U.S. patent application number 10/780149 was filed with the patent office on 2005-04-28 for superficial zone protein-binding molecules and uses thereof.
Invention is credited to Hutchins, Jeff T., Kuettner, Klaus E., Lindley, Kathryn Mason, Schmid, Thomas M., Schumacher, Barbara L., Stimpson, Stephen Anthony, Su, Jui-Lan.
Application Number | 20050089881 10/780149 |
Document ID | / |
Family ID | 9885347 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089881 |
Kind Code |
A1 |
Hutchins, Jeff T. ; et
al. |
April 28, 2005 |
Superficial zone protein-binding molecules and uses thereof
Abstract
The invention provides an antibody or a fragment thereof having
specific binding affinity for superficial zone protein (SZP) or a
variant, fragment, or protein core thereof, wherein the binding
affinity of the antibody or fragment thereof for human superficial
zone protein is the same or greater than the binding affinity for
bovine superficial zone protein in a competitive binding assay,
IAsys analysis, or BIAcore analysis. The present invention further
provides hybidoma cells that produce the monoclonal antibody and
antibody reagent kits comprising the antibody or fragment of the
invention. Further provided by the invention are methods of SZP
detection, methods of diagnosing a degenerative joint condition,
and screening methods related to the use of the antibody or
fragment thereof.
Inventors: |
Hutchins, Jeff T.; (Chapel
Hill, NC) ; Kuettner, Klaus E.; (Chicago, IL)
; Lindley, Kathryn Mason; (Chapel Hill, NC) ;
Schmid, Thomas M.; (Downers Grove, IL) ; Schumacher,
Barbara L.; (Cardiff by the Sea, CA) ; Stimpson,
Stephen Anthony; (Chapel Hill, NC) ; Su, Jui-Lan;
(Chapel Hill, NC) |
Correspondence
Address: |
DAVID J LEVY, CORPORATE INTELLECTUAL PROPERTY
GLAXOSMITHKLINE
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Family ID: |
9885347 |
Appl. No.: |
10/780149 |
Filed: |
February 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10780149 |
Feb 17, 2004 |
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09780718 |
Feb 9, 2001 |
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6720156 |
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60181377 |
Feb 9, 2000 |
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60201989 |
May 3, 2000 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C07K 16/18 20130101;
G01N 2500/00 20130101; G01N 2800/52 20130101; G01N 2800/105
20130101; A61K 2039/53 20130101; G01N 33/6887 20130101; C12N
2799/026 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Goverment Interests
[0002] This invention was made in part with government support
under grant 2P50-AR39239 awarded by the National Institute for
Arthritis and Musculoskeletal Diseases of the National Institutes
of Health. The government has certain rights in the invention.
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2000 |
GB |
0003092.4 |
Claims
What is claimed is:
1. A method of detecting superficial zone protein in a sample,
comprising: (a) contacting the sample with a monoclonal antibody or
fragment thereof having specific binding affinity for superficial
zone protein, wherein the binding affinity of the antibody or
fragment thereof for human superficial zone protein is the same or
greater than the binding affinity for bovine superficial zone
protein in a competitive binding assay, resonant mirror biosensor
analysis or surface plasmon resonance analysis, under conditions in
which an antigen/antibody complex can form; and (b) detecting the
presence of the antigen/antibody complex, wherein the presence of
the antigen/antibody complex indicates the presence of superficial
zone protein in the sample.
2. The method of claim 1, wherein the sample is selected from the
group consisting of synovial fluid, tears, saliva, urine, serum,
plasma, and bone marrow, synovium, tendon, tendon sheath, ligament,
meniscus, intervertebral disk, pericardium, chondrocytes, and
articular cartilage.
3. The method of claim 1, wherein the detecting step comprises an
assay selected from the group consisting of a competition or
sandwich ELISA, a radioimmunoassay, a Western blot assay, an
immunohistological assay, an immunocytochemical assay, a dot blot
assay, a fluorescence polarization assay, a scintillation proximity
assay, a homogeneous time resolved fluorescence assay, a resonant
mirror biosensor analysis, and a surface plasmon resonance
analysis.
4. A method of diagnosing a degenerative joint condition in a
subject, comprising: (a) obtaining a test sample from the subject;
(b) detecting superficial zone protein in the test sample; and (c)
comparing the amount of superficial zone protein in the sample with
an amount present in a control sample; a modulated amount of
superficial zone protein in the test sample indicating the
degenerative joint condition.
5. The method of claim 4, wherein the degenerative joint condition
is an arthritic condition.
6. The method of claim 5, wherein the arthritic condition is
osteoarthritis.
7. The method of claim 5, wherein the arthritic condition is
rheumatoid arthritis.
8. The method of claim 4, wherein the test sample and control
sample are selected from the group consisting synovial fluid,
tears, saliva, urine, serum, plasma, and bone marrow, synovium,
tendon, tendon sheath, ligament, meniscus, intervertebral disk,
pericardium, chondrocytes, and articular cartilage.
9. The method of claim 8, wherein the test sample is synovial fluid
or synovium and wherein the degenerative joint condition is
indicated by an elevated amount of superficial zone protein in the
test sample.
10. The method of claim 4, wherein the superficial zone protein is
detected by contacting the test sample with the antibody or
fragment thereof of claim 1, under conditions in which an
antigen/antibody complex can form, and detecting the level of
antigen/antibody complex in the test sample.
11. The method of claim 8, wherein the test sample is articular
cartilage or chondrocytes and wherein the degenerative joint
condition is indicated by an decrease in the amount of superficial
zone protein.
12. The method of claim 4, wherein the detecting step comprises an
assay selected from the group consisting of a competition or
sandwich ELISA, a radioimmunoassay, a Western blot assay, an
immunohistological assay, an immunocytochemical assay, a dot blot
assay, a fluorescence polarization assay, a scintillation proximity
assay, a homogeneous time resolved fluorescence assay, a resonant
mirror biosensor analysis, and a surface plasmon resonance
analysis.
13. A method of screening for a substance that modulates levels of
superficial zone protein, comprising: (a) contacting a test sample
with the substance to be screened, wherein the test sample contains
superficial zone protein-producing cells; (b) contacting, under
conditions in which an antigen/antibody complex can form, the
superficial zone protein in the test sample with a monoclonal
antibody or a fragment thereof having specific binding affinity for
superficial zone protein, wherein the binding affinity of the
antibody or fragment thereof for human superficial zone protein is
the same or greater than the binding affinity for bovine
superficial zone protein in a competitive binding assay, resonant
mirror biosensor analysis or surface plasmon resonance analysis;
(c) detecting the level of the antigen/antibody complex in the test
sample; and (d) comparing the level of the antigen/antibody complex
in the test sample with the level of antigen/antibody complex in a
control sample, a lower or higher level of the antigen/antibody
complex in the test sample indicating a substance that modulates
levels of superficial zone protein.
14. The method of claim 13, wherein the superficial zone
protein-producing cells are selected from the group consisting of
chondrocytes, synovial cells, pericardial cells, bone marrow cells,
and other connective tissue cells.
15. The method of claim 13, wherein the superficial zone protein
contacted in step (b) is secreted by the superficial zone
protein-producing cells.
16. The method of claim 13, wherein the detecting step comprises an
assay selected from the group consisting of a competition or
sandwich ELISA, a radioimmunoassay, a Western blot assay, an
immunohistological assay, an immunocytochemical assay, a dot blot
assay, a fluorescence polarization assay, a scintillation proximity
assay, a homogeneous time resolved fluorescence assay, an resonant
mirror biosensor analysis, and a Surface plasmon resonance
analysis.
17. The method of claim 13, wherein the test sample is further
contacted with an agent that increases levels of superficial zone
protein and wherein the lower or higher level of the
antigen/antibody complex indicates a substance that attenuates or
potentiates the increase in superficial zone protein.
18. The method of claim 17, wherein the agent that increases levels
of superficial zone protein is a cytokine or growth factor.
19. The method of claim 18, wherein the cytokine or growth factor
is selected from the group consisting of TGF.beta., IGF-1, BMP-1,
BMP-4, and BMP-7.
20. A method of screening for a substance that reduces a
degenerative joint condition in a subject, comprising: (a)
contacting a first test sample from the subject with a monoclonal
antibody or a fragment thereof having specific binding affinity for
superficial zone protein, wherein the binding affinity of the
antibody or fragment thereof for human superficial zone protein is
the same or greater than the binding affinity for bovine
superficial zone protein in a competitive binding assay, resonant
mirror biosensor analysis or surface plasmon resonance analysis,
under conditions in which an antigen/antibody complex can form; (b)
detecting the level of the antigen/antibody complex in the first
test sample; (c) treating the subject with the substance to be
screened; (d) contacting a second test sample from the subject with
the antibody or fragment thereof, under conditions whereby an
antigen/antibody complex can form; (e) detecting the level of the
antigen/antibody complex in the second test sample; and (f)
comparing the level of the antigen/antibody complex in the first
test sample with the level of antigen/antibody complex in the
second test sample, a modulated level of the antigen/antibody
complex in the second test sample indicating a substance that
reduces the degenerative joint condition.
21. The method of claim 20, wherein the degenerative joint
condition is an arthritic condition.
22. The method of claim 21, wherein the arthritic condition is
osteoarthritis.
23. The method of claim 21, wherein the arthritic condition is
rheumatoid arthritis.
24. The method of claim 20, wherein the test samples are selected
from the group consisting of synovial fluid, tears, saliva, urine,
serum, plasma, bone marrow, synovium, tendon, tendon sheath,
ligament, meniscus, intervertebral disk, pericardium, chondrocytes,
and articular cartilage.
25. The method of claim 20, wherein the detecting steps comprise an
assay selected from the group consisting of a competition or
sandwich ELISA, a radioimmunoassay, a Western blot assay, an
immunohistological assay, an immunocytochemical assay, a dot blot
assay, a fluorescence polarization assay, a scintillation proximity
assay, a homogeneous time resolved fluorescence assay, an resonant
mirror biosensor analysis, and a Surface plasmon resonance
analysis.
26. A method of screening for subjects who would benefit from
treatment for a degenerative joint condition, comprising: (a)
obtaining a test sample from each subject; (b) detecting
superficial zone protein in the test samples; and (c) comparing the
amount of superficial zone protein in the test samples with an
amount present in a control sample; a modulated amount of
superficial zone protein in the test sample indicating a subject
that would benefit from treatment for the degenerative joint
condition.
27. The method of claim 26, wherein the degenerative joint
condition is an arthritic condition
28. The method of claim 27, wherein the arthritic condition is
osteoarthritis.
29. The method of claim 27, wherein the arthritic condition is
rheumatoid arthritis.
30. The method of claim 26, wherein the test sample and control
sample are selected from the group consisting synovial fluid,
tears, saliva, urine, serum, plasma, and bone marrow, synovium,
tendon, tendon sheath, ligament, meniscus, intervertebral disk,
pericardium, chondrocytes, and articular cartilage.
31. The method of claim 30, wherein the test sample is synovial
fluid or synovium and wherein the subjects that would benefit from
treatment are indicated by an elevated amount of superficial zone
protein in the test samples.
32. The method of claim 30, wherein the test sample is articular
cartilage or chondrocytes and wherein the subjects that would
benefit from treatment are indicated by a decrease in the amount of
superficial zone protein in the test samples.
33. The method of claim 26, wherein the superficial zone protein is
detected by contacting the test sample with a monoclonal antibody
or fragment thereof having specific binding affinity for
superficial zone protein under conditions in which an
antigen/antibody complex can form and detecting the level of
antigen/antibody complex in the test sample, wherein the binding
affinity of the antibody or fragment thereof for human superficial
zone protein is the same or greater than the binding affinity for
bovine superficial zone protein in a competitive binding assay,
resonant mirror biosensor analysis, or Surface plasmon resonance
analysis.
34. The method of claim 26, wherein the detecting step comprises an
assay selected from the group consisting of a competition or
sandwich ELISA, a radioimmunoassay, a Western blot assay, an
immunohistological assay, an immunocytochemical assay, a dot blot
assay, a fluorescence polarization assay, a scintillation proximity
assay, a homogeneous time resolved fluorescence assay, an resonant
mirror biosensor analysis, and a Surface plasmon resonance
analysis.
35. A method of monitoring a subject's response to a treatment for
a degenerative joint condition, comprising: (a) contacting a first
test sample from the subject with a monoclonal antibody or fragment
thereof having specific binding affinity for superficial zone
protein under conditions in which an antigen/antibody complex can
form, wherein the binding affinity of the antibody or fragment
thereof for human superficial zone protein is the same or greater
than the binding affinity for bovine superficial zone protein in a
competitive binding assay, resonant mirror biosensor analysis, or
Surface plasmon resonance analysis; (b) detecting the level of the
antigen/antibody complex in the first test sample; (c) treating the
subject; (d) contacting a second test sample from the subject with
the antibody or fragment thereof, under conditions whereby an
antigen/antibody complex can form; (e) detecting the level of the
antigen/antibody complex in the second test sample; and (f)
comparing the level of the antigen/antibody complex in the first
test sample with the level of antigen/antibody complex in the
second test sample, a modulated level of the antigen/antibody
complex in the second test sample indicating the subject's response
to the treatment.
36. The method of claim 35, wherein the degenerative joint
condition is an arthritic condition.
37. The method of claim 36, wherein the arthritic condition is
osteoarthritis.
38. The method of claim 36, wherein the arthritic condition is
rheumatoid arthritis.
39. The method of claim 35, wherein the test samples are selected
from the group consisting of synovial fluid, tears, saliva, urine,
serum, plasma, bone marrow, synovium, tendon, tendon sheath,
ligament, meniscus, intervertebral disk, pericardium, chondrocytes,
and articular cartilage.
40. The method of claim 39, wherein the test sample is synovial
fluid or synovium and wherein a reduction in the amount of
superficial zone protein in the second test sample indicates a
positive response to the treatment.
41. The method of claim 39, wherein the test sample is articular
cartilage or chondrocytes and wherein an increase in the amount of
superficial zone protein in the second test sample indicates a
positive response to the treatment.
42. The method of claim 35, wherein the detecting steps comprise an
assay selected from the group consisting of a competition or
sandwich ELISA, a radioimmunoassay, a Western blot assay, an
immunohistological assay, an immunocytochemical assay, a dot blot
assay, a fluorescence polarization assay, a scintillation proximity
assay, a homogeneous time resolved fluorescence assay, an resonant
mirror biosensor analysis, and a Surface plasmon resonance
analysis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from Great Britain
application number 0003092.4 filed Feb. 10, 2000. This application
is a divisional of U.S. application Ser. No. 09/780,718, filed Feb.
9, 2001, which is a continuation in part of and claims the benefit
of U.S. Provisional Application No. 60/181,377, filed Feb. 9, 2000,
the entirety of which is incorporated herein by this reference.
This application is also a continuation in part of and claims the
benefit of U.S. Provisional Application No. 60/201,989, filed May
3, 2000, the entirety of which is incorporated herein by this
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention generally relates to superficial zone
protein-binding molecules and their uses, including therapeutic
uses in the diagnosis, screening, and imaging in degenerative joint
disease.
[0005] 2. Background Art
[0006] Articular cartilage is a highly organized, heterogeneous,
avascular, resilient, weight-bearing tissue that covers the ends of
bones in diarthrodial (synovial) joints. The organizational
arrangement of articular cartilage is marked by zonal differences.
For example, the superficial zone of adult articular cartilage is
distinctly different from the middle, deep, and calcified zones of
the underlying cartilage in cellularity, morphology, matrix and
macromolecular composition (which includes the presence of gene
products made in different zones), macromolecular organization, and
material properties. Among the metabolic differences is the
synthesis of a proteoglycan, called superficial zone protein (SZP),
which is synthesized and secreted by chondrocytes in the
superficial zone of articular cartilage but is not synthesized or
secreted by chondrocytes in the deeper zones of the tissue
(Schumacher et al. (1994) Arch. Biochem. Biophys. 311:144-52).
[0007] SZP, which is homologous to human megakaryocyte stimulating
factor precursor (MSF) and camptodactyly-arthropathy-coxa
vara-pericarditis (CACP) protein, has an apparent molecular weight
of 345 kDa and is substituted with keratan sulfate and chondroitin
sulfate glycosaminoglycan chains (Schumacher et al. (1994) Arch.
Biochem. Biophys. 311:144-52). Removal of the glycosaminoglycan
side chains results in minimal change in molecular weight, which
suggests that SZP has only small glycosaminoglycan chains on its
core protein and that it is not an aggrecan metabolite (Schumacher
et al. (1999), J. Orthop. Res 17:110-120). SZP contains large
(76-78 repeats) and small (6-8 repeats) mucin-like O-linked
oligosaccharide-rich repeat domains flanked by cysteine-rich N- and
C-terminal domains (Flannery et al. (1999) Biochem. Biophys. Res.
Comm. 254:535-541). The protein core contains potential sites for
N-linked oligosaccharide and glycosaminoglycan attachment, and a
putative heparin-binding domain (Id.). The heparin binding domain
is encoded by exon 4 of MSF. Merberg et al. (1993) In: Biology of
Vitronectins and Their Receptors (eds. Pressner et al.), pp. 45-53.
Chondrocytes in the superficial zone and cells of the synovial
lining, in vivo and in vitro, have been shown to synthesize SZP
(Schumacher et al. (1999), J. Orthop. Res 17:110-120). Unlike other
proteoglycan molecules, such as aggrecan, decorin, biglycan, and
fibromodulin, very little SZP is retained in the matrix surrounding
the chondrocytes (Schumacher et al. (1994) Arch. Biochem. Biophys.
311:144-52). The SZP proteoglycan present in synovial fluid has a
lower molecular weight than SZP in the cartilage matrix, suggesting
that either there are differences in glycosylation of SZP produced
by synovial cells as compared to SZP produced by chondrocytes or
that the proteoglycan is partially degraded in the synovial fluid
(Schumacher et al. (1999), J. Orthop. Res 17:110-120).
[0008] SZP forms a thin layer on the surface of adult bovine
articular cartilage but not fetal articular cartilage (Schumacher
et al. (1999), J. Orthop. Res 17:110-120). The thickness of the
stained layer increases gradually near the junction of articular
cartilage with synovium and the synovium also contains SZP (Schmid
et al. (1994) Proceedings of the Orthopedic Research Society, p.
97-17.). This accumulation on adult articular cartilage has been
hypothesized to be due to entrapment of SZP in an acellular
collagenous layer at the surface of articular cartilage (Schumacher
et al. (1999), J. Orthop. Res 17:110-120). The biosynthesis of SZP
by chondrocytes has been shown to be upregulated by certain growth
factors and cytokines, such as TGF.beta. and IGF-1, but down
regulated by others, such as IL-1 (Flannery et al. (1999) Biochem
Biophys. Res. Comm. 254:535-541).
[0009] SZP is thought to play a role in normal articular cartilage
and in degenerative joint conditions. Recently, molecular defects
in human SZP have been identified in individuals with
camptodactyl-arthropathy-coxa vara-pericarditis syndrome (CACP), a
very rare condition that can be marked by a proliferation of
synovial cells, severe limitations in joint range of motion, and
non-inflammatory pericarditis (Marcelino et al. (1999) Nature
Genetics 23:319-322). Accordingly, it is desirable to detect SZP in
various mammalian species, including humans, in order to monitor
modulations in SZP levels, localization, and function.
SUMMARY OF THE INVENTION
[0010] In accordance with the purpose(s) of this invention, as
embodied and broadly described herein, this invention, in one
aspect, relates to a polyclonal or monoclonal antibody or a
fragment thereof having specific binding affinity for superficial
zone protein (SZP), wherein the binding affinity of the antibody or
fragment thereof for human superficial zone protein is the same or
greater than the binding affinity for bovine superficial zone
protein in a competitive binding assay, IA sys analysis, or BIAcore
analysis. Preferably the ligand is SZP or a variant, fragment, or
protein core thereof. The present invention further provides a
hybidoma cell that produces the monoclonal antibody of the
invention. The invention also provides an antibody reagent kit
comprising the antibody or fragment thereof of the invention and
reagents for detecting binding of the antibody or fragment thereof
to a ligand. In one embodiment, the kit further comprises
containers containing the antibody or fragment thereof of the
invention and containers containing the reagents.
[0011] In another aspect, the invention relates to methods of
detecting SZP and methods of diagnosing degenerative conditions,
such as joint, connective tissue, and blood disorders.
Specifically, provided is a method of detecting superficial zone
protein in a sample, comprising contacting the sample with the
antibody or fragment of the present invention, under conditions in
which an antigen/antibody complex can form; and detecting the
presence of the antigen/antibody complex, wherein the presence of
the antigen/antibody complex indicates the presence of superficial
zone protein in the sample. Preferably the sample is selected from
the group consisting of body fluids, such as synovial fluid, tears,
saliva, urine, serum, plasma, and bone marrow, and connective
tissue and components thereof, such as synovium, tendon, tendon
sheath, ligament, meniscus, intervertebral disk, pericardium,
chondrocytes, and articular cartilage. The method of diagnosing a
degenerative joint condition in a subject, as provided by the
present invention, comprises obtaining a test sample from the
subject; detecting superficial zone protein in the test sample; and
comparing the amount of superficial zone protein in the test sample
with an amount present in a control sample; a modulated amount of
superficial zone protein in the test sample indicating the
degenerative joint condition.
[0012] In yet another aspect, the invention relates to screening
methods, including a method of screening for a substance that
modulates levels of superficial zone protein, comprising contacting
a test sample with the substance to be screened, wherein the test
sample contains superficial zone protein-producing cells;
contacting, under conditions in which an antigen/antibody complex
can form, the superficial zone protein in the test sample with the
antibody or a fragment of the invention; detecting the level of the
antigen/antibody complex in the test sample; and comparing the
level of the antigen/antibody complex in the test sample with the
level of antigen/antibody complex in a control sample; a lower or
higher level of the antigen/antibody complex in the test sample
indicating a substance that modulates levels of superficial zone
protein. The superficial zone protein-producing cells are selected
from the group consisting of chondrocytes, synovial cells,
pericardial cells, and bone marrow cells of any species. Further
provided is a method of screening for a substance that reduces a
degenerative condition, such as a degenerative joint condition, in
a subject, comprising contacting a first test sample from the
subject with the antibody or fragment thereof of the invention,
under conditions in which an antigen/antibody complex can form;
detecting the level of the antigen/antibody complex in the first
test sample; treating the subject with the substance to be
screened; contacting a second test sample from the subject with the
antibody or fragment of the invention, under conditions whereby an
antigen/antibody complex can form; detecting the level of the
antigen/antibody complex in the second test sample; and comparing
the level of the antigen/antibody complex in the first test sample
with the level of antigen/antibody complex in the second test
sample, a modulated level of the antigen/antibody complex in the
second test sample indicating a substance that reduces the
degenerative condition. Alternatively, in one embodiment, the test
sample is compared to a known standard or to a control sample from
a second untreated subject with degenerative disease.
[0013] The invention also relates to a method of screening for
subjects who would benefit from treatment for a degenerative joint
condition, comprising the steps of obtaining a test sample from
each subject; detecting superficial zone protein in the test
samples; and comparing the amount of superficial zone protein in
the test samples with an amount present in a control sample, a
modulated amount of superficial zone protein in the test sample
indicating a subject that would benefit from treatment for the
degenerative joint condition. Also provided a method of monitoring
a subject's response to a treatment for a degenerative joint
condition, comprising contacting a first test sample from the
subject to be monitored with the monoclonal antibody or fragment
thereof of the invention, under conditions that allow formation of
an antigen/antibody complex; detecting the level of the
antigen/antibody complex in the first test sample; treating the
subject; contacting a second test sample from the subject with the
antibody or fragment thereof, under conditions whereby an
antigen/antibody complex can form; detecting the level of the
antigen/antibody complex in the second test sample; and comparing
the level of the antigen/antibody complex in the first test sample
with the level of antigen/antibody complex in the second test
sample, a modulated level of the antigen/antibody complex in the
second test sample indicating the subject's response to the
treatment.
[0014] Further related to the invention is a method of imaging an
articular surface and/or synovium of a joint, comprising contacting
the articular surface and/or synovium of the joint with the
antibody or fragment of the invention, under conditions in which an
antigen/antibody complex can form on the articular surface and/or
synovium, wherein the antibody or fragment thereof is detectably
tagged; visualizing the detectable tag in antigen/antibody
complexes in a plurality of locations on the articular surface; the
visualization of detectable tag in antigen/antibody complexes
showing the articular surface of the joint.
[0015] Additional advantages of the invention will be set forth in
part in the description that follows and, in part, will be obvious
from the description or may be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate (one) several
embodiment(s) of the invention and together with the description,
serve to explain the principles of the invention.
[0017] FIG. 1 shows the ELISA data using a monoclonal antibody
(GW4.23) to SZP, four monoclonal antibodies (S6.79, S17.109,
S13.52, S13.233) and a control monoclonal antibody to glutathione
S-transferase. MAb S6.79, derived from SZP-KLH immunization,
S13.233 and S17.109, derived from immunization with a mixture of
SZP and hyaluronic acid (SZP-HA), show strong immunoreactivity
against SZP purified from both synovial fluid (SZP-sf) and
articular cartilage (SZP-ac), with no cross-reactivity against KLH
or lectin. GW4.23 also shows specific but lower immunoreactivity
against SZP from both preparations. S13.52, raised against synovial
fluid derived-SZP-HA complex, is the only monoclonal antibody that
shows differential reactivity against SZP from different sources.
There was no immunoreactivity with the negative control antibody
129R10.
[0018] FIG. 2 shows the results of an SZP sandwich ELISA, using
lectin-S6.79 MAb, with a SZP standard and three samples of synovial
fluids, which are designated samples 115, 120, and 124. The X axis
shows the concentration of SZP. The synovial fluids were diluted by
two-fold serial dilutions starting with a 1:125 dilution.
[0019] FIG. 3 shows ranges of concentrations of SZP in 50 synovial
fluid samples assayed using the SZP sandwich ELISA. The results
show the combined data for patients with degenerative joint disease
and organ donors and the data for patients and donors
separately.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention may be understood more readily by
reference to the following detailed description of preferred
embodiments of the invention and the Examples included therein and
to the Figures and their previous and following description.
[0021] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that this invention is not limited to specific
antibodies, specific hybridomas, or to particular methods, as such
may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to be limiting.
[0022] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "an antibody" includes mixtures of antibodies,
reference to "a pharmaceutical carrier" includes mixtures of two or
more such carriers, and the like.
[0023] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0024] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not. For example, the phrase
"optionally obtained prior to treatment" means obtained before
treatment, after treatment, or not at all.
[0025] The invention provides an antibody (monoclonal or
polyclonal) or a fragment thereof having specific binding affinity
for superficial zone protein (SZP), wherein the binding affinity of
the antibody or fragment thereof for human superficial protein is
the same or greater than the binding affinity for bovine
superficial zone protein in a competitive binding assay, IAsys
analysis, or BIAcore analysis. In a preferred embodiment, the
antibody is a monoclonal antibody. The antibody is raised to SZP
from any species, including, for example, human, pig, guinea pig,
dog, or rabbit.
[0026] Having "the same or greater" binding affinity as compared to
the affinity for bovine SZP means that the antibody has less
affinity for bovine than for human SZP, including, for example,
when the binding for bovine SZP does not exceed background levels
of binding. Thus, the antibody may have no affinity over background
for bovine SZP or it may have the same affinity for bovine SZP as
for human or any amount in between.
[0027] As used throughout, "superficial zone protein" or "SZP"
includes the full length proteoglycan, the full length protein
core, variants of SZP (e.g., alternatively spliced variants),
fusion proteins comprising SZP, and immunogenic fragments of SZP,
which are glycosylated or non-glycosylated SZP and which are
non-reduced or reduced SZP. For example, the antibody binds full
length SZP, a variant of SZP (e.g., an alternatively spliced
variant), the protein core of SZP, a fusion protein, or any epitope
thereon. The SZP to which the antibody is raised is naturally
occurring or recombinant. The antibody can be used in techniques or
procedures such as diagnostics, screening, or imaging.
Anti-idiotypic antibodies and affinity matured antibodies are also
considered to be part of the invention.
[0028] As used herein, the term "antibody" encompasses, but is not
limited to, whole immunoglobulin (i.e., an intact antibody) of any
class. Native antibodies are usually heterotetrameric
glycoproteins, composed of two identical light (L) chains and two
identical heavy (H) chains. Typically, each light chain is linked
to a heavy chain by one covalent disulfide bond, while the number
of disulfide linkages varies between the heavy chains of different
immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced intrachain disulfide bridges. Each heavy chain has
at one end a variable domain (V(H)) followed by a number of
constant domains. Each light chain has a variable domain at one end
(V(L)) and a constant domain at its other end; the constant domain
of the light chain is aligned with the first constant domain of the
heavy chain, and the light chain variable domain is aligned with
the variable domain of the heavy chain. Particular amino acid
residues are believed to form an interface between the light and
heavy chain variable domains. The light chains of antibodies from
any vertebrate species can be assigned to one of two clearly
distinct types, called kappa (k) and lambda (l), based on the amino
acid sequences of their constant domains. Depending on the amino
acid sequence of the constant domain of their heavy chains,
immunoglobulins can be assigned to different classes. There are
five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM,
and several of these may be further divided into subclasses
(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.
The heavy chain constant domains that correspond to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively.
[0029] The term "variable" is used herein to describe certain
portions of the variable domains that differ in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not usually evenly distributed through the variable
domains of antibodies. It is typically concentrated in three
segments called complementarity determining regions (CDRs) or
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of the
variable domains are called the framework (FR). The variable
domains of native heavy and light chains each comprise four FR
regions, largely adopting a b-sheet configuration, connected by
three CDRs, which form loops connecting, and in some cases forming
part of, the b-sheet structure. The CDRs in each chain are held
together in close proximity by the FR regions and, with the CDRs
from the other chain, contribute to the formation of the antigen
binding site of antibodies (see Kabat E. A. et al., "Sequences of
Proteins of Immunological Interest," National Institutes of Health,
Bethesda, Md. (1987)). The constant domains are not involved
directly in binding an antibody to an antigen, but exhibit various
effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity.
[0030] As used herein, the term "antibody or fragments thereof"
encompasses chimeric antibodies and hybrid antibodies, with dual or
multiple antigen or epitope specificities, and fragments, such as
F(ab').sub.2, Fab', Fab and the like, including hybrid fragments.
Thus, fragments of the antibodies that retain the ability to bind
their specific antigens are provided. For example, fragments of
antibodies which maintain SZP binding activity are included within
the meaning of the term "antibody or fragment thereof." Such
antibodies and fragments can be made by techniques known in the art
and can be screened for specificity and activity according to the
methods set forth in the Examples and in general methods for
producing antibodies and screening antibodies for specificity and
activity (See Harlow and Lane. Antibodies, A Laboratory Manual.
Cold Spring Harbor Publications, New York, (1988)).
[0031] Preferably, the antibodies are derived using SZP as an
immunizing agent in one of several forms, including for example,
modified or non-modified SZP. "Modified" forms of SZP include, for
example, superficial zone protein-keyhole limpet hemocyanin
(SZP-KLH) conjugate or a mixture of SZP and hyaluronic acid
(SZP-HA). A summary of examples of various monoclonal antibodies
generated from various forms of SZP is provided as Table I.
1TABLE 1 Summary of SZP Monoclonal Antibodies Immunoreactivity*
Affinity human human human bovine dog g. pig rabbit MAb Antigen
Isotype K.sub.D (M) SF plasma serum SF SF SF SF GW4.23 SZP IgG1 + +
- S6.79 SZP- IgG2b 3.14 .times. 10.sup.-9 +++++ + +/- + ++ +/- ++
KLH S13.52 SZP-HA IgG1 ++++ ++++ +++ S13.233 SZP-HA IgG1 + ++ +
S17.109 SZP-HA IgG2b 1.83 .times. 10.sup.-8 +++++ +/- -
Immunoreactivity*: intensity of immuno-stained bands in Western
blots strongest ++++ > ++++ > +++ > ++ > + > +/-
faint SF: synovial fluid
[0032] Also included within the meaning of "antibody or fragments
thereof" are conjugates of antibody fragments and antigen binding
proteins (single chain antibodies) as described, for example, in
U.S. Pat. No. 4,704,692, the contents of which are hereby
incorporated by reference.
[0033] Optionally, the antibodies are generated in other species
and "humanized" for administration in humans. Humanized forms of
non-human (e.g., murine) antibodies are chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2, or other antigen-binding subsequences of antibodies)
which contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues that are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones et
al., Nature, 321:522-525 (1986); Riechmann et al., Nature,
332:323-327 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596
(1992)).
[0034] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source that is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0035] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important in
order to reduce antigenicity. According to the "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993) and
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0036] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three dimensional models of the parental and
humanized sequences. Three dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the consensus and import sequence so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding (see, WO 94/04679, published 3 Mar.
1994).
[0037] Transgenic animals (e.g., mice) that are capable, upon
immunization, of producing a full repertoire of human antibodies in
the absence of endogenous immunoglobulin production can be
employed. For example, it has been described that the homozygous
deletion of the antibody heavy chain joining region (J(H)) gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge (see,
e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255
(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann
et al., Year in Immuno., 7:33 (1993)). Human antibodies can also be
produced in phage display libraries (Hoogenboom et al., J. Mol.
Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581
(1991)). The techniques of Cote et al. and Boerner et al. are also
available for the preparation of human monoclonal antibodies (Cole
et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991)).
[0038] The present invention further provides a hybidoma cell that
produces the monoclonal antibody of the invention. The term
"monoclonal antibody" as used herein refers to an antibody obtained
from a substantially homogeneous population of antibodies, i.e.,
the individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. The monoclonal antibodies herein
specifically include "chimeric" antibodies in which a portion of
the heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired activity (See, U.S. Pat. No. 4,816,567 and Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
[0039] Monoclonal antibodies of the invention may be prepared using
hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256:495 (1975) or Harlow and Lane. Antibodies, A Laboratory
Manual. Cold Spring Harbor Publications, New York, (1988). In a
hybridoma method, a mouse or other appropriate host animal, is
typically immunized with an immunizing agent to elicit lymphocytes
that produce or are capable of producing antibodies that will
specifically bind to the immunizing agent. Alternatively, the
lymphocytes may be immunized in vitro. Preferably, the immunizing
agent comprises SZP. Traditionally, the generation of monoclonal
antibodies has depended on the availability of purified protein or
peptides for use as the immunogen. More recently DNA based
immunizations have shown promise as a way to elicit strong immune
responses and generate monoclonal antibodies. In this approach,
DNA-based immunization can be used, wherein DNA encoding a portion
of SZP, preferably the N- or C-terminal region, is injected into
the host animal according to methods known in the art and as
described in the examples. An alternate approach to immunizations
with either purified protein or DNA is to use antigen expressed in
baculovirus. The advantages to this system include ease of
generation, high levels of expression, and post-translational
modifications that are highly similar to those seen in mammalian
systems. Use of this system involves expressing domains of proteins
as fusions to the baculovirus surface glycoprotein gp64. The
antigen is produced by inserting a gene fragment in-frame between
the signal sequence and the mature protein domain of the gp64
nucleotide sequence. This results in the display of the foreign
proteins on the surface of the virion. This method allows
immunization with whole virus, eliminating the need for
purification of target antigens.
[0040] Generally, either peripheral blood lymphocytes ("PBLs") are
used in methods of producing monoclonal antibodies if cells of
human origin are desired, or spleen cells or lymph node cells are
used if non-human mammalian sources are desired. The lymphocytes
are then fused with an immortalized cell line using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, "Monoclonal Antibodies: Principles and Practice" Academic
Press, (1986) pp. 59-103). Immortalized cell lines are usually
transformed mammalian cells, including myeloma cells of rodent,
bovine, equine, and human origin. Usually, rat or mouse myeloma
cell lines are employed. The hybridoma cells may be cultured in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
substances prevent the growth of HGPRT-deficient cells.
[0041] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Rockville, Md. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., "Monoclonal Antibody
Production Techniques and Applications" Marcel Dekker, Inc., New
York, (1987) pp. 51-63).
[0042] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against SZP. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art, and are
described further in the Examples below or in Harlow and Lane
"Antibodies, A Laboratory Manual" Cold Spring Harbor Publications,
New York, (1988).
[0043] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution or FACS sorting procedures
and grown by standard methods. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0044] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0045] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells,
plasmacytoma cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. The DNA also may be
modified, for example, by substituting the coding sequence for
human heavy and light chain constant domains in place of the
homologous murine sequences (U.S. Pat. No. 4,816,567) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Optionally, such a non-immunoglobulin polypeptide is substituted
for the constant domains of an antibody of the invention or
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody comprising one antigen-combining site having specificity
for SZP and another antigen-combining site having specificity for a
different antigen.
[0046] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 published Dec. 22, 1994, U.S. Pat. No. 4,342,566,
and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York, (1988). Papain digestion of
antibodies typically produces two identical antigen binding
fragments, called Fab fragments, each with a single antigen binding
site, and a residual Fc fragment. Pepsin treatment yields a
fragment, called the F(ab').sub.2 fragment, that has two antigen
combining sites and is still capable of cross-linking antigen.
[0047] The Fab fragments produced in the antibody digestion also
contain the constant domains of the light chain and the first
constant domain of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxy terminus
of the heavy chain domain including one or more cysteines from the
antibody hinge region. The F(ab').sub.2 fragment is a bivalent
fragment comprising two Fab' fragments linked by a disulfide bridge
at the hinge region. Fab'-SH is the designation herein for Fab' in
which the cysteine residue(s) of the constant domains bear a free
thiol group. Antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0048] An isolated immunogenically specific paratope or fragment of
the antibody is also provided. A specific immunogenic epitope of
the antibody can be isolated from the whole antibody by chemical or
mechanical disruption of the molecule. The purified fragments thus
obtained are tested to determine their immunogenicity and
specificity by the methods taught herein. Immunoreactive paratopes
of the antibody, optionally, are synthesized directly. An
immunoreactive fragment is defined as an amino acid sequence of at
least about two to five consecutive amino acids derived from the
antibody amino acid sequence.
[0049] One method of producing proteins comprising the antibodies
of the present invention is to link two or more peptides or
polypeptides together by protein chemistry techniques. For example,
peptides or polypeptides can be chemically synthesized using
currently available laboratory equipment using either Fmoc
(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)
chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One
skilled in the art can readily appreciate that a peptide or
polypeptide corresponding to the antibody of the present invention,
for example, can be synthesized by standard chemical reactions. For
example, a peptide or polypeptide can be synthesized and not
cleaved from its synthesis resin whereas the other fragment of an
antibody can be synthesized and subsequently cleaved from the
resin, thereby exposing a terminal group which is functionally
blocked on the other fragment. By peptide condensation reactions,
these two fragments can be covalently joined via a peptide bond at
their carboxyl and amino termini, respectively, to form an
antibody, or fragment thereof. (Grant G A (1992) Synthetic
Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky
M and Trost B., Ed. (1993) Principles of Peptide Synthesis.
Springer-Verlag Inc., NY. Alternatively, the peptide or polypeptide
is independently synthesized in vivo as described above. Once
isolated, these independent peptides or polypeptides may be linked
to form an antibody or fragment thereof via similar peptide
condensation reactions.
[0050] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide-.alpha.-thioester with another unprotected
peptide segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site. Application of this
native chemical ligation method to the total synthesis of a protein
molecule is illustrated by the preparation of human interleukin 8
(IL-8) (Baggiolini M et al. (1992) FEBS Lett. 307:97-101;
Clark-Lewis I et al., J. Biol. Chem., 269:16075 (1994); Clark-Lewis
I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al.,
Biochemistry 33:6623-30 (1994)).
[0051] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
[0052] The invention also provides fragments of antibodies which
have bioactivity. The polypeptide fragments of the present
invention can be recombinant proteins obtained by cloning nucleic
acids encoding the polypeptide in an expression system capable of
producing the polypeptide fragments thereof, such as an adenovirus
or baculovirus expression system. For example, one can determine
the active domain of an antibody from a specific hybridoma that can
cause a biological effect associated with the interaction of the
antibody with SZP. For example, amino acids found to not contribute
to either the activity or the binding specificity or affinity of
the antibody can be deleted without a loss in the respective
activity.
[0053] For example, in various embodiments, amino or
carboxy-terminal amino acids are sequentially removed from either
the native or the modified non-immunoglobulin molecule or the
immunoglobulin molecule and the respective activity assayed in one
of many available assays. In another example, a fragment of an
antibody comprises a modified antibody wherein at least one amino
acid has been substituted for the naturally occurring amino acid at
a specific position, and a portion of either amino terminal or
carboxy terminal amino acids, or even an internal region of the
antibody, has been replaced with a polypeptide fragment or other
moiety, such as biotin, which can facilitate in the purification of
the modified antibody. For example, a modified antibody can be
fused to a maltose binding protein, through either peptide
chemistry or cloning the respective nucleic acids encoding the two
polypeptide fragments into an expression vector such that the
expression of the coding region results in a hybrid polypeptide.
The hybrid polypeptide can be affinity purified by passing it over
an amylose affinity column, and the modified antibody receptor can
then be separated from the maltose binding region by cleaving the
hybrid polypeptide with the specific protease factor Xa. (See, for
example, New England Biolabs Product Catalog, 1996, pg. 164.).
Similar purification procedures are available for isolating hybrid
proteins from eukaryotic cells as well.
[0054] The fragments, whether attached to other sequences or not,
include insertions, deletions, substitutions, or other selected
modifications of particular regions or specific amino acids
residues, provided the activity of the fragment is not
significantly altered or impaired compared to the nonmodified
antibody or antibody fragment. These modifications can provide for
some additional property, such as to remove or add amino acids
capable of disulfide bonding, to increase its bio-longevity, to
alter its secretory characteristics, etc. In any case, the fragment
must possess a bioactive property, such as binding activity,
regulation of binding at the binding domain, etc. Functional or
active regions of the antibody may be identified by mutagenesis of
a specific region of the protein, followed by expression and
testing of the expressed polypeptide. Such methods are readily
apparent to a skilled practitioner in the art and can include
site-specific mutagenesis of the nucleic acid encoding the antigen.
(Zoller M J et al. Nucl. Acids Res. 10:6487-500 (1982).
[0055] As used herein, the phrase "specific binding affinity"
refers to a binding reaction which is determinative of the presence
of the SZP in a heterogeneous population of proteins,
proteoglycans, and other biologics. Thus, under designated
conditions, the antibodies or fragments thereof of the present
invention bind to a particular proteoglycan (e.g., human or porcine
SZP or any variant thereof) or protein core, fragment, or variant
thereof and do not bind in a significant amount to other proteins
or proteoglycans present in the subject. Furthermore, the
antibodies of the present invention have a binding affinity for
bovine that is the same as or lower than for human SZP, as
measured, for example, in a competitive binding assay, IAsys
analysis, or BIAcore analysis. Preferably, in an ELISA, the binding
of the antibody of the present invention to bovine SZP is 1-10
times the background level (i.e., comparable to non-specific
binding or slightly above non-specific binding), when the binding
to a comparable amount of human SZP is 10 or more times background.
More preferably, the binding of the antibody of the present
invention to bovine SZP is no more than 5 times the background
level, whereas the binding affinity for human SZP is more than 15
times background. Even more preferably, the binding of the antibody
of the present invention to bovine SZP is no more than 2.5 times
the background level, whereas the binding affinity for human SZP is
more than 18 times background. Thus, the binding affinity of the
antibody for human SZP is preferably at least 3 times greater than
the binding affinity for bovine SZP.
[0056] In one embodiment of the invention, the antibody or fragment
thereof, in addition to binding human SZP, binds SZP from at least
one non-human species selected from the group consisting of dog,
guinea, pig, and rabbit. Thus, in various embodiments the antibody
shows cross-reactivity for SZP derived from various species.
[0057] Selective binding to an antibody under such conditions may
require an antibody that is selected for its specificity for a
particular protein, proteoglycan, or variant, fragment, or protein
core thereof. A variety of immunoassay formats may be used to
select antibodies that selectively bind with a particular protein,
proteoglycan, or variant, fragment, or protein core thereof. For
example, solid-phase ELISA immunoassays are routinely used to
select antibodies selectively immunoreactive with a protein,
proteoglycan, or variant, fragment, or protein core thereof. See
Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring
Harbor Publications, New York, (1988), for a description of
immunoassay formats and conditions that could be used to determine
selective binding. The binding affinity of a monoclonal antibody
can, for example, be determined by the Scatchard analysis of Munson
et al., Anal. Biochem., 107:220 (1980).
[0058] The antibody or fragment of the invention binds either a
glycosylated or a non-glycosylated superficial zone protein, or it
binds both glycosylated and non-glycosylated forms. Also, the
antibody or fragment thereof binds non-reduced (i.e., native)
superficial zone protein. Under certain conditions, the antibody or
fragment thereof may also bind reduced forms.
[0059] The invention also provides an antibody reagent kit
comprising the antibody or fragment thereof of the invention and
reagents for detecting binding of the antibody or fragment thereof
to a ligand. Optionally, the kit further comprises containers
containing the antibody or fragment thereof of the invention and
containers containing the reagents. Preferably the ligand is SZP or
a variant, fragment, or protein core thereof. Particularly, the kit
detects the presence of SZP specifically reactive with the antibody
or an immunoreactive fragment thereof. The kit optionally includes
an antibody bound to a substrate, a secondary antibody reactive
with the antigen and/or a reagent for detecting a reaction of the
secondary antibody with the antigen. In one embodiment, the kit is
an ELISA kit, comprising the substrate, primary and secondary
antibodies when appropriate, and/or any other necessary reagents
such as detectable moieties, enzyme substrates and color reagents
as described above. The diagnostic kit, alternatively, is an
immunoblot kit generally comprising the components and reagents
described herein. Alternatively, the kit is a radioimmunoassay kit,
a Western blot assay kit, an immunohistological assay kit, an
immunocytochemical assay kit, a dot blot assay kit, a fluorescence
polarization assay kit, a scintillation proximity assay kit, a
homogeneous time resolved fluorescence assay kit, an IAsys analysis
kit, or a BIAcore analysis kit.
[0060] As used throughout, methods of detecting SZP or
antigen/antibody complexes, including complexes comprising SZP and
optionally the antibody of the present invention, comprise an ELISA
(competition or sandwich), a radioimmunoassay, a Western blot
assay, an immunohistological assay, an immunocytochemical assay, a
dot blot assay, a fluorescence polarization assay (Jolley (1981);
Jiskoot et al (1991); Seethala et al. (1998); Bicamumpaka et al.
(1998)), a scintillation proximity assay (Amersham Life Science
(1995) Proximity News. Issue 17; Amersham Life Science (1995)
Proximity News. Issue 18; Park et al. (1999)), a homogeneous
time-resolved fluorescence assay (Park et al. (1999); Stenroos et
al. (1988); Morrison, 1988)), a IAsys analysis (Edwards and
Leatherbarrow (1997)), or a BIAcore analysis (Fgerstam et al.
(1992)). Preferably, the antigen/antibody complex is detectably
tagged either directly or indirectly. Any desired tag can be
utilized, such as a fluorescent tag, a radiolabel, a magnetic tag,
or an enzymatic reaction product.
[0061] The invention also provides a method of detecting
superficial zone protein in a sample, comprising contacting the
sample with the antibody or fragment of the present invention,
under conditions in which an antigen/antibody complex can form; and
detecting the presence of the antigen/antibody complex, wherein the
presence of the antigen/antibody complex indicates the presence of
superficial zone protein in the sample. Preferably the sample is
selected from the group consisting of body fluids, such as synovial
fluid, tears, saliva, urine, serum, plasma, and bone marrow, and
connective tissue and components thereof, such as synovium, tendon,
tendon sheath, ligament, meniscus, intervertebral disk,
pericardium, chondrocytes, and articular cartilage. The contacting
step of the present method is either in vivo or in vitro.
[0062] As used throughout, by "subject" is meant an individual.
Preferably, the subject is a mammal such as a primate, and, more
preferably, a human. The term "subject" includes domesticated
animals, such as cats, dogs, etc., livestock (e.g., cattle, horses,
pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse,
rabbit, rat, guinea pig, etc.).
[0063] The present invention also provides a method of diagnosing a
degenerative condition, such as a joint, connective tissue, or
blood disorder, in a subject, comprising obtaining a test sample
from the subject; detecting superficial zone protein in the test
sample; and comparing the amount of superficial zone protein in the
test sample with an amount present in a control sample; a modulated
amount of superficial zone protein in the test sample indicating
the degenerative condition. As used throughout, the terms
"degenerative condition" or "degenerative disease" includes a
variety of blood, connective tissue, and joint diseases, including,
for example, tendonitis, pericarditis, osteoporosis, and
degenerative joint disease. The term "degenerative joint condition"
or "degenerative joint disease" includes a variety of conditions
marked by inflammatory or non-inflammatory joint disease, including
arthritic conditions (e.g., osteoarthritis, rheumatoid arthritis,
gout, psoriatic arthritis, reactive arthritis, viral or post-viral
arthritis, spondylarthritis, juvenile arthritis, and systemic lupus
erythematosus), CACP, osteoporosis, and trauma. Such degenerative
joint diseases are characterized by morphological, compositional,
and metabolic changes in articular cartilage. A subject with a
degenerative joint disease may show clinical or subclinical signs
of the disease, and thus demonstrate either early or late stages of
the disease.
[0064] "Osteoarthritis," as used herein, would include both primary
and secondary degenerative joint disease, and a subject with
osteoarthritis may show any of the early manifestations of
osteoarthritis, including, for example, increased water content of
the cartilage, increased collagen extractability, increased levels
of annexin V, crepitus, and radiologic changes (including joint
space narrowing, subchondral sclerosis or cysts, and osteophyte
formation), or later manifestations, including, for example, joint
pain, joint swelling, joint stiffness, reduced quality and quantity
of cartilage matrix, deformity, chondrocalcinosis, and reduced
range of motion.
[0065] "Rheumatoid arthritis" as used herein refers to inflammatory
joint disease in both early and late stages. Signs and
manifestations of the early stages include, for example, general
fatigue, joint stiffness or aching, synovial inflammation,
excessive synovial fluid, joint effusion, osteoporosis in the ends
of the bones forming the affected joint or joints, edematous
synovial cells, and proliferation of synovial lining cells. In
later stages, additional signs and manifestations are detected,
including joint pain, redness, swelling, and inflammation. Pannus
can be seen in the joints. Cartilage and subchondral bone can be
eroded at the articular surface. Changes in the composition of the
synovial fluid can occur. Laxity in tendons and ligaments, as well
as deformity, can occur and can cause limitations in joint range of
motion and joint instability. Furthermore, Rheumatoid Factor(s) can
be detected in the subject's blood at both early and late stages of
the disease.
[0066] As used throughout, a "test sample" is selected from the
group consisting of body fluids, such as synovial fluid, tears,
saliva, urine, serum, plasma, and bone marrow, and connective
tissue and components thereof, such as synovium, tendon, tendon
sheath, ligament, meniscus, intervertebral disk, pericardium,
chondrocytes, and articular cartilage. The test sample can be
obtained by methods well known in the art, including for example,
by aspiration or biopsy. As used throughout, a "control sample"
comprises either a sample obtained from a control subject (e.g.,
from the same subject before treatment, or from a second subject
without degenerative disease or without treatment) or comprises a
known standard.
[0067] In the method of diagnosing, the amount of superficial zone
protein in the test sample is compared with an amount present in a
control sample by contacting the test sample with the antibody of
the present invention and detecting the antibody/antigen complex.
The contacting step is performed either in vivo or in vitro. One
skilled in the art would know or could readily determine normal
levels of SZP against which to compare the test sample. For
example, SZP (MSF) has been reported to be present in concentration
of less than 1 ng/ml in serum and urine. The concentration of SZP
in biological fluids is measured using, for example, competition
ELISA, RIA, or sandwich ELISA. Optionally, levels are quantified by
competition immunoassays in a homogenous format, such as
Scintillation Proximity Assay (Amersham Life Science (1995)
Proximity News. Issue 17; Amersham Life Science (1995) Proximity
News. Issue 18; Park et al. (1999)), Homogeneous Time-Resolved
Fluorescence Assay (Park et al. (1999); Stenroos et al. (1988);
Morrison, 1988)), and Fluorescence Polarization Assay (Jolley
(1981); Jiskoot et al (1991); Seethala et al. (1998)). A homogenous
assay format does not require separation of antibody-antigen
complex from unbound antibody or antigen, thus eliminating
washing/error prone steps and decreasing experimental variations.
Optionally, the concentration of SZP in biological fluids is
measured when compared to a serial dilution of non-radiolabeled SZP
or SZP fragment. SZP fragment can be a fragment purified from
biological fluid, enzymatic digestion or recombinant truncated SZP
protein.
[0068] By "modulation" or "modulating" is meant either an increase
or a decrease in SZP levels. Whether the levels are increased or
decreased in a subject with a degenerative condition depends on the
particular sample and the status of the disease state. For example,
with the onset of a degenerative joint disease, SZP levels may
transiently decrease in the synovial fluid, only to be followed by
a compensatory increase. Such changes may occur when chondrocyte
synthesis of SZP decreases early in the disease process and is
followed by a compensatory increase in synthesis and/or release by
either chondrocytes or synovial cells. As another example, in the
synovium or synovial fluid, the degenerative joint condition may be
indicated by an elevated amount of superficial zone protein in the
test sample but indicated by a decreased level of SZP in articular
cartilage or on the articular surface. SZP in the test sample is
compared to a control sample by contacting the test sample with the
antibody or fragment of the present invention or other antibody to
SZP, under conditions that allow formation of an antibody/antigen
complex, and the antigen/antibody complex is detected by the
various detection methods mentioned above.
[0069] The invention further provides a method of screening for
subjects who would benefit from treatment for a degenerative joint
condition, comprising the steps of obtaining a test sample from
each subject; detecting superficial zone protein in the test
samples; and comparing the amount of superficial zone protein in
the test samples with an amount present in a control sample, a
modulated amount of superficial zone protein in the test sample
indicating a subject that would benefit from treatment for the
degenerative joint condition. This method is useful in identifying
subjects who are candidates for treatment when the symptoms of the
degenerative joint condition are clinical or subclinical. By
"clinical" or "subclinical" is meant a degenerative joint condition
that may or may not be accompanied by clinical symptoms such as
pain, limited range of motion, radiologic changes in the joint,
etc. Thus, the present method is used to identify subjects with
very early to late manifestations of the degenerative joint
condition so that treatment can be started and further
manifestation of the condition can be prevented or reduced.
Preferably, the test sample and control sample are selected from
the group consisting of synovial fluid, tears, saliva, urine,
serum, plasma, and bone marrow, synovium, tendon, tendon sheath,
ligament, meniscus, intervertebral disk, pericardium, chondrocytes,
and articular cartilage. The modulation in the amount of
superficial zone protein in the test sample that indicates a
subject that would benefit from treatment for the degenerative
joint condition depends upon the test sample selected. For example,
in one embodiment of the invention, when the test sample is
synovial fluid or synovium, the subjects that would benefit from
treatment are indicated by an elevated amount of superficial zone
protein in the test samples, but, in another embodiment, when the
test sample is articular cartilage or chondrocytes, the subjects
that would benefit from treatment are indicated by an decrease in
the amount of superficial zone protein in the test samples.
Preferably, the superficial zone protein is detected by contacting
the test sample with the monoclonal antibody or fragment of the
invention.
[0070] The invention further provides a method of screening for a
substance that modulates levels of superficial zone protein,
comprising contacting a test sample with the substance to be
screened, wherein the test sample contains superficial zone
protein-producing cells; contacting, under conditions in which an
antigen/antibody complex can form, the superficial zone protein in
the test sample with the antibody or a fragment of the invention;
detecting the level of the antigen/antibody complex in the test
sample; and comparing the level of the antigen/antibody complex in
the test sample with the level of antigen/antibody complex in a
control sample; a lower or higher level of the antigen/antibody
complex in the test sample indicating a substance that modulates
levels of superficial zone protein. The superficial zone
protein-producing cells are selected from the group consisting of
chondrocytes, synovial cells, pericardial cells, bone marrow cells,
and connective tissue cells (e.g., cells from tendon, ligament,
meniscus, or intervertebral disk) of any species. Preferably, the
cells are mammalian cells. Even more preferably, the mammalian
cells are human cells.
[0071] The contacting step of the present method is either in vitro
or in vivo. Preferably, the superficial zone protein contacted in
the sample is secreted by the superficial zone protein-producing
cells.
[0072] The method of screening, optionally, further comprises
contacting the test sample with an agent that increases levels of
superficial zone protein, wherein the lower or higher level of the
antigen/antibody complex indicates, respectively, a substance that
attenuates or potentiates the increase in superficial zone protein.
The agent that increases levels of SZP is, for example, a synthetic
agent, a cytokine, or growth factor, such as TGF.beta., IGF-1,
BMP-1, BMP-4, BMP-7 (osteogenic protein-1). The step of contacting
the test sample with the agent optionally is before, after, or
simultaneously with the step of contacting the sample with the
substance to be screened. By "attenuation" is meant a reduction in
the increase in SZP level that occurs upon contact of the SZP
secreting cell with the agent that increases SZP release or
synthesis. By "potentiation" is meant a synergistic or additive
effect between the substance to be tested and the agent that
increases SZP release or synthesis.
[0073] Further provided is a method of screening for a substance
that reduces a degenerative condition, such as a degenerative joint
condition, in a subject, comprising contacting a first test sample
from the subject with the antibody or fragment thereof of the
invention, under conditions in which an antigen/antibody complex
can form; detecting the level of the antigen/antibody complex in
the first test sample; treating the subject with the substance to
be screened; contacting a second test sample from the subject with
the antibody or fragment of the invention, under conditions whereby
an antigen/antibody complex can form; detecting the level of the
antigen/antibody complex in the second test sample; and comparing
the level of the antigen/antibody complex in the first test sample
with the level of antigen/antibody complex in the second test
sample, a modulated level of the antigen/antibody complex in the
second test sample indicating a substance that reduces the
degenerative condition. Alternatively, the test sample is compared
to a known standard or to a control sample from a second untreated
subject with degenerative disease. The contacting step is performed
either in vivo or in vitro. The nature of the observed modulation
will vary depending on the test sample selected and the disease
state. For example, when the test sample is synovium or synovial
fluid, the degenerative joint condition will be indicated by an
elevated amount of superficial zone protein in the test sample; and
when the test sample is chondrocytes, articular cartilage, or the
articular surface, the degenerative joint condition is indicated by
a reduced amount of superficial zone protein in the test
sample.
[0074] Further provided is a method of imaging an articular surface
or synovium of a joint, comprising contacting the articular surface
of the joint with the antibody or fragment of the invention, under
conditions in which an antigen/antibody complex can form on the
articular surface, wherein the antibody or fragment thereof is
detectably tagged; visualizing the detectable tag in
antigen/antibody complexes in a plurality of locations on the
articular surface; the visualization of detectable tag in
antigen/antibody complexes showing the articular surface of the
joint. Such an imaging method is used for the purpose of prognosis,
diagnosis, and monitoring of a degenerative joint condition. Thus,
the invention further provides a method of diagnosing or monitoring
a degenerative joint condition in a subject, comprising imaging one
or more articular surfaces in the subject using the method of the
invention and comparing the articular surface or surfaces of the
subject to a control articular surface. Degenerative changes in the
articular surface or surfaces of the subject indicates the
degenerative joint condition.
[0075] The detectable tag used in the imaging method is, for
example, a radio-opaque substance, radiolabel, a fluorescent label,
or a magnetic label. The detectable tag, optionally, is selected
from the group consisting of gamma-emitters, beta-emitters, and
alpha-emitters, gamma-emitters, positron-emitters, X-ray-emitters
and fluorescence-emitters suitable for localization.
[0076] Fluorescent compounds that are suitable for conjugation to a
monoclonal antibody include fluorescein sodium, fluorescein
isothiocyanate, phycoerythrin, and Texas Red sulfonyl chloride.
See, DeBelder & Wik, 1975, Carbohydrate Research 44:254-257.
Those skilled in the art will know, or will be able to ascertain
with no more than routine experimentation, other fluorescent
compounds that are suitable for labeling monoclonal antibodies.
[0077] Suitable radioisotopes for labeling antibodies include
Iodine-131, Iodine-123, Iodine-125, Iodine-126, Iodine-133,
Bromine-77, Indium-111, Indium-113m, Gallium-67, Gallium-68,
Ruthenium-95, Ruthenium-97, Ruthenium-103, Ruthenium-105,
Mercury-107, Mercury-203, Rhenium-99m, Rhenium-105, Rhenium-101,
Tellurium-121 m, Tellurium-122m, Tellurium-125m, Thulium-165,
Thulium-167, Thulium-168, Technetium-99m and Fluorine-18. The
halogens can be used more or less interchangeably as labels since
halogen-labeled antibody fragments and/or normal immunoglobulin
fragments would have substantially the same kinetics and
distribution and similar metabolism.
[0078] The visualization step optionally comprise a means of
visualization selected from the group consisting of nuclear
magnetic resonance, radioimmunoscintigraphy, X-radiography,
positron emission tomography, computerized axial tomography,
magnetic resonance imaging, and ultrasonography. For visualization,
the subject, for example, is scanned with a gamma ray emission
counting machine such as the axial tomographic scanner commercially
available under the designation CT (80-800 CT/T) from General
Electric Company (Milwaukee, Wis.), or with a positron emission
transaxial tomography scanner.
[0079] The gamma-emitters Indium-111 and Technetium-99m are
detected with a gamma camera and have favorable half lives for
imaging in vivo. The antibody, for example, is labeled with
Indium-111 or Technetium-99m via a conjugated metal chelator, such
as DTPA (diethlenetriaminepentaacetic acid). See Krejcarek et al.,
1977, Biochem. Biophys. Res. Comm. 77:581; Khaw et al., 1980,
Science 209:295; Gansow et al., U.S. Pat. No. 4,472,509; Hnatowich,
U.S. Pat. No. 4,479,930, the teachings of which are incorporated
herein by reference.
[0080] For purposes of imaging the articular surface or synovium,
the antibody is administered by a variety of techniques known in
the art, including orally, intravenously, or intra-articularly by
injection into the joint to be visualized. The antibody optionally
is administred in a carrier pharmaceutically acceptable to the
subject. Suitable carriers and their formulations are described in
Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack
Publishing Co., edited by Oslo et al. Typically, an appropriate
amount of a pharmaceutically-acceptable salt is used in the
formulation to render the formulation isotonic. Examples of the
pharmaceutically-acceptable carrier include saline, Ringer's
solution and dextrose solution. The pH of the solution is
preferably from about 5 to about 8, and more preferably from about
7 to about 7.5. Further carriers include sustained release
preparations such as semipermeable matrices of solid hydrophobic
polymers containing the antibody, which matrices are in the form of
shaped articles, e.g., films, liposomes or microparticles. It will
be apparent to those persons skilled in the art that certain
carriers may be more preferable depending upon, for instance, the
route of administration and concentration of antibody being
administered.
[0081] Suitable carriers for oral administration of the antibody or
fragment thereof include one or more substances which may also act
as flavoring agents, lubricants, suspending agents, or as
protectants. Suitable solid carriers include calcium phosphate,
calcium carbonate, magnesium stearate, sugars, starch, gelatin,
cellulose, carboxypolymethylene, or cyclodextrans. Suitable liquid
carriers may be water, pyrogen free saline, pharmaceutically
accepted oils, or a mixture of any of these. The liquid optionally
also contains other suitable pharmaceutical additions such as
buffers, preservatives, flavoring agents, viscosity or
osmo-regulators, stabilizers or suspending agents. Examples of
suitable liquid carriers include water with or without various
additives, including carboxypolymethylene as a pH-regulated gel.
The antibody or fragment thereof may be contained in enteric coated
capsules that release the agent into the intestine to avoid gastric
breakdown. For parenteral administration of the antibody or
fragment thereof, a sterile solution or suspension is prepared in
saline that may contain additives, such as ethyl oleate or
isopropyl myristate, and can be injected for example, into
subcutaneous or intramuscular tissues, as well as intravenously or
intra-articularly. Alternatively, the antibody or fragment thereof
is microencapsulated with either a natural or a synthetic polymer
into microparticles, which releases the antibody or fragment
thereof.
[0082] The amount of antibody or fragment thereof administered or
the schedule for administration will vary among individuals based
on age, size, weight, condition, the joint to be assessed, mode of
administration, the imaging system, and the degree of degenerative
joint disease. One skilled in the art will realize that dosages are
best optimized by the practicing physician and methods for
determining dosage are described, for example in Remington's
Pharmaceutical Science, 16th ed., 1980, Mack Publishing Co., edited
by Oslo et al. Guidance in selecting appropriate doses for
antibodies is found in the literature on therapeutic uses of
antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et
al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and
pp. 303-357; Smith et al., Antibodies in Human Diagnosis and
Therapy, Haber et al., eds., Raven Press, New York (1977) pp.
365-389. A typical dose of the antibody used alone might range from
about 1 .mu.g/kg to up to 100 mg/kg of body weight or more per day,
and preferably 1 .mu.g/kg to up to 1 mg/kg, depending on the
factors mentioned above. An intravenous injection of the antibody
or fragment thereof, for example, could be 10 ng-1 g of antibody or
fragment thereof, and preferably 10 ng-1 mg depending on the
factors mentioned above. For injection into a joint, a typical
quantity of antibody ranges from 1 pg to 1 mg. Preferably, the
intrarticular injection would be at an antibody concentration of
1-100 .mu.g/ml, and preferably 1-20 .mu.g/ml. Volumes of antibody
and carrier will vary depending upon the joint, but approximately
0.5-10 ml, and preferably 1-5 ml, is injected into a human knee and
approximately 0.1-5 ml, and preferably 1-2 ml into the human ankle.
The delay between administration of the antibody or fragment
thereof and the visualization will be predetermined as the time
sufficient for formation of antigen/antibody complexes and,
preferably, for non-bound antibody to clear from the subject's body
or joint.
[0083] Also provided by the present invention is a method of
monitoring a subject's response to a treatment for a degenerative
joint condition. The method comprises the steps of (a) contacting a
first test sample from the subject to be monitored with the
monoclonal antibody or fragment thereof of the invention, under
conditions that allow formation of an antigen/antibody complex; (b)
detecting the level of the antigen/antibody complex in the first
test sample; (c) treating the subject; (d) contacting a second test
sample from the subject with the antibody or fragment thereof,
under conditions whereby an antigen/antibody complex can form; (e)
detecting the level of the antigen/antibody complex in the second
test sample; and (f) comparing the level of the antigen/antibody
complex in the first test sample with the level of antigen/antibody
complex in the second test sample, a modulated level of the
antigen/antibody complex in the second test sample indicating the
subject's response to the treatment. The test samples are
preferably selected from the group consisting of synovial fluid,
tears, saliva, urine, serum, plasma, bone marrow, synovium, tendon,
tendon sheath, ligament, meniscus, intervertebral disk,
pericardium, chondrocytes, and articular cartilage. The modulation
in the amount of superficial zone protein indicates either a
positive or a negative effect of the treatment, and the nature of
the modulation depends upon the test sample and other factors. For
example, in one embodiment of the invention, when the test sample
is synovial fluid or synovium, a reduction in the amount of
superficial zone protein in the second test sample indicates a
positive response to the treatment. In another embodiment, when the
test sample is articular cartilage or chondrocytes, an increase in
the amount of superficial zone protein in the second test sample
indicates a positive response to the treatment.
[0084] The first test sample optionally may be obtained prior to,
simultaneously with, or after the first treatment. The present
method optionally further comprises contacting one or more
additional test samples (for example, a third, fourth, fifth,
sixths etc. sample) with the antibody or fragment thereof and
detecting the level of the antigen/antibody complex in the
additional test sample(s). The level or levels in the additional
test sample or samples is compared to the control or to the
previous test sample or samples. Thus, in the presence of a single
treatment, the short and long term effects on the levels can be
monitored. Similarly, with an ongoing treatment regimen, the short
and long term effects, as well as the cumulative effect of
treatment can be monitored.
[0085] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
EXAMPLES
Example 1
Isolation and Purification of Human Superficial Zone Protein (SZP)
from Cartilage
[0086] Human tali were obtained through collaboration with The
Regional Organ Bank of Illinois (ROBI) with the approval of the
institutional review board (IRB) of the Medical College of Rush
Presbyterian St. Luke's Medical Center. Individual entire human
tali were submerged in approximately 100-130 ml of medium
consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented
with 5% fetal bovine serum, 25-50 .mu.g/ml ascorbic acid and 20
.mu.Ci of .sup.3H-proline for 18-22 hours in a humidified
atmosphere of 5% CO.sub.2/air at 37.degree. C. with constant
stirring. After the incubation period, the medium (containing
.sup.3H-proline labeled Superficial Zone Protein (SZP) was
harvested and six Complete.TM. mini protease inhibitor cocktail
tablets (Boehinger Mannheim, Gmbh, Germany) were added. Dry
guanidinium hydrochloride (GuHCl) was added to the medium to bring
the concentration of GuHCl to 4 M. The solution was brought to an
initial density of 1.46 gm/ml by the addition of Cesium chloride
(0.57 grams per gram of medium). The solution was subjected to
equilibrium density gradient ultracentrifugation at 33,000 RPM for
40 hours at 10.degree. C. The resulting gradient was fractionated
into five equal portions, designated as D5 at the top to D1 at the
bottom of the gradient solution. The D5 fraction was dialyzed
against water and brought to 8 M urea, 0.005 M EDTA, 0.15 M sodium
chloride, 0.05 M sodium acetate, pH 6.0 by the addition of dry
chemicals and acetic acid. This solution was subjected to anion
exchange chromatography on DEAE Sephacel equilibrated in 8 M urea,
0.15 M sodium chloride, 0.005 M EDTA, 0.05 M sodium acetate at pH
6.0 and the SZP was eluted from the DEAE in a stepwise fashion
using increasing concentrations of sodium chloride of 0.3 M and 0.6
M salt. SZP eluted between 0.3 and 0.6 M sodium chloride. The SZP
containing fraction was dialyzed against water, lyophilized,
dissolved in column buffer and subjected to column chromatography
on Sepharose CL-4B in the presence of 4 M GuHCl, 0.1 M sodium
sulfate, 0.005 M EDTA and 0.05 M sodium acetate, pH 5.8. The eluate
from the column was collected in equal fractions and the fractions
were analyzed for the presence of .sup.3H-proline by scintillation
counting. Putative SZP containing fractions were pooled, dialyzed
against water, lyophilized, dissolved in sample buffer and analyzed
by SDS-PAGE to confirm the presence of SZP. This entire procedure
was repeated without the presence of .sup.3H-proline and the final
lyophilized material was used as antigen for the production of
monoclonal antibodies.
[0087] SZP purified from culture media of human tali had similar
characteristics to bovine SZP for all of the steps in the
purification procedure. SDS-PAGE analysis of purified human SZP
revealed a single broad band as visualized by staining with
Stains-all, suggesting a highly glycosylated protein. SZP had an
apparent molecular mass of 345 kDa compared to globular standards.
Putative SZP was subjected to peptide mapping using endoproteinase
Lys-C. Two of the resulting 4 peptides were sequenced, and
sequences (RGGSIQQYIY (SEQ ID NO:1) and DQYYNIDVPS (SEQ ID NO:2)
matched SZP in the nonredundant protein database.
Example 2
Isolation and Purification of Human Superficial Zone Protein (SZP)
from Media of Cultures of Chondrocytes and from Synovial Fluid
[0088] SZP was purified from culture medium or synovial fluid by a
combination of affinity chromatography, first on a peanut lectin
and then on a monoclonal anti-SZP antibody column. Culture medium
or synovial fluid was made 0.5 M in NaCl and 5 mM in EDTA and
clarified by centrifugation at 10,000 g for 15 minutes. The
supernatant, either 50 ml of culture medium or 5 ml of synovial
fluid, was incubated with 5 ml of peanut lectin-agarose beads
(Sigma, St. Louis, Mo.) with rotation overnight at 4.degree. C. The
lectin beads were washed with 25 ml of 10 mM sodium phosphate, 0.5
M NaCl, 5 mM EDTA, pH 7.5 buffer. The bound SZP was eluted with the
same buffer containing 0.4 M lactose. The lectin beads were
subsequently washed with the washing buffer containing 1.5 M NaCl
and then again with washing buffer containing 0.35 M lactose and
1.5 M NaCl. The majority of the SZP was eluted in the first elution
with 0.4 M lactose. This SZP preparation also contained small
amounts of fibronectin and albumin.
[0089] Like the form of SZP isolated from cartilage slice cultures,
the band was stained by Coomassie blue, Stainsall or silver, but
usually 10-15 .mu.g of protein was necessary for a detection of the
band on the gels. The form of SZP isolated from human synovial
fluid had comparable movility on 5% polyacrylamide gels to the form
of the molecule isolated from cartilage organ culture. The putative
SZP band from the SDS--PAGE gel was excised and subjected to in-gel
digestion using trypsin prior to characterization by tandem mass
spectrometry. All five resulting peptide spectra matched entries in
the nonredundant database for SZP. These were GFGGLTGQIVAALSTAK
(SEQ ID NO:3), ETSLTVNK (SEQ ID NO:4), ETSLTVNKETTVETK (SEQ ID
NO:5), DQYYNIDVPSR (SEQ ID NO:6), and CFESFER (SEQ ID NO:7). This
confirmed the identity of the SZP isolated from human synovial
fluid by the peanut lectin affinity column.
[0090] The SZP preparations were further purified on an anti-SZP
monoclonal antibody affinity column. Five ml of Sepharose CL-2B
(Sigma, St. Louis, Mo.) was activated with CNBr as described by
March et al. (1974)) and incubated with 2.5 mg each of purified
monoclonal antibodies S6.79 and 17.109. Residual reactive sites
were blocked with 0.1 M Tris, pH 9.8 for 1 h and the beads washed
with 2 M urea, followed by 1 M NaCl in PBS buffer. Antibody
conjugation efficiency to the Sepharose beads was greater than 80%.
SZP preparations were made 1 M in NaCl and 1% Triton and incubated
with the anti-SZP beads overnight with rotation at 4.degree. C. The
beads were washed with PBS containing 1 M NaCl and 1% Triton. The
bound SZP was eluted with 2 M guanidine hydrochloride, pH 7.5. The
eluted SZP was dialyzed against 0.5 M NaCl, 10 mM sodium phosphate,
pH 7.5 and stored at -20.degree. C. These preparations yielded a
single band of SZP at 345 kDa by SDS-PAGE.
Example 3
Production of Monoclonal Antibodies to Non-Modified SZP
[0091] A. SZP Immunization
[0092] Purified human SZP, which was prepared as described in
Example 1, was used as the antigen for immunization for antibody
production. Two 8 week old female SJL mice were immunized using
either a RIMMS (Repetitive Immunization Multiple Sites) protocol or
a conventional immunization regime (e.g., Su et al, 1999).
[0093] For RIMMS, one 8-week-old female SJL mouse (Jackson
Laboratories, Bar Harbor, Me.) was immunized on days 0, 3, 5, 7 and
11, following the RIMMS immunization regime (Kilpatrick et al.,
1997). The mouse was anesthetized with isofluorane prior to each
series of immunizations. Twelve sites proximal to the draining
lymph nodes were injected subcutaneously with 50 .mu.l per site of
antigen/adjuvant mixture. Six of the sites received antigen diluted
1:1 with complete Freund's adjuvant (FCA; Life Technologies, Inc.,
Grand Island, N.Y.) and the six juxtaposed sites received antigen
diluted 1:1 in RIBI adjuvant (RIBI ImmunoChem. Research, Inc.,
Hamilton, Mont.).
[0094] One eight week old female SJL mouse was immunized, using a
conventional immunization regimen (e.g., Su et al, 1999), on day 0,
14, 21, and 24. The immunizations on day 0 and 14 were I.P. with
the antigen diluted 1:1 in RIBI adjuvant. The day 21 immunization
was I.V. with the antigen diluted in sterile PBS. The final
immunization was I.P. with the antigen diluted in sterile PBS.
[0095] B. PEG Induced Somatic Fusion Protocols
[0096] Mice were sacrificed, and a single cell suspension was
prepared from either the spleen or the lymph node cells (brachial,
axillary, superficial inguinal and popliteal). These cells were
combined at a ratio of 2.5:1 with the modified myeloma cell line
P3XBcl-2-13 (Kilpatrick et al., 1997). Somatic fusion was performed
using 1 ml of 50% polyethylene glycol 1500 (Boehringer Mannheim,
GmbH, Germany). Pelleted cells were resuspended in media containing
40% Excell-610 (JRH Biosciences, Lenexa, Kans.), 40% RPMI 1640, 10%
FBS, 10% Origen Cloning Factor (Igen, Rockville Md.), 2 mM
L-glutamine, 100 .mu.g/ml penicillin, and 0.01 mM 2-ME, plated out
at 1 ml per well in 24 well plates, and incubated overnight at
37.degree. C. After 24 hours, 1 ml of selection media containing a
2.times. concentration of HAT (0.1 mM hypoxanthine, 0.16 mM
thymidine, and 4 mM aminopterin, GibcoBRL) prepared in the above
media, was added to each well. After one week in culture, media was
changed to contain HT (0.1 mM hypoxanthine, 0.16 mM thymidine,
GibcoBRL).
Example 4
Screening of Potential Monoclonal Antibodies to SZP
[0097] Attempts to generate hybridomas using SZP derived from human
tali conditioned media as immunizing antigen resulted in 7
ELISA-reactive wells out of 144 growth-positive wells. Upon
preliminary ELISA screening, 5 of 7 hybridomas showed low but
specific immunoreactivity against SZP with no reactivity against
aggrecans isolated from either bovine nasal cartilage or rat
chondrosarcoma. Following the primary ELISA, the ELISA-reactive
hybridomas were further screened by chondrocyte immunocytochemistry
using chondrocyte subpopulations, a protocol adapted from a
fluorescence microtiter screening assay to isolate
immunocytochemistry-reactive antibodies (Su, 1997). Uncloned
hybridoma 4.23 was selected based on its immunostaining on the
superficial chondrocytes and negative for the deep zone
chondrocytes. Hybridoma GW4.23, secreting IgG1, was isolated from
limiting dilution cloning of parent hybridoma 4-23.
[0098] A. Primary ELISA Screening
[0099] Aggrecan isolated from bovine nasal cartilage (BNS) and rat
chondrosarcoma (RCS), which served as negative controls, were
resuspended in 0.1M Tris pH 8.0, aliquoted 1 ml/vial, and frozen at
-80.degree. C. The plates were then coated with 3 .mu.g/ml of
either SZP, BNS, or RCS. Antigen was diluted in carbonate coating
buffer, pH 9.2-9.6, and plated 10 .mu.l/well on EIA/RIA plates from
Costar. The plates were incubated for 2 hr at 37.degree. C. Then
blocked with 100 .mu.l/well of TBS containing 5% normal goat serum
and 1 mg/ml PEG for 30 min at 37.degree. C. 100 .mu.l/well of
tissue culture media corresponding to each individual well of the
fusion plates were added and incubated for 1 hr at 37.degree. C.
The plates then were washed 3.times. with 200 .mu.l/well of
1.times.TBS+ 1% Tween 20. 100 .mu.l/well of secondary antibody
G.alpha.M-IgG alkaline phosphatase conjugated diluted 1:1000 in
blocking buffer were added. Plate was then incubated for 1 hr at
37.degree. C. The plates were then developed with Sigma 104
phosphatase substrate and the color change was read at 15 and 30
min.
[0100] All wells were tested for binding, and all were growth
positive. Seven wells were selected as positive from the
conventional immunization and fusion. These seven wells were plated
at 30 cells per plate in a 96-well plate for limiting dilution
cloning. Four wells from the RIMMS fusion that were selected as
positive were also plated to clone by limiting dilution. Aliquots
of the supernatants were further analyzed in the subsequent
assays.
[0101] B. Antibody Staining of Chondrocyte Cultures
[0102] Thin slices of articular cartilage from human tali were
manually dissected from the superficial, middle and deep zones of
the cartilage and placed in DMEM. The slices from the middle zone
were discarded. The cartilage slices from the superficial and deep
zones were treated separately with 0.2% pronase in DMEM
supplemented with 5% fetal bovine serum for 1.5 hours at 37.degree.
C. (Aydelotte and Kuettner, Connect Tissue Res. 18:205-222, 1988;
Aydelotte et al. Connect Tissue Res 18:223-234, 1988; Schumacher et
al., J. Orthop. Res. 17:110-120, 1999). The slices were then rinsed
extensively with DMEM and treated further with 0.025% Collagenase P
for 18-22 hours in DMEM supplemented with 5% fetal bovine serum.
The resulting chondrocyte suspensions were centrifuged at 1000 RPM
for 15 minutes in order to pellet the cells. The chondrocytes were
washed in DMEM three times and centrifuged as stated above to
collect the cells. The number of chondrocytes in each sample was
determined by counting the cells on a hemacytometer. Chondrocytes
from the superficial and deep zones were seeded separately into a
96 well tissue culture plate at high density (250,000
cells/cm.sup.2) in medium consisting of DMEM supplemented with 10%
fetal bovine serum. The cells were allowed to attach overnight and
refed with medium consisting of DMEM supplemented with 10% fetal
bovine serum and 50 .mu.g/ml ascorbic acid. After three days in
culture the cells were refed with medium plus 10 M monensin for
four hours in order to prevent secretion through the Golgi
apparatus. At the end of the incubation period the cells were
rinsed briefly in phosphate buffered saline (PBS) and fixed with a
solution of 4% paraformaldehyde in PBS, pH 7.4 for five minutes at
room temperature. The cells were rinsed in PBS and permeabilized
with a solution of 0.1% Triton-X 100.RTM. (Sigma Chemical Co., St.
Louis, Mo.) for five minutes at room temperature. The cells were
rinsed in PBS and non-specific binding sites were blocked with a
solution of 1% bovine serum albumin (BSA) and 1% normal goat serum
for 20 minutes at room temperature. The cells were rinsed in PBS
and pairs of wells containing cells from the superficial and deep
zones were incubated with different hybridoma media, potentially
containing a monoclonal antibody to SZP for 45 minutes at room
temperature. The cells were rinsed in PBS and incubated with a goat
anti-mouse rhodamine conjugated IgG diluted 1:50 with PBS for 45
minutes at room temperature. The cells were rinsed in PBS and
examined by fluorescence microscopy. Any pair of wells containing
cells from the superficial and deep zone that was positive in the
chondrocytes from the superficial zone and negative in the
chondrocytes from the deep zone was considered as a positive
reaction as a monoclonal antibody to SZP. Four monoclonal
antibodies that were positive for the superficial chondrocytes and
negative for the deep chondrocytes were obtained. They were
designated as GW 3.15, GW 4.10, GW 4.23 and GW 5.15.
[0103] C. Direct ELISA for Human SZP
[0104] A 96 well ELISA plate was coated overnight at 4.degree. C.
with conditioned media from human talar superficial chondrocytes or
deep chondrocytes in the presence of 20 mM NaHCO.sub.3/Na.sub.2CO3,
pH 9.2. All wells were rinsed and incubated with the various
hybridoma media for 1 hour at room temperature. The wells were
rinsed and incubated with a horseradish peroxidase conjugated goat
anti-mouse IgG for 1 hour at room temperature. The wells were
rinsed and color development was achieved using hydrogen peroxide
and o-phenylenediamine as the chromogenic substrate. Plates were
read with an automatic ELISA plate reader. Any pair of wells
containing conditioned media from chondrocytes from the superficial
zone and chondrocytes from the deep zone in which there was a
positive result for the superficial chondrocytes and not the deep
chondrocytes was considered positive for SZP.
[0105] This method was also used to test samples of human synovial
fluids from normal donors and patients with osteoarthritis (OA) and
rheumatoid arthritis (RA). Direct ELISA of samples of synovial
fluids from normal donors, patients with osteoarthritis and
patients with rheumatoid arthritis revealed that SZP is elevated in
synovial fluids from patients with OA or RA compared to normal
synovial fluid.
[0106] D. Immunohistochemistry of Human Knee and Ankle
Cartilage
[0107] Samples from full thickness slices of cartilage and thin
slices from the superficial zone from the articular surface from
human femoral condyle and talar dome cartilage were obtained within
24 hours of the death of the donor. Cartilage samples were fixed in
4% paraformaldehyde/PBS for 30 minutes at room temperature and
rinsed in PBS. Vertical frozen sections and paraffin embedded
sections were obtained from samples of the full thickness of
cartilage from human knee and ankle cartilages. Horizontal frozen
sections and paraffin embedded sections were obtained from the thin
slices of cartilage from the superficial zone. Some cartilage
samples were pre-treated with monensin at a concentration of
10.sup.-6 M for four hours before fixation. Sections of cartilage
were rinsed in PBS, permeabilized in 0.1% Triton-X 1100 (Sigma
Chem. Co., St. Louis, Mo.) for five minutes at room temperature and
rinsed in PBS. Non-specific binding sites were blocked in a
solution of 1% BSA, 1% normal goat serum for 20 minutes at room
temperature. The sections were rinsed in PBS and incubated with the
monoclonal antibody GW 4.23 (MAb GW 4.23) for 45 minutes at room
temperature. The sections were rinsed in PBS and incubated with a
horseradish peroxidase conjugated goat anti mouse IgG for 45
minutes at room temperature. The sections were rinsed in 0.05 M
Tris, pH 7.6 and positive SZP sites were visualized using hydrogen
peroxide and diaminobenzidine as the chromogenic substrate.
Alternatively, sections for immunohistochemistry were tested using
the Pierce Immunopure.RTM. ABC Alkaline Phosphate mouse IgG
staining kit (Pierce, Rockford, Ill.), following all manufacturer's
directions.
[0108] Using MAb GW 4.23, the chondrocytes in the superficial zone
of articular cartilage from knee and ankle samples were positive
for SZP whereas the chondrocytes in the middle and deep zones were
non-reactive. A thin layer of immuno-positive material for SZP was
also observed at the articular surface in vertical sections of
articular cartilage from both knee and ankle samples. Horizontal
sections of the superficial zone also revealed a fine meshwork of
immuno-positive material for SZP at the articular surface.
[0109] Using all three of the screening protocols, MAb GW 4.23 was
the strongest and most specific of all the hybridoma media tested
in this experiment.
Example 5
SDS-PAGE and Western Blotting Using MAb GW 4.23
[0110] SDS-PAGE was performed on 4-10% gradient separating gels,
with a 3.6% stacking gel. Samples for SDS-PAGE were dissolved in
sample buffer consisting of 1% SDS, 0.08 M Tris, pH 6.8 containing
16% ethylene glycol and 0.0006% bromophenol blue. All samples were
run non-reduced and not boiled. Separated proteins were transferred
to nitrocellulose by Western blotting. Western blotting was
performed overnight at 250 mAmps in a buffer consisting of 12 mM
Tris, pH 7.4, 0.03 mM EDTA and 6 mM sodium acetate. Non-specific
binding sites on the nitrocellulose membrane containing the
separated proteins were blocked in a solution of 5% non-fat milk in
PBS for 30 minutes at room temperature and rinsed in PBS. The
nitrocellulose membrane was incubated with MAb GW 4.23 (1:10
dilution) for 1 hour at room temperature. The membrane was rinsed
in PBS and incubated with a horseradish-peroxidase conjugated goat
anti mouse IgG (1:500 dilution) for 1 hour at room temperature. The
membrane was rinsed in 0.5 M Tris pH 7.6 and protein bands specific
for the epitope recognized by MAb GW 4.23 were visualized using
hydrogen peroxide and 4-chloro-1-napthol as the chromogenic
substrate.
[0111] Western blotting of purified SZP or samples of human
synovial fluids showed a prominent band at 345 kD. Western blots of
conditioned medium from slices of the superficial zone from knee
and ankle cartilage also showed a protein band of similar mobility
as compared to the band for purified SZP.
Example 6
Staining of the Articular Surface of Human Tali Using MAb GW
4.23
[0112] Several cylindrical punches of the full thickness of
articular cartilage, 8 mm in diameter, were obtained from the talar
dome of human ankles. These cartilage plugs were fixed in 4%
paraformaldehyde/PBS for 30 minutes at room temperature. The plugs
were rinsed in TBS. Non-specific binding sites were blocked with 1%
BSA, 1% NGS for 20 minutes at room temperature in TBS. The first
antibody, GW 4.23, was applied to different plugs for different
amounts of time. Time points of 0, 5, 15, 30, 60, and 120 minutes
were used. The plugs were rinsed in TBS and a biotinylated second
antibody and avidin-biotin alkaline phosphatase complex applied as
stated above for immunohistochemistry. The plugs were incubated
with NBT/BCIP substrate used at half strength until maximal color
development was achieved. Thirty minutes was chosen as the optimal
time of incubation of the first antibody with the cartilage samples
and was used for subsequent experiments.
[0113] In a different experiment, a matched pair of intact human
ankle joints was obtained and one ankle was injected with GW 4.23
(1:10 dilution in DMEM) into the synovial cavity and the other
ankle was injected with DMEM. Both joints were incubated for 30
minutes at 37.degree. C. in a humidified chamber. At the end of the
incubation times both joints were opened and cylindrical punches of
the full thickness of articular cartilage were taken. The punches
were fixed in 4% paraformaldehyde/PBS for 30 minutes at room
temperature. The punches were rinsed extensively in PBS and then
processed as stated above for immunohistochemistry. The punches
were placed in NBT/BCIP used at half strength until maximal color
development occurred.
[0114] In another experiment, a matched pair of normal intact human
ankle tali (Collin's grade 0) was obtained and one talus was fixed
as stated above and incubated with GW 4.23 (1:10 dilution) for 30
minutes at room temperature. The other talus was fixed and
incubated with mouse IgG used at the same concentration of GW 4.23.
Both tali were processed as stated above for immunohistochemistry.
The tali were placed in NBT/BCIP used at half strength until
maximal color development was achieved.
[0115] One talus with degenerative changes was obtained (Collin's
grade 2). This talus had fissures and a small lesion off to one
side of the talar dome. The talus was fixed and processed as stated
above using GW 4.23 (1:10 dilution). The talus was placed in
NBT/BCIP used at half strength until maximal color development was
achieved.
[0116] A large piece of cartilage from femoral condyle removed
during knee replacement surgery was obtained. The sample was fixed
as stated above and processed as stated above using GW 4.23 (1:10
dilution). The sample was placed in NBT/BCIP used at half strength
until maximal color development was achieved.
[0117] In the experiments outlined here, MAb GW 4.23 was used
successfully to stain the articular surface of cylindrical plugs of
articular cartilage from human tali. Staining of the articular
surface was present at all time points of incubation of MAb GW 4.23
with the tissue samples from as little as 5 minutes incubation with
the antibody to as long as 2 hours incubation with the antibody
with the optimal time of incubation being 30 minutes. There was no
staining of the cartilage when the MAb GW 4.23 was omitted (time
point 0). In these experiments, only the surface of the cartilage
plug was stained. No staining was seen in the cells or at the deep
cut end of the cartilage plug.
[0118] Positive staining of the articular surface of cylindrical
cartilage plugs was observed when MAb GW 4.23 was injected into an
intact human ankle joint prior to fixation or processing of the
tissue. No staining was seen in the cells or at the cut edges of
the cartilage plug.
[0119] Positive staining of the surface of normal human intact
ankle tali was observed when MAb GW 4.23 was used in these
experiments. The staining at the articular surface was smooth and
even and showed no defects at the articular surface. There was also
positive staining observed in the synovial tissue surrounding the
cartilage. There was no staining at the articular surface of the
tali that was treated with the same concentration of a non-specific
IgG control.
[0120] The articular surface of a human talus showing degenerative
changes showed uneven heterogeneous staining when MAb GW 4.23 was
used to stain the tissue. Fissures in the articular surface were
stained darker than surrounding tissue and areas were present at
the surface which were unstained. The lesion site showed very dark
staining.
[0121] When MAb GW 4.23 was used to stain a piece of cartilage
removed from a patient undergoing joint replacement, the surface
stained intensely even though there was no superficial zone
present. Upon closer examination of the tissue it was found that
the staining was due to material deposited at the surface of the
damaged cartilage. There was no cellular staining or staining of
the matrix within the tissue. All the staining material was at the
damaged surface.
[0122] Collectively, these experiments demonstrate that a
monoclonal antibody to the superficial zone protein can
successfully be used to visualize the surface of articular
cartilage in both normal and damaged joints as well as the
surrounding synovial tissue.
Example 7
Production of Monoclonal Antibodies to Modified SZP
[0123] Eight week old, female SJL mice (Jackson Laboratories, Bar
Harbor, Me.) were immunized on days 0, 14 and 24 intraperitoneally
(IP) with purified human SZP (10 .mu.g), conjugated with KLH or
mixed with hyaluronic acid at 1:10 ratio by weight, in RIBI
adjuvant (RIBI, Hamilton, Mont.). Prior to the last IP injection,
on day 21, an intravenous injection of the immunizing antigens
diluted in PBS was administered to each mouse. On day 25, the mice
were sacrificed, splenocytes prepared and somatic fusions were
performed as previously described (Su, 1999). Briefly, splenocytes
and mouse myeloma cells P3X63BCL2-13 (Kilpatrick et al., 1997) at
2.5:1 ratio were fused using polyethylene glycol 1500 (Boehringer
Mannheim GmBH, Germany). Fused cells were resuspended in media
containing equal volume of RPMI 1640 (GibcoBRL, Grand Island, N.Y.)
and EXCELL-610 (JRH Biosciences, Lenexa, Kans.) supplemented with
1.times.Origen Hybridoma Cloning Factor (Igen, Gaithersburg, Md.),
10% fetal bovine serum (Hyclone, Logan, Utah), 2 mM L-glutamine,
and penicillin/streptomycin. Cells were then plated in 24-well
microtiter plates (Costar, Cambridge, Mass.) (1 ml/well), and
cultured at 37.degree. C., 5% CO.sub.2. Twenty four hours later, 1
ml of 2.times.HAT selection media [100 .mu.M hypoxanthine, 0.4
.mu.M aminopterin, 16 .mu.M thymidine (GIBCO, Grand Island, N.Y.)
in the above media] was added to each well. After 10 days of
culture in 1.times.HAT selection media, media was changed to
contain HT (0.1 mM hypoxanthine, 0.16 mM thymidine).
[0124] Limiting dilution cloning was used for the cloning of
hybridomas. Immunoglobulin classes and subclasses were determined
using an ELISA subtyping kits (Southern Biotechnology, Birmingham,
Ala.) following the manufacturer's instructions.
Example 8
Screening or Potential Monoclonal Antibodies to Modified SZP
[0125] When SZP was used as an immunizing antigen without
modification, it may not easily elicit T-cell dependent B-cell
response. Thus, two approaches were used to modify synovial
fluid-derived SZP: (1) conjugation of SZP to a carrier protein,
keyhole limpet hemocyanin (KLH) to increase immunogenicity, and (2)
mixing SZP with hyaluronic acid (HA), a component present at high
concentration (1-3 mg/ml) in synovial fluid, to form complexes,
mimicking the form of the molecule most likely to occur in vivo.
Both approaches resulted in greater than 95% of 294 growth-positive
wells that were positive in ELISA.
[0126] A. ELISA Analysis
[0127] 96-well immunoplates were coated overnight at 4.degree. C.
with purified SZP, KLH, peanut lectin in 0.1M sodium bicarbonate
buffer containing 0.5M NaCl at pH 9.6. After the plates were washed
in PBS, all remaining procedures were carried out at room
temperature. Following 30 minutes incubation of immunoplates with
blocking buffer (1% BSA in PBS), purified antibodies (1 .mu.g/ml)
were added to the wells and incubated for 1 h. Plates were then
washed 3 times with PBS plus 0.1% Triton X-100 (PBST), and goat
anti-mouse IgG conjugated with either alkaline phosphatase or
horseradish peroxidase (Southern Biotechnology, Birmingham, Ala.)
diluted {fraction (1/1000)} in blocking buffer was added for 1 h
incubation. The plates were washed as above and appropriate
substrate (Sigma alkaline phosphate substrate or K-blue substrate)
was added. Following color development immunoreactivity was
measured at 405 nm or 650 nm in a microplate reader (UV max.TM.,
Molecular Devices, Menlo Park, Calif.).
[0128] Ten ELISA-reactive hybridomas were selected from each
immunization and further analyzed by Western blotting and
immunohistochemistry. Subsequently, hybridomas that showed
different immunoreactivity profiles were cloned by limiting
dilution cloning. The ELISA data from GW4.23, a monoclonal antibody
derived from non-modified SZP immunization, and a control antibody
are represented in FIG. 1. MAb S6.79, derived from SZP-KLH
immunization, S13.233 and S17.109, derived from immunization with a
mixture of SZP and hyaluronic acid (SZP-HA), show strong
immunoreactivity against SZP purified from both synovial fluid
(SZP-sf) and articular cartilage (SZP-ac), with no cross-reactivity
against KLH or lectin. GW4.23 also shows specific but lower
immunoreactivity against SZP from both preparations. S13.52, raised
against synovial fluid derived-SZP-HA complex, is the only
monoclonal antibody that shows differential reactivity against SZP
from different sources. There was no immunoreactivity with the
negative control antibody 129R10, a monoclonal antibody against
glutathione S-transferase.
[0129] B. Chondrocyte Immunocytochemistry
[0130] The superficial, middle and deep zones were manually
dissected from the articular cartilage of human tali. The slices
from the middle zone were discarded. The cartilage slices from the
superficial and deep zones were treated separately with 0.2%
pronase in DMEM supplemented with 5% FBS for 1.5 hours at
37.degree. C. The slices were then treated with 0.025% Collagenase
P for 18 hours in DMEM supplemented with 5% FBS. The superficial
and deep zone cells were washed and seeded into the wells of a 96
well tissue culture plate at high density (250,000 cells/cm.sup.2).
The cells were allowed to attach overnight and fed with medium
(DMEM, 10% FBS and 25 .mu.g/ml ascorbic acid). Monensin (10 M) was
added to the cultures for the last four hours of culture. At the
end of the incubation period the cells were rinsed briefly in PBS,
fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X
100' for five minutes at room temperature. The cells were incubated
with 1% bovine serum albumin and 1% normal goat serum for 30
minutes at room temperature in order to block non-specific binding
sites. Pairs of wells containing cells from the superficial and
deep zones were incubated with media from the seven ELISA-positive
hybridoma wells, rinsed and incubated with a goat anti mouse
rhodamine conjugated IgG diluted 1:50 with PBS. The cells were
examined by fluorescence microscopy.
[0131] C. Immunolocalization of SZP in Human Articular
Cartilage
[0132] Full thickness cartilage slices were collected from the
talar dome of organ donors within 24 hours of death. Frozen
sections of the cartilage slices were cut perpendicular to the
articular surface. The sections were fixed with 10% formalin/PBS
for 5 minutes and washed in 50 mM Tris, 0.1 M NaCl, pH 7.5.
Sections were treated with 0.5% testicular hyaluronidase (Sigma),
1% BSA in PBS for 30 minutes to facilitate antibody penetration in
the cartilage tissue and then washed with PBS. They were treated
with purified SZP monoclonal antibodies for 2 hours at room
temperature and washed with PBS-0.05% Tween. The sections were
incubated with a goat-anti-mouse IgG horseradish peroxidase
conjugate (Pierce Chemical Co., Rockford Ill.) for 1 hour and
washed as above with PBS-Tween buffer. Peroxidase activity was
detected with hydrogen peroxide and diaminobenzidine
substrates.
Example 9
SDS-PAGE and Western Blotting using Antibodies to Modified SZP
[0133] Purified human SZP (0.25 .mu.g/lane), synovial fluids (0.25
.mu.l human and 1 .mu.l bovine, dog, guinea pig, or rabbit
samples), human plasma (1 .mu.l) or human serum (1 .mu.l) were
separated by electrophoresis on 3-8% Nu-PAGE Tris acetate gel
(Invitrogen, Carlsbad, Calif.) and transferred onto nitrocellulose
using Nu-PAGE non-reduced gel buffer system following the
manufacturer's instructions. Blots were incubated in blocking
solution [5% non-fat dry milk in Tris-buffered saline-Tween (TBST:
50 mM Tris-HCL pH 7.5, 150 mM NaCl, 0.05% Tween-20)]. After brief
washes with TBST, the filters were then reacted with SZP monoclonal
antibody (1:5 to 1:20 dilution of culture media or 0.25 to 1
.mu.g/ml purified antibody) for 1 hr at room temperature followed
by extensive washes with TBST. Blots were then incubated with Goat
anti-mouse antibody conjugated with horseradish peroxidase
(Southern Biotech, Birmingham, Ala.) for 1 hr, washed with TBST,
and developed using the ECL procedure (Amersham, Arlington Heights,
Ill.). Unlike the MAb GW4.23, the antibodies generated to modified
SZP were able to detect SZP after reduction and boiling; however,
they often had stronger signals on Westerns if the preparations
were not reduced. Western blotting using MAb S6.79 (0.4 .mu.g/ml)
and S17.109 shows strong immunoreactivity against SZP purified from
both cartilage and synovial fluid. S13.233 (at 1 .mu.g/ml) also
gave a similar staining pattern but the intensity was weaker.
[0134] Some of the antibodies were able to detect SZP in plasma and
serum. The antibodies detected at least two different forms of the
SZP molecule, the large form of the molecule at 345 kDa. S6.79,
S17.109, S13.52, S13.233, and GW4.23 all detected a 345 kDa form of
SZP in synovial fluid. S6.79, S13.52 and S13.233 are able to detect
this form of the molecule in plasma and serum. The amount of SZP
appeared to be substantially greater in synovial fluid than in
plasma or serum.
Example 10
Preparation of Proteolytic SZP Fragments and Assignment of
Epitope-Containing Domain
[0135] A modification of the method of Su et al. (Hybridoma 1995;
14(4)383-390) is used to assign the epitope-containing domain of
SZP. Purified SZP is reduced and alkylated by incubating the
protein in 6M guanidine-HCl, 0.5 M Tris-HCl, 10 mM EDTA and 20 mM
dithiothreitol (pH 8.6) for 1 h at 37.degree. C. under nitrogen,
followed by addition of 4-vinylpyridine to 50 mM for 30 min at room
temperature. The pyridylethylated material is desalted by HPLC with
a BU300 column (2.1.times.30 mm, Brownlee, Foster City, Calif.)
using a linear gradient of acetonitrile (16-64%) in 0.1%
trifluoroacetic acid (TFA) over 30 min. The eluted protein is then
digested with sequencing grade Lys-C (Wako, Richmond, Va.) in 0.1M
Tris-HCl (pH 8.5) for 16 h at room temperature, with an
enzyme:substrate ratio of 1:100. The Lys-C generated peptides are
then separated and isolated on the BU300 column using a linear
gradient of acetonitrile (8-64%) in 0.1% TFA over a 40 min period.
Peptide fragments are dried by flushing with nitrogen and are then
resuspended in TBS. Automated Edman degradations are performed
using the Applied Biosystems 477A liquid-pulse sequencer (Applied
Biosystem, Foster City, Calif.) equipped with a 120A PTH analyzer
for the identification of phenylthiohydantoin amino acids.
[0136] The SZP protelytic fragments separated by HPLC are used for
coating an ELISA plate for reaction with anti-SZP antibody. The
fragment that is recognized by anti-SZP is identified as the
epitope-containing domain.
Example 11
Antibody Affinity Measurements
[0137] A. BIAcore Analysis
[0138] BIAcore technology and its use in characterizing
inter-molecular interactions has previously been described
(Fagerstam et al. (1992)). The BIAcore 2000 system, CM5 sensor
chips, P-20 surfactant, the coupling kit which contained
N-hydroxysuccinimide, N-ethyl-N'-(3-diethylaminopropyl)-c-
arbodiimide, ethanolamine hydrochloride pH 8.5, and rabbit
anti-mouse FC-y is from Pharmacia Biosensor AB (Uppsala, Sweden).
All other chemicals are reagent grade. The BIAcore running buffer
used for immobilization and binding studies contains 10 mM HEPES
(pH 7.4), 150 mM NaCl, 0.05% volume of a 10% P-20 surfactant
solution.
[0139] Carboxyl groups of the BIAcore CM5 sensor chip hydrogel
matrix is activated for 7 min with a mixture of 50 mM
N-hydroxysuccinimide and 200 mM
N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide. Rabbit anti-mouse
Fc-y (RAMfc) antibody is diluted to 40 .mu.g/ml in 10 mM sodium
acetate pH 5.0 then is injected onto the sensor chip for 3 min at a
flow rate of 5 .mu.l/min. Unreacted groups are then deactivated
with a 7-min injection of 1 M ethanolamine hydrochloride pH 8.5. To
determine binding constants, antibodies are injected over the RAMfc
at 5 .mu.l/min. for 4 min. The flow is then increased to 40
.mu.l/min and dilutions of human SZP and bovine SZP are injected
for 1 min. The surface is regenerated with 100 mM HCl. Binding
constants are determined using BIAevaluation software.
[0140] B. IAsys Analysis
[0141] The binding characteristics of the anti-SZP monoclonal
antibodies were measured using resonant mirror technology on an
IAsys instrument (Affinity Sensors, Cambridge, England, UK).
Biotin-conjugated cuvettes (Affinity Sensors) were used and all
experiments were performed at 23.degree. C. Sixty .mu.g of SZP was
biotinylated with 300 .mu.g sulfo-N-hydroxysuccinimidyl ester
(Pierce Chemical Co., Rockford, Ill.) in 1 ml of 0.1 M sodium
carbonate buffer pH 9 for 1 hr. The reaction was terminated by the
addition of 0.1 M Tris buffer pH 7.5 and dialyzed against PBS-Tween
0.05% (PBST). Neutraavidin (Pierce Chemical Co., Rockford, Ill.) at
50 .mu.g/ml in PBST was incubated with the biotin-cuvettes for 10
minutes and washed with PBST. Biotinylated SZP (25 .mu.g/ml) was
captured on the neutraavidin-coated biotin cuvettes for 10 minutes
and washed with PBST. Different monoclonal antibodies were tested
in the cuvettes for their ability to bind to the SZP-coated
cuvettes at the following concentrations, 50, 20, 10, 5, 2, and 1
.mu.g/ml. Association experiments were performed for 10 minutes
followed by a dissociation phase for 10 minutes after a PBST wash.
The cuvettes were stripped of residual antibodies between each
antibody binding experiment with 10 mM HCl for 2 minutes and then
washed with PBST. Binding and dissociation kinetics were calculated
using Affinity Sensors FASTfit software. The association rate
constant was calculated as the slope of a linear plot of
association rates (Y) versus antibody concentrations (X). The
dissociation rate constant was the Y intercept from the same line.
The dissociation constant (K.sub.D) for the antibody was calculated
from the dissociation rate constant divided by the association rate
constant (k.sub.dissoc/k.sub.assoc).
Example 12
Quantitation of SZP Using Homogenous Formats
[0142] A. Scintillation Proximity Assay (SPA)
[0143] SZP antibody, radiolabeled (beta emitter) SZP or SZP
fragment and the scintillant-embedded polyvinyl toluene beads
conjugated with anti-mouse or protein A are mixed together. When
radiolabeled SZP or SZP fragment captured by anti-SZP, the beta
emitter are brought to the proximity of scintlillant-embedded
beads, resulting in the emission of light that is measured by a
scintillation counter.
[0144] B. Homogeneous Time-Resolved Fluorescence Assay (HTRFA)
[0145] Biotinlyted SZP or SZP fragment, lanthanide chelate-labeled
anti-SZP (fluorescence energy donor) and streptavidin conjugated
with the energy acceptor are incubated together to allow the energy
donors to be in the proximity of energy acceptors. Donor/acceptor
pairs may include for example, Eu/allophycocyanin (or Cy5) or
Terbium (Tb)/tetramethylrhodamine. Upon excitation of the donor,
the specific energy is transferred from the donor to the acceptor,
and the resultant fluorescent signals is measured by a
time-resolved fluorometer.
[0146] C. Fluorescence Polarization Assay (FPA)
[0147] Fluorescent labeled SZP fragment (<30 kDa) and SZP
antibody are mixed together to allow the antibody binding to SZP
fragment. After the binding reaches equilibrium, the immune
complex, due to increased in mass, tumbles more slowly, thus,
yielding a polarized fluorescence signal that is measured by a
fluorescent polarization meter.
Example 13
DNA-based Immunization for the Production of SZP Monoclonal
Antibodies
[0148] DNA plasmid preparation, DNA/gold particle bullets and
delivery of DNA bullets to mouse epidermis have previously been
reported (Kilpatrick et al., 1998; Eisenbraun et al., 1993; Pertmer
et al., 1996). DNA encoding the N- or the C-terminal region of SZP
is cloned into the Alpha+vector that has human Fc cDNA inclusion
(Kaplan et al., 1997). The Alpha+SZP/Fc plasmid is transfected into
E. coli and DNA is prepared from a selected clone. After DNA/gold
particle bullets are prepared, DNA/gold particles are propelled
into the shaved thorastic and abdominal regions of mice using a
helium-driven Accell gene gun (PowerJet Vaccines, Incorp. 585
Science drive, Suite C, Madison, Wis. 53711). Following the primary
immunization, mice receive one to four booster immunization/s
within 8-11 days. On the day of fusion (day 9-13), lymphocytes
harvested from axillary, brachial and superficial inquinal nodes
are prepared and fused with myeloma cells following a previously
published protocol (Su et al, 1999).
Example 14
Immunization via Recombinant Baculovirus Displaying SZP-Fusion
Proteins for the Production of SZP Monoclonal Antibodies
[0149] A. Generation of SZP Fusion Transfer Plasmids
[0150] The Baculovirus fusion protein is produced using the
BacVector Virus Display system from Novagen (Madison, Wis.).
Cloning, subcloning and sequencing of DNA are carried out using
standard protocols (Sambrook et al., 1989). The amino-terminal
domains of human SZP are amplified and cloned into the Kpn I site
of the pBACsurf I vector. Positive plaques are selected based on
anti-gp64 staining of both native gp64 and gp64 fusion bands in
Western blot analysis (Lindley et al. J. Immunological Methods
2000;234:123-135. The virus is then scaled up to a 150 ml
suspension culture (1.times.10.sup.6 cells/ml), and incubated on a
shaker for 3 days at 27.degree. C. For generation of antigen for
immunizations, 450 ml of Sf9 cells at 1.times.10.sup.6 cells/ml are
infected with relevant virus, at a multiplicity of infection (MOI)
of 0.1, and grown for 3 days at 27.degree. C. To harvest virus, the
culture supernatant is cleared by high-speed centrifugation for 3
hr at 61,000.times.g. The virus pellet is resuspended in phosphate
buffered saline (PBS) and filtered through a 0.2 .mu.M filter.
Virus is diluted in PBS and the mice are immunized as described in
Example 2 using the RIMMS immunization regime detailed below. The
total amount of antigen used for immunizations is approximately 15
.mu.g of the SZP.
[0151] B. ELISA Screening
[0152] Primary ELISA screenings are performed using previously
published procedures (Harlow & Lane 1988). High binding EIA
plates (Corning/Costar Corning, N.Y.) are coated with whole cell
lysates from Sf9 cells infected with either a control virus, or the
SZP-fusion virus to allow subtractive comparisons. Lysates are
prepared from cells infected at a multiplicity of infection (MOI)
of 1 pfu/cell at 48 hr post infection. Infected cells are pelleted
and subjected to repeated freeze-thaw cycles in a dry ice ethanol
bath. The lysates are then diluted 1:10 in carbonate coating buffer
and 100 .mu.l per well are incubated at 37.degree. C. for 1 hr.
Plates are blocked with 100 .mu.l/well of Tris buffered saline
(TBS), containing 5% normal goat serum and 1% PEG for 1 hr at
37.degree. C. Undiluted tissue culture supernatant is added at 100
.mu.l/well and incubated at 37.degree. C. for 1 hr. Plates are
washed with 1.times.TBS+1% Tween 20 (TBS-T). Secondary antibody,
goat-anti-mouse IgG-AP light chain specific (Southern Biotechnology
Associates, Birmingham Ala.), was diluted 1:1000 in blocking
buffer, and 100 .mu.l/well is reacted for 1 hr at 37.degree. C.
Plates are developed with phosphatase substrate (Sigma, St. Louis,
Mo.) at room temperature and readings are taken at 15 and 30
minutes. SZP reactive supernatants are further characterized as
described in examples 3 through 9.
Example 15
Measurement of SZP in Human Synovial Fluid
[0153] An ELISA assay was developed to measure the concentration of
SZP in synovial fluids. Anti-human SZP monoclonal antibody was
purified from the culture medium of hybridoma cultures, as
described above.
[0154] A peanut lectin (Sigma Chemical Co., St. Louis, Mo.) was
used to coat black 96-well plates at a concentration of 1 .mu.g/ml
in 0.1 M NaHCO.sub.3, pH 8.5. Plates were blocked with 1% BSA.
Dilutions of synovial fluid or an SZP standard were made and
incubated with the lectin-coated plates for 2 h. After washing the
plates with PBS-Tween (0.05%), the plates were incubated with an
anti-SZP monoclonal antibody for 1 h, washed and incubated with a
goat-anti-mouse-HRP conjugate (Pierce Chemical Co., Rockford,
Ill.). Bound HRP enzyme activity was detected with a
chemiluminescent substrate (Pierce Chemical Co, Rockford, Ill.) and
a luminometer. Two-fold serial dilutions (1:60 to 1:4000) were
sufficient to measure the concentrations of SZP in synovial fluid.
The assay was able to measure an SZP concentration in the range of
25-5000 ng/ml. See FIG. 2. Fifty samples of human synovial fluids
from organ donors or patients with degenerative joint disease
contained a range of SZP concentration from about 60-600 .mu.g/ml.
The mean value was 286 .mu.g/ml with a standard deviation of 146
.mu.g/ml. The data are shown in FIG. 3.
[0155] Similar results were obtained using SDS-PAGE analysis. The
concentration of SZP in human synovial fluid was also assessed
using Western blotting of two-fold dilutions of synovial fluid
compared to a purified SZP standard. Equivalent amounts of SZP were
separated by SDS-PAGE and transferred to nitrocellulose, then
serial dilutions of synovial fluid were compared to purified SZP to
determine the relative detection limits for both preparations. Such
an experiment showed synovial fluid to have about ten times the
amount of SZP as the purified SZP stock solution with a
concentration of 20 .mu.g/ml. This experiment also estimated the
concentration of SZP in synovial fluid to be about 200
.mu.g/ml.
Example 16
Cross-Reactivity of Antibodies to Bovine, Guinea Pig, and Rabbit
SZPs
[0156] Synovial fluid was collected from various species including
bovine, dog, guinea pig and rabbit for Western analysis. MAb S6.79
showed strong cross-reactivity against proteins at approximately
330-350 kDa in dog and rabbit samples, their staining intensity at
1 .mu.l per sample is close to that of 0.25 .mu.l of human synovial
fluid. MAb S6.79 also reacted with bovine and guinea pig synovial
fluids, but the staining intensity was weaker. Furthermore, in
guinea pig the immunoreactive bands are smaller in size, at
approximately 250 kDa molecular weight. To confirm that the
immunostained bands detected by S6.79 are indeed SZP, an antibody
absorption experiment was performed. MAb S6.79 was pre-incubated
with either BSA or purified SZP (1:25 ratio in weight) before
applying to the synovial fluid-containing blot. Pre-incubation of
antibody with BSA did not change or reduce the staining pattern or
intensity of immunostained bands. As expected, pre-incubation of
antibody with purified SZP almost completely abolished the
staining. These data clearly indicated that the immunostained bands
were SZP.
[0157] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to describe more fully the state of the art to
which this invention pertains.
[0158] Although the present process has been described with
reference to specific details of certain embodiments thereof, it is
not intended that such details should be regarded as limitations
upon the scope of the invention except as and to the extent that
they are included in the accompanying claims.
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Sequence CWU 1
1
7 1 10 PRT Artificial Sequence Description of Artificial
Sequence/note=synthetic construct 1 Arg Gly Gly Ser Ile Gln Gln Tyr
Ile Tyr 1 5 10 2 10 PRT Artificial Sequence Description of
Artificial Sequence/note=synthetic construct 2 Asp Gln Tyr Tyr Asn
Ile Asp Val Pro Ser 1 5 10 3 17 PRT Artificial Sequence Description
of Artificial Sequence/note=synthetic construct 3 Gly Phe Gly Gly
Leu Thr Gly Gln Ile Val Ala Ala Leu Ser Thr Ala 1 5 10 15 Lys 4 8
PRT Artificial Sequence Description of Artificial
Sequence/note=synthetic construct 4 Glu Thr Ser Leu Thr Val Asn Lys
1 5 5 15 PRT Artificial Sequence Description of Artificial
Sequence/note=synthetic construct 5 Glu Thr Ser Leu Thr Val Asn Lys
Glu Thr Thr Val Glu Thr Lys 1 5 10 15 6 11 PRT Artificial Sequence
Description of Artificial Sequence/note=synthetic construct 6 Asp
Gln Tyr Tyr Asn Ile Asp Val Pro Ser Arg 1 5 10 7 7 PRT Artificial
Sequence Description of Artificial Sequence/note=synthetic
construct 7 Cys Phe Glu Ser Phe Glu Arg 1 5
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