U.S. patent application number 14/124439 was filed with the patent office on 2014-08-07 for monoclonal antibody and antigens for diagnosing and treating lung disease and injury.
This patent application is currently assigned to Indiana University Research and Technology Corp.. The applicant listed for this patent is Matthias Clauss, Irina Petrache, Robert Voswinckel. Invention is credited to Matthias Clauss, Irina Petrache, Robert Voswinckel.
Application Number | 20140221607 14/124439 |
Document ID | / |
Family ID | 47296784 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140221607 |
Kind Code |
A1 |
Clauss; Matthias ; et
al. |
August 7, 2014 |
MONOCLONAL ANTIBODY AND ANTIGENS FOR DIAGNOSING AND TREATING LUNG
DISEASE AND INJURY
Abstract
The present invention provides methods for diagnosing a patient
with emphysema, COPD of lung injury caused by tobacco use by
detecting the levels of EMAP II in a sample. Disclosed herein are
the hypervariable regions for a rat monoclonal antibody that binds
to a form of EMAP II. This disclosure also includes a polypeptide
sequence included in EMAP II that is the target for the binding of
the antibody to its target protein. This epitope serves as the
basis for a humanized antibody that can be used to treat patients
that suffer from pathologies that exhibit elevated levels of EMAP
II expression.
Inventors: |
Clauss; Matthias;
(Indianapolis, IN) ; Petrache; Irina;
(Indianapolis, IN) ; Voswinckel; Robert; (Geissen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Clauss; Matthias
Petrache; Irina
Voswinckel; Robert |
Indianapolis
Indianapolis
Geissen |
IN
IN |
US
US
DE |
|
|
Assignee: |
Indiana University Research and
Technology Corp.
Indianapolis
IN
|
Family ID: |
47296784 |
Appl. No.: |
14/124439 |
Filed: |
June 8, 2012 |
PCT Filed: |
June 8, 2012 |
PCT NO: |
PCT/US2012/041722 |
371 Date: |
April 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61494720 |
Jun 8, 2011 |
|
|
|
Current U.S.
Class: |
530/324 ;
530/326; 530/387.3 |
Current CPC
Class: |
C07K 2317/76 20130101;
A61K 2039/505 20130101; C07K 16/2866 20130101; A61P 11/00 20180101;
C07K 16/18 20130101; C07K 2317/73 20130101; A61P 35/00 20180101;
C07K 16/22 20130101 |
Class at
Publication: |
530/324 ;
530/387.3; 530/326 |
International
Class: |
C07K 16/22 20060101
C07K016/22 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0002] This invention was made with government support under
HL090950 awarded by the National Institutes of Health. The U.S.
Government has certain rights in the invention.
Claims
1. An antibody, comprising: a heavy chain variable region, wherein
said heavy chain variable region includes at least a portion of a
first polypeptide according to SEQ. ID. NO. 2; and a light chain
variable region, wherein said light chain variable region includes
at least a portion of a second polypeptide according to SEQ. ID.
NO. 3, wherein said antibody is humanized and the humanized
antibody binds to human EMAPII.
2. The antibody according to claim 1, wherein said first
polypeptide has at least 99 percent homology to SEQ. ID. NO. 2, and
said second polypeptide has at least 99 percent homology to SEQ.
ID. NO. 3.
3. The antibody according to claim 1, wherein said first
polypeptide has at least 95 percent identity to SEQ. ID. NO. 2 and
said second polypeptide has at least 95 percent identity to SEQ.
ID. NO. 3.
4. The antibody according to claim 1, wherein said first
polypeptide has at least 99 percent identity to SEQ. ID. NO. 2 and
said second polypeptide has at least 99 percent identity to SEQ.
ID. NO. 3.
5. The antibody according to claim 1, wherein said first
polypeptide is SEQ. ID. NO. 2 and said second polypeptide is SEQ.
ID. NO. 3.
6. An antibody, comprising: a heavy chain, wherein said heavy chain
includes the heavy chain hypervariable regions CDR1, CDR2 and CDR3,
wherein CDR1 includes at least a portion of the polypeptide
according to SEQ. ID. NO. 5, CDR2 includes at least a portion of
the polypeptide according to SEQ. ID. NO. 6, and CDR3 includes at
least a portion of the polypeptide according to SEQ. ID. NO. 7; and
a light chain, wherein said light chain includes the light chain
hypervariable regions CDR1.sub.L, CDR2.sub.L, and CDR3.sub.L,
wherein CDR1.sub.L includes at least a portion of the polypeptide
according to SEQ. ID. NO. 8, CDR2.sub.L includes at least a portion
of the polypeptide according to SEQ. ID. NO. 9 and CDR3.sub.L
includes at least a portion of the polypeptide according to SEQ.
ID. NO. 10, wherein the heavy chain and the light chain form a
portion of a humanized antibody, that binds to human EMAPII.
7. The humanized antibody according to claim 6, wherein: CDR1 is
SEQ. ID. NO. 5, CDR2 is SEQ. ID. NO. 6, and CDR3 is SEQ. ID. NO. 7;
CDR1.sub.L his SEQ. ID. NO. 8, CDR2.sub.L is SEQ. ID. NO. 9, and
CDR3.sub.L is SEQ. ID. NO. 10.
8. An epitope, comprising: an epitope of human EMAP II, wherein
said epitope includes at least a portion of an isolated polypeptide
according to SEQ. ID. NO. 12.
9. The epitope, according to claim 8, wherein said isolated
polypeptide has at least 95 percent homology to SEQ. ID. NO.
12.
10. The epitope, according to claim 8, wherein said isolated
polypeptide has at least 99 percent homology to SEQ. ID. NO.
12.
11. The epitope, according to claim 8, wherein said isolated
polypeptide has at least 95 percent identity to SEQ. ID. NO.
12.
12. The epitope, according to claim 8, wherein said isolated
polypeptide has at least 99 percent identity to SEQ. ID. NO.
12.
13. The epitope, according to claim 8, wherein said isolated
polypeptide is SEQ. ID. NO. 12.
14. The epitope, according to claim 8, wherein said isolated
polypeptide has at least 95 percent identity to SEQ. ID. NO.
11.
15. The epitope, according to claim 8, wherein said isolated
polypeptide has at least 99 percent identity to SEQ. ID. NO.
11.
16. The epitope, according to claim 8, wherein said isolated
polypeptide is SEQ. ID. NO. 11.
17. A method of making an antibody, comprising the steps of:
producing a synthetic polypeptide wherein at least one portion of
the synthetic polypeptide includes at least a portion of the
polypeptide according to SEQ. ID. NO. 12.
18. The method according to claim 17, wherein said at least one
portion of the synthetic polypeptide has at least 95 percent
homology to SEQ. ID. NO. 12.
19. The method according to claim 17, wherein said at least one
portion of the synthetic polypeptide has at least 99 percent
homology to SEQ. ID. NO. 12.
20. The method according to claim 17, wherein said at least one
portion of the synthetic polypeptide has at least 95 percent
identity to SEQ. ID. NO. 12.
21-29. (canceled)
Description
PRIORITY CLAIM
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/494,720, filed on Jun. 8, 2011 and
incorporated herein by reference in its entirety.
FIELD
[0003] The present invention is directed generally to method for
diagnosing and treating a patient with emphysema or chronic
obstructive pulmonary disease (COPD), and more particularly to
methods for diagnosing and treating a patient with emphysema or
COPD by detecting the presence of endothelial monocyte activating
protein II (EMAP II) and neutralizing EMAP II action.
BACKGROUND
[0004] Over 3.1 million Americans have been diagnosed with
emphysema. Emphysema and chronic bronchitis are the two components
of the syndrome of COPD. COPD is the fourth leading cause of death
in America (See
www.nhlbi.nih.gov/health/public/lung/other/copd_fact.htm#toc). This
disease has no effective treatment that reverses its course or
halts its progression.
[0005] Pulmonary emphysema is a prevalent fatal disease,
characterized by loss of both matrix and cellular elements of the
lung, thus impairing gas exchange between the alveolar space and
the capillary blood. Emphysema is defined as "a condition of the
lung characterized by abnormal, permanent enlargement of airspaces
distal to the terminal bronchiole, accompanied by destruction of
their walls, with or without obvious fibrosis". Report of a
National Heart, Lung, and Blood Institute, Division of Lung
Diseases workshop, Am Rev Respir Dis 132, 182-185. (1985). The
concepts of permanent and destruction are critical in this
definition as they convey the unique and characteristic
distinguishing features of a disease process ultimately leading to
the disappearance of lung tissue.
[0006] Although the environmental inducers in susceptible
individuals have been identified, the mechanisms by which these
initiate a loss of alveoli leading to emphysema are poorly
understood. Over the past decades, inflammation and a
protease/antiprotease imbalance have been proposed to act as
downstream effectors of the lung destruction following chronic
cigarette smoking, which accounts for most cases of emphysema.
Pro-inflammatory stimuli are postulated to recruit and activate
lung inflammatory cells, triggering matrix protease release and
lung remodeling. Shapiro, S. D., J Clin Invest 106, 1309-1310
(2000). However, these models fail to fully account for the
mechanisms behind the eradication of septal structures and the
unique nature of lung destruction as compared to alterations seen
in other inflammatory lung diseases. To account for the permanent
destruction seen in emphysema, excessive apoptosis of structural
alveolar cells have emerged as a second major mechanism of
emphysema. Excessive alveolar endothelial apoptosis is thought to
cause capillary regression, with subsequent loss of alveolar wall.
Tuder, R. M. et al., Am J Respir Cell Mol Biol 28, 551-554 (2003).
However, the coexistence of an excessive lung structural cell
apoptosis with that of an activated inflammatory state in emphysema
and the hierarchy of these two mechanisms have not yet been
explained.
[0007] As can be seen, there is a need for a method for treating
pulmonary emphysema. There is also a need for a method for
diagnosing pulmonary emphysema in the early stages. Early diagnosis
and subsequent treatment may result in more effective treatment of
the disease and a better prognosis for the patient.
SUMMARY
[0008] In one aspect of the present invention there is provided a
method of diagnosing a patient for emphysema or COPD comprising
detecting the overexpression of EMAP II in a patient's biological
sample where the sample may be serum, plasma, lung lavage or lung
biopsy. The EMAP II may be detected by immunological methods such
as enzyme-linked immunosorbent assay (ELISA), sandwich ELISA,
Western blot, or mass spectrometry, for example. The overexpression
of EMAP II may be determined by comparing to a control sample.
[0009] In another aspect of the present invention there is provided
a method of predicting a patient's susceptibility of developing
emphysema or COPD by detecting the presence of EMAP II in a
patient's sample.
[0010] In a further aspect of the present invention there is
provided a method for treating a patient having emphysema or COPD
comprising administering a therapeutically effective amount of an
EMAP II neutralizing compound. The EMAP II neutralizing compound
may be an antibody, an agonist of the CXCR3 receptor, an siRNA or
antisense RNA. The EMAP II neutralizing compound may be
administered systemically or by inhalation.
[0011] Aspects of the invention include antibodies, either
humanized or not-humanized, comprising: a heavy chain variable
region, wherein said heavy chain variable region includes at least
a portion of a first polypeptide according to SEQ. ID. NO. 2; and a
light chain variable region, wherein said light chain variable
region includes at least a portion of a second polypeptide
according to SEQ. ID. NO. 3, wherein the antibodies bind to at
least one form of EMAPII. In some embodiments the first polypeptide
has at least 99 percent homology to SEQ. ID. NO. 2, and said second
polypeptide has at least 99 percent homology to SEQ. ID. NO. 3. In
other embodiments, the first polypeptide has at least 95 percent
identity to SEQ. ID. NO. 2, and said second polypeptide has at
least 95 percent identity to SEQ. ID. NO. 3. In other embodiments
the first polypeptide has at least 99 percent identity to SEQ. ID.
NO. 2, and said second polypeptide has at least 99 percent identity
to SEQ. ID. NO. 3. And in still other embodiments, the first
polypeptide is SEQ. ID. NO. 2, and said second polypeptide is SEQ.
ID. NO. 3. In some embodiments the antibodies bind to at least the
pro form of EMAPII (pro-EMAPII), and in some embodiments the
antibodies bind to EMAPII found in humans and/or in mice and/or in
other mammals.
[0012] Some aspects of the invention include antibodies,
comprising: a heavy chain, wherein said heavy chain includes the
heavy chain hypervariable regions CDR1, CDR2 and CDR3, wherein CDR1
includes at least a portion of the polypeptide according to SEQ.
ID. NO. 5, CDR2 includes at least a portion of the polypeptide
according to SEQ. ID. NO. 6, and CDR3 includes at least a portion
of the polypeptide according to SEQ. ID. NO. 7; and a light chain,
wherein said light chain includes the light chain hypervariable
regions CDR1.sub.L, CDR2.sub.L, and CDR3.sub.L, wherein CDR1.sub.L
includes at least a portion of the polypeptide according to SEQ.
ID. NO. 8, CDR2.sub.L includes at least a portion of the
polypeptide according to SEQ. ID. NO. 9 and CDR3 L includes at
least a portion of the polypeptide according to SEQ. ID. NO. 10,
wherein the heavy chain and the light chain form a portion of a
humanized antibody, that binds to human EMAPII. In some
embodiments, CDR1 is SEQ. ID. NO. 5, CDR2 is SEQ. ID. NO. 6, and
CDR3 is SEQ. ID. NO. 7; and CDR1.sub.L is SEQ. ID. NO. 8,
CDR2.sub.L is SEQ. ID. NO. 9, and CDR3.sub.L is SEQ. ID. NO. 10. In
some embodiments the antibodies bind to at least the pro form of
EMAPII (pro-EMAPII), and in some embodiments the antibodies bind to
EMAPII found in humans and/or in mice and/or in other mammals. In
some embodiments the antibodies are humanized.
[0013] Some aspects of the invention include epitopes, or other
antigenic portions of EMAPII, that give rise to antibodies that
bind to at least one form of mammalian EMAPII, comprising: an
epitope of human EMAP II, wherein the epitope includes at least a
portion of an isolated polypeptide according to SEQ. ID. NO. 12. In
some embodiments, the isolated polypeptide has at least 95 percent
homology to SEQ. ID. NO. 12. In still other embodiments, the
isolated polypeptide has at least 99 percent homology to SEQ. ID.
NO. 12. In yet other embodiments, the isolated polypeptide has at
least 95 percent identity to SEQ. ID. NO. 12, while in some
embodiments, the isolated polypeptide has at least 99 percent
identity to SEQ. ID. NO. 12. In some embodiments, the isolated
polypeptide is SEQ. ID. NO. 12. In some embodiments, the isolated
polypeptide has at least 95 percent identity to SEQ. ID. NO. 11. In
still other embodiments, the isolated polypeptide has at least 99
percent identity to SEQ. ID. NO. 11. In some embodiments, the
isolated polypeptide is SEQ. ID. NO. 11. Some embodiments include
these epitopes, or portions thereof, attached to at least one other
polypeptide. Such co-joined polypeptides may not be naturally
occurring, at least not in the organism that is expressing the
polypeptide.
[0014] Some aspects of the invention include methods for making
antibodies that bind to at least one form EMAPII found in either
humans or in other mammals, these methods may comprise the steps
of: producing a synthetic polypeptide wherein at least one portion
of the synthetic polypeptide includes at least a portion of the
polypeptide according to SEQ. ID. NO. 12. In some embodiments, the
at least one portion of the synthetic polypeptide has at least 95
percent homology to SEQ. ID. NO. 12. In still other embodiments,
that at least one portion of the synthetic polypeptide has at least
99 percent homology to SEQ. ID. NO. 12. In yet other embodiments,
that at least one portion of the synthetic polypeptide has at least
95 percent identity to SEQ. ID. NO. 12. In some embodiments, the at
least one portion of the synthetic polypeptide has at least 99
percent identity to SEQ. ID. NO. 12. In still other embodiments,
the at least one portion of the synthetic polypeptide is SEQ. ID.
NO. 12. In some embodiments, the at least one portion of the
synthetic polypeptide has at least 95 percent homology to SEQ. ID.
NO. 11. In other embodiments, the at least one portion of the
synthetic polypeptide has at least 99 percent homology to SEQ. ID.
NO. 11. In still other embodiments, the at least one portion of the
synthetic polypeptide has at least 95 percent identity to SEQ. ID.
NO. 11. In yet other embodiments, the at least one portion of the
synthetic polypeptide has at least 99 percent identity to SEQ. ID.
NO. 11. In some embodiments, at least one portion of the synthetic
polypeptide is SEQ. ID. NO. 11. The inventive methods may include
the step of contacting a synthetic polypeptide that includes at
least one portion of at least one epitope of EMAPII disclosed
herein with the immune system of a mammal. Some methods may include
the further step of selecting a B-cell from said mammal contacted
with said synthetic polypeptide, wherein said B-cell produces
antibody that binds with high affinity to EMAPII. And in some
embodiments, the antibodies raised to the epitopes disclose herein
are humanized. In some embodiments, the humanized antibodies are
used to treat a lung related disease or injury in humans and/or
other mammals, or to diagnose such conditions in humans and/or
other animals.
[0015] These and other features, aspects and advantages of the
present invention will become better understood with reference to
the following drawings, description and claims.
TABLE-US-00001 SEQUENCE LISTING SEQ. ID. NO. 1
GCGGTGCACCTTGTTGAGTCTGGTGGAGGATTTGT Nucleotide sequence
GCAGCCTACGGAGTCATTGAAAATCTCATGTGCA of the IgG heavy
GCCTCTGGATTCACCTTCAGTGATGCTGCCATGTA chain from rat
CTGGGTCCGCCAGGCTCCAGGAAAGGGTCTGGAA antibody hybridoma
TGGGTTGCTCGCATAAGAACTAAACCTAATAATT clone M7/1.
ATGCAACATATTATGCTGATTCAGTGAAAGGCAG
ATTCACCATCTCCCGAGATGATTCAAAAAGCATG
GTCTACCTACAAATGGATAACTTGAAAACTGAGG
ACACAGCCATGTATTACTGTACATCATGGAGCTA
CGACTTTGATTACTGGGGCCAAGGAGTCATGGTC ACAGTCTCCTCA SEQ. ID. NO. 2
AVHLVESGGGFVQPTESLKISCAASGFTFSDAAMY Polypeptide sequence
WVRQAPGKGLEWVARIRTKPNNYATYYADSVKGR of the IgG heavy
FTISRDDSKSMVYLQMDNLKTEDTAMYYCTSWSY chain from rat DFDYWGQGVMVTVSS
antibody hybridoma clone M7/1. SEQ. ID. NO. 3
DIVMTQGALPNPVPSGESASITCQSSKSLLHSSGKTY Polypeptide sequence
LNWYLQRPGQSPHLLIYWMSTRASGVSDRLSGSGS of the IgG light chain
GTDFTLKISSVEAEDVGVYYCQQFLEYPLTFGSGTK from rat antibody LEIK
hybridoma clone M7/1. SEQ. ID. NO. 4
GATATTGTGATGACCCAGGGTGCACTCCCCAACC Nucleotide sequence
CTGTCCCCTCTGGAGAGTCAGCTTCCATCACCTGC of the IgG light chain
CAGTCTAGTAAGAGTCTGCTGCACAGCAGTGGCA from rat antibody
AGACATACTTGAATTGGTATCTGCAGAGGCCAGG hybridoma clone
ACAGTCTCCTCATCTCCTGATCTATTGGATGTCCA M7/1.
CCCGTGCATCAGGAGTCTCAGACAGGCTCAGTGG
CAGTGGGTCAGGAACAGATTTCACACTGAAAATC
AGCAGCGTGGAGGCTGAGGATGTGGGTGTGTATT
ACTGTCAGCAATTTCTAGAGTATCCTCTCACGTTC GGTTCTGGGACCAAGCTGGAGATCAAAC
SEQ. ID. NO. 5 GFTFSDAA Polypeptide CDR1 from IgG heavy chain of
rat antibody hybridoma clone M7/1. SEQ. ID. NO. 6 IRTKPNNYAT
Polypeptide CDR2 from IgG heavy chain of rat antibody hybridoma
clone M7/1. SEQ. ID. NO. 7 TSWSYDFDY Polypeptide CDR3 from IgG
heavy chain of rat antibody hybridoma clone M7/1. SEQ. ID. NO. 8
KSLLHSSGKTY Polypeptide CDR1 from IgG light chain of rat antibody
hybridoma clone M7/1. SEQ. ID. NO. 9 WMS Polypeptide CDR2 from IgG
light chain of rat antibody hybridoma clone M7/1. SEQ. ID. NO. 10
QQFLEYPLT Polypeptide CDR3 from IgG light chain of rat antibody
hybridoma clone M7/1. SEQ. ID. NO. 11
QQSIAGSADSKPIDVSRLDLRIGCIITARKHPDADSLY Polypeptide sequence
VEEVDVGEIAPRTVVSGLVNHVPLEQMQNRM identified in human EMAPII as the
portion of the protein that is protected from trypsin digestion by
the binding of rat antibody hybridoma clone M7/1. SEQ. ID. NO. 12
QQSIAGSADSKPIDVSR Polypeptide sequence from human EMAPII that
interacts with rat antibody hybridoma clone M7/1. SEQ. ID. NO. 13
KHPDADSLYVEEVDVGE Polypeptide sequence from human EMAPII that does
not appear to interact strongly with rat antibody hybridoma clone
M7/1. SEQ. ID. NO. 14 VLKRLEQKGAEADQIIE Random, synthetic
polypeptide sequence that does not interact with rat antibody
hybridoma clone M7/1. SEQ. ID. NO. 15
MLPAVAVSEPVVLRFMIFCRLLAKMANNDAVLKRL Polypeptide sequence
EQKGAEADQIIEYLKQQVSLLKEKAILQATLREEKK of human EMAPII.
LRVENAKLKKEIEELKQELIQAEIQNGVKQIPFPSGT
PLHANSMVSENVIQSTAVTTVSSGTKEQIKGGTGDE
KKAKEKIEKKGEKKEKKQQSIAGSADSKPIDVSRLD
LRIGCIITARKHPDADSLYVEEVDVGEIAPRTVVSGL
VNHVPLEQMQNRMVILLCNLKPAKMRGVLSQAMV
MCASSPEKIEILAPPNGSVPGDRITFDAFPGEPDKELN
PKKKIWEQIQPDLHTNDECVATYKGVPFEVKGKGV CRAQTMSNSGIK
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. A bar graph illustrating an increase in secreted
EMAP II expression in humans in the broncho-alveolar lavage (BAL)
of smokers compared to non-smokers.
[0017] FIG. 2A. A bar graph showing the effect of cigarette smoke
(CS) exposure on the activity levels of caspase-3 in mouse
lungs.
[0018] FIG. 2B. A bar graph showing the effect of cigarette smoke
on the levels of pro-apoptotic ceramide levels in mouse lungs.
[0019] FIG. 2C. A bar graph of the alveolar size in mice exposed to
cigarette smoke for 6 months.
[0020] FIG. 3A. A bar graph that illustrates the effect of
cigarette smoke exposure on the levels of EMAP II expression.
[0021] FIG. 3B. A Western blot showing the kinetics of EMAP II
secretion in BAL from mice exposed to cigarette smoke (CS) or air
(AC).
[0022] FIG. 3C. A Western blot showing VEGF receptor inhibition
with SU5416.
[0023] FIG. 4A. A bar graph and Western blot that illustrates the
effect of cigarette smoke exposure on EMAP II levels in lung
lysates.
[0024] FIG. 4B. Photomicrographs that show the effect of cigarette
smoke exposure on the amount of inflammatory cells in lung
tissue.
[0025] FIG. 5A. A Western blot showing the induction of EMAP II in
mice after 24 hours of tetracycline treatment.
[0026] FIG. 5B. Photomicrographs of a lung section showing the
alveolar after tetracycline treatment for 3 months.
[0027] FIG. 5C. A bar graph showing the mean linear intercept of
lung tissue of mice treated with tetracycline for 3 months and
controls.
[0028] FIG. 5D. Photomicrographs of a lung section showing the
alveolar after tetracycline treatment for 6 months.
[0029] FIG. 5E. A bar graph showing the volume weighted mean volume
of lung tissue of mice treated with tetracycline for 6 months and
controls.
[0030] FIG. 6A. A graph showing the caspase-3 activity in lung
lysates of single or EMAP II double transgenic mice after 3
months.
[0031] FIG. 6B. A graph showing caspase-3 activity in lung lysates
from single or EMAP II double transgenic mice after 6 months.
[0032] FIG. 6C. A graph showing caspase-3 activity in lungs of
single or EMAP II double transgenic mice treated with nonspecific
control IgG and neutralizing EMAP II antibody.
[0033] FIG. 7A. A bar graph showing the number of cells in the
lungs of mice overexpressing EMAP II compared to a control.
[0034] FIG. 7B. A bar graph showing the quantification MMP-9- and
TNF.alpha.-positive cells.
[0035] FIG. 8A. A bar graph showing the effect of EMAP II
overexpression on caspase-3 activity.
[0036] FIG. 8B. A graph showing the effect of treatment of lung
microvascular endothelial cells with recombinant proteins
comprising the pro- and mature isoforms of EMAP II on
apoptosis.
[0037] FIG. 8C. A bar graph showing the expression levels of CXCR3
in cells cultured with low serum.
[0038] FIG. 8D. A bar graph showing the expression levels of CXCR3
in cells treated with acellular BAL from mice exposed to cigarette
smoke (CS) or air (AC).
[0039] FIG. 8E. A bar graph showing the effect of anti-CXCR3
antibody on caspase-3 activity.
[0040] FIG. 9. A bar graph showing the effect of CXCR3-targeting
siRNA on CXCR3 expression.
[0041] FIG. 10A. An immunoblot showing the effect of cigarette
smoke exposure on EMAP II expression in the mouse lung.
[0042] FIG. 10B. A bar graph showing EMAP II expression in the lung
parenchyma of DBA2 mice exposed to cigarette smoke for 4 weeks.
[0043] FIG. 10C. An immunoblot showing lung EMAP II expression in a
mouse model of apoptosis-dependent emphysema.
[0044] FIG. 10D. A bar graph showing lung macrophage accumulation
in pulmonary parenchyma in response to cigarette smoke
exposure.
[0045] FIG. 10E. A bar graph showing lung apoptosis as measured by
caspase-3 activity assay in lung lysates following cigarette smoke
exposure.
[0046] FIG. 11A. Fluorescent microscope images showing inhibition
of EMAP II-induced apoptosis in endothelial cells with neutralizing
antibody M 7/1 compared to control rat IgG.
[0047] FIG. 11B. A bar graph showing the ratio of apoptotic cells
to total cells for pro-EMAPII with neutralizing antibody M 7/1
compared to control rat IgG.
[0048] FIG. 11C. A bar graph showing the ratio of apoptotic cells
to total cells for mature EMAPII with neutralizing antibody M 7/1
compared to control rat IgG.
[0049] FIG. 12A. Graph of EMAPII (pro and mature forms) in lung
lysates from mice exposed to Cigarette Smoke (CS) for 4 weeks
compared with EMAPII levels in the lungs of mice that were not
exposed to CS (ambient air control group, AC); EMAPII levels were
assessed by Western blots (mean densitometry units [DUs] normalized
to vinculin.+-.SEM; *P<0.05 versus control; n=5/group).
[0050] FIG. 12B. Photomicrographs of mouse lung tissue stained for
EMAPII; tissue from mice exposed to CS and from exposed to ambient
air (AC).
[0051] FIG. 12C. Schematic representation of treatment
protocol.
[0052] FIG. 12D. Graph showing apoptosis detected by caspase-3
activity measured in lung lysates (caspase unites normalized by
protein; mean.+-.SEM; *P<0.05, ANOVA).
[0053] FIG. 12E. Graph showing the number of cells in BALF.
[0054] FIG. 12F. Graph showing lung static compliance (mean.+-.SEM;
*P<0.01, ANOVA).
[0055] FIG. 12G. Representative H&E-stained lung sections
(scale bar: 100 .mu.m) showing simplification of lung alveolar
structures in response to CS but perseved alveolar architecture
when treated with neutralizing EMAPII,
[0056] FIG. 12H. Morphometric measurement of MLI (mean.+-.SEM:
*P<0.05, ANOVA: n=5-12.
[0057] FIG. 13. Agarose gel showing PCR amplification products.
[0058] FIG. 14. Summary of results from sequences of rat
antibody.
[0059] FIG. 15. Sequence data for the variable regions of the rat
antibody.
[0060] FIG. 16. Scheme of EMAP II protein sequence. A range, which
is protected from proteolytic degradation by binding to M7/1
antibody is highlighted.
[0061] FIG. 17. Binding competition of one peptide out of the
protected area which is capable of competing with M7/1 antibody.
Recombinant pro-EMAP II was submitted to Western blotting using
control IgG and EMAP II neutralizing M7/1 antibody in the
presence/absence of a 300 fold molar excess of peptide
hexadecamers. Only Peptide 2 (QQSIAGSADSKPIDVSR) but not Peptide 1
(KHPDADSLYVEEVDVGE) or Peptide 3 (as a control) was able to compete
with M7/1. Arrows indicate the position of molecular weight
standards (in rel kDa).
DESCRIPTION
[0062] The following detailed description is of the best currently
contemplated modes of carrying out the invention. The description
is not to be taken in a limiting sense, but is made merely for the
purpose of illustrating the general principles of the invention,
since the scope of the invention is best defined by the appended
claims. As used herein, unless explicitly stated otherwise or
clearly implied otherwise, the term `about` refers to a range of
values plus or minus 10 percent, e.g. about 1.0 encompasses values
from 0.9 to 1.1.
[0063] As used herein, unless explicitly stated otherwise or
clearly implied otherwise, the terms `therapeutically effective
dose,` `therapeutically effective amounts,` and the like, refer to
a portion of a compound that has a net positive effect on the
health and well being of a human or other animal. Therapeutic
effects may include an improvement in longevity, quality of life
and the like, and may also include a reduced susceptibility to
developing disease or deteriorating health or well being. The
effects may be immediate realized after a single dose and/or
treatment or they may be cumulative and realized after a series of
doses and/or treatments.
[0064] As used herein, unless explicitly stated otherwise or
clearly implied otherwise, the term `homology` as applied to
polynucleotides refers to 3 nucleic acid long Condons that, while
not identical to one another, encode the same information when
transcribed into proteins. For a further discussion of this term as
it is used in regards to polynucleotides, please see, Elliot and
Elliot, Biochemistry and Molecular Biology, pages 293-295,
published in 1997 by Oxford University Press, New York, N.Y., this
portion of which is hereby incorporated herein by reference in its
entirety.
[0065] As used herein, unless explicitly stated otherwise or
clearly implied otherwise, the term `homology` as applied to
polypeptides refers to amino acids commonly found in living
organisms that are considered to be similar to one another in size,
structure, and chemical reactivity. For a further discussion of
this term as it is used in regards to polypeptides, please see,
Stryer, L., Biochemistry, 2.sup.nd edition, pages 13-17, copyright
1981, published by W. H. Freeman and Company, San Francisco,
Calif., this portion of which is hereby incorporated herein by
reference in its entirety.
[0066] Broadly, the present invention provides methods for
diagnosing or treating a patient with emphysema or COPD comprising
detecting the presence of EMAP II in a biological sample from a
patient or treating with a therapeutically effective amount of an
EMAP II neutralizing compound. The same method may also be used to
determine if a patient is susceptible to developing emphysema or
COPD. EMAP II is a cytokine induced by conditions present in
emphysematous lungs including oxidative, apoptotic, and hypoxic
cellular stresses. EMAP II is released from cells as either a 43 kD
pro-form or a 23 kDa "mature" protein upon proteolytic cleavage by
proteases including caspases and matrix metalloproteinases (MMPs),
which are known to participate in COPD. Given the potent
pro-apoptotic effect of EMAP II on lung endothelial cells, coupled
with its ability to recruit pro-inflammatory monocytes, excessive
EMAP II release in response to cigarette smoking may engage both
lung endothelial cell apoptosis and accumulation of lung
macrophages, and therefore may be a key molecular mediator of
pulmonary emphysema. It has now been discovered by the inventors
that smoke-induced emphysema is preceded by robust EMAP II
production and apoptosis in mice and that lung-specific increases
in EMAP II are sufficient to cause lung apoptosis and emphysema.
Moreover, increased levels of EMAP II have now been measured in the
lungs of emphysema patients and EMAP II has been found to be
robustly upregulated in the BAL of smokers (FIG. 1). Therefore,
EMAP II may be a biomarker for emphysema and COPD, allowing for
earlier detection and treatment of these conditions.
[0067] In one embodiment a method is provided for diagnosing
whether or not a patient has emphysema or COPD where the method may
comprise the step of detecting EMAP II in a biological sample from
a patient. It has been found that expression of EMAP II is
significantly elevated by at least 2-fold in samples from patients
who have emphysema or COPD. The method may further comprise
comparing the EMAP II detected in the patient's sample with a
control and diagnosing the patient as either having emphysema or
COPD. The control may be a sample from a patient who does not have
emphysema or COPD and, more specifically, from a patient who does
not smoke. Control levels of EMAP II may be defined by a number of
samples from control patients wherein the expression levels of EMAP
II. It will be appreciated that the more control samples available,
the better the comparison. The comparison may be a visual
comparison observing elevated EMAP II levels or the amount of EMAP
II in the sample and/or control may be quantified and then
compared.
[0068] In one embodiment, the biological sample may be serum,
plasma, BAL, or lung biopsy. Obtaining such samples is routine in
the art. The overexpression of EMAP II in a biological sample may
be assessed at the protein or nucleic acid level. In an
illustrative embodiment, immunocytochemistry techniques are
provided that utilize antibodies to detect the overexpression EMAP
II in biological samples. In this aspect of the invention, at least
one antibody directed to EMAP II may be used. Overexpression of
EMAP II may also be detected by nucleic acid-based techniques,
including, for example, hybridization and RT-PCR. Kits comprising
reagents for practicing the methods of the invention are further
provided.
[0069] Methods for detecting EMAP II may comprise any methods that
determine the quantity or the presence of EMAP II either at the
nucleic acid or protein level. Such methods are well known in the
art and include but are not limited to Western blots, northern
blots, southern blots, ELISA, immunoprecipitation,
immunofluorescence, flow cytometry, immunocytochemistry, nucleic
acid hybridization techniques, nucleic acid reverse transcription
methods, and nucleic acid amplification methods. In illustrative
embodiments, overexpression of EMAP II may be detected on a protein
level using, for example, antibodies that are directed against
specific biomarker proteins. The antibodies may be, but are not
limited to, polyclonal and monoclonal antibodies. Examples of
monoclonal antibodies are provided herein as well as in U.S. Pat.
No. 5,641,867, which is incorporated by reference herein. These
antibodies can be used in various methods such as Western blot,
ELISA, immunoprecipitation, or immunocytochemistry techniques.
[0070] In one embodiment, EMAP II overexpression may be determined
on the protein level. Antibodies specific for EMAP II may be
utilized to detect the overexpression of a biomarker protein in a
body sample. The method comprises obtaining a body sample from a
patient, contacting the body sample with at least one antibody
directed to EMAP II, and detecting antibody binding to determine if
EMAP II is overexpressed in the patient sample. Overexpression of
EMAP II may be determined by comparing the results to a control
sample.
[0071] In an alternate embodiment, EMAP II overexpression may be
detected at the nucleic acid level. Nucleic acid-based techniques
for assessing expression are well known in the art and include, for
example, determining the level of biomarker mRNA in a body sample.
Many expression detection methods use isolated RNA. Any RNA
isolation technique that does not select against the isolation of
mRNA can be utilized for the purification of RNA from cervical
cells (see, e.g., Ausubel et al., ed., Current Protocols in
Molecular Biology, John Wiley & Sons, New York, 1987-1999).
Additionally, large numbers of tissue samples can readily be
processed using techniques well known to those of skill in the art,
such as, for example, the single-step RNA isolation process of U.S.
Pat. No. 4,843,155, which is incorporated by reference herein.
[0072] Isolated mRNA may be used in hybridization or amplification
assays that include, but are not limited to, Southern or Northern
analyses, polymerase chain reaction analyses and probe arrays. One
method for the detection of mRNA levels involves contacting the
isolated mRNA with a nucleic acid molecule (probe) that can
hybridize to the mRNA encoded by the gene being detected. The
nucleic acid probe may be, for example, a full-length cDNA, or a
portion thereof, such as an oligonucleotide of at least 7, 15, 30,
50, 100, 250 or 500 nucleotides in length and sufficient to
specifically hybridize under stringent conditions to an mRNA or
genomic DNA encoding a biomarker of the present invention. The
polynucleotide sequence of EMAP II is known in the art (i.e., U.S.
Pat. No. 6,013,483, which is incorporated by reference herein), and
nucleic acid probes may be selected without undue experimentation.
Hybridization of an mRNA with the probe indicates that the
biomarker in question is being expressed.
[0073] In another embodiment, methods are provided for determining
a patient's susceptibility to developing emphysema or COPD.
Although no symptoms may be present, those who smoke or were
habitual smokers in the past have a significantly higher risk of
developing emphysema than those who never smoked. Therefore, it may
be desirable to determine the susceptibility of a patient who is a
smoker to develop emphysema. Early detection may lead to a better
treatment regime. The method may comprise the step of detecting
EMAP II in a patient's sample as described above. The method may
further comprise comparing the EMAP II in the patient's sample with
a control as described above.
[0074] In yet another embodiment, kits for practicing the methods
of the present invention are further provided. The kit may comprise
at least one reagent (e.g., an antibody, a nucleic acid probe,
etc.) for specifically detecting the expression of EMAP II. The
kits may also comprise positive and/or negative controls to
validate the activity and correct usage of reagents employed in
accordance with the invention. Controls may include biological
samples, such as lung tissue or lung lavage samples from control
patients (negative control). EMAP II may be added to the control
samples to provide positive controls.
[0075] In a further embodiment, methods are provided for treating a
patient having emphysema or COPD comprising the step of
administering a therapeutically effective amount of at least one
EMAP II neutralizing compound. The neutralizing compound may be any
compound or molecule that decreases or inhibits the activity or
action of EMAP II in the patient. In one embodiment the
neutralizing compound may be an anti-EMAP II antibody where the
antibody may be a polyclonal or monoclonal antibody, antibody
fragments, humanized or chimeric antibodies that retain the
combining region that specifically binds to EMAP II.
[0076] In an alternate embodiment, the neutralizing compound may be
an agonist of the CXCR3 receptor. The agonist may be a peptide,
peptidomimetic or any other compound that disrupts the interaction
between EMAP II and the CXCR3 receptor. In an illustrative
embodiment, the neutralizing compound is an EMAP II analog.
Interruption of the binding of EMAP II to CXCR3 may interfere with
the detrimental action of EMAP II in lung tissue.
[0077] In yet another embodiment, the neutralizing compound may be
a compound or molecule that decreases the expression of EMAP II.
Non-limiting examples may be siRNA or antisense RNA targeted to
EMAP II RNA or DNA. Alternatively, the neutralizing compound may be
a compound or molecule such as, but not limited to, siRNA or
antisense RNA, that interferes and decreases the expression of
CXCR3. As shown in FIG. 9, when human lung microvascular
endothelial cells were electroporated in the presence of
CXCR3-targeting siRNA, CXCR3 expression levels showed reductions of
about 60% to about 80%. As the nucleotide sequences are known for
both EMAP II and CXCR3, one skilled in the art would be able to
select siRNA and/or antisense RNA sequences for EMAP II and/or
CXCR3 without undue experimentation. Examples of compounds and
compositions for modulating the expression of EMAP II are disclosed
in U.S. Patent Application Publication No. 2004/0110114 and U.S.
Pat. No. 5,665,593, both of which are expressly incorporated by
reference herein.
[0078] In one embodiment, protocols for the administration of the
EMAP II neutralizing compounds are similar to the protocols for the
administration of any other agent typically administered for a lung
disorder. As a general guideline, protocols developed for the
administration of any agent for the treatment of lung disease form
a starting point for the administration of the EMAP II neutralizing
compounds of the present invention. Thus, the EMAP II neutralizing
compounds and compositions are administered via an inhalant or any
other mechanism by which a disorder such as asthma is treated. In
one embodiment of the invention, the active compounds or
pharmaceutical formulations of the invention are administered
directly to the lungs of the subject by any suitable means, but are
preferably administered by administering an aerosol suspension of
respirable particles comprised of the active compound, which the
subject inhales. The active compound can be aerosolized in a
variety of forms, such as, but not limited to, dry powder
inhalants, metered dose inhalants, or liquid/liquid suspensions.
The respirable particles may be liquid or solid. Alternatively,
EMAP II neutralizing compounds may be administered systemically,
either intravenously or through other means known in the art.
[0079] Any of the protocols, formulations, routes of administration
and the like that have previously been used in the treatment of
lung disorders may readily be modified for use in the present
invention. In some cases, mechanical ventilation is appropriate.
Such ventilation may include high-frequency oscillatory ventilation
(HFOV) or other unconventional forms of mechanical ventilation.
Theoretically, partial liquid ventilation (PLV) offers the
advantage of lung lavage combined with ventilator support.
[0080] In another embodiment, the dosages are determined using an
animal model, such as the EMAP II double transgenic models known to
those of skill in the art, and modified and adapted to use in
higher mammals. The total dose of therapeutic agent is administered
in multiple doses or in a single dose. In certain embodiments, the
compositions are administered alone, and in other embodiments the
compositions are administered in conjunction with other
therapeutics directed to the disease or directed to other symptoms
thereof.
[0081] Regardless of the route of administration of the active
compounds or formulations of the invention, the therapeutically
effective dosage of any one active compound, the use of which is in
the scope of present invention, will vary somewhat from compound to
compound, and patient to patient, and will depend upon factors such
as the age, weight and condition of the patient, and the route of
delivery. Such dosages can be determined in accordance with routine
pharmacological procedures known to those skilled in the art. In
one exemplary embodiment, a dosage from about 0.1 to about 50 mg/kg
will have therapeutic efficacy, with all weights being calculated
based upon the weight of the active compound. Toxicity concerns at
the higher level may restrict intravenous dosages to a lower level
such as up to about 10 mg/kg. A dosage from about 10 mg/kg to about
50 mg/kg may be employed for oral administration. Typically, a
dosage from about 0.5 mg/kg to 5 mg/kg may be employed for
intramuscular injection. Preferred dosages are 1 .mu.mol/kg to 50
.mu.mol/kg, and more preferably 22 .mu.mol/kg and 33 .mu.mol/kg of
the compound for intravenous or oral administration.
[0082] In another exemplary embodiment, dosages of the compounds of
the present invention, for antisense oligonucleotides the dosage is
preferably one which produces intracellular concentrations of the
oligonucleotide of from 0.05 to 50 .mu.M. Typically the dosage to a
human will be from about 0.01, 0.1 or 1 mg/Kg up to 50, 100, or 150
mg/Kg. In an additional example, for antibodies the dosage is
typically 0.01, 0.05 or 0.1 mg/Kg up to 20, 40 or 60 mg/Kg.
[0083] When administration of the active compounds or
pharmaceutical formulations is via inhalation, the dosage of active
compound will also vary depending on the condition being treated
and the state of the subject, but generally may be an amount
sufficient to achieve dissolved concentrations of active compound
on the airway surfaces of the subject of from about 10.sup.-9 to
about 10.sup.-1 Moles/liter, and more preferably from about
10.sup.-6 to about 10.sup.-4 Moles/liter.
[0084] Methods of formulating antibodies, peptides or other
compounds for therapeutic administration are known to those of
skill in the art. Methods of formulating siRNA or antisense RNA are
also known in the art. Administration of these compositions
according to the present invention will be via any common route so
long as the target tissue is available via that route. Most
commonly, these compositions are formulated for oral
administration, such as by an inhalant. However, other conventional
routes of administration (e.g., by subcutaneous, intravenous,
intradermal, intramusclar, intramammary, intraperitoneal,
intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term
release), aerosol, sublingual, nasal, anal, vaginal, or transdermal
delivery, or by surgical implantation at a particular site) are
also used, particularly when oral administration is problematic.
The treatment may consist of a single dose or a plurality of doses
over a period of time.
[0085] It will be appreciated by those skilled in the art that the
compounds of the present invention can be employed in a wide
variety of pharmaceutical forms; the compound can be employed neat
or admixed with a pharmaceutically acceptable carrier or other
excipients or additives. Generally speaking, the compound will be
administered orally or intravenously. It will be appreciated that
therapeutically acceptable salts of the compounds of the present
invention may also be employed. The selection of dosage,
rate/frequency and means of administration is well within the skill
of the artisan and may be left to the judgment of the treating
physician. The method of the present invention may be employed
alone or in conjunction with other therapeutic regimens.
[0086] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as inhalents, injectable
solutions, drug release capsules and the like. For parenteral
administration in an aqueous solution, for example, the solution is
suitably buffered if necessary and the liquid diluent first
rendered isotonic with sufficient saline or glucose. These
particular aqueous solutions are especially suitable for
intravenous, intramuscular, subcutaneous and intraperitoneal
administration.
[0087] The frequency of dosing will depend on the pharmacokinetic
parameters of the agents and the routes of administration. The
optimal pharmaceutical formulation will be determined by one of
skill in the art depending on the route of administration and the
desired dosage. Such formulations may influence the physical state,
stability, rate of in vivo release and rate of in vivo clearance of
the administered agents. Depending on the route of administration,
a suitable dose is calculated according to body weight, body
surface areas or organ size. The availability of animal models is
particularly useful in facilitating a determination of appropriate
dosages of a given therapeutic. Further refinement of the
calculations necessary to determine the appropriate treatment dose
is routinely made by those of ordinary skill in the art without
undue experimentation, especially in light of the dosage
information and assays disclosed herein as well as the
pharmacokinetic data observed in animals or human clinical
trials.
[0088] Typically, appropriate dosages are ascertained through the
use of established assays for determining blood levels in
conjunction with relevant dose response data. The final dosage
regimen will be determined by the attending physician, considering
factors which modify the action of drugs (e.g., the drug's specific
activity, severity of the damage and the responsiveness of the
patient, the age, condition, bodyweight, sex and diet of the
patient, the severity of any infection, time of administration and
other clinical factors). As studies are conducted, further
information will emerge regarding appropriate dosage levels and
duration of treatment for specific diseases and conditions.
[0089] In one embodiment of the present invention methods are
provided for monitoring the effectiveness of treatment of a patient
for emphysema and/or COPD and undergoing treatment by determining
the expression levels of EMAP II. The method may comprise the step
of detecting EMAP II in a patient's sample as described above. The
method may further comprise comparing the EMAP II in the patient's
sample with a control as described above. Alternatively, the EMAP
II expression levels may be compared to a sample from the same
patient before treatment (i.e., from diagnosis) and/or samples from
earlier in the treatment. In an illustrative embodiment, a method
is provided comprising the steps of diagnosing a patient for
emphysema and/or COPD by determining the expression level of EMAP
II, treating the patient if the diagnosis was positive and
monitoring the effectiveness of the treatment by determining the
expression level of EMAP II during the treatment.
Example 1
Methods
[0090] Reagents and Antibodies.
[0091] All chemical reagents were purchased from Sigma-Aldrich (St.
Louis, Mo.), unless otherwise stated. EMAP II antiserum was
produced as recently described (Knies, U. E., Kroger, S., and
Clauss, M. 2000. Expression of EMAP II in the developing and adult
mouse. Apoptosis 5:141-151). Other antibodies employed were of
commercial source, including MAC-3 (Becton Dickinson Biosciences,
Franklin Lakes, N. J.), CXCR3 (R&D systems, Minneapolis,
Minn.), and MMP-12 (R&D).
[0092] Cells.
[0093] Human lung microvascular endothelial cells (HLMVEC) were
obtained from Lonza (Allendale, N.J.) and maintained in culture
medium consisting of EMB-2, 10% FBS, 0.4% hydrocortisone, 1.6%
hFGF, 1% VEGF, 1% IGF-1, 1% ascorbic acid, 1% hEGF, 1% GA-100, and
1% heparin. All primary cell cultures were maintained at 37.degree.
C. in 5% CO.sub.2 and 95% air. Experiments were performed up to
passage 10 with cells at 80-100% confluence.
[0094] Monoclonal Anti-EMAP II Antibody.
[0095] The rat monoclonal neutralizing antibody M7 against mouse
EMAP II was developed by immunizing Lewis rats with recombinant
murine pro-EMAP II. Lymphocytes isolated from the spleen and lymph
nodes of immunized rats were fused with the mouse myeloma SP2/0,
and Clones were selected by testing hybridoma supernatants in ELISA
for binding both pro- and mature EMAP II. The clones most active in
ELISA were further characterized by Western blotting and
neutralization of EMAP II-induced endothelial apoptosis in tissue
culture experiments (manuscript in preparation). For purification
of MoAbs for in vivo studies, hybridomas were grown in protein-free
hybridoma medium (GIBCO-BRL) and antibodies were purified with
protein G-Sepharose (Pharmacia, Uppsala, Sweden).
[0096] Animal Studies.
[0097] C57/B16 mice were purchased from Jacksons Lab. A
lung-specific inducible EMAP II transgenic mouse was generated by
crossing the EMAP II responder mouse with homozygous transgenic
mice containing the transactivator controlled by the lung
epithelium specific CCSP. The EMAP II responder transgenic mouse
contained the secreted (mature) form of EMAP II under a minimal
promoter containing tetracycline-inducible sequences. Therefore the
murine mature EMAP II cloned from meth mouse tumor cells (Knies, U.
E., Behrensdorf, H. A., Mitchell, C. A., Deutsch, U., Risau, W.,
Drexler, H. C., and Clauss, M. 1998. Regulation of endothelial
monocyte-activating polypeptide II release by apoptosis. Proc Natl
Acad Sci USA 95:12322-12327) and fused to a signal peptide derived
from INFb was inserted into the tet-repeat containing plasmid
pUD10-3 by using Sac II and Xho I insertion sites. The resulting
plasmid was injected into oocytes for implantation into foster mice
and a transgenic line was established. After crossing of the
resulting responder mice with the rtTA transactivator mice, the
first generation of mice heterozygous for the EMAP II responder
transgene were compared to the CCSP transactivator with CCSP
transactivator-only transgenic mice. Of note, only the EMAP II/CCSP
transactivator but not the CCSP transactivator-only transgene can
induce EMAP II expression. With this design, CCSP transactivator
background effects and tetracycline effects can be ruled out, as
both groups can be treated with tetracycline. Transgenic mice were
bred in an AAALAC accredited animal facility. Double transgenic
EMAP II/CCSP-rtTA and single transgenic CCSP-rtTA mice were
maintained on regular water until 3 to 4 month of age. Thereafter,
the mice were placed on doxycycline treatment for up to 6 months.
At the end of experiments, the mice were euthanized and the tissue
was processed as described (Petrache, I., Natarajan, V., Zhen, L.,
Medler, T. R., Richter, A. T., Cho, C., Hubbard, W. C., Berdyshev,
E. V., and Tuder, R. M. 2005. Ceramide upregulation causes
pulmonary cell apoptosis and emphysema-like disease in mice. Nat
Med 11:491-498). In addition, mice underwent BAL with 0.6 ml of PBS
thrice. BAL cells were sedimented via centrifugation and the
acellular fluid was then snap-frozen in liquid nitrogen and stored
at -80.degree. C. for further analysis.
[0098] Cigarette Smoke Exposure.
[0099] Cigarette smoke exposure was performed as previously
described (Cavarra, E., Bartalesi, B., Lucattelli, M., Fineschi,
S., Lunghi, B., Gambelli, F., Ortiz, L. A., Martorana, P. A., and
Lungarella, G. 2001. Effects of cigarette smoke in mice with
different levels of alpha(1)-proteinase inhibitor and sensitivity
to oxidants. Am J Respir Crit Care Med 164:886-890). Mice (C57/B16
mice, female, age 12 weeks; n=5-10 per group) were exposed to
cigarette smoke or ambient air for up to 24 weeks. In a separate
experiment, double transgenic EMAP II/CCSP transactivator or single
transgenic CCSP transactivator control littermates, male and
female, age 12 weeks; n=5-10 per group were exposed to cigarette
smoke or ambient air by a similar protocol as above. Prior to (for
the duration indicated) and during the cigarette smoke exposure,
all transgenic mice received water with doxycycline. In a separate
experiment, mice (DBA2, female, age 12 weeks; n=5-12 per group)
were exposed to cigarette smoke as described above or ambient air
for four months; during the third month of cigarette smoke
exposure, two groups of mice exposed to cigarette smoke received
either EMAP II antibody by nebulization or isotype IgG control, and
one group exposed to ambient air received isotype IgG control. The
day following the end of the cigarette smoking schedule in all
experiments mice were euthanized and lung processing was performed
as previously described (Petrache, I., Natarajan, V., Zhen, L.,
Medler, T. R., Richter, A. T., Cho, C., Hubbard, W. C., Berdyshev,
E. V., and Tuder, R. M. 2005. Ceramide upregulation causes
pulmonary cell apoptosis and emphysema-like disease in mice. Nat
Med 11:491-498).
[0100] VEGF Receptor Blockade.
[0101] VEGF receptor blockade was performed as previously described
(Petrache, I., Natarajan, V., Zhen, L., Medler, T. R., Richter, A.
T., Cho, C., Hubbard, W. C., Berdyshev, E. V., and Tuder, R. M.
2005. Ceramide upregulation causes pulmonary cell apoptosis and
emphysema-like disease in mice. Nat Med 11:491-498). Mice
(n=4-6/group) were injected with SU5416 (Calbiochem; 20 mg/kg,
subcutaneously) or vehicle (carboxymethylcellulose) and the mice
were euthanized at the indicated time.
[0102] Morphometric analysis was performed on coded slides as
described, using a macro developed by R.M.T. for Metamorph (Tuder,
R. M., Zhen, L., Cho, C. Y., Taraseviciene-Stewart, L., Kasahara,
Y., Salvemini, D., Voelkel, N. F., and Flores, S. C. 2003.
Oxidative stress and apoptosis interact and cause emphysema due to
vascular endothelial growth factor receptor blockade. Am J Respir
Cell Mol Biol 29:88-97; Aherne, W. A., and Dunnill, M. S. 1982.
Morphometry. London: E. Arnold. xiv, 205 pp).
[0103] Human Lung Tissue.
[0104] Human lung tissue consisted of sections from fixed, paraffin
embedded explanted lung tissue from COPD patients and patients
without lung disease (collected at the Johns Hopkins University).
The specimen collection and storage were approved by the
Institutional Research Board from the Johns Hopkins University.
[0105] Apoptosis was detected in lysates (Petrache, I., Natarajan,
V., Zhen, L., Medler, T. R., Richter, A. T., Cho, C., Hubbard, W.
C., Berdyshev, E. V., and Tuder, R. M. 2005. Ceramide upregulation
causes pulmonary cell apoptosis and emphysema-like disease in mice.
Nat Med 11:491-498) or inflated fixed lung sections enabling focus
on alveoli, rather than large airways and vessels (Tuder, R. M.,
Zhen, L., Cho, C. Y., Taraseviciene-Stewart, L., Kasahara, Y.,
Salvemini, D., Voelkel, N. F., and Flores, S. C. 2003. Oxidative
stress and apoptosis interact and cause emphysema due to vascular
endothelial growth factor receptor blockade. Am J Respir Cell Mol
Biol 29:88-97), via active caspase-3 IHC (Abcam and Cell Signaling)
or in situ labeling of apoptotic DNA on murine lung, using rat
serum as negative control. The immunostaining for both active
caspase-3 and TUNEL was followed by DAPI (Molecular Probes) nuclear
counter-staining. Executioner caspase (caspase-3 and/or -7)
activity was measured with ApoONE Homogeneous Caspase-3/7 assay kit
(Promega, Madison, Wis.). Human recombinant caspase-3 (Calbiochem)
was utilized as positive control.
[0106] Lipid Extraction and Ceramide Species Measurement by Tandem
Mass Spectroscopy.
[0107] Cellular or lung tissue lipids were extracted and lipid
content was assessed by measurements of total lipid phosphorus
(P.sub.i) (Petrache, I., Natarajan, V., Zhen, L., Medler, T. R.,
Richter, A. T., Cho, C., Hubbard, W. C., Berdyshev, E. V., and
Tuder, R. M. 2005. Ceramide upregulation causes pulmonary cell
apoptosis and emphysema-like disease in mice. Nat Med 11:491-498).
After lipid extraction, the following individual molecular species
of ceramides were monitored: 14:0, 16:0, 18:0, 18:1, 20:0, 24:0,
and 24:1-ceramides and utilizing C.sub.17 ceramide as internal
standard, ceramides were measured by combined liquid
chromatography-tandem mass spectrometry (LC-MS/MS).
[0108] IHC.
[0109] Paraffin sections were blocked with 10% rabbit (or goat
serum if secondary antibody from goat) and incubated with
antibodies or control antibodies. Polyclonal rabbit antiserum
included EMAP II (1:500 dilute), caspase-3 (Cell signaling) and
anti-MMP-12 (1:100, Sigma). Bound antibody was detected according
to the manufacturer's instructions or a biotin-conjugated goat
anti-rat IgG secondary antibody (Dianova, 1:100) and
Streptavidin-coupled phycoerythrin (Dianova, 1:1000). For some
application (anti-CD144, Pharmingen) cryosections were used.
Sections were counterstained with DAPI and mounted with Mowiol 488
(Calbiochem). Microscopy was performed on either a Nicon Eclipse
(TE200S) inverted fluorescent or a combined confocal/multi-photon
(Spectraphysics laser, BioRad MRC1024MP) inverted system. Images
and quantitative intensity (expression) data were processed by
MetaMorph Imaging software (Universal).
[0110] Western Blotting.
[0111] Lung tissue was homogenized in RIPA buffer with protease
inhibitors on ice and proteins were isolated by centrifugation at
10,000 g for 10 minutes at 4.degree. C. BAL supernatants from
transgenic mice or patients were collected and proteins were
concentrated and precipitated by addition of trichloroacetic acid.
Proteins were loaded in equal amounts (10 mg, unless otherwise
noted) as determined by BCA protein concentration assay (Pierce,
Rockville, Ill.). Total proteins were separated by SDS-PAGE using
Novex gels (Invitrogen, Carlsbad, Calif.), followed by
immunoblotting for EMAP II as previously described (Knies, U. E.,
Behrensdorf, H. A., Mitchell, C. A., Deutsch, U., Risau, W.,
Drexler, H. C., and Clauss, M. 1998. Regulation of endothelial
monocyte-activating polypeptide II release by apoptosis. Proc Natl
Acad Sci USA 95:12322-12327). Briefly, samples were mixed with
Laemmli buffer, boiled at 95.degree. C. for 10 min and loaded onto
15% SDS/PAGE gels. Proteins were separated by electrophoresis and
blotted onto nitrocellulose (Pierce) using a semidry blotting
apparatus. Unspecific binding was reduced by blocking the membrane
in TBS/0.1% Tween 20/5% nonfat dry milk. The primary antibody
(rabbit anti-EMAP II antiserum SA 2847, diluted 1:1000 in TBS/0.1%
Tween 20/5% BSA) was applied overnight at 4.degree. C. After
washing, the membranes were incubated in a peroxidase-coupled goat
anti-rabbit IgG (Dianova/Jackson Immuno Research; diluted 1:3500 in
blocking buffer) for 1 h at room temperature and developed using an
enhanced chemilluminescence kit (Amersham Pharmacia Biotech)
Immunoblotting for EMAP II in lung lysates or BAL was performed by
incubation with EMAP II-specific antibody (rabbit serum, produced
as described above) in a 1:250 dilution in TBST for 1 h at room
temperature. The chemiluminescent signals were quantified by
densitometry (ImageQuant; Amersham, Piscataway, N.J.) and
normalized by housekeeping proteins (actin, GAPDH, or
vinculin).
[0112] Statistical analysis was performed with SigmaStat software
using ANOVA with Student-Newman-Keuls post hoc test. Statistical
difference was accepted at p<0.05.
Example 2
Effect of Cigarette Smoke Exposure or VEGF Receptor Inhibition on
EMAP II Expression in the Mouse Lung
[0113] To test the hypothesis that smoking induces cellular stress
causing release of EMAP II, the effect of smoking on EMAP II
protein production was measured. The extent of apoptosis induced by
cigarette smoking in the mouse lung was also assessed. To more
specifically address the correlation between endothelial cell death
and EMAP II overproduction, the lung EMAP II expression in mice
treated with a VEGF receptor blocker, which induces endothelial
cell apoptosis was tested.
[0114] Mice susceptible to cigarette smoke-induced emphysema were
exposed to cigarette smoke for various periods of time, from 4 days
to 6 months. EMAP II expression was measured in lung lysates by
Western blotting and apoptosis by caspase-3 activity and ceramide
production. Finally, lungs from mice treated with VEGF receptor
blocker SU5416 (20 mg/kg subcutaneously) were tested for EMAP II
expression by Western blotting at 3 weeks, a time when lungs
typically show morphometric changes of emphysema.
[0115] Cigarette smoke CS exposure for 4 days increased caspase-3
activity in lungs, and thus increased apoptotic activity as early
as 1 week after cigarette smoke exposure in C57/B16 mice (FIG. 2A),
long preceding the increases in airspaces typical of emphysema that
occurred at 6 months of cigarette smoke exposure (FIG. 2C). At 1
month the lung content of ceramide increased in DBA 2 mice (FIG.
2B). These early increases in apoptotic activity were paralleled by
an increase in both the pro- and mature forms of EMAP II expression
(FIGS. 3A and 3B). Similarly, in another experimental model of
apoptosis-dependent emphysema, SU5416 induced a robust EMAP II
expression at 4 weeks in the C57/B16 mouse lung (FIG. 3C).
[0116] These results suggest an increase in apoptotic rates and
EMAP II production in the emphysematous lungs of mice, including
those exposed to cigarette smoke. While not wishing to be bound by
theory, the increase in EMAP II may result from direct cell stress,
or from apoptosis-activated caspases. Furthermore, EMAP II release
may itself be responsible for inducing further lung endothelial
cell apoptosis.
Example 3
Effect of Elevated Lung EMAP II Levels on the Severity of Cigarette
Smoke-Induced Injury in the Mouse Lung
[0117] To test whether increases in EMAP II have an additive or a
synergistic effect with cigarette smoking in the lung, EMAP II
expression in the lungs was induced for 8 weeks prior to cigarette
smoke exposure. The conditional transgenic overexpression system is
presented in more detail in Example 4.
[0118] An increase in baseline EMAP II levels in the lung followed
by a 4 week cigarette smoke exposure profoundly elevated the levels
of mature EMAP II and increased the number of inflammatory cells in
the inter-alveolar/interstitial tissue consistent with a further
increase in parenchymal inflammation compared to smoking alone.
[0119] These results suggest that EMAP II contributes to cigarette
smoke-induced lung injury and may independently worsen or
predispose the lung to a more severe inflammatory response to
smoke.
Example 4
Transgenic Induction of EMAP II in the Lung Causes Emphysema-Like
Disease in Mice
[0120] To study the mechanism by which increased lung levels of
EMAP II trigger emphysema, a transgenic murine model of inducible
expression of EMAP II in the lung was established using the
tetracycline inducible transactivator (TTA) controlled by the lung
epithelium-specific CCSP promoter. Although both EMAP II forms were
available as inducible constructs, the mature EMAP II was initially
assessed since it has been classically involved in the apoptosis
and inflammatory effects of EMAP II. Furthermore, the pro-EMAP II
is usually easily cleaved to generate mature EMAP II, making it
difficult to assess its specific, mature-form-independent
effects.
[0121] The transgenic mouse tet EMAP II (responder mouse) contained
the mature form of EMAP II under a minimal promoter containing
tetracycline-inducible sequences. This mouse line does not express
elevated levels of EMAP II because it lacks the transactivator gene
product. The responder mouse was crossed with homozygous transgenic
mice containing the transactivator controlled by the lung
epithelium specific CCSP promoter (CCSP mouse line), which in this
form targets gene expression predominately in alveolar type II
cells versus in Clara cells. Clark, J. C., et al. Am J Physiol Lung
Cell Mol Physiol 280, L705-715 (2001); Li, Y., et al. Cancer Res
67, 8494-8503 (2007). The first generation of mice heterozygous for
the EMAP II responder transgene and the CCSP transactivator with
CCSP transactivator-only transgenic mice were compared. Of note,
this CCSP transactivator-only transgene cannot induce EMAP II
overexpression. With this design, CCSP transactivator background
effects as described recently (Sisson, T. H., et al. Am J Respir
Cell Mol Biol 34, 552-560 (2006)) and tetracycline effects can be
ruled out, as both groups were treated with tetracycline.
Furthermore, the tetracycline concentration used in this induction
system is insufficient to ameliorate any inflammation and MMP
activities. Expression was analyzed by Western of BAL and lung
lysates and by IHC of lung sections using EMAP II antiserum. To
determine whether long term EMAP II over-expression in the lung
induces an emphysema-like phenotype, double transgenic mice with
tetracycline in the drinking water were treated for up to 6
months.
[0122] Transgenic induction of EMAP II caused high EMAP II
secretion into the lungs of double transgenic mice after as early
as 24 h (FIGS. 3B, 4B and 5A). Of note, the EMAP II expression
pattern in the lung parenchyma resembled typical staining pattern
for alveolar type II cells, which is in line with the reported
selectivity for this transgenic promoter. EMAP II double transgenic
mice treated for 3 or 6 months with tetracycline to induce EMAP II
expression displayed significant emphysema-like increase in
airspace (FIG. 7A). This was measured both by the mean linear
intercept and the recently established method of volume-weighted
mean airspace volume. Morphological parameters for emphysema appear
to increase proportional to the duration of EMAP II induction,
which is reflected by morphometry: the volume-weighted mean
airspace volume was 1.36E+08.+-.0.15, n=5 in control mice;
1.56E+08.+-.0.3 in EMAP II transgenic mice induced for 3 months;
and 1.91E+08.+-.0.3, n=6, in those induced for 6 months;
p=0.027)
[0123] Increased EMAP II production in the lungs leads to formation
of emphysema-like morphological changes. This is the first evidence
that excessive levels of a protein causing endothelial cell death
leads to emphysema.
Example 5
Excessive EMAP II Production in the Lung Causes Pulmonary Cell
Apoptosis
[0124] To address the hypothesis that EMAP II over-production
promotes emphysema via endothelial cell apoptosis, apoptosis in the
lungs of EMAP II-overexpressing mice was assessed. To determine the
EMAP II-specificity of apoptosis, and to test in vivo the efficacy
of an EMAP II-neutralizing antibody, the anti-EMAP antibody was
administered to a group of EMAP II transgenic animals.
[0125] Fluorescent microscopy with specific active caspase-3
antibody of lung sections from EMAP II/CCSP double transgenic (EMAP
II tg) or CCSP control transgenic animals (ctl) was used to detect
the presence and localization of apoptosis in the lung.
Anti-VE-cadherin antibody was used to test for colocalization of
apoptosis with endothelial cells. In addition, lung lysates were
tested for caspase-3 activity by fluorimetric enzymatic assay
(Promega). For the neutralization experiment, EMAP II tg (induced
for 48 h before harvesting the lungs) received anti-EMAP II rat
monoclonal antibody or isotype IgG control, by a single injection
i.p., 12 h after the induction.
[0126] EMAP II significantly increased the number of caspase-3
positive cells in the lung parenchyma of EMAP II tg versus ctl (-6
fold, p=0.003, by fluorescence quantitation using Metamorph on
blinded slides) as early as 3 weeks after induction. The increased
lung apoptosis persisted after 3 months and 6 months of EMAP II
inductions as assessed by both IHC and caspase-3 activity from lung
lysates (FIGS. 6A and 6B). The majority of caspase-3 positive cells
were endothelial cells. There was a trend for decreased apoptosis
in mice receiving neutralizing EMAP II antibody (FIG. 6C).
[0127] It is thought that changes by in situ detection of activated
caspase-3 were more dramatically significant due to the higher
signal to noise ratio in lysates resulting from having many other
non-dying cells other than endothelial cells. Finally, although not
yet statistically significant, the neutralizing effects of
anti-EMAP II antibody are extremely encouraging in that apoptosis
observed is EMAP II dependent and that the neutralizing antibody is
effective in vivo. Taken together these data support the conclusion
that endothelial cell apoptosis may be a key event in EMAP
II-induced emphysema formation.
Example 6
Effect of Lung-Specific EMAP II Overexpression on the Monocyte
Recruitment in the Lung
[0128] It was previously shown that EMAP II attracted and activated
monocytes in a dose-dependent manner, caused inflammation when
locally injected, and triggered leukostasis in the lung upon
systemic application. Kao, J., et al., J Biol Chem 269, 25106-25119
(1994); Kao, J., et al., J Biol Chem 269, 9774-9782 (1994). The
chemotactic effect of EMAP II on monocytes may be important in the
inflammatory responses associated with emphysema.
[0129] Confocal imaging of fluorescent immunostaining of markers
for lung macrophage accumulation and activation in lung sections
from EMAP II/CCSP double transgenic vs. CCSP single transgenic
animals was performed using MAC-3-(macrophage marker) as well as
TNF.alpha.-, MMP-9-, and MMP-12-specific antibodies.
[0130] The lung specific overexpression of mature EMAP II
dramatically increased the numbers of MAC-3-expressing cells along
with staining for TNF.alpha.-, MMP-9, MMP-12 in the lung (FIGS. 7A
and 7B). The vast majority of TNF.alpha.-, MMP-9, MMP-12 and MAC-3
positive cells displayed a large nuclear phenotype, characteristic
for macrophages, whereas MMP-12-positivity colocalized not only
with Mac-3 (FIG. 7A), but also with other cells within the alveolar
wall, possibly epithelial cells.
[0131] The increase in Mac-3 positive cells was most likely due to
recruitment of monocytes form the circulation to the lung, as the
proliferation capacity of already resident lung macrophages is
extremely low. These macrophages may be a source of inflammatory
activation in the lungs of EMAP II transgenic.
Example 7
Both Pro- and Mature EMAP II-Induce Significant Apoptosis in Human
Primary Microvascular Lung Endothelial Cells
[0132] Situations associated with stress can induce both forms of
EMAP II. It is not known which form is more potent in inducing
endothelial cell apoptosis and whether the mechanism by which this
occurs is form-dependent. These detailed mechanistic assays can
only be done in cell cultures. However to increase their
significance, only primary lung microvascular endothelial cells of
human origin, commercially obtained (Lonza) were tested.
[0133] Primary human lung microvascular endothelial cells were
treated with recombinant pro- or mature-EMAP II at 10-16 .mu.g/ml.
Apoptosis was measured by caspase-3 activity and Annexin/PI
staining by flow cytometry. Treatment with both forms of EMAP II
resulted in increased apoptosis as measured by caspase-3 activity
(FIG. 8A) and Annexin/PI staining (FIG. 8E).
[0134] Both the pro- and mature EMAP II forms appeared equally
potent at inducing endothelial cell apoptosis in culture
conditions.
Example 8
The Stress-Sensitive CXCR3 Receptor Mediates EMAP II-Induced Lung
Endothelial Cell Apoptosis
[0135] To investigate whether the CXCR3 receptor mediates EMAP
II-induced lung endothelial cell apoptosis, its expression on
primary human lung microvascular endothelial cells was initially
assessed and secondly, its function was inhibited by specific
blocking antibodies.
[0136] Primary human lung microvascular endothelial cells were
cultured in normal growth media, as well as in media containing low
serum concentration (2%), or even treated with acellular BAL from
smoked or control mice. The BAL was concentrated (50-fold) and
cells were incubated with a volume representing 10% of the original
undiluted cellular BAL. CXCR3 was detected by using labeled
anti-CXCR3 antibody detected by FACS. To assess the role of the
CXCR3 caspase-3 activation in lung microvascular endothelial cells,
cells with blocking anti-CXCR3 antibodies were pretreated (1 ng/ml,
pretreated for 30 min).
[0137] Primary human lung microvascular endothelial cells express
CXCR3 at low levels. Stressful conditions such as serum starvation,
treatment with BAL from smoked but not from non-smoked mice, or
even electroporation (FIG. 9) increased significantly its
expression (FIGS. 8A-8D). Anti-CXCR3 antibodies, but not isotype
IgG antibodies significantly reduced mature EMAP II-induced
endothelial cell death (FIGS. 8A-8D).
[0138] These results are strong evidence that EMAP II-induced
endothelial cell apoptosis in the lung may be mediated primarily by
the CXCR3 receptor. This implies that CXCR3 mediates the functional
effects of EMAP II on both endothelial cells and monocytes and may
be important for the development of cigarette smoke emphysema.
Example 9
Cigarette Smoke Increased the Expression of Both EMAP II Forms in
the Mouse Lung
[0139] Based on previous findings that mature EMAP II is released
by apoptosis and the proform upon stress, the induction of EMAP II
in the lung in vivo upon exposure to cigarette smoke was
investigated. Therefore, EMAP II expression was measured in two
inbred mouse strains, C57/B16 and DBA2, which reportedly develop
emphysema after chronic exposure to cigarette smoke for 6 or 4
months, respectively. Cigarette smoke exposure (CSE) (for up to 24
weeks) profoundly increased both the pro- and mature forms of EMAP
II (approximately 8- and 2-fold, respectively) secreted in the BAL
and detected by Western blotting (FIG. 10A). Equal volume (100
.mu.l) of acellular BAL from each mouse was pooled (n=5 per time
point), then equally concentrated (10.times.) and equally loaded
(10 .mu.l) in each lane. Specific EMAP II antibody (1:250) detected
both the pro- and the mature forms of the EMAP II in the lavage.
BAL from the EMAP II overexpressing transgenic (Tg) mice was
utilized as positive (Pos) control. Similar increases in the two
forms of EMAP II expression were noted in the lung parenchyma of
DBA2 mice exposed to cigarette smoke for 4 weeks (FIG. 10B).
[0140] Interestingly, in a distinct experimental model of
apoptosis-dependent murine emphysema which develops secondary to
VEGF receptor inhibition, EMAP II expression was also markedly
upregulated in the lungs of mice which developed airspace
enlargement compared to control mice, but predominantly in the
pro-form (FIG. 10C). FIG. 10C shows EMAP II expression in the lung
parenchyma of C57/B16 mice at four weeks after treatment with the
VEGF receptor inhibitor (VEGFR-inh). Each lane was loaded with 40
mg lung lysate from individual mice treated with vehicle
(carboxymethyl cellulose) or the VEGFR-inh SU5416 (20 mg/kg,
subcutaneous). Vinculin was immunoblotted as loading control. The
kinetics of EMAP II elevation in response to cigarette smoking
demonstrated that the increase in lung EMAP II secretion preceded
that of alveolar macrophage accumulation, first noted at 4 weeks,
but not 2 weeks of cigarette smoke exposure (FIG. 10D). The kinetic
relationship of the EMAP II increase with the caspase-3 activation
in the lung was more complex, as significant caspase-3 activation
was noted throughout the time course of the EMAP II increases in
response to cigarette smoking in mice (FIG. 10E). Since EMAP II's
biological properties include monocyte chemoattraction and
apoptosis of proliferative and hypoxic endothelial cells, EMAP II
could play an important role in the inflammatory and apoptotic
responses in the lung in response to cigarette smoke exposure.
Example 10
Neutralization of Pro and Mature-EMAP II-Induced Endothelial Cell
Apoptosis
[0141] Because mature EMAP II has been shown to induce endothelial
apoptosis, it was investigated whether a rat antibody hybridoma
clone M7/1 (M/71 antibody) was also able to neutralize apoptosis
induced by EMAP II. In particular it was investigated whether this
M7/1 antibody was able to neutralize pro-apoptotic activities of
both pro- and mature EMAP II.
[0142] EMAP II induced apoptosis was assessed by quantification of
TUNEL-positive cells (FIG. 11A). Endothelial cells incubated with
pro-EMAPII protein (50 .mu.g/ml) or mature-EMAPII protein (50
.mu.g/ml) demonstrated a significant apoptosis (arrows) as shown by
TUNEL (*p<0.01). Pretreatment of these cells with the
neutralizing M 7/1 antibody (10 .mu.g/ml), but not with control rat
IgG, significantly (**p<0.03) inhibited apoptosis induced by
both pro and mature EMAPII as shown from representative fluorescent
microscope images following TUNEL assay. Quantification of TUNEL
positive cells by MetaMorph software normalized to total DAPI
nuclear positive cells is also shown for pro-EMAPII (FIG. 11B) and
mature EMAPII (FIG. 11C). Data shown are from a representative
experiment performed in triplicates and repeated independently two
additional times with similar results. Scale bar=50 .mu.m.
[0143] Thus, EMAP II induced apoptosis was significantly
(p<0.03) blocked by the anti-EMAP II M 7/1 antibody, but not by
control rat IgG (FIGS. 11A-11C). Interestingly, it was observed
that pro-EMAP II at the same molar concentrations as mature EMAP II
was also a strong inducer of endothelial apoptosis. Again, the M
7/1 antibody was able to completely neutralize this activity
(p<0.01). These data demonstrate that the M7/1 antibody can
effectively neutralize the pro-apoptotic function of both EMAP II
forms and may be a suitable tool to inhibit pathophysiological
activities of this protein in mice. (Rajashekhar, G. et al, A
monoclonal rat anti-mouse EMAP II antibody that functionally
neutralizes pro- and mature-EMAP II in vitro, J Immunol Methods.
2009 Oct. 31; 350(1-2): 22-28).
Example 11
Neutralization of EMAPII Levels Markedly Reduces CS-Induced
Lungemphysema in Mice
[0144] Because EMAPII has been shown to be produced and released by
apoptosis, hypoxia, and cellular stress, it was investigated
whether EMAPII is induced in the lung in vivo upon exposure to
cigarette smoke (CS). EMAPII protein expression was measured in the
DBA/2 mouse strain, which develops emphysema after chronic exposure
to CS as early 16 weeks, exhibiting a 20% increase in airspace
size, compared with only a 9% increase measured in the C57BL/6
strain at this time point, respectively. CS exposure for only 4
weeks significantly increased the pro and mature forms of EMAPII
expression in the lung parenchyma of DBA/2 mice compared with that
in control mice exposed to ambient air (air control [AC]), measured
by immunoblotting (FIG. 12A).
[0145] Next, the cellular localization of EMAPII expression in
normal and CS-exposed mice was investigated by coimmunofluorescence
with EMAPII antiserum, CD11b antibody, and DAPI. Under ambient air
conditions, lungs of control mice showed sparse EMAPII expression
that colocalized mostly with CD11b-labeled alveolar macrophages
(FIG. 12B, left panel). By contrast, cigarette smoking robustly
increased both intracellular and extracellular EMAPII production,
which colocalized with both macrophages (FIG. 12B, middle panel)
and alveolar septal cells (FIG. 12B, right panel).
[0146] The M7/1 antibody from Example 10 was used to functionally
assess the role of the secreted EMAPII in CS-induced lung injury
and emphysema. The M7/1 antibodies (50 .mu.g/application) were
administered directly to the lung via inhalation of a nebulized
solution, which showed effective deposition in the lung parenchyma
at 15 minutes by fluorescence microscopy of the lung and at 4 hours
by immune adsorption analysis of recovered biotinylated antibody
from plasma. This method of administration has the advantages of
targeting the local EMAPII pool and has been previously shown to
allow the use of lower antibody doses compared with the systemic
route. The timing of M7/1 antibody delivery was chosen to follow
the increases in EMAPII detected in response to CS exposure, while
the duration of antibody M7/1 treatment was limited to 4 weeks to
minimize or avoid nonspecific immunological side effects. DBA/2
mice were first exposed to CS alone for 8 weeks, followed by
targeting EMAPII with neutralizing M7/1 antibodies between weeks 9
to 12 and 4 additional weeks of CS exposure (FIG. 12C).
[0147] The administration of EMAPII-neutralizing M7/1antibody
significantly decreased lung apoptosis measured by caspase-3
activity in tissue lysates (FIG. 12D). In addition, this treatment
decreased the number of inflammatory cells retrieved in the BALF
(FIG. 12E), particularly alveolar macrophages and neutrophils, and
reduced the number of neutrophils in the lung parenchyma.
Furthermore, anti-EMAPII M7/1 antibodies significantly improved the
lung static compliance (FIG. 12F) by almost 40%. Importantly,
consistent with these functional data, neutralization of EMAPII
abolished the CS-induced airspace enlargement measured as a 19.4%
increase in MLI compared with that in air-exposed mice, which is in
a typical range for CS-induced emphysema mouse models (FIGS. 2G and
2H). Interestingly, neutralizing EMAPII antibodies had no effect on
CS-induced large airway epithelial remodeling but restored the
thickness of the epithelial layer of small airways (smaller than
150 .mu.m in diameter), which was significantly reduced by CS
exposure. (Clauss, M. et al., Lung endothelial monocyte-activating
protein 2 is a mediator of cigarette smoke-induced emphysema in
mice, J Clin Invest doi:10.1172/JCI43881).
Example 12
Lung-Specific EMAP II Overexpression Induced Emphysema-Like
Pathology of the Lung
[0148] Endothelial cell death, alveolar macrophage accumulation and
MMP-12 expression are implicated in emphysema pathogenesis.
Lung-specific EMAP II overexpression for up to 6 months
significantly increased airspace diameters, consistent with
simplification of alveolar structures (FIGS. 5B-5E). The airspace
enlargement was progressive, noted on hematoxyllin-eosin stained
lung sections and measured by the volume-weighted mean airspace
volume, which significantly increased from 1.36E+08 (.+-.0.15, n=5)
in control mice to 1.56E+08 (.+-.0.3 SD, n=6) at 3 months (not
shown) and 1.91E+08 (.+-.0.3, n=6) at 6 months of EMAP II lung
overexpression (p=0.027) (FIG. 5E). The loss of alveolar septae was
further supported by an increase in the mean linear intercept in
the mice overexpressing EMAP II for 3 months compared to control
mice (FIG. 5C). Note that the bar in FIGS. 5B and 5D represents 300
.mu.m. These data suggest that EMAP II increase alone may be
sufficient to trigger emphysema-like airspace enlargement.
Example 13
Specific Neutralization of Secreted EMAP II Inhibits Cigarette
Smoke-Induced Airspace Enlargement in Mice
[0149] To investigate whether an excess of secreted EMAP II is also
necessary for the pathogenesis of airspace enlargement in response
to cigarette smoking, EMAP II was neutralized by administration of
specific monoclonal antibodies in mice exposed to cigarette
smoking. The DBA2 mice, which develop significant airspace
enlargement after 4 months of cigarette smoke exposure, were first
exposed to cigarette smoke for 2 months. For the following 1 month
of exposure, specific EMAP II antibodies or isotype IgG (1 mg/kg)
were administered thrice weekly via nebulization. At the end of the
4 month of total cigarette smoke exposure, lung morphometry
demonstrated significant increase in airspace size consistent with
simplification of alveolar structure, reminiscent of emphysema, in
response to smoking but not ambient air (FIG. 12G, left panel and
middle panel, bar is 100 .mu.m). While inhaled IgG did not have an
inhibitory effect on cigarette smoke-induced airspace size (not
shown), treatment of mice with inhaled EMAP II antibody
significantly inhibited the airspace enlargement induced by
cigarette smoking (FIG. 12G, right panel, and FIG. 12H). These data
suggest that application of neutralizing antibodies can reduce
emphysema development even after a considerable time of smoke
exposure.
Example 14
Synergistic Effects of EMAP II and Cigarette Smoke Exposure in the
Lung
[0150] Having shown that EMAP II is both sufficient and necessary
in smoke induced emphysema, it was next asked whether enhanced
levels of baseline EMAP II in the lung sensitize the lungs to
cigarette smoke-induced injury, specifically apoptosis and
macrophage inflammation. Increased lung levels of EMAP II were
achieved in the double transgenic mice by tetracycline
administration for 8 weeks. Double transgenic (EMAP II
overexpressing) or single transgenic control mice were then exposed
to cigarette smoking daily, five times a week, for 4 weeks. Lungs
were then assessed for levels of apoptosis by extracting and
measuring whole lung apoptosis-signaling ceramides, as reported
previously (Petrache, I., Natarajan, V., Zhen, L., Medler, T. R.,
Richter, A. T., Cho, C., Hubbard, W. C., Berdyshev, E. V., and
Tuder, R. M. 2005. Ceramide upregulation causes pulmonary cell
apoptosis and emphysema-like disease in mice. Nat Med 11:491-498).
At this time point of cigarette smoke exposure, lungs of wild-type
mice express only modest increases in ceramides (Petrache, I.,
Medler, T. R., Richter, A. T., Kamocki, K., Chukwueke, U., Zhen,
L., Gu, Y., Adamowicz, J., Schweitzer, K. S., Hubbard, W. C., et
al. 2008. Superoxide dismutase protects against apoptosis and
alveolar enlargement induced by ceramide. Am J Physiol Lung Cell
Mol Physiol 295:L44-53). Interestingly, there was a dramatic
increase in ceramides in the lungs of mice overexpressing EMAP II
prior to cigarette smoking compared to either EMAP II
overexpression or cigarette smoking alone (FIG. 4A). Similarly the
number of lung macrophages measured by IHC using F4/80 antibody
increased synergistically in the mice overexpressing EMAP II prior
to cigarette smoking compared to mice exposed for the same duration
to either stimulus alone. Levels of lung ceramide (FIG. 2C), a
marker of alveolar apoptosis elevated in emphysema were measured by
tandem mass spectrometry and levels were normalized for lipid
phosphorus (Pi) content. Horizontal lines represents median and
whiskers depict the 5.sup.th and 95.sup.th percentile. Groups were
compared by ANOVA; *p=0.01 vs control; **P=<0.006 vs. control
and vs. control+cigarette smoke. H&E staining showed increased
inflammatory cells in CS-exposed mice which is further aggravated
in Tg mice exposed to CS. These data provide evidence for the
hypothesis that EMAP II may be a predictor and mediator of
emphysema formation.
Example 15
EMAP II Elevations in Human Lungs with COPD and in the
Broncho-Alveolar Lavage of Smokers
[0151] To investigate the relevance of increased lung EMAP II
levels for human emphysema, EMAP II in subjects diagnosed with
emphysema was assessed. Immunostaining (IHC) of lung samples
obtained from patients with emphysema at the time of lung
transplantation with specific EMAP II antibody demonstrated
markedly increased EMAP II staining compared with non-diseased
lungs. Interestingly, variable levels of EMAP II expression were
noted in individuals without a diagnosis of COPD at the time of
tissue sampling. This variability may be related to smoking status,
as the BAL obtained from active smokers without a COPD diagnosis
exhibited increased EMAP II levels compared to nonsmokers (FIG. 1).
Secreted EMAP II (mature form) expression in the BAL acellular
fluid of smokers was compared to non-smokers, as measured by
Western blotting with a specific EMAP II antibody. Levels measured
by densitometry of EMAP II expression in individual BAL samples.
(Mean.+-.SEM,*p=<0.01).
Example 16
Extraction of Total RNA from Hybridomas
[0152] First-round of RT-PCT. QIAGEN.RTM. OneStep RT-PCR Kit (Cat
No. 210210) was used. RNA was isolation using a Qiagen kit
according to standard methods in conformity with the manufacture's
and the instructions. Briefly, RT-PCR was performed with primer
sets specific for the heavy and light chains. For each RNA sample,
12 individual heavy chain and 11 light chain RT-PCR reactins were
set up using degenerate forward primer mixtures covering the leader
sequences of variable regions. Reverse primers are located in the
constant regions of heavy and light chains. No restriction sites
were engineered into the primers.
[0153] Second-round semi-nested PCR. The RT-PCR products from the
first-round reactions were further amplified in the second-round
PCR. 12 individual heavy chain and 11 light chain RT-PCR reactions
were set up using sem-nested primer sets specific for antibody
variable regions.
[0154] Referring now to FIG. 13. After PCR was finished, a PCR
reaction was run and samples from the PCR reaction were run onto an
agarose gel to visualize the DNA fragments amplified. The correct
antibody variable region DNA fragments should have a size between
400-500 base pair.
[0155] Referring now to FIGS. 14 and 15. After sequencing more than
15 DNA fragments amplified by nested RT-PCR, several antibody heavy
and light chains were cloned. The protein sequence and alignment
and CDR analysis identified one heavy chain and one light chain
Example 17
EMAP II Epitope Peptide Sequence Identification
[0156] Referring now to FIGS. 16 and 17. Based on the protocol of
Parker and Tomer, tryptic digestion-derived peptides of protein
bound to another compound (such as an antibody) maybe protected
from digestion at the binding site. (Parker, C. et al.,
MALDI/MS-based epitope mapping of antigens bound to immobilized
antibodies, Molecular Biotechnology, Volume 20, Number 1 (2002),
49-62). Accordingly, the portion of a protein bound to a
sepharose-immobilized M7/1 antibody would likely be protected from
proteolysis.
[0157] A binding competition was performed using human recombinant
pro-EMAP II and the M7/1 antibody. Referring now to FIG. 17.
Recombinant pro-EMAP II was submitted to Western blotting using
control IgG and EMAP II neutralizing M7/1 antibody in the
presence/absence of a 300 fold molar excess of peptide
hexadecamers. Only Peptide 2 (QQSIAGSADSKPIDVSR) but not Peptide 1
(KHPDADSLYVEEVDVGE) or Peptide 3 (as a control) was able to compete
with M7/1. Arrows indicate the position of molecular weight
standards (in rel kDa).
[0158] Peptides in the pull-down fraction were identified by liquid
chromatography tandem mass spectrometry (LC-MS/MS). By analyzing
the sequences bound to protein G sepharose immobilized M7/1
antibody, protected peptides ranging over the sequence
QQSIAGSADSKPIDVSRLDLRIGCIITARKHPDADSLYVEEVDVGEIAPRTVVS
GLVNHVPLEQMQNRM (SEQ. ID NO. 11) were identified. From this peptide
sequences 2 hexadecamer peptides randomly chosen for competition in
M7/1 Western blotting: Peptide 1: KHPDADSLYVEEVDVGE (SEQ. ID NO.
13) and Peptide 2: QQSIAGSADSKPIDVSR (SEQ. ID NO. 12). A Western
blotting competition assay was used in order to determine which
polypeptide is the best epitope. In this assay, M7/1 antibody
binding to recombinant pro-EMAP II was performed in the presence of
a 300-fold excess of hexadecamer Peptides 1 or 2 or a control
Peptide 3: VLKRLEQKGAEADQIIE (SEQ. ID NO. 14). Peptide 2 competed
strongly for the M7/1 antibody binding as indicated by the absence
of a Western blot band for M7/1 staining, whereas the other
identified Peptide 1 and the control Peptide 3 had no effect.
[0159] It should be understood, of course, that the foregoing
relates to exemplary embodiments of the invention and that
modifications may be made without departing from the spirit and
scope of the invention as set forth in the following claims.
[0160] While the novel technology has been illustrated and
described in detail in the figures and foregoing description, the
same is to be considered as illustrative and not restrictive in
character, it being understood that only the preferred embodiments
have been shown and described and that all changes and
modifications that come within the spirit of the novel technology
are desired to be protected. As well, while the novel technology
was illustrated using specific examples, theoretical arguments,
accounts, and illustrations, these illustrations and the
accompanying discussion should by no means be interpreted as
limiting the technology. All patents, patent applications, and
references to texts, scientific treatises, publications, and the
like referenced in this application are incorporated herein by
reference in their entirety.
Sequence CWU 1
1
151354DNARattus rattus 1gcggtgcacc ttgttgagtc tggtggagga tttgtgcagc
ctacggagtc attgaaaatc 60tcatgtgcag cctctggatt caccttcagt gatgctgcca
tgtactgggt ccgccaggct 120ccaggaaagg gtctggaatg ggttgctcgc
ataagaacta aacctaataa ttatgcaaca 180tattatgctg attcagtgaa
aggcagattc accatctccc gagatgattc aaaaagcatg 240gtctacctac
aaatggataa cttgaaaact gaggacacag ccatgtatta ctgtacatca
300tggagctacg actttgatta ctggggccaa ggagtcatgg tcacagtctc ctca
3542118PRTRattus rattus 2Ala Val His Leu Val Glu Ser Gly Gly Gly
Phe Val Gln Pro Thr Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Ala Met Tyr Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg
Thr Lys Pro Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met 65 70 75 80
Val Tyr Leu Gln Met Asp Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85
90 95 Tyr Cys Thr Ser Trp Ser Tyr Asp Phe Asp Tyr Trp Gly Gln Gly
Val 100 105 110 Met Val Thr Val Ser Ser 115 3112PRTRattus rattus
3Asp Ile Val Met Thr Gln Gly Ala Leu Pro Asn Pro Val Pro Ser Gly 1
5 10 15 Glu Ser Ala Ser Ile Thr Cys Gln Ser Ser Lys Ser Leu Leu His
Ser 20 25 30 Ser Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Arg Pro
Gly Gln Ser 35 40 45 Pro His Leu Leu Ile Tyr Trp Met Ser Thr Arg
Ala Ser Gly Val Ser 50 55 60 Asp Arg Leu Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Ser Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Gln Gln Phe 85 90 95 Leu Glu Tyr Pro Leu
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110
4337DNARattus rattus 4gatattgtga tgacccaggg tgcactcccc aaccctgtcc
cctctggaga gtcagcttcc 60atcacctgcc agtctagtaa gagtctgctg cacagcagtg
gcaagacata cttgaattgg 120tatctgcaga ggccaggaca gtctcctcat
ctcctgatct attggatgtc cacccgtgca 180tcaggagtct cagacaggct
cagtggcagt gggtcaggaa cagatttcac actgaaaatc 240agcagcgtgg
aggctgagga tgtgggtgtg tattactgtc agcaatttct agagtatcct
300ctcacgttcg gttctgggac caagctggag atcaaac 33758PRTRattus rattus
5Gly Phe Thr Phe Ser Asp Ala Ala 1 5 610PRTRattus rattus 6Ile Arg
Thr Lys Pro Asn Asn Tyr Ala Thr 1 5 10 79PRTRattus rattus 7Thr Ser
Trp Ser Tyr Asp Phe Asp Tyr 1 5 811PRTRattus rattus 8Lys Ser Leu
Leu His Ser Ser Gly Lys Thr Tyr 1 5 10 93PRTRattus rattus 9Trp Met
Ser 1 109PRTRattus rattus 10Gln Gln Phe Leu Glu Tyr Pro Leu Thr 1 5
1169PRTHomo sapiens 11Gln Gln Ser Ile Ala Gly Ser Ala Asp Ser Lys
Pro Ile Asp Val Ser 1 5 10 15 Arg Leu Asp Leu Arg Ile Gly Cys Ile
Ile Thr Ala Arg Lys His Pro 20 25 30 Asp Ala Asp Ser Leu Tyr Val
Glu Glu Val Asp Val Gly Glu Ile Ala 35 40 45 Pro Arg Thr Val Val
Ser Gly Leu Val Asn His Val Pro Leu Glu Gln 50 55 60 Met Gln Asn
Arg Met 65 1217PRTHomo sapiens 12Gln Gln Ser Ile Ala Gly Ser Ala
Asp Ser Lys Pro Ile Asp Val Ser 1 5 10 15 Arg 1317PRTHomo sapiens
13Lys His Pro Asp Ala Asp Ser Leu Tyr Val Glu Glu Val Asp Val Gly 1
5 10 15 Glu 1417PRTRandom 14Val Leu Lys Arg Leu Glu Gln Lys Gly Ala
Glu Ala Asp Gln Ile Ile 1 5 10 15 Glu 15672PRTHomo sapiens 15Met
Leu Pro Ala Val Ala Val Ser Glu Pro Val Val Leu Arg Phe Met 1 5 10
15 Ile Phe Cys Arg Leu Leu Ala Lys Met Ala Asn Asn Asp Ala Val Leu
20 25 30 Lys Arg Leu Glu Gln Lys Gly Ala Glu Ala Asp Gln Ile Ile
Glu Tyr 35 40 45 Leu Lys Gln Gln Val Ser Leu Leu Lys Glu Lys Ala
Ile Leu Gln Ala 50 55 60 Thr Leu Arg Glu Glu Lys Lys Leu Arg Val
Glu Asn Ala Lys Leu Lys 65 70 75 80 Lys Glu Ile Glu Glu Leu Lys Gln
Glu Leu Ile Gln Ala Glu Ile Gln 85 90 95 Asn Gly Val Lys Gln Ile
Pro Phe Pro Ser Gly Thr Pro Leu His Ala 100 105 110 Asn Ser Met Val
Ser Glu Asn Val Ile Gln Ser Thr Ala Val Thr Thr 115 120 125 Val Ser
Ser Gly Thr Lys Glu Gln Ile Lys Gly Gly Thr Gly Asp Glu 130 135 140
Lys Lys Ala Lys Glu Lys Ile Glu Lys Lys Gly Glu Lys Lys Glu Lys 145
150 155 160 Lys Gln Gln Ser Ile Ala Gly Ser Ala Asp Ser Lys Pro Ile
Asp Val 165 170 175 Ser Arg Leu Asp Leu Arg Ile Gly Cys Ile Ile Thr
Ala Arg Lys His 180 185 190 Pro Asp Ala Asp Ser Leu Tyr Val Glu Glu
Val Asp Val Gly Glu Ile 195 200 205 Ala Pro Arg Thr Val Val Ser Gly
Leu Val Asn His Val Pro Leu Glu 210 215 220 Gln Met Gln Asn Arg Met
Val Ile Leu Leu Cys Asn Leu Lys Pro Ala 225 230 235 240 Lys Met Arg
Gly Val Leu Ser Gln Ala Met Val Met Cys Ala Ser Ser 245 250 255 Pro
Glu Lys Ile Glu Ile Leu Ala Pro Pro Asn Gly Ser Val Pro Gly 260 265
270 Asp Arg Ile Thr Phe Asp Ala Phe Pro Gly Glu Pro Asp Lys Glu Leu
275 280 285 Asn Pro Lys Lys Lys Ile Trp Glu Gln Ile Gln Pro Asp Leu
His Thr 290 295 300 Asn Asp Glu Cys Val Ala Thr Tyr Lys Gly Val Pro
Phe Glu Val Lys 305 310 315 320 Gly Lys Gly Val Cys Arg Ala Gln Thr
Met Ser Asn Ser Gly Ile Lys 325 330 335 Met Leu Pro Ala Val Ala Val
Ser Glu Pro Val Val Leu Arg Phe Met 340 345 350 Ile Phe Cys Arg Leu
Leu Ala Lys Met Ala Asn Asn Asp Ala Val Leu 355 360 365 Lys Arg Leu
Glu Gln Lys Gly Ala Glu Ala Asp Gln Ile Ile Glu Tyr 370 375 380 Leu
Lys Gln Gln Val Ser Leu Leu Lys Glu Lys Ala Ile Leu Gln Ala 385 390
395 400 Thr Leu Arg Glu Glu Lys Lys Leu Arg Val Glu Asn Ala Lys Leu
Lys 405 410 415 Lys Glu Ile Glu Glu Leu Lys Gln Glu Leu Ile Gln Ala
Glu Ile Gln 420 425 430 Asn Gly Val Lys Gln Ile Pro Phe Pro Ser Gly
Thr Pro Leu His Ala 435 440 445 Asn Ser Met Val Ser Glu Asn Val Ile
Gln Ser Thr Ala Val Thr Thr 450 455 460 Val Ser Ser Gly Thr Lys Glu
Gln Ile Lys Gly Gly Thr Gly Asp Glu 465 470 475 480 Lys Lys Ala Lys
Glu Lys Ile Glu Lys Lys Gly Glu Lys Lys Glu Lys 485 490 495 Lys Gln
Gln Ser Ile Ala Gly Ser Ala Asp Ser Lys Pro Ile Asp Val 500 505 510
Ser Arg Leu Asp Leu Arg Ile Gly Cys Ile Ile Thr Ala Arg Lys His 515
520 525 Pro Asp Ala Asp Ser Leu Tyr Val Glu Glu Val Asp Val Gly Glu
Ile 530 535 540 Ala Pro Arg Thr Val Val Ser Gly Leu Val Asn His Val
Pro Leu Glu 545 550 555 560 Gln Met Gln Asn Arg Met Val Ile Leu Leu
Cys Asn Leu Lys Pro Ala 565 570 575 Lys Met Arg Gly Val Leu Ser Gln
Ala Met Val Met Cys Ala Ser Ser 580 585 590 Pro Glu Lys Ile Glu Ile
Leu Ala Pro Pro Asn Gly Ser Val Pro Gly 595 600 605 Asp Arg Ile Thr
Phe Asp Ala Phe Pro Gly Glu Pro Asp Lys Glu Leu 610 615 620 Asn Pro
Lys Lys Lys Ile Trp Glu Gln Ile Gln Pro Asp Leu His Thr 625 630 635
640 Asn Asp Glu Cys Val Ala Thr Tyr Lys Gly Val Pro Phe Glu Val Lys
645 650 655 Gly Lys Gly Val Cys Arg Ala Gln Thr Met Ser Asn Ser Gly
Ile Lys 660 665 670
* * * * *
References