCCTCGAGGGCTTGCAGGGCAAATAT-3'' [reverse]) or rat specific
(5'-CAGGATCCGTCAGTCCTACATTTCAA-3' [forward];
5'-TCCTCGAGCGCTTCCAAAGCGAATA- T-3' [reverse]) primers. The
resulting PCR amplified products were purified, ligated into
pGEX4t-i (Pharmacia), and transformed into competent DH5.alpha.
cells (Life Technologies). Ampicillin resistant colonies were
screened for the expression of the .about.45 kDa glutathione
S-transferase-I domain fusion protein. The sequences from inserts
of plasmid DNA of clones that were selected for further
characterization were confirmed by DNA sequencing.
[0104] A rat/human chimeric .alpha.1-I domain (R.DELTA.H) was
generated (MORPH Mutagenesis kit; 5 prime-3 prime), exchanging the
rat residues G92, R93, Q94, and L97 (FIG. 4) for the corresponding
human residues, V, Q, R, and R, respectively. Clones harboring the
R.DELTA.H I domain were identified by the loss of a diagnostic Stu
1 restriction enzyme site, and the inserts confirmed by DNA
sequencing. The amino acid sequence of the human .alpha.1-I domain
is shown in FIG. 5.
Example 4
[0105] Generation of mAbs specific to the -.alpha.1 I domain.
Monoclonal antibodies have proved to be very useful probes in
studying the relationship between structure and function of
integrin subunits. For example, mAbs were used extensively to study
regions of the .beta.1 subunit associated with an activated
conformation (Qu, A., and Leahy, D. J. (1996) Structure 4,
931-942). Thus, to identify potential probes for conformational
changes of the .alpha.1-I domain, we generated a panel of mAbs to
the human .alpha.1-I domain.
[0106] Generation of anti-.alpha.1 I domain Monoclonal Antibodies.
Female Robertsonian mice (Jackson Labs) were immunized
intraperitoneally (i.p.) with 25 .mu.g of purified human
.alpha.1.beta.1 (Edwards et al. (1995) J. Biol. Chem. 270,
12635-12640) emulsified with complete Fruend's adjuvant (Life
Technologies). They were boosted three times i.p. with 25 .beta.g
of .alpha.1.beta.1 emulsified with incomplete Freunds's adjuvant
(Life Technologies). The mouse with the highest anti-.alpha.1-I
domain titer was boosted i.p. with 100 .mu.g of .alpha.1.beta.1
three days prior to fusion, and intravenously with 50 .mu.g of
.alpha.1.beta.1 one day prior to fusion. Spleen cells were fused
with FL653 myeloma cells at a 1:6 ratio and were plated at 100,000
and 33,000 per well into 96 well tissue culture plates.
[0107] Supernatants were assessed for binding to the
.alpha.1.beta.1 integrin by single color FACS. Prior to FACS
analysis, supernatants were incubated with untransfected K562 cells
to eliminate IgG that bound solely to the .beta. subunit.
Subsequently, 3-5 .times.10.sup.4 K562 cells transfected with the
.alpha.1 integrin subunit (K562-.alpha.1) suspended in FACS buffer
(1% fetal calf serum (FCS) in PBS containing 0.5% NaN.sub.3) were
incubated with supernatant for 45 minutes at 40.degree. C., washed
and incubated with anti-mouse IgG conjugated to phycoerythrin.
After washing twice with FACS buffer, cells were analyzed in a
Becton Dickinson Flow Cytometer.
[0108] Supernantants from the resulting hybridomas were screened
for binding to the .alpha.1-I domain. Briefly, 50 .mu.l of 30
.mu.g/ml human .alpha.1-I domain-GST fusion in PBS was coated onto
wells of a 96 -well plate (Nunc) overnight at 4.degree. C. The
plates were washed with PBS, blocked with 1% BSA in PBS and the
hybridoma supernatant was incubated with the I domain at room
temperature for 1 hour. After extensive washing with PBS containing
0.03% Tween 20, alkaline phosphatase linked anti-mouse IgG (Jackson
ImmunoResearch) was added for an additional hour. After a final
wash, 1 mg/ml p-nitrophenylphosphate (pNPP) in 0.1 M glycine, 1 mM
ZnCl.sub.2, and 1 mM MgCl.sub.2 was added for 30 minutes at room
temperature, and the plates were read at O.D. 405.
[0109] Selected supernatants were tested for their ability to
inhibit K562-.alpha.1 dependent adhesion to Collagen IV.
K562-.alpha.1 cells were labeled with 2 mM 2',7'
(bis-2-carboxyethyl-5 and 6) carboxyfluorescein penta
acetoxymethylester (BCECF; Molecular Probes) in DMEM containing
0.25% BSA at 37.degree. C. for 30 minutes. Labeled cells were
washed with binding buffer (10 mM Hepes, pH 7.4; 0.9% NaCl; and 2%
glucose) and resuspended in binding buffer plus 5 mM MgCl.sub.2 at
a final concentration of 1.times.10.sup.6 cells/ml. 50 .mu.l of
supernatant was incubated with an equal volume of 2.times.10.sup.5
K562-.alpha.1 cells in wells of a 96 well plate. The plate was then
centrifuged and the supernatants removed. Cells were resuspended in
binding buffer and transferred to wells of a collagen-coated plate
and incubated for 1 hour at 37.degree. C. Following incubation, the
non-adherent cells were removed by washing three times with binding
buffer. Attached cells were analyzed on a Cytofluor
(Millipore).
[0110] We initially identified 19 hybridomas, the supernatants of
which bound to human leukemia K562 cells expressing the alp
integrin (K562-.alpha.1) and to the .alpha.1-I domain. The
immunoglobulins were purified from each of these hybridomas and
tested for the ability to block either K562-.alpha.1 or .alpha.1-I
domain binding to collagen IV. The mAbs fall into two classes:
those that block and those that do not block .alpha.1.beta.1
function. For example, while the mAbs produced by clones AEF3, BGC5
and AJH10 bind the .alpha.1-I domain (FIG. 2A, data not shown for
BGC5), only mAb AJH10 inhibits .alpha.1-I domain-dependent (FIG.
2B) or K562-.alpha.1 (FIG. 2C) adhesion to collagen IV.
[0111] Sequencing of the Complementarity Determining Regions. To
establish the clonal origin of this panel of mAbs, we amplified by
PCR and sequenced the CDRs from 12 of the 19 antibodies (data not
shown).
[0112] 2 .mu.g of mRNA, isolated from 10.sup.7 hybridomas (
FastTrack mRNA isolation kit, Invitrogen), was reverse transcribed
(Ready-To-Go You Prime First Strand Kit, Pharmacia Biotech) using
25 pM each of the following primers: heavy chain VH1FOR-2
(Michishita et al. (1993) Cell 72, 857-867); light chain, VK4FOR,
which defines four separate oligos (Kern et al. (1994) J. Biol.
Chem. 269, 22811-22816). For each hybridoma, heavy and light chains
were amplified in four separate PCR reactions using various
combination of the following oligos: 1) Heavy chain: VH1FR1K
(Kamata et al. (1995) J. of Biol. Chem. 270, 12531-12535), VH1
BACK, VH1BACK (Baldwin et al.(1998) Structure 6, 923-935),
V.sub.Hfr1a, V.sub.Hfr1b, V.sub.Hfr1e, V.sub.Hfr1f, V.sub.Hfr1g
(Ignatius et al. (1990) J. Cell Biol. 111, 709-720), or VH1FOR-2
(Michishita, M., Videm, V., and Arnaout, M. A. (1993) Cell 72,
857-867); 2) Light chain: VK1BACK (Baldwin et al. (1998) Structure
6, 923-935), VK4FOR, VK2BACK oligos (Kem et al. (1994) J. Biol.
Chem. 269, 22811-22816), or V.sub.Kfr1a, V.sub.Hfr1c, V.sub.Hfr1e,
V.sub.Hfr1f (Ignatius et al. (1990) J. Cell Biol. 111, 709-720).
Products were amplified (5 min at 95.degree. C., 50 cycles of 1 min
at 94.degree. C., 2 min at 55.degree. C., 2 min at 72.degree. C.,
and a final cycle of 10 min at 72.degree. C.), gel purified
(QIAquick, Qiagen), and sequenced directly using various of the
listed oligos on an ABI 377 Sequencer.
[0113] Sequences from clones producing function-blocking mAbs were
nearly identical across all the complementarity-determining regions
(CDRs) and the intervening framework regions suggesting that these
hybridomas are clonally related.
Example 5
[0114] Immunoblotting and FACS Analysis. Sequences of the variable
regions of the non-blocking antibodies were markedly different from
the clonally related family of sequences found for the blocking
antibodies. As the blocking antibodies appear to originate from a
single clone, we chose one (AJH 10) to characterize further.
[0115] Immunoblotting The smooth muscle cell layer dissected from
sheep aorta, and K562-.alpha.1 cells were extracted with 1% Triton
X-100 in 50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM
phenylmethylsulfonyl flouride (PMSF), 20 .mu.g/ml aprotinin, 10
.mu.g/ml leupeptin, 10 mM ethylenediaminetetraacefic acid (EDTA).
Samples were subjected to 4-20% gradient SDS-PAGE, and
electroblotted onto nitrocellulose membranes. The blots were
blocked with 5% dry milk in TBS; washed in TBS containing 0.03%
Tween-20, and incubated with antibodies in blocking buffer
containing 0.05% NaN.sub.3 for 2 hours. Blots were then washed as
before, incubated with horseradish peroxidase conjugated anti-mouse
IgG for one hour, washed again and then treated with ECL reagent
(Amersham). Blots were then exposed to film (Kodak) for 30 to 60
seconds, and developed.
[0116] Immunoblotting (FIG. 3A) and FACS analysis (FIG. 3B)
demonstrate that AJH10 reacts with human, rabbit, and sheep, but
not rat .alpha.1.beta.1 integrin suggesting that the blocking mAbs
bind to an evolutionarily conserved, linear epitope. The
non-blocking mAbs were neither efficient at immunoblotting nor did
they react with species other than human.
Example 6
[0117] Binding of the .alpha.1-I domain to collagen is divalent
cation-dependent
[0118] A. Purification of the .alpha.1-I domains.
[0119] The .alpha.1-I domains were expressed in E. coli as GST
(glutathione-S-transferase) fusion proteins containing a thrombin
cleavage site at the junction of the sequences. The clarified
supernatant from cells lysed in PBS was loaded onto a glutathione
Sepharose 4B column (Pharmacia) which was washed extensively with
PBS. The .alpha.1-I domain-GST fusion protein was eluted with 50 mM
Tris-HCl, pH 8.0, 5 mM glutathione (reduced). For denaturation
studies, the I domain was cleaved with thrombin in 50 mM Tris, pH
7.5, and purified from the GST fusion partner. DTT was added to 2
mM and the sample was loaded on a glutathione Sepharose 4B column.
The flow-through and wash fractions were pooled and loaded onto a Q
Sepharose FF column (Pharmacia). The .alpha.1-I domain was eluted
with 50 mM Tris HCl, pH 7.5, 10 mM 2-mercaptoethanol, 75 mM NaCl.
The purified I domain displayed its predicted mass (Lee et al.
(1995) Structure 3, 1333-1340, 871 Da) by electrospray
ionization-mass spectrometry (ESI-MS), migrated as a single band by
SDS-PAGE, and the protein eluted as a single peak of appropriate
size by size exclusion chromotography on a Superose 6 FPLC column
(Pharmacia).
[0120] B. Functional Analysis
[0121] 96 well plates were coated overnight at 4.degree. C. with 1
.mu.g/ml collagen IV (Sigma) or collagen Type I (Collaborative
Biomedical), washed with Triton buffer (0.1% Triton X-100; 1 mM
MnCl.sub.2; 25 mM Tris-HCl; 150 mM NaCl), and blocked with 3%
bovine serum albumin (BSA) in 25 mM Tris-HCl; 150 mM NaCl (TBS).
Serial dilutions of the .alpha.1-I domain-GST fusion protein in TBS
containing 1 mM MnCl.sub.2 and 3% BSA were incubated on the coated
plates at room temperature for 1 hour, and washed in Triton buffer.
Bound .alpha.1-I domain was detected with serial additions of 10
.mu.g/ml biotinylated anti-GST polyclonal antibody (Pharmacia);
ExtrAvidin-horseradish peroxidase (Sigma) diluted 1:3000 in TBS
containing 1 mM MnCl.sub.2 and 3% BSA, and 1-Step ABTS
(2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]; Pierce). Plates
were read at O.D. 405 on a microplate reader (Molecular
Devices).
[0122] Results.
[0123] The human and rat (95% identity to human) .alpha.1-I domains
were expressed in E. coli as GST-fusion proteins and purified over
glutathione sepharose. Both proteins were examined for binding to
collagen I and IV using a variation of an ELISA-based assay
previously described (Qu, A., and Leahy, D. J. (1995) Proc. Natl.
Acad. Sci USA 92, 10277-10281). The human .alpha.1-I domain binds
collagen IV with better efficiency than collagen I (FIG. 1A). An
antibody specific to the.alpha.1-I domain, but not an antibody
specific to the .alpha.2-I domain (FIG. 1B) abrogated binding to
both ligands (data for collagen I is not shown). Both Mn.sup.2+ and
Mg.sup.2+ stimulated binding, and EDTA reduced binding to
background levels (FIG. 1C). No measurable differences in ligand
binding were detected between the human and rat .alpha.1-I domains
suggesting that the sequence differences between species are not
functionally relevant (data not shown). Thus, the .alpha.1-I
domain, specifically, require cation for efficient ligand
binding.
Example 7
[0124] A Cation-Dependent Epitope Resides near the MIDAS motif. We
exploited the observation that AJH10 recognizes the human, but not
the rat .alpha.1-I domain sequences to map the epitope for the
.alpha.1.beta.1 function-blocking mAbs. The human and rat sequences
differ by only 12 amino acids, 4 of which lie in a stretch of 6
amino acids (aa 92-97, FIG. 4A) adjacent to the critical threonine
(FIG. 4A, aa 98) within the MIDAS motif To test the hypothesis that
the 6 amino acid residues, Val-Gln-Arg-Gly-Gly-Arg, comprise the
epitope for the blocking mAbs, we constructed a chimeric I domain
(R.DELTA.H), exchanging the rat residues G92, R93, Q94, and L97 for
the corresponding human residues, V, Q, R, and R, respectively. AJH
10, along with all the function-blocking mAbs, recognizes the
chimeric I domain (R.DELTA.H; FIG. 4B).
[0125] To orient these residues with respect to the MIDAS domain in
the tertiary structure of the .alpha.1-I domain, we modeled the
.alpha.1-I domain using the coordinates of the crystal structure of
the .alpha.2 I domain.
[0126] A homology model of the human .alpha.1 I-domain was built
using the X-ray crystal structure of the human .alpha.2 I-domain
(Ward et al. (1989) Nature 341, 544-546). The model was built using
the homology modeling module of Insight II (version 2.3.5; Biosym
Technologies). The program CHARMM (Clackson et al. (1991) Nature
352, 624-628) was used with the all-hydrogen parameter set 22 with
a distant dependent dielectric constant of two times the atom
separation distance. We first did 1000 steps of steepest descent
minimization with mass-weighted harmonic positional constraints of
1 kcal/(mol .ANG..sup.2) on all atoms of the .alpha.1-I domain.
This minimization was followed by another 1000 steps of steepest
descent and 5000 steps of Adopted-Basis Newton Raphson with
constraints of 0.1 kcal/( mol .ANG..sup.2) on the C-.alpha. atoms
of the .alpha.1-I domain to avoid significant deviations from the
.alpha.2-I domain X-ray crystal structure.
[0127] The .alpha.1.beta.1 and .alpha.2.beta.1 integrin sequences
exhibit 51% identity with no insertions or deletions, suggesting
that the overall structure of the two I domains will be similar.
The metal coordination site is predicted to be the same in the
.alpha.1-I domain as in the .alpha.2-I domain, and the residues
that comprise the epitope for the blocking mAbs lie on a loop
between helix .alpha.3 and helix .alpha.4 which contains the
threonine within the MIDAS motif critical for cation binding. The
.alpha.1-I domain model predicts that the amide nitrogen of Q92
(FIG. 4A) hydrogen bonds with the carbonyl group of I133, the
residue adjacent to S32. Thus, the loop that contains the epitope
may play a functional role in stabilizing the MIDAS region.
[0128] The proximity to and the potential interaction of the loop
containing the epitope with the MIDAS motif suggested that the
epitope, itself, might be sensitive to the presence of divalent
cation. Initial ELISA-based experiments confirmed that binding of
AJH10, but not AEF3 (FIG. 6A) to purified .alpha.1.beta.1 integrin
increases in the presence of cations. Binding of AJH10 to cell
surface-expressed .alpha.1.beta.1 is also enhanced by the addition
of cation (FIG. 6B). To further analyze this observation, we
measured the relative binding affinities of the blocking mAbs, in
the presence or absence of divalent cations, using a surface
plasmon resonance (SPR) biosensor. Monitoring the reversible
binding of the mAbs to the recombinant .alpha.1-I domain in real
time allows the derivation of the association (ka) and dissociation
rates (kd), as well as the corresponding apparent dissociation
constants (K.sub.D) The addition of cation decreased the K.sub.D of
blocking mAb AJH10 from 400 to 20 nM (data not shown). The addition
of cation had no effect on the K.sub.D of non-blocking, control mAb
AEF3 (data not shown). Analysis of the ka and kd associated with
binding reveals that the increase in affinity is primarily
attributable to a decrease in the rate of dissociation (data not
shown). For example, in the absence of cation, AJH10 has a
dissociation rate constant of 1.65.times.10.sup.-3/sec. Addition of
Mn.sup.2+ decreases the dissociation rate constant by a factor of 8
to 2.12.times.10.sup.-4/sec (data not shown) while increasing the
association rate by only a factor of 2 (3.9.times.10.sup.3
M.sup.-s.sup.-1 to 8.0.times.10.sup.3 M.sup.-1 s.sup.-1). Thus, the
addition of divalent cation appears to stabilize the epitope rather
than unmask a cryptic site, consistent with the proximity of the
epitope to the MIDAS region.
Example 8
[0129] Cation is required for I domain Stability. One
interpretation of the effect Mn.sup.2+ and Mg.sup.2+ have on
epitope expression is that divalent cations are required to
stabilize the MIDAS region, or the entire .alpha.1-I domain. Thus,
we looked at the stability of the .alpha.1-I domain in the presence
or absence of cations under denaturing conditions.
[0130] The presence of divalent cations had a stabilizing effect on
the .alpha.1-I domain structure readily detected by measuring the
susceptibility of the protein to denaturation by urea (FIG. 7).
[0131] The denaturation of the .alpha.1-I domain as a function of
urea was measured by fluorescence spectroscopy in an Aminco-Bowman
series 2 Luminescence Spectrometer. Samples containing 0.6 .mu.M
.alpha.1-I domain in 50 mM Tris HCl pH 7.0, 0.15 mM DTT with no
addition, 1 mM CaCl.sub.2, or 1 mM MnCl.sub.2 and with the varying
amounts of urea were analyzed at 25.degree. C. using an excitation
wavelength of 280 nm. Emission spectra from 300-400 nm were
collected. Fluorescence data at 350 nm were plotted as a function
of urea and standardized using the change in fluorescence from 0 to
9 M urea for each of the test conditions as a measure of the total
fraction folded.
[0132] As described above, the denaturation of the .alpha.1-I
domain was assessed by monitoring the change in intrinsic
fluorescence that results from the exposure of buried tryptophan
and tyrosine residues to the aqueous environment as the protein
unfolds. Denaturation produced both an increase in fluorescence
intensity and a red shift in the emission spectrum. The maximal
effect was seen at 360 nm where denaturation of the .alpha.1-I
domain resulted in a greater than 4-fold increase in intrinsic
fluorescence intensity. In the absence of divalent cation, the
.alpha.1-I domain was sensitive to the presence of low
concentrations of urea and the amount needed to produce a half
maximal change in fluorescence intensity was 3.4 M urea. In the
presence of Mn.sup.2+, half maximal denaturation shifted to 6.3 M
urea, indicating a substantial stabilization of the .alpha.1-I
domain.
[0133] The output of the spectrophotometric data discussed above is
determined primarily by the fluorescence of a single buried
tryptophan, which lies within the MIDAS region of the .alpha.1-I
domain (W36, FIG. 4A). Thus, the spectrophotometric data only
provide a view of the MIDAS region and not of the entire
I-domain.
Example 9
[0134] Circular dichroism. To distinguish between local and
possible wide range effects on structure, we determined, by
measuring circular dichroism spectra in the presence or absence of
Mn.sup.2+ and Mg.sup.2+, the temperature at which the I domain
denatured, and the effect of denaturation on secondary structure of
the protein. The circular dichroism measurements described below
revealed that cation binding effects not just the local expression
of the epitope, but stabilizes the secondary structure of the
.alpha.1-I domain.
[0135] Circular dichroism spectra were recorded using a J-710
spectropolarimeter (JASCO, Japan) equipped with a programmable
temperature water bath (CTC-345, JASCO). Far-UV (185-250 nm) and
temperature-dependent measurements were performed using U-type
cells of path-length 0.0148 cm and volume 0.045 ml with .alpha.1-I
domain in 20 mM HEPES, 1mM EDTA, 1 mM DTT, pH 7.5 in the absence of
divalent cations, or the presence of either 2 mM Mg.sup.2+ or
Mn.sup.2+ at a protein concentration of 55 .mu.M. CD spectra were
recorded using a scan speed of 20 nm/min, a response time of 2 s
and a band-width of 2 nm. Temperature-dependent measurements were
performed in the range 10-80.degree. C. The continuous temperature
scan at fixed wavelength (222 nm) in the far-UV range was done
using a scan rate of 50.degree. C./hr and a response time of 8 s.
Data are presented as molar ellipticity per residue.
[0136] Near and far UV CD spectra for the .alpha.1-I domain, in the
presence and absence of cation at room temperature, were
indistinguishable (data not shown). In contrast, large,
cation-dependent differences were seen in the susceptibility of the
I domain to thermal denaturation. In the absence of divalent
cations, the I domain denatured at T.sub.m=49.5.degree. C. Both
Mn.sup.2+ (T.sub.m=58.6.degree. C.) and Mg.sup.2+
(T.sub.m=54.6.degree. C.) stabilized the I domain as indicated by
increases in T.sub.m (FIG. 8). Heat denaturation in the apo state
was accompanied by a 20-25% decrease in ordered secondary structure
at 65.degree. C. Decreases of 45% were observed for the Mn.sup.2+
state at 70.degree. C. and of 34% for the Mg.sup.2+ state at
80.degree. C. For the apo state, CD spectrum at 65.degree. C. and
80.degree. C. have minima, which are characteristic of a high
content of a helical structure whereas in the presence of divalent
cations, CD spectra at 70-80.degree. C. have shape that are
characteristic for "aggregational" .beta.-structure. These data
suggest that, in addition to the local stabilizing effect cations
have on the MIDAS region, the presence of cations has a wide
ranging effect on the secondary structure of the .alpha.1-I domain.
It is interesting to note the Mn.sup.2+ is more stabilizing than
Mg.sup.2+ as evidenced by a greated shift in T.sub.m. Since
Mn.sup.2+ is more effective at promoting ligand binding to the
.alpha.1.beta.1 integrin (Bridges et al. (1995) Mol. Immunol 32,
1329-1338), the stabilizing effects of Mn.sup.2+ may be related to
the increased affinity of the I domain for ligand.
[0137] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent to those skilled in the art
that certain changes and modifications will be practiced.
Therefore, the description and examples should not be construed as
limiting the scope of the invention, which is delineated by the
appended claims.
Sequence CWU 1
1
10 1 26 DNA Homo sapien 1 caggatccgt cagccccaca tttcaa 26 2 26 DNA
Homo sapien 2 tcctcgaggg cttgcagggc aaatat 26 3 26 DNA Homo sapien
3 caggatccgt cagtcctaca tttcaa 26 4 26 DNA Homo sapien 4 tcctcgagcg
cttccaaagc gaatat 26 5 214 PRT Rat 5 Val Ser Pro Thr Phe Gln Val
Val Asn Ser Phe Ala Pro Val Gln Glu 1 5 10 15 Cys Ser Thr Gln Leu
Asp Ile Val Ile Val Leu Asp Gly Ser Asn Ser 20 25 30 Ile Tyr Pro
Trp Glu Ser Val Ile Ala Phe Leu Asn Asp Leu Leu Lys 35 40 45 Arg
Met Asp Ile Gly Pro Lys Gln Thr Gln Val Gly Ile Val Gln Tyr 50 55
60 Gly Glu Asn Val Thr His Glu Phe Asn Leu Asn Lys Tyr Ser Ser Thr
65 70 75 80 Glu Glu Val Leu Val Ala Ala Lys Lys Ile Gly Arg Gln Gly
Gly Leu 85 90 95 Gln Thr Met Thr Ala Leu Gly Ile Asp Thr Ala Arg
Lys Glu Ala Phe 100 105 110 Thr Glu Ala Arg Gly Ala Arg Arg Gly Val
Lys Lys Val Met Val Ile 115 120 125 Val Thr Asp Gly Glu Ser His Asp
Asn Tyr Arg Leu Lys Gln Val Ile 130 135 140 Gln Asp Cys Glu Asp Glu
Asn Ile Gln Arg Phe Ser Ile Ala Ile Leu 145 150 155 160 Gly His Tyr
Asn Arg Gly Asn Leu Ser Thr Glu Lys Phe Val Glu Glu 165 170 175 Ile
Lys Ser Ile Ala Ser Glu Pro Thr Glu Lys His Phe Phe Asn Val 180 185
190 Ser Asp Glu Leu Ala Leu Val Thr Ile Val Lys Ala Leu Gly Glu Arg
195 200 205 Ile Phe Ala Leu Glu Ala 210 6 214 PRT Homo sapien 6 Val
Ser Pro Thr Phe Gln Val Val Asn Ser Ile Ala Pro Val Gln Glu 1 5 10
15 Cys Ser Thr Gln Leu Asp Ile Val Ile Val Leu Asp Gly Ser Asn Ser
20 25 30 Ile Tyr Pro Trp Asp Ser Val Thr Ala Phe Leu Asn Asp Leu
Leu Lys 35 40 45 Arg Met Asp Ile Gly Pro Lys Gln Thr Gln Val Gly
Ile Val Gln Tyr 50 55 60 Gly Glu Asn Val Thr His Glu Phe Asn Leu
Asn Lys Tyr Ser Ser Thr 65 70 75 80 Glu Glu Val Leu Val Ala Ala Lys
Lys Ile Val Gln Arg Gly Gly Arg 85 90 95 Gln Thr Met Thr Ala Leu
Gly Thr Asp Thr Ala Arg Lys Glu Ala Phe 100 105 110 Thr Glu Ala Arg
Gly Ala Arg Arg Gly Val Lys Lys Val Met Val Ile 115 120 125 Val Thr
Asp Gly Glu Ser His Asp Asn His Arg Leu Lys Lys Val Ile 130 135 140
Gln Asp Cys Glu Asp Glu Asn Ile Gln Arg Phe Ser Ile Ala Ile Leu 145
150 155 160 Gly Ser Tyr Asn Arg Gly Asn Leu Ser Thr Glu Lys Phe Val
Glu Glu 165 170 175 Ile Lys Ser Ile Ala Ser Glu Pro Thr Glu Lys His
Phe Phe Asn Val 180 185 190 Ser Asp Glu Leu Ala Leu Val Thr Ile Val
Lys Thr Leu Gly Glu Arg 195 200 205 Ile Phe Ala Leu Glu Ala 210 7 6
PRT Rat 7 Gly Arg Gln Gly Gly Leu 1 5 8 6 PRT Homo sapien 8 Val Gln
Arg Gly Gly Arg 1 5 9 214 PRT Homo sapien 9 Val Ser Pro Thr Phe Gln
Val Val Asn Ser Ile Ala Pro Val Gln Glu 1 5 10 15 Cys Ser Thr Gln
Leu Asp Ile Val Ile Val Leu Asp Gly Ser Asn Ser 20 25 30 Ile Tyr
Pro Trp Asp Ser Val Thr Ala Phe Leu Asn Asp Leu Leu Lys 35 40 45
Arg Met Asp Ile Gly Pro Lys Gln Thr Gln Val Gly Ile Val Gln Tyr 50
55 60 Gly Glu Asn Val Thr His Glu Phe Asn Leu Asn Lys Tyr Ser Ser
Thr 65 70 75 80 Glu Glu Val Leu Val Ala Ala Lys Lys Ile Val Gln Arg
Gly Gly Arg 85 90 95 Gln Thr Met Thr Ala Leu Gly Thr Asp Thr Ala
Arg Lys Glu Ala Phe 100 105 110 Thr Glu Ala Arg Gly Ala Arg Arg Gly
Val Lys Lys Val Met Val Ile 115 120 125 Val Thr Asp Gly Glu Ser His
Asp Asn His Arg Leu Lys Lys Val Ile 130 135 140 Gln Asp Cys Glu Asp
Glu Asn Ile Gln Arg Phe Ser Ile Ala Ile Leu 145 150 155 160 Gly Ser
Tyr Asn Arg Gly Asn Leu Ser Thr Glu Lys Phe Val Glu Glu 165 170 175
Ile Lys Ser Ile Ala Ser Glu Pro Thr Glu Lys His Phe Phe Asn Val 180
185 190 Ser Asp Glu Leu Ala Leu Val Thr Ile Val Lys Thr Leu Gly Glu
Arg 195 200 205 Ile Phe Ala Leu Glu Ala 210 10 7 PRT homo sapien 10
Val Gln Arg Gly Gly Arg Gln 1 5
* * * * *