U.S. patent application number 14/331803 was filed with the patent office on 2015-02-05 for lung cancer markers and uses thereof.
The applicant listed for this patent is Celera Corporation. Invention is credited to Charles BIRSE, Marcia LEWIS, Mehdi MESRI, Steve RUBEN.
Application Number | 20150037820 14/331803 |
Document ID | / |
Family ID | 40668079 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150037820 |
Kind Code |
A1 |
BIRSE; Charles ; et
al. |
February 5, 2015 |
LUNG CANCER MARKERS AND USES THEREOF
Abstract
Methods and compositions are provided for assessing (e.g.,
diagnosing), treating, and preventing diseases, especially cancer,
and particular lung cancer, using lung cancer markers (LCM).
Individual LCM and panels comprising multiple LCM are provided for
these and other uses. Methods and compositions are also provided
for determining or predicting the effectiveness of a treatment or
for selecting a treatment using LCM. Methods and compositions are
further provided for modulating cell function using LCM. Also
provided are compositions that modulate LCM (e.g., antagonists or
agonists), such as antibodies, proteins, small molecule compounds,
and nucleic acid agents (e.g., RNAi and antisense agents), as well
as pharmaceutical compositions thereof. Further provided are
methods of screening for agents that modulate LCM, and agents
identified by these screening methods.
Inventors: |
BIRSE; Charles; (Danville,
CA) ; RUBEN; Steve; (Brookeville, MD) ; LEWIS;
Marcia; (Cohasset, MD) ; MESRI; Mehdi; (North
Potomac, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Celera Corporation |
Alameda |
CA |
US |
|
|
Family ID: |
40668079 |
Appl. No.: |
14/331803 |
Filed: |
July 15, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13005031 |
Jan 12, 2011 |
8808997 |
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14331803 |
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12273994 |
Nov 19, 2008 |
7892760 |
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13005031 |
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61003767 |
Nov 19, 2007 |
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Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
C12Q 2600/158 20130101;
A61P 11/00 20180101; G01N 33/57423 20130101; G01N 2333/8114
20130101; C12Q 2600/136 20130101; G01N 33/57488 20130101; C12Q
1/6886 20130101; G01N 33/57492 20130101; C12Q 2600/106 20130101;
C12Q 2600/118 20130101; G01N 2800/56 20130101; G01N 2333/96486
20130101; A61P 35/00 20180101; C12Q 2600/112 20130101 |
Class at
Publication: |
435/7.92 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Claims
1. A method of detecting lung cancer in an individual, the method
comprising detecting the level of TFPI in a sample from said
individual, wherein an elevated level of TFPI indicates that said
individual has lung cancer.
2. The method of claim 1, further comprising detecting the level of
at least one marker selected from the group consisting of Cyfra,
SLPI, TIMP1, SCC, CEACAM5, MMP2, OPN, and MDK in a sample from said
individual, wherein an elevated level of the marker indicates that
said individual has lung cancer.
3. The method of claim 2, wherein the method comprises detecting
the levels of the markers Cyfra, SLPI, TIMP1, CEACAM5, and MDK,
wherein elevated levels of a plurality of said markers indicate
that said individual has lung cancer.
4. The method of claim 3, wherein the method further comprises
detecting the levels of the markers SCC, MMP2, and OPN, wherein
elevated levels of a plurality of said markers indicate that said
individual has lung cancer.
5. A method of detecting lung cancer in an individual, the method
comprising detecting the levels of a panel of markers selected from
the group consisting of the panels provided in Table 5, wherein
elevated levels of a plurality of said markers indicate that said
individual has lung cancer.
6-17. (canceled)
18. The method of claim 4, wherein the detecting comprises
detecting the marker by immunoassay.
19. The method of claim 18, wherein the immunoassay is ELISA.
20-21. (canceled)
22. The method of claim 2, wherein the detecting comprises
contacting the sample with an antibody that selectively binds to
the marker and detecting the binding of the antibody to the
marker.
23-27. (canceled)
28. A kit comprising at least one container and detection reagents
for detecting a plurality of lung cancer markers comprising Cyfra,
SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK, wherein the
detection reagents are stored in one or more of the containers.
29. The kit of claim 28, wherein the detection reagents are
antibodies or antigens for use in an immunoassay.
30. The kit of claim 28, wherein at least one of the detection
reagents is labeled with a detectable moiety.
31. The kit of claim 29, wherein the immunoassay is an ELISA.
32. The kit of claim 28, wherein the detection reagents are
antibodies, and wherein the antibodies are at least one of
monoclonal, polyclonal, fully human, humanized, chimeric,
single-chain, and anti-idiotypic antibodies.
33. The kit of claim 28, wherein the detection reagents are
antibodies, and wherein the antibodies comprise an antibody
fragment selected from the group consisting of: a) an Fab fragment;
b) an F(ab').sub.2 fragment; and c) an Fv fragment.
34. The method of claim 4, wherein the sample is a serum
sample.
35. The method of claim 4, further comprising the step of providing
a prognosis to the individual.
36. The method of claim 4, wherein the method is carried out
following lung cancer treatment.
37. The method of claim 4, further comprising selecting a lung
cancer treatment for administration to an individual based on the
level of a plurality of said markers, and administering the
treatment to the individual.
38. The method of claim 4, further comprising selecting said
individual for inclusion in a clinical trial of a therapeutic
agent.
39-43. (canceled)
44. The method of claim 17, wherein the method is carried out in
conjunction with lung cancer treatment.
45. The method of claim 44, wherein the earlier time point is
before the lung cancer treatment and the later time point is after
the lung cancer treatment.
46. The method of claim 44, wherein the lung cancer treatment
comprises at least one of administration of a therapeutic agent,
surgical resection of at least a portion of a lung tumor, or
radiation therapy.
47-50. (canceled)
51. The method of claim 5, wherein said elevated levels are either
greater than predetermined cutoff levels, or greater than or equal
to predetermined cutoff levels.
52. (canceled)
53. The method of claim 5, wherein said panel is selected from the
group consisting of the following panels: panels comprising the
9-marker panel (Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN,
and MDK); and panels comprising the 6-marker subset of the 9-marker
panel (Cyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK).
54. The method of claim 53, wherein the panel further comprises at
least one marker selected from the group consisting of the markers
provided in Table 4.
55. The method of claim 2, wherein the method comprises detecting
the levels of the markers TFPI, Cyfra, SCC, and CEACAM5.
56. The method of claim 55, further comprising detecting the levels
of at least one marker selected from the group consisting of TIMP1
and OPN.
57. The method of claim 56, further comprising detecting the level
of MDK.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/005,031, filed on Jan. 12, 2011, which is a divisional of
U.S. application Ser. No. 12/273,994, filed on Nov. 19, 2008 (and
issued as U.S. Pat. No. 7,892,760 on Feb. 22, 2011), which claims
the benefit of U.S. provisional application Ser. No. 61/003,767,
filed on Nov. 19, 2007, the content of each of which are hereby
incorporated by reference in their entirety into this
application
FIELD OF THE INVENTION
[0002] This invention relates to the field of disease assessment
and therapy. The invention provides compositions and methods for
assessing and treating diseases, especially cancer, and
particularly lung cancer.
BACKGROUND OF THE INVENTION
[0003] Cancer is one of the leading causes of death worldwide, and
cancer, especially lung cancer, is difficult to diagnose and treat
effectively. Accordingly, there is a need in the art for new
compositions and methods for assessing and treating various
cancers, particularly lung cancer.
[0004] Lung Cancer
[0005] Lung cancer is the second most prevalent type of cancer for
both men and women in the United States and is the most common
cause of cancer death in both men and women. The five-year survival
rate for lung cancer continues to be poor at only about 8-15%. This
low survival is because lung cancer is commonly not detected until
it has spread beyond the lungs. Only 16% of new lung cancer cases
in the United States are detected at the earliest stage, when the
cancer is still localized to the lungs. At this early stage,
survival is considerably higher, with estimates as high as 70-80%.
Therefore, procedures for detecting lung cancer are of critical
importance to the outcome of a patient since these procedures have
the potential to reduce mortality. Thus, there is a need for new
diagnostic compositions and methods that are more sensitive and
specific for detecting early lung cancer.
[0006] Furthermore, there is also a need for new diagnostic
compositions and methods for determining the stage of a patient's
disease. Stage determination has potential prognostic value and
provides criteria for designing optimal therapy. Biomarkers that
are indicative of different stages of lung cancer would be useful
to facilitate the staging of lung cancer.
[0007] Lung cancer patients are typically monitored following
initial therapy and during adjuvant therapy to determine their
response to therapy and to detect persistent or recurrent disease
or metastasis. Thus, there is clearly a need for lung cancer
markers that are more sensitive and specific in detecting lung
cancer, its recurrence, and progression.
[0008] Although imaging modalities, such as computed tomography
(CT) screening, are being studied to aid in the early detection of
lung cancer, controversy remains as to the ability of these methods
to impact mortality (1-ELCAP Investigators, NEJM 2006 (355):1763-71
and Bach et al. 2007. JAMA 297:953-961). In addition, the most
advanced imaging technologies under study are expensive and not
widely available. These CT imaging tests may lead to over-diagnosis
of lung cancer, resulting in significant expenses to the health
care system to manage patients with pulmonary nodules observed
through these CT imaging tests. Furthermore, there is significant
morbidity associated with the management of the pulmonary nodules
in an effort to ascertain whether the nodules are malignant or
benign. It is estimated that 10-50% of smokers in a high risk group
have pulmonary nodules upon imaging studies (CHEST 2007
Supplement--Evidence for the Treatment of Patients With Pulmonary
Nodules: When Is It Lung cancer?: AACP Evidence-Based Clinical
Practice Guidelines). Thus, there is a significant need for novel
diagnostics that can be used either independently or with imaging
modalities for early diagnosis and improved management of patients
with lung cancer. For example, a blood test for biomarkers that has
high performance (e.g., high sensitivity and specificity) for
detecting lung cancer could provide a low cost complement to CT
testing for early detection of cancer. If the performance of a
biomarker test were sufficiently high, such a test could serve as a
lower cost alternative to CT or X-ray testing. For example, only
those patients that tested positive in a biomarker test may then
need to undergo more expensive imaging tests. Furthermore, a
biomarker test could be used, for example, in a yearly screening
regimen for lung cancer.
[0009] Although there have been reports of circulating tumor
markers and antigens with potential use in lung cancer (see
Schneider, J. 2006. Advances in Clin Chem, 42: 1-41 for a review),
markers currently used generally suffer from low sensitivity and
less than desirable specificity, especially among smokers
(Schneider, 2006), and are typically only used to monitor for
recurrence of lung cancer. Thus, there is a need in the art for a
panel of markers with high sensitivity (and varying specificities,
depending on the clinical indication), such as for detecting lung
cancer. Furthermore, there is also a need for novel markers that
are useful individually or as part of a panel for detecting lung
cancer. Such markers, and panels of markers, would facilitate
management of patients with lung cancer, for example.
[0010] For a further review of lung cancer diagnostics, including
the use of tumor biomarkers as well as CT screening, see the
following citations: Schneider, "Tumor markers in detection of lung
cancer", Adv Clin Chem. 2006; 42:1-41; Bach et al., "Computed
tomography screening and lung cancer outcomes", JAMA. 2007 Mar. 7;
297(9):953-61; and International Early Lung Cancer Action Program
Investigators et al., "Survival of patients with stage I lung
cancer detected on CT screening", N Engl J. Med. 2006 Oct. 26;
355(17):1763-71. Also see Pepe et al., "Phases of biomarker
development for early detection of cancer", J Nat'l Cancer Inst.
2001. 93(14):1054-1061
[0011] Description of Tables 1-2
[0012] Tables 1 and 2 provide further information for lung cancer
markers ("LCM"), including their names, symbols (alternative
symbols are indicated in parentheses), Genbank protein accession
numbers, and an exemplary protein sequence for each marker (except
for the carbohydrate antigens CA 242, CA 19-9, and CA 72-4, for
which representative journal citations are provided for each).
Exemplary LCM protein sequences are provided as SEQ ID NOS:1-65
(additionally, the carbohydrate antigens CA 242, CA 19-9, and CA
72-4 are also provided). Nucleic acid sequences (e.g., mRNA
transcript sequences and genomic DNA) and alternative protein
sequences for each marker are well known in the art and can readily
be derived using the information provided in Tables 1-2, for
example.
[0013] The LCM provided in Table 1 are as follows (alternative
names/symbols are indicated in parentheses): SLPI, MIF, TIMP1,
TFPI, ENO2 (NSE), CEA (CEACAM5), MMP2, AMBP, Cyfra 21-1 (Cyfra,
KRT19), SCC (SERPINB3), OPN, defensin (DEFA1, HNP-1, HNP1-3), CA
242, CA 19-9, CA 72-4, MN/CAIX (CA9), ProGRP (GRP), KRT18 (TPS),
ECAD (CDH1), TIMP2, CD44, LGALS3BP, ERBB2 (HER-2), UPA (PLAU), DKK
(DKK1), CHGA, VEGF, KITLG, PBEF (visfatin), SORT1 (sortilin), MDK
(midkine), IGFBP3, IGFBP4, CTSC, ICAM3, CTGF, LCN2, EGFR, BGN,
TIMP3, HGF, MUC16 (CAl25), NCAM, CRP, SERPINA1 (ATT), PKM2, RBP,
KLK11, KLK13, SAA, and APOC3.
[0014] The LCM provided in Table 2 (which are particularly useful
as autoantibody markers) are as follows (alternative names/symbols
are indicated in parentheses): TP53 (p53), KLKB1, CFL1 (CFLN),
EEF1G, HSP90.alpha. (HSP90AA1), RTN4, ALDOA, GLG1, PTK7, EFEMP1,
SLC3A2 (CD98), CHGB, CEACAM1, ALCAM, HSPB1 (HSP27), LGALS1, and
B7H3.
[0015] Elevated levels of each of these LCM are indicative of lung
cancer, except for sortilin (SORT1), for which low levels are
indicative of lung cancer.
[0016] Description of Tables 3-12
[0017] Table 3 provides 35 different panels of 11 markers (each row
of 11 markers represents a panel) that have at least 98%
specificity and 82% sensitivity for detecting lung cancer. The
total number of occurrences of each marker in these 35 11-marker
panels is indicated at the bottom of Table 3. Seven markers (SLPI,
TIMP1, TFPI, SCC, OPN, CEA and CA242) appear in all 35 of these
panels, GRP appears in 33 of these 35 panels, MIF appears in 29 of
these 35 panels, and NSE and HNP-1 each appear in 15 of these 35
panels. AMBP, Cyfra, MMP2, Ca72-4, Ca19-9, and CAIX each appear in
7-9 of these panels, as indicated in Table 3.
[0018] Table 4 provides markers the can be included in any of the
panels disclosed herein. For example, the markers in Table 4 can be
added to any of the panels disclosed herein and/or can replace one
or more members of any of the panels disclosed herein. As a
specific example, the markers in Table 4 can be added to any of the
panels disclosed in Table 5 and/or can replace one or more members
of any of the panels disclosed in Table 5. The markers disclosed in
Table 4 are also disclosed in Table 2.
[0019] Tables 5-12 provide data for the analysis of various panels
in various lung cancer uses, such as distinguishing lung cancer
samples versus normal samples such as for diagnosing/detecting lung
cancer (Tables 5-6 and 11-12, for example), as well as certain
specific uses (these specific uses, which may be referred to herein
as "indications" or as determining or assessing lung cancer
"characteristics", are provided in Tables 7-10, for example). In
Tables 5-12, each row represents a panel (a panel may comprise an
individual marker). For each panel in Tables 5-12, data are
presented based on logistic regression and/or split point analysis
(as indicated in each table). Area under the curve (AUC),
sensitivity at 95% specificity, and specificity at 95% sensitivity
are indicated. "Size" (second column) indicates the number of
markers in the given panel. Further information regarding
characteristics of the sample sets (the "54.times.53",
"50.times.50", and "44.times.44" sample sets) used in each of the
analyses is provided in FIG. 16 (the "104.times.103" sample set
used in Table 8 is the "54.times.53" and "50.times.50" sample sets
combined). In Tables 5-12, and elsewhere herein, "trained" refers
to the sample set (which may be referred to as the "training set")
which was used to formulate cutoff levels, and "tested" refers to
the sample set (which may be referred to as the "testing set") to
which these cutoff levels were applied (such as to classify a
sample as a lung tumor or normal sample, or other specific use,
based on whether marker levels were above or below the cutoff
levels established from the training set).
[0020] Table 5 provides data for logistic regression and
split-point analysis of the 9-marker panel of Cyfra, SLPI, TIMP1,
SCC, TFPI, CEACAM5, MMP2, OPN, and MDK, and all subcombinations
thereof (including individual markers), in distinguishing lung
tumor samples versus normal (i.e., control/healthy) samples, such
as for diagnosing/detecting lung cancer. For each panel in Table 5,
data are presented based on logistic regression and split point
analysis and based on analysis of either training and testing on
the same 54.times.53 (54 controls and 53 cases) sample set, or
training on the 54.times.53 sample set and testing on the
50.times.50 (50 controls.times.50 cases) sample set (see FIG. 16
for characteristics of these sample sets). Area under the curve
(AUC), sensitivity at 95% specificity, and specificity at 95%
sensitivity are indicated. The panels are sorted based on the AUC
indicated in the third column. "Size" (second column) indicates the
number of markers in the given panel. Thus, Table 5 provides the
9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK, and all panel subcombinations thereof, including each
of these nine markers individually (each row represents a
panel).
[0021] Table 6 provides data for split-point analysis of panels
(including individual markers) that include any of the nine markers
in the panels provided in Table 5 and/or various other markers
(which are not in the panels provided in Table 5) in distinguishing
lung tumor samples versus normal (i.e., control/healthy) samples,
such as for diagnosing/detecting lung cancer.
[0022] Table 7 provides data for logistic regression analysis of
the 9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK, and subcombinations thereof (including individual
markers), in distinguishing adenocarcinoma versus squamous cell
carcinoma types of lung cancer.
[0023] Table 8 provides data for split-point analysis of the
9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK, and subcombinations thereof (including individual
markers), in distinguishing stage I versus stage III lung cancer.
In addition to their utility in distinguishing between early and
late stage lung cancer (e.g., stage I or II versus stage III or
IV), the panels provided in Table 8 are also useful for
distinguishing between any other stages of lung cancer (e.g., any
of stages I, II, III, and IV).
[0024] Table 9 provides data for split-point analysis of various
panels in distinguishing small cell lung cancer (SCLC) versus other
types of lung cancer (e.g., non-small cell lung cancer, NSCLC). In
the left-side of Table 9, marker levels are higher in NSCLC (as
compared to SCLC). In the right-side of Table 9, marker levels are
higher in SCLC (as compared to NSCLC).
[0025] Table 10 provides data for split-point analysis of the
9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK, and subcombinations thereof (including individual
markers), in distinguishing malignant lung tumors versus benign
lung lesions.
[0026] Table 11 provides data for split-point analysis of various
panels in distinguishing small cell lung cancer (SCLC) versus
normal (i.e., control/healthy) samples.
[0027] Table 12 provides data for split-point analysis of various
panels in distinguishing lung cancer (including both small cell
lung cancer (SCLC) and non-small cell lung cancer (NSCLC)) versus
normal (i.e., control/healthy) samples.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1. Shows relative expression levels for exemplary lung
cancer markers (LCM) screened by ELISA in a sample set of 12 lung
tumor and 12 normal serum samples. The left portion of the table
shown in FIG. 1 provides tumor samples identified by histology and
tumor state (histology abbreviations for tumor samples are
"AS"=adenosquamous, "A"=adenocarcinoma, "SC"=squamous cell
carcinoma, and "BAC"=bronchioalveolar carcinoma), and the right
portion of the table shows normal samples (identified as "N" for
histology). The table is based on mean concentration values of each
sample and uses 2 standard deviations (2SD) above normal mean as
the cutoff; the value is expressed as fold change from normal mean
(thus, any fold change with 2SD above normal mean is above the
cutoff). The column labeled "CRA MS" is a summary of mass
spectrometry data that indicates the number of differentially
expressed lung tumor samples and the median mass spec ratio of
these samples (numerical representation of over-expression is
indicated by 2.0 or more, whereas numerical representation of
under-expression is indicated by 0.5 or less) (lung tumor sample
abbreviations for mass spectrometry are "CL LU"=lung cancer cell
lines, "TS LU"=lung cancer tissues, and "CM LU"=lung cancer
conditioned medium).
[0029] FIG. 2. Shows relative expression levels based on ELISA
screening of a sample set of 12 lung tumor (upper section) and 12
normal (lower section) serum samples for the eight markers TFPI,
SCC (interchangeably referred to as SSC), CEA, CA242, MNCAIX, OPN,
Cyfra 21-1, and MIF (as also shown in FIG. 1). The table is based
on mean concentration values of each sample and uses 2 standard
deviations (2SD) above normal mean as the cutoff; the value is
expressed as fold change from normal mean (thus, any fold change
with 2SD above normal mean is above the cutoff). Any value below
the cut-off is recoded as 0. Any or all of these eight markers may
be used in combination as a panel for lung cancer assessment, and
the panel may optionally include additional markers.
[0030] FIG. 3. Shows the performance of the eight marker panel of
TFPI, SCC, CEA, CA242, MNCAIX, OPN, Cyfra 21-1, and MIF. Using an
algorithm in which markers greater than or equal to two standard
deviations were scored "positive", this panel of eight markers had
a sensitivity of 92% and specificity of 100% among the 12 sera from
lung cancer patients and 12 sera from healthy controls ("FP"=false
positives, "TP"=true positives, "FN"=false negatives, and "TN"=true
negatives)
[0031] FIG. 4. Shows relative expression levels based on ELISA
screening of a sample set of 12 lung tumor (left portion; histology
("Hist") abbreviations are "AS"=adenosquamous, "A"=adenocarcinoma,
"SC"=squamous cell carcinoma, "OC"=oat cell carcinoma, and
"BAC"=bronchioalveolar carcinoma) and 12 normal (right portion;
identified as "N" for histology) serum samples for alternate panels
of LCM, including a panel of the markers SLPI, TFPI, OPN, MIF,
TIMP1, and MMP2. Any or all of these markers can also be used in
any combination with any or all of the following markers: CA242,
SCC, CEA, NSE, CA72-4, CA19-9, Cyfra 21-1, and MN/CAIX, as shown in
FIG. 4. The table is based on mean concentration values of each
sample and uses two standard deviations (2SD) above normal mean as
the cut-off; the value is expressed as fold change from normal mean
(thus, any fold change with 2SD above normal mean is above the
cutoff). Any value below the cut-off is recoded as 0.
[0032] FIG. 5. Shows scatter plots of ELISA data for the six
markers CEA, TFPI, MIF, TIMP1, OPN, and Cyfra 21-1 in 44 normal and
44 lung tumor samples, with exemplary cut-offs indicated (dotted
lines). Cut-offs can be applied that maximize sensitivity while not
compromising specificity of the panel, for example.
[0033] FIG. 6. Shows results of ELISA analysis for the 11 markers
Cyfra 21-1, MIF, TIMP1, TFPI, CEA, OPN, SCC, SLPI, HNP-1, GRP, and
CA242 in 39 control (normal) samples (left portion, labeled
"Control") and 39 lung tumor samples (right portion, labeled
"Tumor"). Values shown are concentration (ng/mL). Manually defined
cut-offs are indicated immediately below each marker name. The
columns labeled "#> cutoff" indicate the total number of markers
with elevated expression (i.e., a concentration greater than the
manually defined cut-offs) in a given serum sample. "Stage"
indicates lung cancer stage, and "Hist Type" indicates histology
type. Any or all of these 11 markers may be used in combination as
a panel for lung cancer assessment, and the panel may optionally
include additional markers.
[0034] FIG. 7. Shows performance of exemplary panels of markers,
demonstrating that increased sensitivity can be achieved by
including additional markers. The marker CEA provides 55%
sensitivity and 90% specificity, the two markers CEA and OPN
provide 60% sensitivity and 90% specificity, the three markers
TFPI, CEA, and OPN provide 67% sensitivity and 90% specificity, and
the four markers TIMP1, TFPI, CEA, and OPN provide 69% sensitivity
and 90% specificity. The score is the sum of the log.sub.2 of the
ratios of the tumor concentration to the mean concentration in
normal serum. ROC curves can be constructed by varying the cut-off
of the score needed to call a sample a tumor.
[0035] FIG. 8. Shows examples of applying a cut-off for various
markers (Cyfra 21-1, MIF, SLPI, TIMP1, SCC, NSE, TFPI, CEA, MMP2,
OPN, and AMBP are shown) that provides desirable performance for
that marker. The circles show the approximate location of an
exemplary cut-off for each marker which is the point on the curve
that is closest to the upper-left corner. Different criteria can
also be used, for instance false negatives could be weighted more
heavily than false positives.
[0036] FIGS. 9-10. Shows AUC (area under curve)=0.8543 for markers
MIF, TIMP1, TFPI, CEA, and OPN (FIG. 9), and AUC=0.8518 for markers
TIMP1, NSE, CEA, and OPN. Score is the number of markers greater
than the cutoff that best separates tumor samples from normal
samples for each marker. ROC curve can be constructed by varying
the cut-off of the score needed to call a sample a tumor.
[0037] FIG. 11. Shows results of analysis for certain autoantibody
markers (bottom table), as well as certain other lung cancer
markers (top table). In the bottom table (autoantibody markers),
the column labeled "Lung MS data" indicates a summary of where
differential expression has been observed by mass spectrometry
(CL=cell lines, TS=tissues, CM=conditioned medium, and
IP=immunoprecipitation), the column labeled "SEREX data" indicates
autoantibody markers that overlap with the Serological Expression
(SEREX) database which identifies markers that elicit a high-titer
IgG antibodies, and the column labeled "Rec Protein" indicates the
source of recombinant protein used for autoantibody analysis
("vendor" indicates an external commercial source and "CRA"
indicates an internal source). Histology abbreviations for tumor
samples in the top table are "AS"=adenosquamous,
"A"=adenocarcinoma, "SC"=squamous cell carcinoma, and "SM"=small
cell carcinoma.
[0038] FIG. 12. Shows exemplary autoantibody LCM, which can be used
alone or in combination with other LCM. Certain of these
autoantibody LCM are also provided in Table 2 along with other
autoantibody LCM.
[0039] FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D. Shows
autoantibody responses observed in lung cancer and normal serum
samples for the autoantibody markers KLKB1 (FIG. 13B), cofilin
(FIG. 13C), and LGALS1 (FIG. 13D), as well as p53 (FIG. 13A). Along
the horizontal axis, T1 through T12 indicate tumor samples and N1
through N12 indicate normal samples.
[0040] FIG. 14. Shows autoantibody detection in lung cancer and
normal serum samples for the autoantibody markers CAl2, KLKB1,
CFLN, LGALS1, and EEF1G, as well as p53. Autoantibody responses
were detected for CAl2, KLKB1, and CFLN (cofilin). The table is
based on mean concentration values of each sample and uses 2SD
above normal mean as the cut-off. Any value below the cutoff is
recoded as 0. p53 showed 0 response in normal sera, therefore
absolute titers are listed for p53 (positive antibody-dependent
values).
[0041] FIG. 15. Shows three additional LCM: visfatin (PBEF),
sortilin (SORT-1), and midkine (MDK). Any or all of these three LCM
can be implemented in a panel of markers for lung cancer diagnosis,
for example. FIG. 15 shows abundance levels (in ng/mL) of these
three markers in 12 normal lung and 12 lung tumor samples based on
ELISA analysis. For sortilin (SORT-1), abundance levels (by
relative copy number) of this marker based on mRNA expression
analysis of 22 normal lung and 23 lung tumor samples is also
provided. For sortilin, lung tumor samples have a decreased
abundance level of this marker compared with normal lung
samples.
[0042] FIG. 16. Shows clinicopathological characteristics of lung
cancer serum samples used in various analyses disclosed herein.
[0043] FIG. 17. Shows results of ELISA analysis for the 9-marker
panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK
in 50 control (normal) samples (left portion, labeled "Normal") and
50 lung tumor samples (right portion, labeled "Tumor"), using
split-point analysis applying manually defined cut-offs. The
manually defined cut-offs are indicated immediately below each
marker name. Values shown are concentration (ng/mL). The columns
labeled ".gtoreq. cut off" indicate the total number of markers
with elevated expression (i.e., a concentration greater than or
equal to the manually defined cut-offs) in a given serum sample.
"Histology" indicates histology type ("adeno"=adenocarcinoma,
"squ"=squamous cell carcinoma, "nsm" or "n-sm"=non-small cell
carcinoma, "bro"=bronchioloalveolar carcinoma, "LG"=large cell
carcinoma, and "neuro"=neuroendocrine). Any or all of these nine
markers may be used in combination as a panel for lung cancer
assessment, and the panel may optionally include additional
markers.
[0044] FIG. 18. Describes the analysis of markers to monitor lung
tumor regression/recurrence, with CEA and Cyfra as examples. In
particular, levels of biomarkers in patient serum 2-4 weeks
following surgery were compared to pre-surgical marker levels.
[0045] FIG. 19. Shows an analysis of markers to monitor for lung
tumor regression/recurrence. Percentage change in levels of
biomarkers in patient serum 2-4 weeks post-surgery as compared to
pre-surgical levels is indicated.
[0046] FIG. 20. Shows an analysis of the expression levels of the
9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK in the following co-morbid lung diseases: asthma,
bronchitis, and benign lung diseases. Values shown are
concentration (ng/mL). The column labeled "#> cut off" indicates
the total number of markers with elevated expression (i.e., a
concentration greater than the manually defined cut-offs) in a
given serum sample. The manually defined cut-offs are indicated
immediately below each marker name (in the row labeled
"Cut-off").
[0047] FIG. 21. Shows an analysis of integrating an exemplary
supplemental biomedical parameter (smoking history) with an
exemplary LCM panel (TIMP1, TFPI, CEACAM5, and Ca72-4) plus pack
years ("pack year": number of cigarettes smoked per day multiplied
by number of years of smoking at this rate). The left-side graph
shows that performance of a 5-marker panel (represented by the line
that includes a vertical portion at 50 of the x-axis) is enhanced
with addition of smoking history (pack years) (represented by the
line that includes a vertical portion at about 43 of the x-axis).
Sensitivity increases from 71.5% to 84.6%. The right-side graph
shows split-point analysis performed following the addition of
smoking history (split at about 45 of the x-axis).
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
[0048] The invention will best be understood by reference to the
following detailed description of the exemplary embodiments, taken
in conjunction with the accompanying table(s) and/or figure(s). The
discussion below is exemplary and is not to be taken as limiting
the scope defined by the claims.
[0049] Exemplary embodiments of the invention provide the following
markers (see Tables 1-2), combinations of these markers, and
methods of using these markers, particularly for lung
cancer-related uses, and especially for lung cancer diagnostics
(alternative names/symbols are indicated in parentheses): SLPI,
MIF, TIMP1, TFPI, ENO2 (NSE), CEA (CEACAM5), MMP2, AMBP, Cyfra 21-1
(Cyfra, KRT19), SCC (SERPINB3), OPN, defensin (DEFA1, HNP-1,
HNP1-3), CA 242, CA 19-9, CA 72-4, MN/CAIX (CA9), ProGRP (GRP),
KRT18 (TPS), ECAD (CDH1), TIMP2, CD44, LGALS3BP, ERBB2 (HER-2), UPA
(PLAU), DKK (DKK1), CHGA, VEGF, KITLG, PBEF (visfatin), SORT1
(sortilin), MDK (midkine), IGFBP3, IGFBP4, CTSC, ICAM3, CTGF, LCN2,
EGFR, BGN, TIMP3, HGF, MUC16 (CAl25), NCAM, CRP, SERPINA1 (ATT),
PKM2, RBP, KLK11, KLK13, SAA, APOC3, TP53 (p53), KLKB1, CFL1
(CFLN), EEF1G, HSP90.alpha. (HSP90AA1), RTN4, ALDOA, GLG1, PTK7,
EFEMP1, SLC3A2 (CD98), CHGB, CEACAM1, ALCAM, HSPB1 (HSP27), LGALS1,
and B7H3, which are collectively referred to herein as "LCM" ("lung
cancer markers"). Elevated levels of each of these LCM are
indicative of lung cancer, except for sortilin (SORT1), for which
low levels are indicative of lung cancer. Tables 1 and 2 provide
further information for each of these LCM, including their names,
symbols, Genbank protein accession numbers, and an exemplary
protein sequence for each marker (except for the carbohydrate
antigens CA 242, CA 19-9, and CA 72-4, for which representative
journal citations are provided for each). Exemplary LCM protein
sequences are provided as SEQ ID NOS:1-65 (additionally, the
carbohydrate antigens CA 242, CA 19-9, and CA 72-4 are also
provided). Nucleic acid sequences (e.g., mRNA transcript sequences
and genomic DNA) and alternative protein sequences for each marker
are well known in the art and can readily be derived using the
information provided in Tables 1-2, for example. The markers
provided in Table 2 are particularly useful as autoantibody
markers.
[0050] Certain embodiments of the invention provide combinations
comprising, consisting of, and consisting essentially of the
following nine LCM, and subcombinations thereof (these nine LCM may
be referred to herein as the "9-marker panel", which is shown in
FIG. 17 and Table 5, for example): Cyfra, SLPI, TIMP1, SCC, TFPI,
CEACAM5, MMP2, OPN, and MDK. Certain embodiments of the invention
provide compositions based on this 9-marker panel and
subcombination thereof, and methods of using this 9-marker panel,
particularly for uses related to lung cancer (such as detecting
lung cancer). In certain embodiments, one or more members of this
9-marker panel is replaced by one or more markers shown in Table 4
and/or one or more markers shown in Table 4 is added to this
9-marker panel. With respect to the nine markers Cyfra, SLPI,
TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK, elevated levels are
indicative of lung cancer (for all of the LCM disclosed herein,
elevated levels are indicative of lung cancer, except for sortilin
(SORT1), for which low levels are indicative of lung cancer). In
certain embodiments, if the levels of two or more markers (i.e., a
"plurality") of the 9-marker panel are elevated in a sample (e.g.,
a serum sample) from an individual, this indicates that the
individual has lung cancer. In various other embodiments, if the
levels of one or more, three or more, four or more, five or more,
six or more, seven or more, eight or more, or all nine markers of
the 9-marker panel are elevated in a sample from an individual,
this indicates that the individual has lung cancer. In certain
embodiments, a marker is classified as being elevated if its level
is greater than (or greater than or equal to) a predetermined
cutoff level.
[0051] Furthermore, certain embodiments of the invention provide
combinations comprising, consisting of, and consisting essentially
of the following six LCM (each of which is also contained in the
above 9-marker panel), and subcombinations thereof (these six LCM
may be referred to herein as the "6-marker subset of the 9-marker
panel", which is shown in Table 5): Cyfra, SLPI, TIMP1, TFPI,
CEACAM5, and MDK. Certain embodiments of the invention provide
compositions based on this 6-marker subset of the 9-marker panel
and subcombination thereof, and methods of using this 6-marker
subset of the 9-marker panel, particularly for uses related to lung
cancer (such as detecting lung cancer). In certain embodiments, one
or more members of this 6-marker subset of the 9-marker panel is
replaced by one or more markers shown in Table 4 and/or one or more
markers shown in Table 4 is added to this 6-marker subset of the
9-marker panel. With respect to the six markers Cyfra, SLPI, TIMP1,
TFPI, CEACAM5, and MDK, elevated levels are indicative of lung
cancer (for all of the LCM disclosed herein, elevated levels are
indicative of lung cancer, except for sortilin (SORT1), for which
low levels are indicative of lung cancer). In certain embodiments,
if the levels of two or more markers (i.e., a "plurality") of the
6-marker subset of the 9-marker panel are elevated in a sample
(e.g., a serum sample) from an individual, this indicates that the
individual has lung cancer. In various other embodiments, if the
levels of one or more, three or more, four or more, five or more,
or all six markers of the 6-marker subset of the 9-marker panel are
elevated in a sample from an individual, this indicates that the
individual has lung cancer. In certain embodiments, a marker is
classified as being elevated if its level is greater than (or
greater than or equal to) a predetermined cutoff level.
[0052] Exemplary embodiments of the invention provide LCM and
combinations of LCM (combinations of LCM may be interchangeably
referred to herein as panels), and uses thereof, particularly uses
related to lung cancer. For example, exemplary embodiments of the
invention provide methods and compositions for assessing (e.g.,
diagnosing/detecting, prognosing, or predicting drug response),
treating, and preventing diseases, especially cancer, and
particularly lung cancer, using LCM. Furthermore, the compositions
and methods of the invention may be suitable for other types of
cancer, particularly other epithelial cell-related cancers and
solid tumors, as well as other lung diseases.
[0053] LCM proteins and fragments thereof (LCM peptides), LCM
carbohydrate antigens and fragments thereof, and LCM nucleic acid
molecules and fragments thereof encoding LCM proteins and peptides,
are collectively referred to as "LCM" or "markers" (which may be
interchangeably referred to as "biomarkers", "antigens", or
"targets").
[0054] The terms "protein" and "polypeptide" are used herein
interchangeably. Furthermore, references herein to
proteins/polypeptides may also typically encompass carbohydrate
antigens ("CA"); for example, references to LCM
proteins/polypeptides may also typically encompass the carbohydrate
antigens CA 242, CA 19-9, and CA 72-4. Exemplary LCM
protein/polypeptide sequences are provided as SEQ ID NOS:1-65
(additionally, carbohydrate antigens CA 242, CA 19-9, and CA 72-4
are also provided). A "peptide" typically refers to a fragment of a
protein/polypeptide. Thus, peptides are interchangeably referred to
as fragments. References herein to proteins, peptides, carbohydrate
antigens, nucleic acid molecules, and antibodies typically are not
limited to the full-size or full-length molecule, but also can
encompass fragments of these molecules (unless a particular
sequence or structure is explicitly stated).
[0055] As used herein, a "lesion" (e.g., a lung lesion) may be
interchangeably referred to as a "nodule" (e.g., a lung nodule),
and "lung" may be interchangeably referred to as "pulmonary".
[0056] As used herein, "subcombinations" (of LCM) may be
interchangeably referred to as "subsets" (of LCM).
[0057] "Abundance level" may be interchangeably referred to herein
as "expression level", or just "level" or "abundance".
Determination of LCM levels may be referred to herein as
"quantifying" LCM, or "quantification" of LCM.
[0058] A "differential" abundance level is a level of a marker
(e.g., LCM protein or nucleic acid) in a test sample (e.g., a
disease sample) either above or below the normal abundance level of
the same marker in a corresponding control or normal sample or
group of control/normal samples (e.g., a sample set or population).
Thus, for example, a "differential" abundance level can encompass
either a "high" (or "increased") or "low" (or "decreased")
abundance level. An example of a normal abundance level for a LCM
is the mean abundance level of the marker in individuals who do not
have lung cancer, which may be the mean abundance of the marker in,
for example, a particular control sample set or population of
individuals who do not have lung cancer. The normal abundance may
also be the typical abundance level of a marker in a normal cell
(e.g., a normal lung cell) compared with the typical abundance
level of the marker in a corresponding disease cell (e.g., a lung
cancer cell).
[0059] An example of a "high", "increased", or "elevated" (these
terms are used herein interchangeably) abundance level for a LCM is
an abundance level that is at least two standard deviations above
the normal abundance level of the marker (e.g., the mean abundance
level of the marker in individuals who do not have lung cancer). An
example of a "low" or "decreased" abundance level for a LCM is an
abundance level that is at least two standard deviations below the
normal abundance level of the marker (e.g., the mean abundance
level of the marker in individuals who do not have lung cancer).
Thus, in this particular example, an abundance level that is
between 2 standard deviations above and 2 standard deviations below
the mean abundance level of the marker in individuals who do not
have lung cancer may be considered within a normal abundance level
range. These are merely exemplary cut-offs which can be used to
label an abundance level of a marker as "high"/"increased" or
"low"/"decreased".
[0060] In alternative exemplary embodiments, the cut-offs for a
"high"/"increased" or "low"/"decreased" abundance can be an
abundance level that is greater that one standard deviation above
or below the normal abundance level, or greater that three standard
deviations above or below the normal abundance level, or any other
desired standard deviation. In further alternative exemplary
embodiments, the cut-offs for a "high"/"increased" or
"low"/"decreased" abundance can be based directly on the expression
ratio or fold difference, for example, a 2-fold increase/decrease,
3-fold increase/decrease, or 4-fold increase/decrease, or any other
desired degree of increase/decrease. Further, the normal abundance
level can be based on, for example, either the mean or median
abundance level (e.g., of a given control sample set). Other
exemplary methods for developing cut-offs for "high"/"increased" or
"low"/"decreased" abundance levels include determining a normal
abundance level range (such as by testing a panel of markers in a
control sample set of normal lung tissue samples), and classifying
any test samples above or below this normal range (or above/below a
desired threshold relative to this normal range, such as outside a
particular percentage of samples within this normal range such as
above or below 95% of samples within the normal range) as
"high"/"increased" or "low"/"decreased", respectively.
[0061] A wide variety of further cut-offs for classifying the
abundance level of a marker as "high"/"increased" or
"low"/"decreased", and methods for formulating these cut-offs, are
known in the art and/or can be implemented by one of ordinary skill
in the art. For a given marker or panel of markers, various
cut-offs can be applied, such as cut-offs that maximize sensitivity
while maintaining a desired specificity, for example, or that
maximize specificity while maintaining a desired sensitivity. For
example, the classification of a sample as a tumor sample or normal
sample can be accomplished using a variety of methods that may
involve using a set of training data to produce a model that can
then be used to classify a test sample (such as to diagnose lung
cancer, for example). Tumor/normal cut-offs can be selected by
manual inspection of multiple markers from the training data set,
and these cut-offs can be applied to classifying test samples (such
as to characterize patient samples with respect to lung cancer).
Exemplary methods include, but are not limited to, split-point
analysis (e.g., Mor et al., "Serum protein markers for early
detection of ovarian cancer", Proc Natl Acad Sci USA. 2005 May 24;
102(21):7677-82, incorporated herein by reference), logistic
regression analysis (e.g., Planque et al., "A multiparametric serum
kallikrein panel for diagnosis of non-small cell lung carcinoma",
Clin Cancer Res. 2008 Mar. 1; 14(5):1355-62, incorporated herein by
reference), Naive Bayes, multivariate analysis, decision tree
modeling (e.g., Patz et al., "Panel of serum biomarkers for the
diagnosis of lung cancer", J Clin Oncol (2007), 25, 5578-5583), and
other classification methods (see, for example, Dudoit et al.,
"Classification in Microarray Experiments", Statistical Analysis of
Gene Expression Microarray Data, 2003, Chapman & Hall/CRC:
93-158, incorporated herein by reference).
[0062] The terms "sensitivity" and "specificity" are used herein
with respect to the ability of one or more markers to correctly
classify a sample as a tumor sample or a non-tumor sample (a
non-tumor sample may be interchangeably referred to as a "normal",
"control", or "healthy" sample), respectively. "Sensitivity"
indicates the performance of the marker(s) with respect to
correctly classifying tumor samples. "Specificity" indicates the
performance of the marker(s) with respect to correctly classifying
non-tumor samples. For example, 98% specificity and 85% sensitivity
for a panel of markers used to test a set of control and tumor
samples indicates that 98% of the control samples were correctly
classified as control samples by the panel, and 85% of the tumor
sample were correctly classified as tumor samples by the panel.
[0063] Area under the curve (AUC) refers to the area under the
curve of a receiver operating characteristic (ROC) curve, which are
well known in the art (see, e.g., Planque et al., "A
multiparametric serum kallikrein panel for diagnosis of non-small
cell lung carcinoma", Clin Cancer Res. 2008 Mar. 1; 14(5):1355-62,
incorporated herein by reference). AUC measures are useful for
comparing the accuracy of a classification algorithm across the
complete data range. Classification algorithms with a greater AUC
have a greater capacity to classify unknowns correctly between two
groups of interest (e.g., lung cancer samples and normal samples).
ROC curves are useful for plotting the performance of a particular
feature (e.g., an LCM and/or a supplemental biomedical parameter)
in distinguishing between two populations (e.g., cases having lung
cancer and controls without lung cancer). Typically, the feature
data across the entire population (e.g., the cases and controls)
are sorted in ascending order based on the value of a single
feature. Then, for each value for that feature, the true positive
and false positive rates for the data are calculated. The true
positive rate is determined by counting the number of cases above
the value for that feature and then dividing by the total number of
cases. The false positive rate is determined by counting the number
of controls above the value for that feature and then dividing by
the total number of controls. Although this definition refers to
scenarios in which a feature is elevated in cases compared to
controls, this definition also applies to scenarios in which a
feature is lower in cases compared to the controls (in such a
scenario, samples below the value for that feature would be
counted). ROC curves can be generated for a single feature as well
as for other single outputs, for example, a combination of two or
more features can be mathematically combined (e.g., added,
subtracted, multiplied, etc.) to provide a single sum value, and
this single sum value can be plotted in a ROC curve. Additionally,
any combination of multiple features, in which the combination
derives a single output value, can be plotted in a ROC curve. These
combinations of features may comprise a test. The ROC curve is the
plot of the true positive rate (sensitivity) of a test against the
false positive rate (specificity) of the test.
[0064] Exemplary embodiments of the invention, which are discussed
in greater detail below, provide antibodies, proteins, carbohydrate
antigens, immunogenic peptides (e.g., peptides which induce a
T-cell response), or other biomolecules, as well as small
molecules, nucleic acid agents (e.g., RNAi and antisense nucleic
acid agents), and other compositions that modulate the markers
(e.g., agonists and antagonists), such as by binding to or
otherwise interacting with or affecting the markers. These
compositions can be used for assessing, treating, and preventing
diseases, especially cancer, and particularly lung cancer, as well
as other uses. Moreover, the invention provides methods for
assessing, treating, and preventing diseases such as lung cancer,
particularly by using these compositions. Further provided are
methods of screening for agents that modulate LCM, such as by
affecting the function, activity, and/or expression level of LCM,
and agents identified by these screening methods.
[0065] Exemplary embodiments of the invention also provide methods
of modulating cell function, especially lung cell function. In
particular, the invention provides methods of modulating cell
proliferation and/or apoptosis. For example, for cancer/tumor
cells, the invention provides methods of inhibiting cell
proliferation and/or stimulating apoptosis. Such methods can be
applied to the treatment of diseases, especially cancer, and
particularly lung cancer. In certain exemplary embodiments, the
invention provides methods of treating lung cancer by targeting LCM
to thereby inhibit proliferation of lung cancer cells and/or
stimulate apoptosis of lung cancer cells.
[0066] Exemplary embodiments of the invention further provide
methods of determining or predicting effectiveness or response to a
particular treatment, and methods of selecting a treatment for an
individual, particularly a lung cancer treatment. For example,
markers that are differentially expressed by cells (e.g., lung
cancer cells) that are more or less responsive (sensitive) or
resistant to a particular treatment, such as a cancer treatment,
are useful for determining or predicting effectiveness or response
to the treatment or for selecting a treatment for an
individual.
[0067] Exemplary embodiments of the invention also provide methods
of selecting individuals for a clinical trial of a therapeutic
agent, particularly a clinical trial for lung cancer or other
cancer. For example, the markers can be used to identify
individuals for inclusion in a clinical trial who are more likely
to respond to a particular therapeutic agent. Alternatively, the
markers can be used to exclude individuals from a clinical trial
who are less likely to respond to a particular therapeutic agent or
who are more likely to experience toxic or other undesirable side
effects from a particular therapeutic agent. Furthermore, such
individuals who are determined to be less likely to respond to a
particular therapeutic agent can be selected for inclusion in a
clinical trial of a different therapeutic agent that may
potentially benefit them.
[0068] In certain exemplary embodiments, the various individual LCM
and LCM panels described herein are provided as compositions. For
example, in certain embodiments, each of the members of an LCM
panel, and/or reagents for detecting each of these members, are
provided as individual compositions, such as in the form of
reagents for detecting each member of an LCM panel by ELISA assays
(which may be referred to herein as "ELISA reagents"). Furthermore,
in certain embodiments, compositions that comprise multiple members
of a panel or an entire panel (and/or reagents for detecting each
of these multiple members), are provided, such as in the form of
kits that contain reagents (such as ELISA reagents) for detecting
multiple members of a panel or an entire panel. Other compositions
of the invention include arrays or other platforms that have
multiple LCM, or multiple reagents (e.g., antibodies) for detecting
multiple LCM, coupled to a substrate. In various compositions of
the invention, the LCM, or reagents for detecting LCM (e.g.,
antibodies), are labeled with a detectable moiety (such as a
fluorescent label).
[0069] Exemplary LCM Combinations/Panels
[0070] For example, using a panel of sera from 12 lung cancer
patients and 12 healthy control individuals, a group of 8 markers
made up of TFPI, SCC, CEA, CA242, MN/CAIX, OPN, Cyfra 21-1, and MIF
(FIG. 2) detected all the cancer samples except a bronchioalveolar
cancer sample (which is biologically distinct from other samples in
the panel), and only a few of these markers were detected at levels
above the threshold in the healthy control samples. When a simple
algorithm was applied (i.e., markers greater than or equal to two
standard deviations were scored "positive", using the criterion
stated above), this group of eight markers had a sensitivity of 92%
and specificity of 100% among the 12 sera from lung cancer patients
and 12 sera from healthy controls (no false positives, 11 true
positives, 1 false negative, and 12 true negatives) (FIG. 3).
[0071] An alternate panel was configured that was made up of
following markers: SLPI, TFPI, OPN, MIF, TIMP1, and MMP2 (FIG. 4).
Any or all of these markers can also be used in any combination
with any or all of the following markers: CA242, SCC, CEA, NSE,
CA72-4, CA19-9, Cyfra 21-1, and MN/CAIX (FIG. 4).
[0072] Further, a six-marker panel made-up of Cyfra 21-1, TIMP-1,
MIF, TFPI, CEA, and OPN was also configured (FIG. 5). This
six-marker panel, when tested on a larger group of 44 lung tumor
sera and 44 normal sera, resulted in 75% sensitivity at 95%
specificity.
[0073] Further, an 11-marker panel made-up of Cyfra, MIF, TIMP1,
TFPI, CEA, OPN, SCC, SLPI, HNP-1, GRP, and CA242 was also
configured (FIG. 6). This 11-marker panel, when tested on a group
of 39 lung tumor sera and 39 normal sera, resulted in 98%
specificity for controls (38/39 controls) and 85% sensitivity for
tumor sera (33/39 tumors) (FIG. 6).
[0074] Table 3 shows further examples of various 11-marker panels.
Specifically, Table 3 provides 35 different panels of 11 markers
(each row of 11 markers represents a panel) that have at least 98%
specificity and 82% sensitivity for detecting lung cancer. Seven
markers (SLPI, TIMP1, TFPI, SCC, OPN, CEA and CA242) appear in all
35 of these panels, GRP appears in 33 of the 35 panels, MIF appears
in 29 of the 35 panels, and NSE and HNP-1 each appear in 15 of the
35 panels. AMBP, Cyfra, MMP2, Ca72-4, Ca19-9, and CAIX each appear
in 7-9 of the panels, as indicated in Table 3.
[0075] Further, a 9-marker panel made-up of Cyfra, SLPI, TIMP1,
SCC, TFPI, CEACAM5, MMP2, OPN, and MDK was also configured (e.g.,
FIG. 17 and Table 5). This 9-marker panel demonstrated 98%
specificity (49/50 controls) and 96% sensitivity (48/50 tumors)
(FIG. 17). Additionally, a 6-marker subset of this 9-marker panel
was also configured that was made-up of Cyfra, SLPI, TIMP1, TFPI,
CEACAM5, and MDK (Table 5).
[0076] Other markers, which are also referred to herein as LCM and
which may be used either alone or in combination with any of the
other LCM described herein in any combination, include a group of
antigens to which "self-made" or "autoantibodies" are often found
in the circulation of patients with various diseases, particularly
cancer (Table 2 and FIGS. 11-14). Examples of these autoantibody
markers include the following: KLKB1, CFL1, LGAGS1, EEF1G, RTN4,
ALDOA, HSPCA, PABPC4, NAGK, CFHL1, CSF1R, and RANBP2 (FIG. 12), and
other autoantibody markers as shown in Table 2. Detection of
autoantibody LCM such as these may complement other LCM and enhance
the performance of LCM panels, particularly for assessing lung
cancer.
[0077] The following are exemplary panels of LCM. Various exemplary
embodiments of the invention provide, for example, compositions
based on these panels and methods of using these panels,
particularly for uses related to lung cancer such as diagnosis of
lung cancer (e.g., differential levels, such as elevated or low
levels as compared to control/normal levels, of a plurality of
markers in a panel, or all markers in panel, can indicate the
presence of lung cancer). These exemplary panels may consist of,
consist essentially of, or comprise the following combinations of
markers: [0078] 1) Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK (which may be referred to herein as the "9-marker
panel" and is shown in FIG. 17 and Table 5). [0079] 2) Cyfra, SLPI,
TIMP1, TFPI, CEACAM5, and MDK (which may be referred to herein as
the "6-marker subset of the 9-marker panel" and is shown in Table
5). [0080] 3) Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN,
and MDK (the "9-marker panel"), which may optionally be in
combination with one or more other markers (which may be added to
this 9-marker panel and/or replace one or more members of this
9-marker panel), wherein these other markers may optionally be
selected from the group consisting of the markers shown in Tables
1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9,
and CA 72-4), Table 4, FIG. 1, and FIG. 12, particularly those
markers that are shown in Table 4. [0081] 4) Cyfra, SLPI, TIMP1,
TFPI, CEACAM5, and MDK (the "6-marker subset of the 9-marker
panel"), which may optionally be in combination with one or more
other markers (which may be added to this 6-marker subset of the
9-marker panel and/or replace one or more members of this 6-marker
subset of the 9-marker panel), wherein these other markers may
optionally be selected from the group consisting of the markers
shown in Tables 1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens
CA 242, CA 19-9, and CA 72-4), Table 4, FIG. 1, and FIG. 12,
particularly those markers that are shown in Table 4. [0082] 5) Any
of the panels (which may include single markers) provided in Table
5 (which provides the 9-marker panel and all subcombinations
thereof; each row of Table 5 represents a different panel), which
may optionally be in combination with one or more other markers
(which may be added to any panel in Table 5 and/or replace one or
more members of any panel in Table 5), wherein these other markers
may optionally be selected from the group consisting of the markers
shown in Tables 1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens
CA 242, CA 19-9, and CA 72-4), Table 4, FIG. 1, and FIG. 12,
particularly those markers that are shown in Table 4. [0083] 6) Any
of the panels provided in Table 5 or Table 6 (particularly the
9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK, as well as subsets thereof, and panels comprising
this 9-marker panel or subsets thereof that further include one or
more additional markers such as those panels set forth in Table 6),
particularly for use in methods for distinguishing lung tumor
samples versus normal (i.e., control/healthy) samples. These panels
are particularly useful for determining whether an individual has
lung cancer (i.e., detecting lung cancer), for example. [0084] 7)
Any of the panels provided in Table 7 (particularly the 9-marker
panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and
MDK, as well as subsets thereof), particularly for use in methods
for distinguishing adenocarcinoma versus squamous cell carcinoma.
These panels are particularly useful for determining whether an
individual's lung cancer is adenocarcinoma or squamous cell
carcinoma, for example. [0085] 8) Any of the panels provided in
Table 8 (particularly the 9-marker panel of Cyfra, SLPI, TIMP1,
SCC, TFPI, CEACAM5, MMP2, OPN, and MDK, as well as subsets
thereof), particularly for use in methods for distinguishing
between any stages of lung cancer (e.g., any of stages I, II, III,
and IV), particularly between early stage (stage I or II) and late
stage (stage III or IV) lung cancer, and especially between stage I
and stage III lung cancer. These panels are particularly useful for
determining the stage of an individual's lung cancer, for example.
[0086] 9) Any of the panels provided in Table 9, particularly for
use in methods for distinguishing SCLC versus other types of lung
cancer (e.g., NSCLC). These panels are particularly useful for
determining whether an individual's lung cancer is SCLC or NSCLC,
for example. [0087] 10) Any of the panels provided in Table 10
(particularly the 9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI,
CEACAM5, MMP2, OPN, and MDK, as well as subsets thereof),
particularly for use in methods for distinguishing malignant lung
tumors versus benign lung lesions. These panels are particularly
useful for determining whether a lung lesion identified in an
individual (such as by CT screening) is a malignant tumor or a
benign lesion, for example. [0088] 11) Any of the panels provided
in Table 11, particularly for use in methods for distinguishing
SCLC versus normal (i.e., control/healthy) samples. These panels
are particularly useful for determining whether an individual has
SCLC, for example. [0089] 12) Any of the panels provided in Table
12, particularly for use in methods for distinguishing lung cancer
(including both SCLC and NSCLC) versus normal (i.e.,
control/healthy) samples. These panels are particularly useful for
determining whether an individual has lung cancer such as SCLC or
NSCLC, for example. [0090] 13) Panels that include any or all of
the 11 markers provided in FIG. 19 (and subsets thereof, as well as
panels comprising these 11 markers or subsets thereof that further
include one or more additional markers), particularly for use in
methods of monitoring for lung tumor regression and/or recurrence.
These panels are particularly useful for monitoring for lung tumor
regression and/or recurrence, for example. [0091] 14) Cyfra 21-1,
MIF, TIMP1, TFPI, CEA, OPN, SCC, SLPI, HNP-1, GRP, and CA242 (which
may be referred to herein as the "11-marker panel" and is shown in
FIG. 6). [0092] 15) TFPI, CEA, MIF, TIMP1, OPN, and Cyfra 21-1
(which may be referred to herein as the "6-marker panel" and is
shown in FIG. 5). [0093] 16) TFPI, SCC, CEA, CA242, MN/CAIX, OPN,
Cyfra 21-1 and MIF (which may be referred to herein as the
"8-marker panel" and is shown in FIGS. 2-3). [0094] 17) TFPI, SLPI,
OPN, MIF, TIMP1, and MMP2, any or all of which can optionally be
used in combination with any or all of the following additional
markers: CA242, SCC, CEA, NSE, CA724, CA199, Cyfra 21-1, and
MN/CAIX (see FIG. 4). [0095] 18) TFPI, TIMP1, CEA, and OPN (see
FIG. 7). [0096] 19) TFPI, CEA, and OPN (see FIG. 7). [0097] 20) CEA
and OPN (see FIG. 7). [0098] 21) TFPI, MIF, TIMP1, CEA, and OPN
(see FIG. 9). [0099] 22) TIMP1, NSE, CEA, and OPN (see FIG. 10).
[0100] 23) TFPI, CEA, TIMP-1, NSE, SLPI, SCC, Cyfra 21-1, and MIF,
any or all of which can optionally be in combination with any or
all of the following autoantibody markers: p53, KLKB1, LGALS1,
CFLN, EEF1G, HSP90.alpha., RTN4, ALDOA, GLG1, PTK7, EFEMP1, CD98,
CHGB, B7H3, and CEACAM1 (see FIG. 11). [0101] 24) TFPI, SLPI,
TFPI2, CEA, and TIMP1. [0102] 25) TFPI, SLPI, and TIMP1. [0103] 26)
KLKB1 and cofilin (CFLN) (see FIG. 13). [0104] 27) KLKB1, cofilin
(CFLN), and CAl2 (see FIG. 14). [0105] 28) TFPI, either alone or in
combination with one or more other markers, which may optionally be
selected from the group consisting of the markers shown in Tables
1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9,
and CA 72-4), Table 4, FIG. 1, and FIG. 12. [0106] 29) One or more
markers selected from the group consisting of defensin (DEFA1,
HNP-1), ICAM3, CTGF, LCN2, biglycan, and HGF, either alone or in
combination with one or more other markers, which may optionally be
selected from the group consisting of the markers shown in Tables
1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9,
and CA 72-4), Table 4, FIG. 1, and FIG. 12. [0107] 30) Two or more
markers selected from the group consisting of TFPI, defensin,
ICAM3, CTGF, LCN2, biglycan, and HGF, either alone or in
combination with one or more other markers, which may optionally be
selected from the group consisting of the markers shown in Tables
1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9,
and CA 72-4), Table 4, FIG. 1, and FIG. 12. [0108] 31) One or more
markers shown in Table 1 (SEQ ID NOS:1-38 and 56-65 and the
carbohydrate antigens CA 242, CA 19-9, and CA 72-4) and/or FIG. 1,
in combination with one or more autoantibody markers shown in Table
2 (SEQ ID NOS:39-55) and/or FIG. 12. [0109] 32) Any of the
11-marker panels provided in Table 3 (each row of Table 3
represents a different 11-marker panel). [0110] 33) SLPI, TIMP1,
TFPI, SCC, OPN, CEA, and CA242, which may optionally be in
combination with GRP and/or MIF (see Table 3). [0111] 34) SLPI,
TIMP1, TFPI, SCC, OPN, CEA, and CA242, which may optionally be in
combination with GRP and/or MIF, and which may optionally further
be in combination with HNP-1 and/or NSE (see Table 3). [0112] 35)
SLPI, TIMP1, TFPI, SCC, OPN, CEA, and CA242, which may optionally
be in combination with any or all of GRP, MIF, HNP-1, and NSE, and
which may optionally further be in combination with any or all of
CAIX, Ca19-9, Ca72-4, MMP2, Cyfra 21-1, and AMBP (see Table 3).
[0113] 36) SLPI, TIMP1, TFPI, SCC, OPN, CEA, and CA242, which may
optionally be in combination with any or all of GRP, MIF, HNP-1,
NSE, and Cyfra 21-1. [0114] 37) One or more markers selected from
the group consisting of visfatin, sortilin, and midkine, either
alone or in combination with one or more other markers, which may
optionally be selected from the group consisting of the markers
shown in Tables 1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens
CA 242, CA 19-9, and CA 72-4), Table 4, FIG. 1, and FIG. 12.
[0115] Exemplary Uses of LCM
[0116] Certain exemplary embodiments of the invention relate to
methods of detecting the presence of lung cancer in an individual
by measuring the amounts of circulating LCM, such as in serum, by
immunological methods or other methods. These LCM are, for example,
differentially expressed (over- or under-expressed) in individuals
with lung cancer as compared to individuals without lung cancer
(individuals without lung cancer are interchangeably referred to
herein as "normal", "control", or "healthy" individuals). Detection
of variation from a "normal" expression level, or differential
expression, can be used for, for example, early diagnosis of lung
cancer, distinguishing between a benign and malignant lung lesion
(such as a lesion observed on a CT scan), monitoring lung cancer
recurrence, or other clinical indications.
[0117] LCM may be used in a variety of clinical indications for
lung cancer, including, but not limited to, detection of lung
cancer (such as in a high-risk individual or population),
characterizing lung cancer (e.g., determining lung cancer type,
sub-type, or stage) such as distinguishing between non-small cell
lung cancer (NSCLC) and small cell lung cancer (SCLC) and/or
between adenocarcinoma and squamous cell carcinoma (or otherwise
facilitating histopathology), determining whether a lung lesion is
a benign lesion or a malignant lung tumor, lung cancer prognosis,
monitoring lung cancer progression or remission, monitoring for
lung cancer recurrence, monitoring metastasis, treatment selection,
monitoring response to a therapeutic agent or other treatment,
stratification of patients for computed tomography (CT) screening
(e.g., identifying those patients at greater risk of lung cancer
and thereby most likely to benefit from spiral-CT screening, thus
increasing the positive predictive value of CT), combining LCM
testing with supplemental biomedical parameters such as smoking
history, etc., or with nodule size, morphology, etc. (such as to
provide an assay with increased diagnostic performance compared to
CT testing or LCM testing alone), facilitating the diagnosis of a
pulmonary nodule as malignant or benign, facilitating clinical
decision making once a lung cancer lesion is observed on CT (e.g.,
ordering repeat CT scans if the lesion is deemed to be low risk,
such as if an LCM-based test is negative, with or without
categorization of lesion size, or considering biopsy if the lesion
is deemed medium to high risk, such as if an LCM-based test is
positive, with or without categorization of lesion size), and
facilitating decisions regarding clinical follow-up (e.g., whether
to implement repeat CT scans, fine needle biopsy, or thoracotomy
after observing a non-calcified lesion on CT). LCM testing may
improve positive predictive value (PPV) over CT screening alone. In
addition to their utilities in conjunction with CT screening, LCM
can also be used in conjunction with any other imaging modalities
used for lung cancer, such as chest X-ray. Furthermore, LCM may
also be useful for enabling certain of these uses to be achieved
before indications of lung cancer are detected by imaging
modalities or other clinical correlates, or before symptoms
appear.
[0118] As examples of how LCM may be useful for diagnosing lung
cancer, a high or low abundance level (i.e., a "differential"
abundance level) of one or more LCM in an individual who is not
known to have lung cancer may indicate that the individual has lung
cancer, thereby enabling early detection of lung cancer at an early
stage of the disease when treatment is most effective, perhaps
before the lung cancer is detected by other means or before
symptoms appear. An increase in the abundance of one or more LCM
during the course of lung cancer may be indicative of lung cancer
progression, e.g., a lung tumor is growing and/or metastasizing
(and thus a poor prognosis), whereas a decrease in the abundance of
one or more LCM may be indicative of lung cancer remission, e.g., a
lung tumor is shrinking (and thus a good prognosis). Similarly, an
increase in the abundance of one or more LCM during the course of
lung cancer treatment may indicate that the lung cancer is
progressing and therefore indicate that the treatment is
ineffective, whereas a decrease in the abundance of one or more LCM
during the course of lung cancer treatment may be indicative of
lung cancer remission and therefore indicate that the treatment is
working successfully. Additionally, an increase or decrease in the
abundance of one or more LCM after an individual has apparently
been cured of lung cancer may be indicative of lung cancer
recurrence. In a situation such as this, for example, the
individual can be re-started on therapy (or the therapeutic regimen
modified such as to increase dosage amount and/or frequency, if the
patient has maintained therapy) at an earlier stage than if the
recurrence of lung cancer was not detected until later.
Furthermore, a differential abundance level of one or more LCM in
an individual may be predictive of the individual's response to a
particular therapeutic agent. In monitoring for lung cancer
recurrence or progression, changes in LCM levels may indicate the
need for repeat imaging (e.g., repeat CT scanning), such as to
determine lung cancer activity, or the need for changes in
treatment.
[0119] Detection of LCM may be particularly useful following, or in
conjunction with, lung cancer treatment, such as to evaluate the
success of the treatment or to monitor lung cancer remission,
recurrence, and/or progression (including metastasis) following
treatment. Lung cancer treatment may include, for example,
administration of a therapeutic agent to a patient, surgery (e.g.,
surgical resection of at least a portion of a lung tumor),
radiation therapy, or any other type of lung cancer treatment used
in the art, and any combination of these treatments. For example,
LCM may be detected at least once after treatment or may be
detected multiple times after treatment (such as at periodic
intervals), or may be detected both before and after treatment. A
differential abundance level of LCM, such as an increase or
decrease in the abundance level of LCM after treatment compared
with the abundance level of LCM before treatment, or an increase or
decrease in the abundance level of LCM at a later time point after
treatment compared with the abundance level of LCM at an earlier
time point after treatment, or a differential abundance level of
LCM at a single time point after treatment compared with normal
levels of LCM, may be indicative of lung cancer progression,
remission, or recurrence.
[0120] As a specific example, ELISA analysis of LCM levels in
pre-surgery and post-surgery (e.g., 2-4 weeks after surgery) serum
samples can be carried out. An increase in the level of LCM in the
post-surgery sample compared with the pre-surgery sample can
indicate progression of lung cancer (e.g., unsuccessful surgery),
whereas a decrease in the level of LCM in the post-surgery sample
compared with the pre-surgery sample can indicate regression of
lung cancer (e.g., the surgery successfully removed the lung
tumor). Similar analyses of LCM levels can be carried out before
and after other forms of treatment, such as before and after
radiation therapy or administration of a therapeutic agent or
cancer vaccine.
[0121] In addition to the utilities of testing LCM levels as
stand-alone screening tests, testing of LCM levels can also be done
in conjunction with CT screening. For example, LCM may facilitate
the medical and economic justification for implementing CT
screening, such as to screen large asymptomatic populations at risk
for lung cancer (e.g., smokers). For example, a "pre-CT" test of
LCM levels could be used to stratify high-risk individuals for CT
screening, such as to identify those who are at highest risk for
lung cancer based on their LCM levels and who should be prioritized
for CT screening. If a CT test is implemented, LCM levels (e.g., as
determined by immunoassay of serum samples) of one or more LCM can
be measured and the scores added to scores for supplemental
biomedical parameters (e.g., tumor parameters determined by CT
testing) to create a combined score, such as to enhance positive
predictive value (PPV) over CT or LCM testing alone. A "post-CT"
immunoassay panel for determining LCM levels can be used to
determine the likelihood that a pulmonary lesion observed by CT (or
other imaging modality) is malignant or benign.
[0122] Detection of LCM may be useful for post-CT testing. For
example, LCM testing may eliminate a significant number of false
positive tests over CT alone. Further, LCM testing may facilitate
treatment of patients. As an example, if a lung tumor is less than
5 mm in size, results of LCM testing may move patients from "watch
and wait" to biopsy at an earlier time, if a lung tumor is 5-9 mm,
LCM testing may eliminate biopsy or thoracotomy on false positive
scans, and if a lung tumor is larger than 10 mm, LCM testing may
eliminate surgery for sub-population of these patients with benign
lesions. Eliminating the need for biopsy in some patients based on
LCM testing would be beneficial because there is significant
morbidity associated with nodule biopsy and difficulty in obtaining
nodule tissue depending on location of nodule. Similarly,
eliminating the need for surgery in some patients, such as those
whose lesions are actually benign, would avoid unnecessary risks
and costs associated with surgery.
[0123] In addition to testing LCM levels in conjunction with CT
screening (e.g., assessing LCM levels in conjunction with size or
other characteristics of a lung nodule observed on a CT scan),
information regarding LCM can also be evaluated in conjunction with
other types of data, particularly data that indicates an
individual's risk for lung cancer (e.g., patient clinical history,
symptoms, family history of cancer, risk factors such as whether or
not the individual is a smoker, and/or status of other biomarkers,
etc.). These various data can be assessed by automated methods,
such as a computer program/software, which can be embodied in a
computer or other apparatus/device.
[0124] The various methods described herein, such as correlating
the level of LCM in an individual with an altered (e.g., increased
or decreased) risk (or no altered risk) for lung cancer, can be
carried out by automated methods such as by using a computer (or
other apparatus/devices such as biomedical devices, laboratory
instrumentation, or other apparatus/devices having a computer
processor) programmed to carry out any of the methods described
herein. For example, computer software (which may be
interchangeably referred to herein as a computer program) can
perform the step of correlating the level of LCM in an individual
with an altered (e.g., increased or decreased) risk (or no altered
risk) of lung cancer for the individual. Accordingly, certain
embodiments of the invention provide a computer (or other
apparatus/device) programmed to carry out any of the methods
described herein.
[0125] LCM may also be used in imaging tests. For example, an
imaging agent can be coupled to an LCM, which can be used to aid in
lung cancer diagnosis, to monitor disease progression/remission or
metastasis, to monitor for disease recurrence, or to monitor
response to therapy, among other uses.
[0126] LCM can be detected using a variety of platforms. For
example, LCM may be detected using singleplex ELISAs,
ultrasensitive detection technologies, multiplex formats, and/or
automated immuno analyzers.
[0127] In addition to detecting LCM in serum, LCM may also be
detected in, for example, plasma and bronchial lavage.
[0128] LCM may be used for pharmacoproteomic or pharmacogenomic
applications; for example, detection of LCM may be used for
treatment selection or stratification. Differential expression of
LCM in, for example, tumor cells that are resistant to a treatment
(e.g., a particular therapeutic agent) and tumor cells that are
sensitive to a treatment can be used to predict resistance or
sensitivity of an individual's lung cancer to the treatment. As
specific examples, CTGF is secreted at elevated levels by cell
lines that are resistant to the chemotherapeutic agent Topotecan.
In contrast, TIMP1, TFPI, and TIMP2 are secreted at elevated levels
by cell lines that are sensitive to the chemotherapeutic agent
Iressa. LCM may also be used as treatment response markers for a
particular therapeutic agent. For example, certain LCM may be used
as surrogate markers of cisplatin or Iressa treatment response.
[0129] Thus, the LCM profile of an individual having lung cancer
can be used to determine which treatment(s) are best suited for
that particular individual. For example, treatments to which an
individual's lung cancer is predicted to be sensitive can be
selected for the individual rather than treatments to which the
individual's lung cancer is predicted to be resistant. As a further
example, LCM levels can be used by a medical practitioner to
distinguish between types of lung cancer (e.g., non-small cell lung
cancer (NSCLC) versus small cell lung cancer (SCLC), adenocarcinoma
versus squamous cell carcinoma, different stages of lung cancer, or
other lung cancer characteristics) in order to adjust therapy
options (e.g., to select a particular therapeutic agent or a
particular form of treatment, such as chemotherapy, surgery, or
radiation therapy, that is best suited for that particular subtype
of lung cancer).
[0130] Tables 5-6 and 11-12 provide panels that are particularly
well-suited for diagnosing/detecting lung cancer, among other lung
cancer-related uses. For example, Tables 5 and 6 provides LCM
panels that are particularly well-suited for distinguishing lung
tumor samples versus normal (i.e., control/healthy) samples, Table
11 provides LCM panels that are particularly well-suited for
distinguishing SCLC versus normal samples, Table 12 provides LCM
panels that are particularly well-suited for distinguishing lung
cancer (including both SCLC and NSCLC) versus normal samples.
[0131] Any of the LCM and the various exemplary LCM panels
disclosed herein (such as any of the panels provided in Tables 5-6,
such as the 9-marker panel or the 6-marker subset of this 9-marker
panel, as well as any LCM provided in Tables 1-2 (SEQ ID NOS:1-65
and the carbohydrate antigens CA 242, CA 19-9, and CA 72-4), Table
4, FIG. 1, and FIG. 12, and any panels that include one or more of
these LCM, particularly panels that include one or more markers
provided in Table 4) may be used for any of the various lung
cancer-related uses disclosed herein. However, certain LCM panels
are particularly well-suited for certain specific lung
cancer-related uses ("indications"); these specific uses may be
referred to herein as determining or assessing various
"characteristics" of lung cancer. Examples of such LCM panels that
are particularly well-suited for certain specific lung
cancer-related uses are provided in Tables 7-10 and FIG. 19. For
example, Table 7 provides LCM panels that are particularly
well-suited for distinguishing adenocarcinoma versus squamous cell
carcinoma types of lung cancer, Table 8 provides LCM panels that
are particularly well-suited for distinguishing between different
stages of lung cancer (such as between early-stage and late-stage
lung cancer such as stage I versus stage III lung cancer, or
between any other of stages I, II, III, and IV) such as to
determine the stage of lung cancer in a patient, Table 9 provides
LCM panels that are particularly well-suited for distinguishing
SCLC versus other types of lung cancer (e.g., NSCLC), Table 10
provides LCM panels that are particularly well-suited for
distinguishing malignant lung tumors versus benign lung lesions,
and FIG. 19 provides LCM that are particularly well-suited for
monitoring for lung tumor regression and/or recurrence.
[0132] Tables 5-12 provide a variety of exemplary LCM panels,
together with performance characterisitics for each panel (AUC,
sensitivity, and specificity). In certain embodiments, LCM panels
are provided that have at least 70% sensitivity at 95% specificity,
or at least 70% specificity at 95% sensitivity. In certain
embodiments, LCM panels are provided that have at least 85%
sensitivity at 95% specificity, or at least 85% specificity at 95%
sensitivity. In further embodiments, LCM panels are provided that
have at least 90% sensitivity or at least 90% specificity, or that
have at least 95% sensitivity or at least 95% specificity. In yet
further embodiments, LCM panels are provided that have at least 70,
75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% (or any
other percentage in-between) sensitivity and 70, 75, 80, 85, 90,
91, 92, 93, 94, 95, 96, 97, 98, or 99% (or any other percentage
in-between) specificity. In yet further embodiments, LCM panels are
provided that have at least 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92,
0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99 (or any other value
in-between) AUC values. Any of these panels are particularly useful
for lung-cancer related uses such as those described herein (e.g.,
diagnosing lung cancer), such as in clinical practice. However, the
desired performance for clinical use of an assay may vary depending
on such factors as the particular use, point of implementation, or
other factors.
[0133] Distinguishing NSCLC and SCLC
[0134] The following panels of LCM are particularly useful for
distinguishing (which may be interchangeably referred to as
"resolving") non-small cell lung carcinoma (NSCLC) and small cell
lung carcinoma (SCLC) from each other and/or from normal (i.e.,
control/healthy) samples. These panels may consist of, consist
essentially of, or comprise the following combinations of markers:
[0135] 1) Any of the panels provided in Table 9, particularly for
distinguishing NSCLC versus SCLC. [0136] 2) Any of the panels
provided in Table 11, particularly for distinguishing SCLC versus
normal samples. [0137] 3) Any of the panels provided in Table 12,
particularly for distinguishing SCLC and NSCLC versus normal
samples. [0138] 4) OPN (either alone or in combination with one or
more other markers), particularly for distinguishing NSCLC from
SCLC and for distinguishing SCLC from NSCLC. [0139] 5) SCC, OPN,
AMBP, and Ca72-4, particularly for distinguishing NSCLC from SCLC
(levels of these LCM are higher in NSCLC as compared to SCLC).
[0140] 6) ENO2, MMP2, Ca19-9, CAIX, and GRP, particularly for
distinguishing SCLC from NSCLC (levels of these LCM are higher in
SCLC as compared to NSCLC). [0141] 7) Cyfra, SLPI, TIMP1, TFPI,
CEACAM5, MMP2, and CA242, particularly for distinguishing SCLC from
normal samples (particularly by using split-point analysis). [0142]
8) SLPI, TIMP1, TFPI, CEACAM5, MMP2, OPN, and CA242, particularly
for distinguishing SCLC from normal samples (particularly by using
split-point analysis). [0143] 9) Cyfra, TIMP1, ENO2, TFPI, CEACAM5,
MMP2, OPN, and DEFA1, particularly for distinguishing NSCLC and
SCLC from normal samples (particularly by using split-point
analysis). [0144] 10) Cyfra, MIF, TIMP1, SCC, TFPI, CEACAM5, OPN,
and DEFA1, particularly for distinguishing NSCLC and SCLC from
normal samples (particularly by using split-point analysis). [0145]
11) TIMP1, ENO2, TFPI, CEACAM5, MMP2, OPN, AMBP, and DEFA1,
particularly for distinguishing NSCLC and SCLC from normal samples
(particularly by using split-point analysis).
[0146] Supplemental Biomedical Parameters
[0147] The term "supplemental biomedical parameters" refers to one
or more assessments of an individual, other than LCM, that are
associated with lung cancer risk. "Supplemental biomedical
parameters" include, but are not limited to, physical descriptors
of a patient, physical descriptors of a pulmonary nodule observed
by CT imaging, the height and/or weight of a patient, the gender of
a patient, smoking history, occupational history, exposure to
carcinogens, exposure to second-hand smoke, family history of lung
cancer (or other cancer), the presence of pulmonary nodules, size
of nodules, location of nodules, morphology of nodules (e.g.,
nodules may be observed by CT imaging), etc. Smoking history is
usually quantified in terms of "pack years", which refers to the
number of years a person has smoked multiplied by the average
number of packs smoked per day. For example, a person who has
smoked, on average, one pack of cigarettes per day for 35 years is
referred to as having 35 pack years of smoking history.
Supplemental biomedical parameters can be obtained from an
individual using routine techniques known in the art, such as from
the individual themselves by use of a routine patient questionnaire
or health history questionnaire, etc., or from a medical
practitioner, etc. Alternately, supplemental biomedical parameters
can be obtained from routine imaging techniques including CT
imaging (e.g., low-dose CT imaging) and X-ray.
[0148] Testing of LCM in combination with an assessment of
supplemental biomedical parameters may, for example, improve
sensitivity, specificity, and/or AUC for detecting lung cancer (or
other lung cancer-related uses) as compared to LCM testing alone or
assessing supplemental biomedical parameters alone (e.g., CT
imaging alone).
[0149] Accordingly, any of the LCM, and panels of LCM, can be used
in combination with supplemental biomedical parameters.
Furthermore, supplemental biomedical parameters may serve to
replace one or more markers of a panel, such as to enable the use
of smaller panels (i.e., panels with fewer biomarkers) while
retaining similar performance (e.g., sensitivity, specificity,
and/or AUC for detecting lung cancer). Thus, supplemental
biomedical parameters can be used in addition to a panel, or in
addition to one or more markers of a panel, or as a substitute for
one or more markers of a panel. As a specific example, one or more
supplemental biomedical parameters can be used in addition to the
9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK or the 6-marker subset of this 9-marker panel (Cyfra,
SLPI, TIMP1, TFPI, CEACAM5, and MDK). As another specific example,
one or more supplemental biomedical parameters can replace one or
more members of the 9-marker panel of Cyfra, SLPI, TIMP1, SCC,
TFPI, CEACAM5, MMP2, OPN, and MDK or the 6-marker subset of this
9-marker panel (Cyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK).
Furthermore, one or more supplemental biomedical parameters can be
used in addition to any of the panels provided in Table 5 and/or
can replace one or more members of any of the panels provided in
Table 5. Moreover, one or more supplemental biomedical parameters
can be used in addition to any of the markers or panels provided
herein and/or can replace one or more members of any of the panels
provided herein, including the markers provided in Tables 1-2 (SEQ
ID NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9, and CA
72-4), Table 4, FIGS. 1, 12, and 19, and the panels provided in
Tables 3 and 6-12. Furthermore, supplemental biomedical parameters
can be incorporated into algorithms and scoring
systems/classifiers, together with biomarker assessments (e.g.,
biomarker levels), for assessing lung cancer (e.g., diagnosing lung
cancer).
[0150] Examples of supplemental biomedical parameters include, but
are not limited to, any of the following. Any or all of these
supplemental biomedical parameters can be used, in any combination,
with any of the LCM and LCM panels disclosed herein. For example,
any of the LCM and LCM panels disclosed herein can be assessed
alone (without considering supplemental biomedical parameters) or
can be assessed in combination with CT results (for example), or
can be assessed in combination with any other supplemental
biomedical parameters, or can be assessed in combination with CT
results plus any other supplemental biomedical parameters, or any
other combination of supplemental biomedical parameters can be
assessed in combination with any of the LCM and LCM panels
disclosed herein. Any of these supplemental biomedical parameters
can be assessed as part of an algorithm or scoring
system/classifier, together with biomarker assessments (e.g.,
biomarker levels), such as for assessing lung cancer (e.g.,
diagnosing lung cancer).
[0151] 1) age, gender, and/or ethnicity;
[0152] 2) family history of lung cancer or other type of
cancer;
[0153] 3) smoking history (e.g., whether or not an individual
previously and/or currently smokes);
[0154] 4) smoking level (e.g., "pack year": number of cigarettes
smoked per day multiplied by number of years of smoking at this
rate);
[0155] 5) size of lesion;
[0156] 6) location of lesion;
[0157] 7) lesion morphology (ground glass opacity (GGO), solid,
non-solid);
[0158] 8) edge characteristics of lesion (smooth, lobulated, sharp
and smooth, spiculated, infiltrating);
[0159] 9) any other parameters determined from computed tomography
(CT) screening;
[0160] 10) exposure to second-hand smoke; and
[0161] 10) any known carcinogen exposure (including, but not
limited to, exposure to any of asbestos, radon gas, chemicals,
smoke from fires, and air pollution, which can include emissions
from stationary or mobile sources such as industrial/factory or
auto/marine/aircraft emissions).
[0162] Exemplary methods of combining LCM with supplemental
biomedical parameters can comprise the steps of obtaining a value
for at least one supplemental biomedical parameter (e.g., smoking
history) from an individual, comparing the value of each of the
supplemental biomedical parameter(s) to one or more predetermined
cutoffs, assigning a score for each supplemental biomedical
parameter based on said comparison, combining the assigned score
for each supplemental biomedical parameter with the assigned score
for each LCM to obtain a total score for said individual, comparing
the total score with a predetermined total score cutoff, and
classifying said individual as having or not having lung cancer (or
the likelihood thereof) based on whether the individual's total
score is above or below (or equal to) the predetermined total score
cutoff. In certain embodiments, if the individual's total score is
above (or equal to) the predetermined total score cutoff, then the
individual is classified as having lung cancer.
[0163] Further exemplary methods can comprise the steps of:
[0164] a) obtaining a value for at least one supplemental
biomedical parameter of an individual;
[0165] b) comparing the value of each supplemental biomedical
parameter against one or more predetermined cutoffs and assigning a
score for each supplemental biomedical parameter based on said
comparison;
[0166] c) quantifying in a test sample obtained from the
individual, the levels of one or more LCM or LCM panels (e.g., the
9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK, or the 6-marker subset of Cyfra, SLPI, TIMP1, TFPI,
CEACAM5, and MDK);
[0167] d) comparing the amount of each LCM quantified to a
predetermined cutoff and assigning a score for each LCM based on
said comparison;
[0168] e) combining the assigned score for each supplemental
biomedical parameter determined in step b with the assigned score
for each LCM determined in step d to obtain a total score for said
individual;
[0169] f) comparing the total score determined in step e with a
predetermined total score cutoff; and
[0170] g) classifying the individual (or the test sample from the
individual) as having or not having lung cancer (or the likelihood
thereof) based on whether the individual's total score is above or
below (or equal to) the predetermined total score cutoff (in
certain embodiments, if the individual's total score is above (or
equal to) the predetermined total score cutoff, then the individual
is classified as having lung cancer).
[0171] In the above exemplary methods, the supplemental biomedical
parameter obtained from the individual can be, for example, the
individual's smoking history, age, carcinogen exposure, gender,
nodule size, nodule morphology, and/or nodule location (nodule
characteristics, such as size, morphology, and/or location, may be
determined by CT imaging, as well as X-ray or other imaging
methods). Preferably, the supplemental biomedical parameter is
related to nodule morphology.
[0172] Exemplary Scoring Systems and Cutoffs
[0173] A variety of methodologies can be used to classify a sample
based on assaying one or more LCM disclosed herein. Classifying a
sample can be based on a score derived from assessing one or more
LCM disclosed herein, optionally in combination with one or more
supplemental biomedical parameters (including, but not limited to,
the supplemental biomedical parameters disclosed in the preceding
section). A score or other classification system can be based on,
for example, determining whether the level of one or more LCM is
above or below a cutoff level (which may be referred to as a
"cutoff value" or just "cutoff"), or is above or below a cutoff
value by a certain amount (e.g., by a certain number of standard
deviations such as two standard deviations), or the
magnitude/extent of how high or low the level of one or more LCM is
(which may optionally be in relation to a cutoff value). A wide
variety of scoring systems and methodologies for establishing
cutoff values are known in the art, and one of ordinary skill in
the art would know how to implement a known scoring system or
method of establishing cutoff values (or devise a new scoring
system or method for establishing cutoff values) that is best
suited for the intended use, such as assessing lung cancer based on
one or more LCM or LCM panels (optionally in combination with one
or more supplemental biomedical parameters). Accordingly, one of
ordinary skill in the art could establish and adjust cutoff values
to suit the intended use, and could incorporate these cutoff values
into any desirable scoring system. For example, cutoff values can
be adjusted based on whether increased sensitivity (for detecting
tumor samples and avoiding false-negatives) or increased
specificity (for avoiding false-positives) is considered more
important. For example, cutoffs can be selected such as to achieve
at least 70% sensitivity at 95% specificity, or at least 70%
specificity at 95% sensitivity, or at least 85% sensitivity at 95%
specificity, or at least 85% specificity at 95% sensitivity, or at
least 90% or 95% sensitivity, or at least 90% or 95% specificity,
or any other desired sensitivity and/or specificity (such as the
sensitivity and specificity values described above). As another
example, cutoffs can be set lower while requiring more markers in a
panel to be above the cutoff levels in order to classify a sample
as a tumor sample, or cutoffs can be set higher while requiring
fewer markers in a panel to be above the cutoff levels in order to
classify a sample as a tumor sample. When a cutoff value is set and
applied to testing, it may be interchangeably referred to herein as
a "predetermined" or "established" cutoff value. Furthermore,
various analysis methods can be applied, including, but not limited
to, split-point analysis (such as for setting discrete cutoffs),
logistic regression analysis (such as for factoring in the
magnitude/extent by which a marker level is elevated or low), Naive
Bayes, multivariate analysis, decision tree modeling, etc.
[0174] A representative example is shown in FIG. 17 for the
9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,
OPN, and MDK. In FIG. 17, exemplary cutoffs for each of these
9-markers are shown just below each marker symbol, as follows
(levels/concentrations, in ng/ml, were determined by ELISA):
Cyfra=1.20 ng/ml, CEA=5.00 ng/ml, SLPI=52 ng/ml, OPN=32 ng/ml,
MDK=0.15 ng/ml, TFPI=150 ng/ml, TIMP1=385 ng/ml, MMP2=210 ng/ml,
and SCC=2.2 ng/ml. These cutoff values were established by
examining the levels of these markers in both normal (control)
samples and lung tumor samples in the 50.times.50 sample set and
determining appropriate cutoff values that would best distinguish
lung tumor versus normal samples (e.g., cutoff values were selected
for which the levels of a majority of lung tumor samples are above
and the levels of a majority of normal samples are below, so as to
maximize sensitivity and specificity). These cutoffs were then
applied to the same 50.times.50 sample set (i.e., the 50.times.50
sample set was used for both training and testing in this example).
In this example, if the levels of two or more of the nine markers
was greater than or equal to the established cutoff value for each
marker, then the sample was classified as a lung tumor sample
(thus, if the levels of none, or only one, of the nine markers was
greater than or equal to the established cutoff value for each
marker, then the sample was classified as a normal sample). Using
this exemplary scoring system in this exemplary sample set, 48 out
of 50 tumor samples were correctly classified, whereas the
42.sup.nd and 43.sup.rd-listed samples were mis-classified as not
being tumor samples since the level of only one of the nine markers
(rather than the minimum of two or more) in each of these two
sample sets was greater than or equal to the cutoff level (96%
sensitivity; right-side of FIG. 17). Similarly, using this
exemplary scoring system in this exemplary sample set, 49 out of 50
normal (control) samples were correctly classified, whereas the
2.sup.nd-listed sample was mis-classified as being a tumor sample
since the levels of three of the nine markers (which meets the
minimum of two or more) in this sample set were greater than or
equal to the cutoff levels (98% specificity; left-side of FIG. 17).
However, one of skill in the art would appreciate that no assay
would be expected to correctly classify every single sample;
rather, some misclassification is expected in the art. The goal is
generally to minimize, rather than eliminate, misclassifications.
Further, an assay (such as an assay of LCM levels) can be combined
with other types of tests (such as CT screening) to further
minimize misclassifications.
[0175] In other exemplary scoring systems, if the levels of one or
more, three of more, four or more, five of more, six or more, seven
or more, eight or more, or all nine markers of the 9-marker panel
of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK are
greater than or equal to the established cutoff value for each
marker, then the sample can be classified as a lung tumor sample.
In certain exemplary scoring systems, if the level of a marker is
greater than or equal to the established cutoff value for that
marker, it can be assigned a value of one (for example) and if the
level of a marker is below the established cutoff value for that
marker, it can be assigned a value of zero (for example). However,
any desired values can be assigned to the various outcomes.
Furthermore, these values can be added together (or otherwise
combined) to determine a total score for a sample, and a
classification of the sample as a tumor or normal sample (for
example) can be assigned based on this total score. For example, in
the example described above and depicted in FIG. 17, a score of two
or greater can be used to classify a sample as a tumor sample (and
a score below two could be used to classify a sample as a normal
sample) if the level of each marker that is greater than or equal
to its established cutoff value is assigned a value of one. Any
other scoring system can be used, and one of ordinary skill in the
art would know how to select or devise a scoring system best suited
for the intended use.
[0176] A scoring system and cutoff values such as those exemplified
in FIG. 17, or any other desirable scoring system and cutoff
values, can be applied to any of the other LCM and LCM panels
disclosed herein, such as the 6-marker subset of the 9-marker panel
(Cyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK) and any of the other
panels provided in Table 5, and can optionally incorporate any
supplemental biomedical parameters. For example, any supplemental
biomedical parameters can be assigned a value in a scoring system
(e.g., a history of smoking can be assigned a value of one or other
value, and no smoking history can be assigned a value of zero,
negative one, or other value), and such values can be combined with
the values assigned to marker levels being above a predetermined
cutoff level (for example), such as to generate a total score for
classifying a sample as a lung tumor or normal sample. Furthermore,
these various scoring systems and cutoff values can be applied to
any of the lung cancer-related uses disclosed herein, including
specific uses such as those disclosed in Tables 7-10.
[0177] Any of the scoring systems disclosed herein or known in the
art, or which may be devised by one of ordinary skill in the art,
can be incorporated into a computer program, and such a computer
program can be embodied on computer readable medium. For example, a
computer program can generate a total score from a sample based on,
for example, the number of markers in a panel for which the levels
are above predetermined cutoff levels, together with parameters
from CT screening (e.g., tumor volume/size, tumor morphology, tumor
location, and/or other tumor characteristics, etc.) and/or other
supplemental biomedical parameters (e.g., smoking history, age of
the individual, etc.), and this total score can be used to classify
a sample as a lung tumor or normal sample, for example. A single
total score can be generated that represents the combination of
multiple different types of assessments (e.g., a combination of LCM
levels and supplemental biomedical parameters), or multiple
individual scores can be generated for evaluation individually
(e.g., a score based on assessment of LCM levels, and one or more
separate scores based on supplemental biomedical parameters). An
example of this type of integrated approach of combining LCM levels
(using the exemplary panel of TIMP1, TFPI, CEACAM5, and Ca72-4)
with a supplemental biomedical parameter (smoking history, as
indicated by "pack years") is shown in FIG. 21.
[0178] Kits
[0179] Any combination of LCM and LCM panels (as well as
supplemental biomedical parameters) can be provided in the form of
kits, such as for use in performing the methods disclosed herein.
Furthermore, any kit can contain one or more detectable labels
(e.g., detectably labeled reagents such as antibodies), such as a
fluorescent moiety, etc.
[0180] For example, a kit can comprise (a) reagents comprising at
least one antibody for quantifying one or more LCM in a test
sample, wherein said LCM comprise: Cyfra, SLPI, TIMP1, SCC, TFPI,
CEACAM5, MMP2, OPN, and MDK (or just Cyfra, SLPI, TIMP1, TFPI,
CEACAM5, and MDK; or any other LCM or LCM panels disclosed herein,
such as the panels disclosed in Tables 5-12), and optionally (b)
one or more algorithms or computer programs for performing the
steps of comparing the amount of each LCM quantified in the test
sample to one or more predetermined cutoffs and assigning a score
for each LCM quantified based on said comparison, combining the
assigned score for each LCM quantified to obtain a total score,
comparing the total score with a predetermined total score, and
using said comparison to determine whether an individual has lung
cancer. Alternatively, rather than one or more algorithms or
computer programs, one or more instructions for manually performing
the above steps by a human can be provided.
[0181] In certain embodiments, a kit can contain: (a) reagents
comprising at least one antibody for quantifying one or more LCM in
a test sample, wherein said LCM are Cyfra, SLPI, TIMP1, SCC, TFPI,
CEACAM5, MMP2, OPN, and MDK, and (b) reagents containing one or
more LCM for quantifying at least one antibody in a test sample;
wherein said antibodies are: TP53 (p53), KLKB1, CFL1 (CFLN), EEF1G,
HSP90.alpha. (HSP90AA1), RTN4, ALDOA, GLG1, PTK7, EFEMP1, SLC3A2
(CD98), CHGB, CEACAM1, ALCAM, HSPB1 (HSP27), LGALS1, and B7H3, and
optionally (c) one or more algorithms or computer programs for
performing the steps of comparing the amount of each LCM and
antibody quantified in the test sample to one or more predetermined
cutoffs and assigning a score for each LCM and antibody quantified
based on said comparison, combining the assigned score for each LCM
and antibody quantified to obtain a total score, comparing the
total score with a predetermined total score, and using said
comparison to determine whether an individual has lung cancer.
Alternatively, rather than one or more algorithms or computer
programs, one or more instructions for manually performing the
above steps by a human can be provided.
[0182] Translating LCM Assessments to Lung Cancer Assessments, and
Systems Therefor
[0183] An assessment of LCM in an individual, such as LCM levels
determined by assaying a serum sample (or other sample) from the
individual, can be translated to an assessment of lung cancer for
the individual. For example, the levels of multiple LCM (such as
each of the LCM in the 9-marker panel of Cyfra, SLPI, TIMP1, SCC,
TFPI, CEACAM5, MMP2, OPN, and MDK) can be translated to a score or
other identifier that indicates whether an individual has lung
cancer (or that indicates the likelihood that the individual has
lung cancer), for example. Similarly, the score or other identifier
may indicate a specific type of lung cancer assessment, such as the
assessments of various lung cancer characteristics described
herein, including (but not limited to), determination of whether an
individual's lung cancer is adenocarcinoma or squamous cell
carcinoma, determination of the stage of an individual's lung
cancer (such as distinguishing between stage I and stage III lung
cancer), determination of whether an individual's lung cancer is
SCLC or NSCLC, determining whether a lung lesion identified in an
individual (such as by CT screening) is a malignant tumor or a
benign lesion, and determining lung tumor regression and/or
recurrence. Any of these determinations may be expressed in a
discrete (e.g., absolute) or continuous (e.g., likelihood) manner,
for example.
[0184] Furthermore, the assessment of LCM in an individual, such as
LCM levels, can be translated to a tangible report. Thus, a score
or other identifier that indicates the lung cancer assessment can
be provided in the form of a tangible report.
[0185] Additionally, the translation, such as the translation of
LCM levels to a lung cancer assessment (such as a score or other
identifier), can be performed by a computer. Furthermore, certain
embodiments provide computer readable medium having a computer
program code embodied thereon for translating LCM levels to a lung
cancer assessment.
[0186] In certain embodiments, the invention provides systems for
assessing one or more LCM, particularly the levels of multiple LCM,
and translating this LCM assessment to an assessment of lung
cancer, such as a determination of whether an individual has (or is
likely to have) lung cancer (which may be indicated by a score or
other identifier). In certain embodiments, these systems include
one or more computers to receive an LCM assessment, translate the
LCM assessment to a lung cancer assessment, and output the lung
cancer assessment (e.g., as a score or other identifier). These
systems may optionally comprise multiple computers that communicate
via the internet (or any other mode of communication used in the
art for inter-computer communication).
[0187] Accordingly, certain embodiments of the invention provide
methods of translating an assessment of LCM (e.g., LCM levels) to
an assessment of lung cancer (e.g., a score or other indication of
whether an individual has lung cancer, or their likelihood of
having lung cancer, or other specific lung cancer assessment). In
certain embodiments, this assessment of lung cancer is provided in
the form of a tangible report. In certain embodiments, the
translation is performed by a computer. Furthermore, certain
embodiments of the invention provide computers programmed to
translate an LCM assessment to a lung cancer assessment. Certain
embodiments provide computer readable medium having a computer
program code embodied thereon for translating LCM levels to a lung
cancer assessment. In certain embodiments, a system is provided for
receiving an LCM assessment, translating the LCM assessment to a
lung cancer assessment, and outputing the lung cancer assessment
(e.g., as a score or other identifier). In various embodiments, the
system comprises one or more computers (which may optionally
communicate via the internet or other mode of communication).
[0188] Reports, Transmission of Reports, and Programmed
Computers
[0189] The results of a test (e.g., a diagnosis of lung cancer for
an individual based on the level, or other assay, of one or more
LCM disclosed herein, or assessment of tumor
progression/regression/recurrence, lung cancer stage, type of lung
cancer such as NSCLC versus SCLC or adenocarcinoma versus squamous
cell carcinoma, malignant tumor versus benign lung lesion, etc.),
and/or any other information pertaining to a test (e.g., the levels
of one or more LCM disclosed herein in a sample from an individual,
which may optionally be provided in the absence of explicit disease
or diagnostic information), may be referred to herein as a
"report". A tangible report can optionally be generated as part of
a testing process (which may be interchangeably referred to herein
as "reporting", or as "providing" a report, "producing" a report,
or "generating" a report).
[0190] Examples of tangible reports may include, but are not
limited to, reports in paper (such as computer-generated printouts
of test results) or equivalent formats and reports stored on
computer readable medium (such as a CD, USB flash drive or other
removable storage device, computer hard drive, or computer network
server, etc.). Reports, particularly those stored on computer
readable medium, can be part of a database, which may optionally be
accessible via the internet (such as a database of patient records
or biomedical information stored on a computer network server,
which may be a "secure database" that has security features that
limit access to the report, such as to allow only the patient and
the patient's medical practitioners to view the report while
preventing other unauthorized individuals from viewing the report,
for example). In addition to, or as an alternative to, generating a
tangible report, reports can also be displayed on a computer screen
(or the display of another electronic device or instrument).
[0191] A report can further be "transmitted" or "communicated"
(these terms may be used herein interchangeably), such as to the
individual who was tested, a medical practitioner (e.g., a doctor,
nurse, clinical laboratory practitioner, etc.), a healthcare
organization, a clinical laboratory, and/or any other party or
requester intended to view or possess the report. The act of
"transmitting" or "communicating" a report can be by any means
known in the art, based on the format of the report. Furthermore,
"transmitting" or "communicating" a report can include delivering a
report ("pushing") and/or retrieving ("pulling") a report. For
example, reports can be transmitted/communicated by various means,
including being physically transferred between parties (such as for
reports in paper format) such as by being physically delivered from
one party to another, or by being transmitted electronically or in
signal form (e.g., via e-mail or over the internet, by facsimile,
and/or by any wired or wireless communication methods known in the
art) such as by being retrieved from a database stored on a
computer network server, etc.
[0192] In certain exemplary embodiments, the invention provides
computers (or other apparatus/devices such as biomedical devices or
laboratory instrumentation) programmed to carry out the methods
described herein. For example, in certain embodiments, the
invention provides a computer programmed to receive (i.e., as
input) the levels of one or more LCM disclosed herein and provide
(i.e., as output) a lung cancer diagnosis or other result (e.g.,
assessment of tumor progression/regression/recurrence, lung cancer
stage, type of lung cancer such as NSCLC versus SCLC or
adenocarcinoma versus squamous cell carcinoma, malignant tumor
versus benign lung lesion, etc.) based on the levels of one or more
LCM. Such output (e.g., communication of lung cancer diagnosis,
etc.) may be, for example, in the form of a report on computer
readable medium, printed in paper form, and/or displayed on a
computer screen or other display.
[0193] Further exemplary embodiments of the invention are described
in greater detail below.
[0194] 1. LCM Proteins
[0195] Exemplary embodiments of the invention provide LCM proteins
that consist of, consist essentially of, or comprise the amino acid
sequences of SEQ ID NOS:1-65 (additionally, carbohydrate antigens
CA 242, CA 19-9, and CA 72-4 are also provided, which may also be
encompassed by references herein to proteins/polypeptides), as well
as all known variants and fragments of these proteins, and nucleic
acid molecules that are within the art to make and use. Examples of
such obvious variants include, but are not limited to,
naturally-occurring allelic variants, pre-processed or mature
processed forms of a protein, non-naturally occurring
recombinantly-derived variants, orthologs, and paralogs. Such
variants can readily be generated using art-known techniques in the
fields of recombinant nucleic acid technology and protein
biochemistry.
[0196] A protein is said to be "isolated" or "purified" when it is
substantially free of cellular material or free of chemical
precursors or other chemicals. LCM proteins can be purified to
homogeneity or other degrees of purity. The level of purification
can be based on the intended use. The primary consideration is that
the preparation allows for the desired function of the protein,
even if in the presence of considerable amounts of other
components.
[0197] In some uses, "substantially free of cellular material"
includes preparations of a protein having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the protein is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0198] The language "substantially free of chemical precursors or
other chemicals" includes preparations of a protein in which the
protein is separated from chemical precursors or other chemicals
that are involved in the protein's synthesis. In one embodiment,
the language "substantially free of chemical precursors or other
chemicals" includes preparations of a LCM protein having less than
about 30% (by dry weight) chemical precursors or other chemicals,
less than about 20% chemical precursors or other chemicals, less
than about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0199] Isolated LCM proteins can be purified from cells that
naturally express it, purified from cells that have been altered to
express it (recombinant), or synthesized using known protein
synthesis methods (e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., (2001)). For example, a nucleic acid
molecule encoding a LCM protein can be cloned into an expression
vector, the expression vector introduced into a host cell, and the
protein expressed in the host cell. The protein can then be
isolated from the cells by an appropriate purification scheme using
standard protein purification techniques.
[0200] A LCM protein or fragment thereof can be attached to
heterologous sequences to form chimeric or fusion proteins. Such
chimeric and fusion proteins comprise a protein operatively linked
to a heterologous protein having an amino acid sequence not
substantially homologous to the protein. "Operatively linked"
indicates that the protein and the heterologous protein are fused
in-frame. The heterologous protein can be fused to the N-terminus
or C-terminus of the protein.
[0201] In some uses, the fusion protein does not affect the
activity of the protein per se. For example, the fusion protein can
include, but is not limited to, beta-galactosidase fusions, yeast
two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged,
and Ig fusions. Such fusion proteins, particularly poly-His
fusions, can facilitate the purification of recombinant LCM
proteins. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a protein can be increased by using
a heterologous signal sequence.
[0202] A chimeric or fusion LCM protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
different protein sequences can be ligated together in-frame in
accordance with conventional techniques. In another embodiment, a
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers that give
rise to complementary overhangs between two consecutive gene
fragments that can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (Ausubel et al., Current
Protocols in Molecular Biology, 1992-2006). Moreover, many
expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST protein). A LCM-encoding nucleic acid
can be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the LCM protein.
[0203] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences can be
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In an
exemplary embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%
or more of the length of a reference sequence can be aligned for
comparison purposes. The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions can then
be compared. When a position in the first sequence is occupied by
the same amino acid residue or nucleotide as the corresponding
position in the second sequence, then the molecules are identical
at that position (as used herein, amino acid or nucleic acid
"identity" is equivalent to amino acid or nucleic acid "homology").
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, that are
introduced for optimal alignment of the two sequences.
[0204] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., Stockton Press, New York, 1991). In an
exemplary embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package, using either a
Blossom 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12,
10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In
another exemplary embodiment, the percent identity between two
nucleotide sequences can be determined using the GAP program in the
GCG software package (Devereux et al., Nucleic Acids Res. 12(1):387
(1984)) using a NWSgapdna. CMP matrix and a gap weight of 40, 50,
60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. In
another exemplary embodiment, the percent identity between two
amino acid or nucleotide sequences is determined using the
algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12, and a gap
penalty of 4.
[0205] The sequences of the proteins and nucleic acid molecules of
the invention can further be used as a "query sequence" to perform
a search against sequence databases to, for example, identify other
protein family members or related sequences. Such searches can be
performed using the NBLAST and XBLAST programs (version 2.0) of
Altschul et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide
searches can be performed with the NBLAST program, score=100,
wordlength=12, to obtain nucleotide sequences homologous to the
query nucleic acid molecule. BLAST protein searches can be
performed with the XBLAST program, score=50, wordlength=3, to
obtain amino acid sequences homologous to the query proteins. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0206] As used herein, two proteins (or a region or domain of the
proteins) have significant homology/identity (also referred to as
substantial homology/identity) when the amino acid sequences are
typically at least about 70-80%, 80-90%, 90-95%, 96%, 97%, 98%, or
99% identical A significantly homologous amino acid sequence can be
encoded by a nucleic acid molecule that hybridizes to a LCM
protein-encoding nucleic acid molecule under stringent conditions,
as more fully described below.
[0207] Orthologs of a LCM protein typically have some degree of
significant sequence homology to at least a portion of a LCM
protein and are encoded by a gene from another organism. Preferred
orthologs are isolated from mammals, preferably non-human primates,
for the development of human therapeutic markers and agents. Such
orthologs can be encoded by a nucleic acid molecule that hybridizes
to a LCM protein-encoding nucleic acid molecule under moderate to
stringent conditions, as more fully described below, depending on
the degree of relatedness of the two organisms yielding the
proteins.
[0208] Non-naturally occurring variants of the LCM proteins can
readily be generated using recombinant techniques. Such variants
include, but are not limited to, deletions, additions, and
substitutions in the amino acid sequence of the LCM protein. For
example, one class of substitutions is conserved amino acid
substitutions. Such substitutions are those that substitute a given
amino acid in a LCM protein by another amino acid of like
characteristics. Typically seen as conservative substitutions are
the replacements, one for another, among the aliphatic amino acids
Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr; exchange of the acidic residues Asp and Glu; substitution
between the amide residues Asn and Gln; exchange of the basic
residues Lys and Arg; and replacements among the aromatic residues
Phe and Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).
[0209] Variant LCM proteins can be fully functional or can lack
function in one or more activities, e.g., ability to bind
substrate, ability to phosphorylate substrate, ability to mediate
signaling, etc. Fully functional variants typically contain only
conservative variations or variation in non-critical residues or in
non-critical regions.
[0210] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncations, or a substitution, insertion,
inversion, or deletion in a critical residue or critical
region.
[0211] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity
or in assays such as in vitro proliferative activity. Sites that
are critical for binding partner/substrate binding can also be
determined by structural analysis such as crystallization, nuclear
magnetic resonance, or photoaffinity labeling (Smith et al., J.
Mol. Biol. 224:899-904 (1992); de Vos et al., Science 255:306-312
(1992)).
[0212] LCM of the invention include fragments of LCM, and peptides
that comprise and consist of such fragments. Such fragments of LCM
may be naturally-occurring in the human body. An exemplary fragment
typically comprises at least about 5, 6, 8, 10, 12, 14, 16, 18, 20
or more contiguous amino acid residues of a LCM protein. Such
fragments can be chosen based on the ability to retain one or more
of the biological activities of LCM or can be chosen for the
ability to perform a function, e.g., bind a substrate or act as an
immunogen. Particularly important fragments are biologically active
fragments, such as peptides that are, for example, about 8 or more
amino acids in length. Such fragments can include a domain or motif
of a LCM, e.g., an active site, a transmembrane domain, or a
binding domain. Further, possible fragments include, but are not
limited to, soluble peptide fragments and fragments containing
immunogenic structures. Domains and functional sites can readily be
identified, for example, by computer programs well known and
readily available to those of skill in the art (e.g., PROSITE
analysis).
[0213] Proteins can contain amino acids other than the 20 amino
acids commonly referred to as the 20 naturally-occurring amino
acids. Further, many amino acids, including the terminal amino
acids, can be modified by natural processes, such as processing and
other post-translational modifications, or by chemical modification
techniques well known in the art. Common modifications that occur
naturally in proteins are well known to those of skill in the
art.
[0214] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, tRNA-mediated addition of
amino acids to proteins such as arginylation, and
ubiquitination.
[0215] Such modifications are well known to those of skill in the
art and have been described in the scientific literature. Several
particularly common modifications, glycosylation, lipid attachment,
sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation, for instance, are described in
most basic texts, such as Proteins-Structure and Molecular
Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company,
New York (1993). Many detailed reviews are available on this
individual, such as by Wold (Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New
York 1-12 (1983)); Seifter et al. (Meth. Enzymol. 182: 626-646
(1990)); and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62
(1992)).
[0216] Accordingly, exemplary LCM proteins and fragments thereof of
the invention can also encompasses derivatives or analogs in which,
for example, a substituted amino acid residue is not one encoded by
the genetic code, in which a substituent group is included, in
which a mature LCM is fused with another composition, such as a
composition to increase the half-life of a LCM (e.g., polyethylene
glycol or albumin), or in which additional amino acids are fused to
a mature LCM, such as a leader or secretory sequence or a sequence
for purification of a mature LCM or a pro-protein sequence.
[0217] 2. Antibodies to LCM Proteins
[0218] Exemplary embodiments of the invention provide antibodies to
LCM proteins, including, for example, monoclonal and polyclonal
antibodies; chimeric, humanized, and fully human antibodies; and
antigen-binding fragments and variants thereof, as well as other
embodiments.
[0219] Antibodies that selectively bind to a LCM protein can be
made using standard procedures known to those of ordinary skills in
the art. The term "antibody" is used in the broadest sense, and
specifically covers, for example, monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies),
chimeric antibodies, humanized antibodies, fully human antibodies,
and antibody fragments (e.g., Fab, F(ab').sub.2, Fv and
Fv-containing binding proteins), so long as they exhibit
LCM-binding activity. Antibodies (Ab's) and immunoglobulins (Ig's)
are glycoproteins typically having the same structural
characteristics. Antibodies can be of the IgG, IgE, IgM, IgD, and
IgA class or subclass thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1
and IgA2). Antibodies may be interchangeably referred to as
"LCM-binding molecules".
[0220] The term "monoclonal antibody", as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are substantially identical except for possible
naturally occurring mutations that may be present in minor amounts.
Monoclonal antibodies are highly specific and are typically
directed against a single antigenic site. Furthermore, in contrast
to polyclonal antibody preparations, which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is typically directed against
a single determinant on an antigen. In addition to their
specificity, monoclonal antibodies are advantageous in that
substantially homogenous antibodies can be produced by a hybridoma
culture which is uncontaminated by other immunoglobulins or
antibodies. The modifier "monoclonal" antibody indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, monoclonal antibodies can be made by hybridoma methods
such as described by Kohler and Milstein, Nature 256: 495-497
(1975), by recombinant methods (e.g., as described in U.S. Pat. No.
4,816,567), or can be isolated from phage antibody libraries such
as by using the techniques described in Clackson et al., Nature
352: 624-628 (1991) or Marks et al., J. Mol. Biol. 222: 581-597
(1991).
[0221] "Humanized" forms of non-human (e.g., murine or rabbit)
antibodies are chimeric immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Typically,
humanized antibodies are human immunoglobulins (a recipient
antibody) in which residues from a complementarity determining
regions ("CDR") of the recipient are replaced by residues from a
CDR of a non-human species (a donor antibody) such as mouse, rat,
or rabbit having the desired specificity, affinity, and capacity.
In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, a humanized antibody may comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework region (FR) sequences. These modifications can be made to
further refine and optimize antibody performance. In general, a
humanized antibody can comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDRs correspond to those of a non-human immunoglobulin
and all or substantially all of the FRs are those of a human
immunoglobulin consensus sequence. A humanized antibody can also
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further details
concerning humanized antibodies, see: Jones et al., Nature
321:522-525 (1986); Reichmann et al., Nature 332:323-327 (1988);
Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Queen et al.,
U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370; and
Winter, U.S. Pat. No. 5,225,539.
[0222] Antibodies, as used herein, include antibody fragments,
particularly antigen-binding fragments, as well as other modified
antibody structures and antigen-binding scaffolds (such as modified
antibody structures that are smaller or have less than all domains
or chains compared with a typical naturally occurring, full-size
human antibody). Examples of antibody fragments and other modified
antibody structures and antigen-binding scaffolds are known in the
art by such terms as minibodies (e.g., U.S. Pat. No. 5,837,821),
Nanobodies (llama heavy chain antibodies; Ablynx, Ghent, Belgium),
Adnectins (fibronectin domains; Adnexus Therapeutics, Waltham,
Mass.), Affibodies (protein-binding domain of Staphylococcus aureus
protein A; Affibody, Stockholm, Sweden), peptide aptamers
(synthetic peptides; Aptanomics, Lyon, France), Avimers (A-domains
derived from cell surface receptors; Avidia, Mountain View, Calif.
(acquired by Amgen)), Transbodies (transferrin; BioRexis
Pharmaceuticals, King of Prussia, Pa. (acquired by Pfizer)),
trimerized tetranectin domains (Borean Pharma, Aarhus, Denmark),
Domain antibodies (heavy or light chain antibodies; Domantis,
Cambridge, UK (acquired by GlaxoSmithKline)), Evibodies (derived
from V-like domains of T-cell receptors CTLA-4, CD28 and inducible
T-cell costimulator; EvoGenix Therapeutics, Sydney, Australia),
scFV fragments (stable single chain antibody fragments; ESBATech,
Zurich, Switzerland), Unibodies (monovalent IgG4 mAbs fragments;
Genmab, Copenhagen, Denmark), BiTEs (bispecific, T-cell activating
single-chain antibody fragments; Micromet, Munich, Germany),
DARPins (designed ankyrin repeat proteins; Molecular Partners,
Zurich, Switzerland), Anticalins (derived from lipocalins; Pieris,
Freising-Weihenstephan, Germany), Affilins (derived from human lens
protein gamma crystalline; Scil Proteins, Halle, Germany), and
SMIPs (small modular immunopharmaceuticals; Trubion
Pharmaceuticals, Seattle, Wash.) (Sheridan, Nature Biotechnology,
2007 April; 25(4):365-6).
[0223] An "isolated" or "purified" antibody is one that has been
identified and separated and/or recovered from a component of the
environment in which it is produced. Contaminant components of its
production environment are materials that would interfere with
diagnostic or therapeutic uses for the antibody, and may include
enzymes, hormones, and other proteinaceous or nonproteinaceous
solutes. In exemplary embodiments, the antibody can be purified as
measurable by any of at least three different methods: 1) to
greater than 95% by weight of antibody as determined by the Lowry
method, preferably more than 99% by weight; 2) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal
amino acid sequence by use of a spinning cup sequenator; or 3) to
homogeneity by SDS-PAGE under reducing or non-reducing conditions
using Coomasie blue or silver stain. Isolated antibody can include
an antibody in situ within recombinant cells since at least one
component of the antibody's natural environment will not be
present. Ordinarily, however, an isolated antibody can be be
prepared by at least one purification step.
[0224] An "antigenic region", "antigenic determinant", or "epitope"
includes any protein determinant capable of specific binding to an
antibody. This is the site on an antigen to which each distinct
antibody molecule binds. Epitopic determinants can be active
surface groupings of molecules such as amino acids or sugar side
chains and may have specific three-dimensional structural
characteristics or charge characteristics.
[0225] "Antibody specificity" refers to an antibody that has a
stronger binding affinity for an antigen from a first individual
species than it has for a homologue of that antigen from a second
individual species. Typically, an antibody "binds specifically" to
a human antigen (e.g., has a binding affinity (Kd) value of no more
than about 1.times.10.sup.-7 M, preferably no more than about
1.times.10.sup.-8 M, and most preferably no more than about
1.times.10.sup.-9 M) but has a binding affinity for a homologue of
the antigen from a second individual species which is at least
about 50-fold, or at least about 500-fold, or at least about
1000-fold, weaker than its binding affinity for the human antigen.
The antibodies can be of any of the various types of antibodies as
described herein, such as humanized or fully human antibodies.
[0226] An antibody "selectively" or "specifically" binds a marker
protein when the antibody binds the marker protein and does not
significantly bind to unrelated proteins. An antibody can still be
considered to selectively or specifically bind a marker protein
even if it also binds to other proteins that are not substantially
homologous with the marker protein as long as such proteins share
homology with a fragment or domain of the marker protein. In this
case, it would be understood that antibody binding to the marker
protein is still selective despite some degree of
cross-reactivity.
[0227] Exemplary embodiments of the invention provide an "antibody
variant", which refers to an amino acid sequence variant of an
antibody wherein one or more of the amino acid residues have been
modified. Such variants necessarily have less than 100% sequence
identity with the amino acid sequence of the antibody, and have at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid
sequence identity with the amino acid sequence of either the heavy
or light chain variable domain of the antibody.
[0228] The term "antibody fragment" refers to a portion of a
full-length antibody, including the antigen binding or variable
region or the antigen-binding portion thereof. Examples of antibody
fragments include Fab, Fab', F(ab').sub.2 and Fv fragments. Papain
digestion of antibodies typically produces two identical antigen
binding fragments, called the Fab fragment, each with a single
antigen binding site, and a residual "Fc" fragment. Pepsin
treatment typically yields an F(ab').sub.2 fragment that has two
antigen binding fragments which are capable of crosslinking
antigen, and a residual other fragment (which is termed pFc').
Examples of additional antigen-binding fragments can include
diabodies, triabodies, tetrabodies, single-chain Fv, single-chain
Fv-Fc, SMIPs, and multispecific antibodies formed from antibody
fragments. A "functional fragment", with respect to antibodies,
typically refers to an Fv, F(ab), F(ab').sub.2 or other
antigen-binding fragments comprising one or more CDRs that has
substantially the same antigen-binding specificity as an
antibody.
[0229] An "Fv" fragment is an example of an antibody fragment that
contains a complete antigen recognition and binding site. This
region typically consists of a dimer of one heavy and one light
chain variable domain in a tight, non-covalent association
(V.sub.H-V.sub.L dimer). It is in this configuration that the three
CDRs of each variable domain interact to define an antigen-binding
site on the surface of the V.sub.H-V.sub.L dimer. Collectively, the
six CDRs confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three CDRs specific for an antigen) has the ability to
recognize and bind antigen.
[0230] An "Fab" fragment (also designated as "F(ab)") also contains
the constant domain of the light chain and the first constant
domain (CH1) of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxyl
terminus of the heavy chain CH1 domain, including one or more
cysteines from the antibody hinge region. Fab'-SH is the
designation for Fab' in which the cysteine residue(s) of the
constant domains have a free thiol group. F(ab') fragments are
produced by cleavage of the disulfide bond at the hinge cysteines
of the F(ab').sub.2 pepsin digestion product. Additional chemical
couplings of antibody fragments are known to those of ordinary
skill in the art.
[0231] A "single-chain Fv" or "scFv" antibody fragment contains
V.sub.H and V.sub.L domains, wherein these domains are present in a
single polypeptide chain. Typically, the Fv polypeptide further
comprises a polypeptide linker between the V.sub.H and V.sub.L
domains that enables the scFv to form the desired structure for
antigen binding. For a review of scFv, see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). A single
chain Fv-Fc is an scFv linked to a Fc region.
[0232] A "diabody" is a small antibody fragment with two
antigen-binding sites, which fragments comprise a variable heavy
domain (V.sub.H) connected to a variable light domain (V.sub.L) in
the same polypeptide chain. By using a linker that is too short to
allow pairing between the two domains on the same chain, the
domains are forced to pair with the complementary domains of
another chain and create two antigen-binding sites. Diabodies are
described more fully in, for example, EP 0 404 097; WO 93/11161;
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448
(1993). Triabodies, tetrabodies and other antigen-binding antibody
fragments have been described by Hollinger and Hudson, 2005, Nature
Biotechnology 23:1126.
[0233] A "small modular immunopharmaceutical" (or "SMIP") is a
single-chain polypeptide including a binding domain (e.g., an scFv
or an antigen binding portion of an antibody), a hinge region, and
an effector domain (e.g., an antibody Fc region or a portion
thereof). SMIPs are described in published U.S. Patent Application
No. 20050238646.
[0234] Many methods are known for generating and/or identifying
antibodies to a given marker protein. Several such methods are
described by Kohler et al., 1975, Nature 256: 495-497; Lane, 1985,
J. Immunol. Meth. 81:223-228; Harlow et al., 1988, Antibodies: A
Laboratory Manual. Cold Spring Harbor Laboratory Press; Harlow et
al., 1998, Using Antibodies, Cold Spring Harbor Press; Zhong et
al., 1997, J. Indust. Microbiol. Biotech. 19(1):71-76; and Berry et
al., 2003, Hybridoma and Hybridomics 22(1): 23-31.
[0235] Polyclonal antibodies can be prepared by any known method or
modifications of these methods, including obtaining antibodies from
patients. In certain exemplary methods for generating antibodies
such as polyclonal antibodies, an isolated protein can be used as
an immunogen which is administered to a mammalian organism, such as
a rat, rabbit, or mouse. For example, a complex of an immunogen
such as a LCM protein (or fragment thereof) and a carrier protein
can be prepared and an animal immunized by the complex. Serum or
plasma containing antibodies against the protein can be recovered
from the immunized animal and the antibodies separated and purified
(in the same manner as for monoclonal antibodies, for example). The
gamma globulin fraction or the IgG antibodies can be obtained, for
example, by use of saturated ammonium sulfate or DEAE SEPHADEX, or
other techniques known to those skilled in the art. The antibody
titer in the antiserum can be measured in the same manner as in the
supernatant of a hybridoma culture.
[0236] A marker such as a full-length LCM protein, an antigenic
peptide fragment, a fusion protein thereof, or a carbohydrate
antigen or fragment thereof, can be used as an immunogen. A marker
used as an immunogen is not limited to any particular type of
immunogen. In one aspect, antibodies can be prepared from regions
or discrete fragments (e.g., functional domains, extracellular
domains, or portions thereof) of a LCM. Antibodies can be prepared
from any region of a marker as described herein. In particular, the
markers can be selected from the group consisting of SEQ ID
NOS:1-65, the carbohydrate antigens CA 242, CA 19-9, and CA 72-4,
and fragments thereof. An antigenic fragment can typically comprise
at least 8, 10, 12, 14, 16, or more contiguous amino acid residues,
for example. Such fragments can be selected based on a physical
property, such as fragments that correspond to regions located on
the surface of a marker (e.g., hydrophilic regions) or can be
selected based on sequence uniqueness.
[0237] Antibodies can also be produced by inducing production in a
lymphocyte population or by screening antibody libraries or panels
of highly specific binding reagents, such as disclosed in Orlandi
et al. (Proc. Natl. Acad. Sci. 86:3833-3837 (1989)) or Winter et
al. (Nature 349:293-299 (1991)). A protein can be used in screening
assays of phagemid or B-lymphocyte immunoglobulin libraries to
identify antibodies having a desired specificity. Numerous
protocols for competitive binding or immunoassays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art (e.g., Smith, Curr. Opin. Biotechnol. 2:
668-673 (1991)).
[0238] Antibodies can also be generated using various phage display
methods known in the art. In representative phage display methods,
functional antibody domains are displayed on the surface of phage
particles which carry nucleic acid molecules that encode the
antibody domains. In particular, such phage can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds an antigen of
interest can be selected or identified with the antigen, e.g.,
using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in methods such as these can typically
be filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv, or disulfide stabilized Fv antibody
domains recombinantly fused to either the phage gene III or gene
VIII protein. Examples of phage display methods that can be used to
make antibodies include methods described in Brinkman et al., J.
Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods
184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et
al., Advances in Immunology 57:191-280 (1994); PCT application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0239] Antibodies, antigen binding fragments, and/or antibody
variants can be produced by recombinant and genetic engineering
methods well known in the art. For example, methods of expressing
heavy and light chain genes in E. coli are described in PCT
publication numbers WO901443, WO901443, and WO9014424, and in Huse
et al., 1989 Science 246:1275-1281. When using recombinant
techniques, such as to produce an antibody variant, the antibody
variant can be produced intracellularly, in the periplasmic space,
or directly secreted into the medium. If an antibody variant is
produced intracellularly, as a first step, the particulate debris,
either host cells or lysed fragments, can be removed, for example,
by centrifugation or ultrafiltration. Carter et al. (Bio/Technology
10: 163-167 (1992)) describe a procedure for isolating antibodies
that are secreted to the periplasmic space of E. coli. Briefly,
cell paste can be thawed in the presence of sodium acetate (pH
3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
minutes. Cell debris can be removed by centrifugation. Where an
antibody variant is secreted into the medium, supernatants from
such expression systems can first be concentrated using a
commercially available protein concentration filter (e.g., an
Amicon or Millipore PELLICON ultrafiltration unit). A protease
inhibitor such as PMSF can be included in any of the foregoing
steps to inhibit proteolysis, and antibiotics can be included to
prevent the growth of contaminating microorganisms.
[0240] An antibody composition prepared from cells can be purified
using, for example, affinity chromatography, hydroxylapatite
chromatography, gel electrophoresis, and/or dialysis. The
suitability of protein A as an affinity ligand typically depends on
the species and isotype of the immunoglobulin Fc domain of an
antibody. Protein A can be used to purify antibodies that are based
on human delta1, delta2, or delta4 heavy chains (Lindmark et al.,
J. Immunol. Meth. 62: 1-13 (1983)). Protein G can be used for all
mouse isotypes and for human delta3 (Guss et al., EMBO J. 5:
1567-1575 (1986)). The matrix to which the affinity ligand is
attached can be, for example, agarose or mechanically stable
matrices such as controlled pore glass or
poly(styrenedivinyl)benzene. Where the antibody comprises a CH3
domain, the BAKERBOND ABX.TM. resin (J. T. Baker, Phillipsburg,
N.J.) can be used for purification. Other exemplary techniques for
antibody purification include, but are not limited to,
fractionation on an ion-exchange column, ethanol precipitation,
reverse phase HPLC, chromatography on silica, chromatography on
heparin hepharos, chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation.
[0241] Following any preliminary purification step(s), contaminants
in a mixture containing an antibody of interest can be removed by
low pH hydrophobic interaction chromatography using an elution
buffer at a pH between about 2.5-4.5, preferably performed at low
salt concentrations (e.g., from about 0-0.25M salt).
[0242] Full-length antibodies, as well as antibody fragments, can
also be expressed and isolated from bacteria such as E. coli, such
as described in Mazor et al., "Isolation of engineered, full-length
antibodies from libraries expressed in Escherichia coli", Nat.
Biotechnol. 2007 May; 25(5):563-5 and Sidhu, "Full-length
antibodies on display", Nat. Biotechnol. 2007 May; 25(5):537-8.
[0243] Further details regarding antibodies are set forth in the
following U.S. Pat. No. 6,248,516 (Winter et al.); U.S. Pat. No.
6,291,158 (Winter et al.); U.S. Pat. No. 5,885,793 (Griffiths et
al.); U.S. Pat. No. 5,969,108 (McCafferty et al.); U.S. Pat. No.
5,939,598 (Kucherlapati et al.); U.S. Pat. No. 4,816,397 (Boss et
al.); U.S. Pat. No. 4,816,567 (Cabilly et al.); U.S. Pat. No.
6,331,415 (Cabilly et al.); U.S. Pat. No. 5,770,429 (Lonberg et
al.); U.S. Pat. No. 5,639,947 (Hiatt et al.); and U.S. Pat. No.
5,260,203 (Ladner et al.), each of which is incorporated herein by
reference, and in the following published U.S. patent applications:
US20040132101 (Lazar et al.), US20050064514 (Stavenhagen et al.),
US20040261148 (Dickey et al.), and US20050014934 (Hinton et al.),
each of which is incorporated herein by reference. Antibody
engineering is further described in Jain et al., "Engineering
antibodies for clinical applications", Trends Biotechnol. 2007
July; 25(7):307-16.
[0244] 3. Antibody-Drug Conjugates to LCM Proteins
[0245] An antibody against LCM can be coupled (e.g., covalently
bonded) to a suitable therapeutic agent (as further discussed
herein) either directly or indirectly (e.g., via a linker group). A
direct reaction between an antibody and a therapeutic agent is
possible when each possesses a substituent capable of reacting with
the other. For example, a nucleophilic group, such as an amino or
sulfhydryl group, on one molecule may be capable of reacting with a
carbonyl-containing group, such as an anhydride or an acid halide,
or with an alkyl group containing a good leaving group (e.g., a
halide) on the other molecule.
[0246] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0247] A variety of bifunctional or polyfunctional reagents, both
homo- and hetero-functional (such as those described in the catalog
of the Pierce Chemical Co., Rockford, Ill.), can be employed as the
linker group. Coupling can be effected, for example, through amino
groups, carboxyl groups, sulfhydryl groups, or oxidized
carbohydrate residues (e.g., U.S. Pat. No. 4,671,958).
[0248] Where a therapeutic agent is more potent when free from the
antibody portion of an immunoconjugate, it may be desirable to use
a linker group that is cleavable during or upon internalization
into a cell. A number of different cleavable linker groups have
been described. Mechanisms for the intracellular release of an
agent from these linker groups include cleavage by reduction of a
disulfide bond (e.g., U.S. Pat. No. 4,489,710), by irradiation of a
photolabile bond (e.g., U.S. Pat. No. 4,625,014), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045),
by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.
4,671,958), by protease cleavable linker (e.g., U.S. Pat. No.
6,214,345), and by acid-catalyzed hydrolysis (e.g., U.S. Pat. No.
4,569,789).
[0249] It may be desirable to couple more than one agent to an
antibody. Multiple molecules of an agent can be coupled to one
antibody molecule, and more than one type of agent can be coupled
to the same antibody. For example, about 1, 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, or 22 (or any other number in-between) molecules of
therapeutic agents can be coupled to an antibody. The average
number or quantitative distribution of therapeutic agent molecules
per antibody molecule in a preparation of conjugation reactions can
be determined by conventional means such as mass spectroscopy,
ELISA, or HPLC. Separation, purification, and characterization of
homogeneous antibody-drug conjugates having a certain number of
therapeutic agents conjugated thereto can be achieved by means such
as reverse phase HPLC or electrophoresis (see, e.g., Hamblett et
al., Clinical Cancer Res. 10:7063-70 (2004).
[0250] Examples of suitable therapeutic agents that can be
conjugated to an antibody include, but are not limited to,
chemotherapeutic agents (e.g., cytotoxic or cytostatic agents or
immunomodulatory agents), radiotherapeutic agents, therapeutic
antibodies, small molecule drugs, peptide drugs, immunomodulatory
agents, differentiation inducers, and toxins.
[0251] Examples of useful classes of cytotoxic or immunomodulatory
agents include, but are not limited to, antitubulin agents,
auristatins, DNA minor groove binders, DNA replication inhibitors,
alkylating agents (e.g., platinum complexes such as cis-platin,
mono(platinum), bis(platinum) and tri-nuclear platinum complexes
and carboplatin), anthracyclines, antibiotics, antifolates,
antimetabolites, chemotherapy sensitizers, duocarmycins,
etoposides, fluorinated pyrimidines, ionophores, lexitropsins,
nitrosoureas, platinols, pre-forming compounds, purine
antimetabolites, puromycins, radiation sensitizers, steroids,
taxanes, topoisomerase inhibitors, vinca alkaloids, and the
like.
[0252] Examples of individual cytotoxic or immunomodulatory agents
include, but are not limited to, androgen, anthramycin (AMC),
asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan,
buthionine sulfoximine, calicheamicin or calicheamicin derivatives,
camptothecin or camptothecins derivatives, carboplatin, carmustine
(BSNU), CC-1065, chlorambucil, cisplatin, colchicine,
cyclophosphamide, cytidine arabinoside (cytarabine), cytochalasin
B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin,
decarbazine, docetaxel, doxorubicin, etoposide, estrogen,
5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D,
hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU),
maytansine, mechlorethamine, melphalan, 6-mercaptopurine,
methotrexate, mithramycin, mitomycin C, mitoxantrone,
nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine,
rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA,
topotecan, vinblastine, vincristine, vinorelbine, VP-16, and
VM-26.
[0253] Examples of other suitable cytotoxic agents include, but are
not limited to, DNA minor groove binders (e.g., enediynes and
lexitropsins, a CBI compound; see also U.S. Pat. No. 6,130,237),
duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins,
vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin,
rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin,
netropsin, epothilone A and B, estramustine, cryptophysins,
cemadotin, a maytansinoid, discodermolide, eleutherobin, and
mitoxantrone.
[0254] Examples of other suitable agents include, but are not
limited to, radionuclides, differentiation inducers, drugs, toxins,
and derivatives thereof. Exemplary radionuclides include .sup.90Y,
.sup.123I, .sup.125I, .sup.131I, .sup.186Re, .sup.188Re,
.sup.211At, and .sup.212Bi. Exemplary drugs include methotrexate,
and pyrimidine and purine analogs. Exemplary differentiation
inducers include phorbol esters and butyric acid. Exemplary toxins
include ricin, abrin, diptheria toxin, cholera toxin, gelonin,
Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral
protein.
[0255] In some embodiments, the therapeutic agent used in an
antibody-drug conjugate is an anti-tubulin agent. Examples of
anti-tubulin agents include, but are not limited to, taxanes (e.g.,
Taxol.RTM. (paclitaxel), Taxotere.RTM. (docetaxel)), T67 (Tularik)
and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and
vinorelbine). Other antitubulin agents include, for example,
baccatin derivatives, taxane analogs (e.g., epothilone A and B),
nocodazole, colchicine and colcimid, estramustine, cryptophysins,
cemadotin, maytansinoid, combretastatins, discodermolide, and
eleutherobin.
[0256] In certain embodiments, the cytotoxic agent is a
maytansinoid, another group of anti-tubulin agents. For example, in
specific embodiments, the maytansinoid is maytansine, DM-1
(ImmunoGen, Inc.; see also Chari et al., Cancer Res. 52:127-131
(1992)) or DM-4. In some embodiments, the therapeutic agent is an
auristatin, such as auristatin E (also known in the art as
dolastatin-10) or a derivative thereof. Typically, an auristatin E
derivative is, e.g., an ester formed between auristatin E and a
keto acid. For example, auristatin E can be reacted with paraacetyl
benzoic acid or benzoylvaleric acid to produce AEB and AEVB,
respectively. Other typical auristatin derivatives include AFP,
MMAF, and MMAE. The synthesis and structure of auristatin
derivatives are described in U.S. Patent Application Publication
Nos. 2003-0083263, 2005-0238649 and 2005-0009751; PCT Publication
Nos WO 04/010957 and WO 02/088172, and U.S. Pat. Nos. 6,323,315;
6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483;
5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024;
5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444;
and 4,486,414.
[0257] 4. LCM Nucleic Acid Molecules
[0258] Exemplary isolated LCM nucleic acid molecules of the
invention consist of, consist essentially of, or comprise a
nucleotide sequence that encodes a LCM protein of the invention, an
allelic variant thereof, or an ortholog or paralog thereof, for
example. As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5 kilobases (KB), 4 KB, 3 KB, 2 KB, or 1 KB or
less, particularly contiguous protein-encoding sequences and
protein-encoding sequences within the same gene but separated by
introns in the genomic sequence, and flanking nucleotide sequences
that contain regulatory elements. The primary consideration is that
the nucleic acid is isolated from remote and unimportant flanking
sequences such that it can be individualed to the specific
manipulations described herein such as recombinant expression,
preparation of probes and primers, and other uses specific to the
nucleic acid molecules. Moreover, an "isolated" nucleic acid
molecule, such as a transcript/cDNA molecule, can be substantially
free of other cellular material, or culture medium when produced by
recombinant techniques, or chemical precursors or other chemicals
when chemically synthesized.
[0259] A nucleic acid molecule can be fused to other coding or
regulatory sequences and still be considered isolated. Isolated
nucleic acid molecules can include heterologous nucleotide
sequences, such as heterologous nucleotide sequences that are fused
to a nucleic acid molecule by recombinant techniques. For example,
recombinant DNA molecules contained in a vector are considered
isolated. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells, or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of isolated DNA molecules. Isolated nucleic acid molecules further
include such molecules produced synthetically.
[0260] Isolated nucleic acid molecules can encode a mature protein
plus additional amino or carboxyl-terminal amino acids, or amino
acids interior to the mature protein (when the mature form has more
than one peptide chain, for instance). Such sequences may play a
role in processing of a protein from precursor to a mature form,
facilitate protein trafficking, prolong or shorten protein
half-life, or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0261] Isolated nucleic acid molecules include, but are not limited
to, sequences encoding a LCM protein alone, sequences encoding a
mature protein with additional coding sequences (such as a leader
or secretory sequence (e.g., a pre-pro or pro-protein sequence)),
and sequences encoding a mature protein (with or without additional
coding sequences) plus additional non-coding sequences (e.g.,
introns and non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding, and/or stability of mRNA). In addition, nucleic
acid molecules can be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0262] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form of DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. Nucleic acid molecules, especially DNA,
can be double-stranded or single-stranded. Single-stranded nucleic
acid can be the coding strand (sense strand) or the non-coding
strand (anti-sense strand).
[0263] Exemplary embodiments of the invention further provide
isolated nucleic acid molecules that encode fragments of a LCM
protein as well as nucleic acid molecules that encode obvious
variants of a LCM protein. Such nucleic acid molecules may be
naturally occurring, such as allelic variants (same locus),
paralogs (different locus), and orthologs (different organism), or
can be constructed by recombinant DNA methods or by chemical
synthesis. Such non-naturally occurring variants can be made by
mutagenesis techniques, including those applied to nucleic acid
molecules, cells, or organisms. Accordingly, nucleic acid molecule
variants can contain nucleotide substitutions, deletions,
inversions, and/or insertions. Variations can occur in either or
both the coding and non-coding regions, and variations can produce
conservative and/or non-conservative amino acid substitutions.
[0264] A fragment of a nucleic acid molecule typically comprises a
contiguous nucleotide sequence at least 8, 10, 12, 15, 16, 18, 20,
22, 25, 30, 40, 50, 100, 150, 200, 250, 500 (or any other number
in-between) or more nucleotides in length. The length of a fragment
can be based on its intended use. For example, a fragment can
encode epitope bearing regions of a protein, or can be used as DNA
probes and primers. Isolated fragments can be produced by
synthesizing an oligonucleotide probe using known techniques, for
example, and can optionally be labeled and used to screen a cDNA
library, genomic DNA, or mRNA, for example. Primers can be used in
PCR reactions to clone specific regions of a gene.
[0265] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. An oligonucleotide
typically comprises a nucleotide sequence that hybridizes under
stringent conditions to at least about 8, 10, 12, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 (or any other number
in-between) or more contiguous nucleotides.
[0266] Allelic variants, orthologs, and homologs can be identified
using methods well known in the art. These variants can comprise a
nucleotide sequence encoding a protein that is typically 60-70%,
70-80%, 80-90%, 90-95%, 96%, 97%, 98%, or 99% homologous to the
nucleotide sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to a nucleotide sequence shown in the Sequence Listing
or a fragment thereof.
[0267] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a protein at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in, for example, Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y. (1989-2006), 6.3.1-6.3.6. One
example of stringent hybridization conditions is hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. Examples of moderate to low stringency
hybridization conditions are well known in the art.
[0268] Exemplary embodiments of the invention also include kits for
detecting the presence of LCM nucleic acid (e.g., DNA or mRNA) in a
biological sample. For example, a kit can comprise reagents such as
a labeled or labelable nucleic acid and/or other agents capable of
detecting LCM nucleic acid in a biological sample; means for
determining the amount of LCM nucleic acid in the sample; and means
for comparing the amount of LCM nucleic acid in the sample with a
standard. The nucleic acid and/or other agent can be packaged in
one or more suitable containers. The kit can further comprise
instructions for using the kit to detect LCM nucleic acid.
[0269] 5. Vectors and Host Cells
[0270] Exemplary embodiments of the invention also provide vectors
containing LCM nucleic acid molecules. The term "vector" refers to
a vehicle, such as a nucleic acid molecule, which can transport the
LCM nucleic acid molecules. When the vector is a nucleic acid
molecule, the LCM nucleic acid molecules are covalently linked to
the vector nucleic acid. A vector can be, for example, a plasmid,
single or double stranded phage, a single or double stranded RNA or
DNA viral vector, or artificial chromosome, such as a BAC, PAC,
YAC, OR MAC.
[0271] A vector can be maintained in a host cell as an
extrachromosomal element where it replicates and produces
additional copies of the LCM nucleic acid molecules. Alternatively,
a vector can integrate into a host cell genome and produce
additional copies of the LCM nucleic acid molecules when the host
cell replicates.
[0272] Exemplary embodiments of the invention provide vectors for
maintenance (cloning vectors) and vectors for expression
(expression vectors) of the nucleic acid molecules, for example.
Expression vectors can express a portion of, or all of, a protein
sequence. Vectors can function in prokaryotic or eukaryotic cells
or in both (shuttle vectors). Vectors also include insertion
vectors, which integrate a nucleic acid molecule into another
nucleic acid molecule, such as into the cellular genome (such as to
alter in situ expression of a gene and/or gene product). For
example, an endogenous protein-coding sequence can be entirely or
partially replaced via homologous recombination with a
protein-coding sequence containing one or more specifically
introduced mutations.
[0273] Expression vectors can contain cis-acting regulatory regions
that are operably-linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. The separate nucleic acid
molecule may provide, for example, a trans-acting factor
interacting with the cis-regulatory control region to allow
transcription of the nucleic acid molecules from the vector.
Alternatively, a trans-acting factor may be supplied by a host
cell. Additionally, a trans-acting factor can be produced from a
vector itself. It is understood, however, that transcription and/or
translation of nucleic acid molecules can occur in cell-free
systems.
[0274] Regulatory sequences to which LCM nucleic acid molecules can
be operably linked include, for example, promoters for directing
mRNA transcription. These include, but are not limited to, the left
promoter from bacteriophage, the lac, TRP, and TAC promoters from
E. coli, the early and late promoters from SV40, the CMV immediate
early promoter, the adenovirus early and late promoters, and
retrovirus long-terminal repeats.
[0275] In addition to control regions that promote transcription,
expression vectors can also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0276] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region, a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. Numerous regulatory
sequences useful in expression vectors are well known in the art
(e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual.
3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (2001)).
[0277] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (2001).
[0278] A regulatory sequence can provide constitutive expression in
one or more host cells (e.g., tissue specific) or can provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factors such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known in the art.
[0279] Nucleic acid molecules can be inserted into vector nucleic
acid by well-known methodology. For example, the DNA sequence that
will ultimately be expressed can be joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known in the art.
[0280] A vector containing a nucleic acid molecule of interest can
be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells (e.g., DG44 or CHO-s), and plant cells.
[0281] As described herein, it may be desirable to express a
protein as a fusion protein. Accordingly, exemplary embodiments of
the invention provide fusion vectors that allow for the production
of fusion proteins. Fusion vectors can, for example, increase the
expression of a recombinant protein; increase the solubility of a
recombinant protein, and/or aid in the purification of a protein
such as by acting as a ligand for affinity purification. A
proteolytic cleavage site can be introduced at the junction of the
fusion moiety so that the desired protein can ultimately be
separated from the fusion moiety. Proteolytic enzymes include, but
are not limited to, factor Xa, thrombin, and enteroenzyme. Typical
fusion expression vectors include pGEX (Smith et al., Gene 67:31-40
(1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to a
recombinant marker protein. Examples of suitable inducible
non-fusion E. coli expression vectors include pTrc (Amann et al.,
Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185:60-89 (1990)).
[0282] Recombinant protein expression can be maximized in host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990), pp.
119-128). Alternatively, the sequence of a nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, such as E. coli (Wada et al., Nucleic Acids
Res. 20:2111-2118 (1992)).
[0283] LCM nucleic acid molecules can, for example, be expressed by
expression vectors in a yeast host. Examples of vectors for
expression in yeast (e.g., S. cerevisiae) include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell 30:933-943 (1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
Nucleic acid molecules can also be expressed in insect cells using,
for example, baculovirus expression vectors. Baculovirus vectors
available for expression of proteins in cultured insect cells
(e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell.
Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al.,
Virology 170:31-39 (1989)). Nucleic acid molecules can also be
expressed in mammalian cells using mammalian expression vectors.
Examples of mammalian expression vectors include pCDM8 (Seed, B.
Nature 329:840 (1987)), pMT2PC (Kaufman et al., EMBO J. 6:187-195
(1987)), and CHEF (U.S. Pat. No. 5,888,809).
[0284] The expression vectors listed herein are provided by way of
example only of well-known vectors available to those of ordinary
skill in the art that would be useful to express LCM nucleic acid
molecules. The person of ordinary skill in the art would be aware
of other vectors suitable for maintenance, propagation, and/or
expression of LCM nucleic acid molecules (e.g., Sambrook et al.
Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
[0285] Exemplary embodiments of the invention also encompasses
vectors in which LCM nucleic acid molecules are cloned into a
vector in reverse orientation, but operably linked to a regulatory
sequence that permits transcription of antisense RNA. Thus, an
antisense transcript can be produced to all, or to a portion, of a
LCM nucleic acid molecule, including coding and non-coding regions.
Expression of this antisense RNA may be individual to each of the
parameters described above in relation to expression of the sense
RNA (e.g., regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0286] Exemplary embodiments of the invention provide recombinant
host cells containing the vectors described herein. Host cells
include, for example, prokaryotic cells, lower eukaryotic cells
such as yeast, other eukaryotic cells such as insect cells, and
higher eukaryotic cells such as mammalian cells.
[0287] Recombinant host cells can be prepared by introducing vector
constructs, such as described herein, into cells by techniques
readily available to a person of ordinary skill in the art. These
techniques include, but are not limited to, calcium phosphate
transfection, DEAE-dextran-mediated transfection, cationic
lipid-mediated transfection, electroporation, transduction,
infection, lipofection, microinjection, and other techniques such
as those found in Sambrook, et al. (Molecular Cloning: A Laboratory
Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (2001).
[0288] For example, using techniques such as these, a retroviral or
other viral vector can be introduced into mammalian cells. Examples
of mammalian cells into which a retroviral vector can be introduced
include, but are not limited to, primary mammalian cultures or
continuous mammalian cultures, COS cells, NIH3T3, 293 cells (ATCC
#CRL 1573), and dendritic cells.
[0289] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, nucleic acid molecules of interest can be
introduced either alone or with other unrelated nucleic acid
molecules such as those providing trans-acting factors for
expression vectors. When more than one vector is introduced into a
cell, the vectors can be introduced independently, co-introduced,
or joined to the nucleic acid molecule vector.
[0290] Bacteriophage and viral vectors can be introduced into cells
as packaged or encapsulated virus by standard procedures for
infection and transduction. Viral vectors can be
replication-competent or replication-defective. If viral
replication is defective, replication can occur in host cells that
provide functions that complement the defects.
[0291] Vectors can include selectable markers that enable the
selection of a subpopulation of cells that contain the recombinant
vector constructs. Markers can be contained in the same vector that
contains the nucleic acid molecules of interest or can be on a
separate vector. Exemplary markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells, and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait can be used.
[0292] While mature proteins can be produced in bacteria, yeast,
mammalian cells, and other cells under the control of appropriate
regulatory sequences, cell-free transcription and translation
systems can also be used to produce these proteins using RNA
derived from the DNA constructs described herein.
[0293] If secretion of a protein is desired, appropriate secretion
signals can be incorporated into a vector. The signal sequence can
be endogenous or heterologous to the protein.
[0294] If a protein is not secreted into a medium, the protein can
be isolated from a host cell by standard disruption procedures,
including freeze/thaw, sonication, mechanical disruption, use of
lysing agents, and the like. A protein can then be recovered and
purified by well-known purification methods including, for example,
ammonium sulfate precipitation, acid extraction, anion or cationic
exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0295] It is also understood that, depending upon the host cell
used in recombinant production of a protein, proteins can have
various glycosylation patterns or can be non-glycosylated, such as
when produced in bacteria. In addition, proteins can include an
initial modified methionine in some instances as a result of a
host-mediated process.
[0296] Recombinant host cells that express a LCM protein have a
variety of uses. For example, such host cells are useful for
producing LCM proteins, which can be further purified to produce
desired amounts of the protein or fragments thereof. Thus, host
cells containing expression vectors are useful for protein
production.
[0297] Host cells are also useful for conducting cell-based assays
involving a LCM protein or fragments thereof. For example, a
recombinant host cell expressing a LCM protein can be used to assay
compounds that stimulate or inhibit the protein's function.
[0298] Host cells are also useful for identifying mutant LCM
proteins in which the protein's function is affected. Host cells
expressing mutant proteins are useful for assaying compounds that
have a desired effect on the mutant proteins (e.g., stimulating or
inhibiting function), particularly if the mutant proteins naturally
occur and give rise to a pathology.
[0299] 6. Diagnosis and Treatment in General
[0300] The following terms, as used in the present specification
and claims, are intended to have the meaning as defined below,
unless indicated otherwise.
[0301] As used herein, a "biological sample" (or just "sample") can
comprise, for example, tissue, blood, sera, cells, cell lines, or
biological fluids such as plasma, interstitial fluid, urine,
cerebrospinal fluid, and the like. A biological sample is
typically, although not necessarily, obtained from an individual by
a medical practitioner.
[0302] As used herein, a "individual" can be a mammalian individual
or non-mammalian individual, preferably a mammalian individual. A
mammalian individual can be a human or non-human, preferably a
human. The terms "individual", "individual", and "patient" are used
herein interchangeably.
[0303] A "healthy" or "normal" individual or biological sample is a
individual or biological sample in which the disease of interest
(e.g., lung cancer) is not detectable, as ascertained by using
conventional diagnostic methods (such a biological sample can
interchangeably be referred to as a "control" sample).
[0304] As used herein, "disease(s)" include cancer, especially
aerodigestive cancers, and particularly lung cancer, as well as
associated diseases and pathologies, such as other lung
diseases.
[0305] The term "diagnose" (or "diagnosing", etc.) refers to
determining the current state or status (e.g., the presence/absence
or characteristics) of a disease condition, such as initially
detecting the presence of a disease, characterizing/classifying a
disease, or detecting disease progression, remission, or
recurrence.
[0306] The term "prognose" (or "prognosing", etc.) refers to
predicting the future course of a disease in a patient who has the
disease (e.g., predicting patient survival).
[0307] The term "assess" (or "assessing", etc.) can encompass
"diagnose" and "prognose" but can also encompass making future
determinations/predictions about the disease in an individual who
does not have the disease or determining/predicting the likelihood
that a disease will recur in an individual who apparently has been
cured of the disease. The term "assess" can also encompass making
assessments of an individual's response to a therapy, such as
predicting whether an individual is likely to respond favorably to
a therapeutic agent or is unlikely to respond to a therapeutic
agent (or will experience toxic or other undesirable side effects,
for example), selecting a therapeutic agent for administration to
an individual, or monitoring or determining an individual's
response to a therapy that has been administered to the
individual.
[0308] Thus, "assessing" lung cancer can include, for example,
prognosing the future course of lung cancer; predicting recurrence
of lung cancer in an individual who apparently has been cured of
lung cancer; and/or determining or predicting an individual's
response to a lung cancer treatment or selecting a lung cancer
treatment to administer to an individual based on the individual's
LCM profile (i.e., the differential abundance level of one or more
LCM in the individual).
[0309] The following examples may be referred to as either
"diagnosing" or "assessing" lung cancer: initially detecting the
presence of lung cancer; determining a specific stage, type or
sub-type, or other classification or characteristic of lung cancer;
determining whether a lung lesion is a benign lesion or a malignant
lung tumor; and/or detecting/monitoring lung cancer progression
(e.g., monitoring lung tumor growth or metastatic spread),
remission, or recurrence.
[0310] LCM are therefore useful as "prognostic markers" (e.g.,
predicting disease progression) and "predictive markers" (e.g.,
predicting drug response), among other uses.
[0311] "Treat", "treating", or "treatment" of a disease includes:
(1) inhibiting the disease, i.e., arresting or reducing the
development of the disease or its clinical symptoms, or (2)
relieving the disease, i.e., causing regression of the disease or
its clinical symptom(s).
[0312] The term "prophylaxis" is used to distinguish from
"treatment," and to encompass both "preventing" and "suppressing."
It is not always possible to distinguish between "preventing" and
"suppressing," as the ultimate inductive event or events may be
unknown, latent, or the patient is not ascertained until well after
the occurrence of the event or events. Therefore, the term
"protection", as used herein, is meant to include
"prophylaxis."
[0313] A "therapeutically effective amount" means the amount of an
agent that, when administered to a individual for treating a
disease, is sufficient to effect such treatment for the disease.
The "therapeutically effective amount" can vary depending on such
factors as the agent, the disease and its severity, and the age,
weight, etc., of the individual to be treated.
[0314] Exemplary embodiments of the invention provide methods for
treating diseases, especially cancer, and particularly lung cancer,
comprising administering to a patient a therapeutically effective
amount of an antagonist, agonist, or a pharmaceutical composition
thereof. Exemplary embodiments of the invention further provide
agonists and antagonists to LCM proteins, as well as pharmaceutical
compositions that comprise an agonist or antagonist with a suitable
carrier such as a pharmaceutically acceptable excipient.
[0315] Exemplary agonists or antagonists include antibodies that
specifically bind to a LCM protein. Antibodies can be used alone or
in combination with one or more other therapeutic agents (e.g., as
an antibody-drug conjugate or a combination therapy). Further
examples of molecules that can be used as antagonists include, but
are not limited to, small molecules that inhibit the function or
abundance level of LCM, and inhibitory nucleic acid molecules such
as RNAi or antisense nucleic acid molecules that specifically
hybridize to LCM nucleic acid.
[0316] Exemplary embodiments of the invention further encompass
novel agents identified by screening assays using LCM, such as the
screening assays described herein, as well as methods of using
these agents, such as for treatment or diagnostic purposes. For
example, an agent identified as described herein (e.g., a
LCM-modulating agent, a LCM-specific nucleic acid molecule such as
an RNAi or antisense molecule, a LCM-specific antibody, a
LCM-specific antibody-drug conjugate, or a LCM-binding partner) can
be used in an animal or other model, such as to determine efficacy,
toxicity, or side effects of treatment with the agent.
[0317] Modulators of LCM protein activity, such as modulators
identified according to the drug screening assays described herein,
can be used to treat a individual with a disorder mediated by a
LCM, e.g., by treating cells or tissues that express LCM at a
differential level. Methods of treatment can include the step of
administering a modulator of LCM activity in a pharmaceutical
composition to a individual in need of such treatment.
[0318] In certain exemplary embodiments, if decreased expression or
activity of a protein is desired, an antibody to the protein or an
inhibitor/antagonist and the like, or a pharmaceutical agent
containing one or more of these molecules, can be administered to
an individual. In other exemplary embodiments, if increased
expression or activity of a protein is desired, the protein itself
or an agonist/enhancer and the like, or a pharmaceutical agent
containing one or more of these molecules, can be administered.
Administration can be effected by methods well known in the art and
may include delivery by an antibody specifically targeted to the
protein. Neutralizing antibodies, which inhibit dimer formation,
can be used when decreased expression or activity of a protein is
desired.
[0319] Although modulating agents can be administered in a pure or
substantially pure form, modulating agents can also be administered
as pharmaceutical compositions, formulations, or preparations with
a carrier. Exemplary formulations of the invention, such as for
human or veterinary use, comprise a suitable active LCM-modulating
agent, together with one or more pharmaceutically acceptable
carriers and, optionally, other therapeutic ingredients. The
carrier(s) are "acceptable" in the sense of being compatible with
other ingredients of a formulation and not deleterious to the
recipient thereof. The formulations can be presented in unit dosage
form and can be prepared by any method known to the skilled
artisan.
[0320] Examples of suitable pharmaceutical carriers include
proteins such as albumins (e.g., U.S. Pat. No. 4,507,234), peptides
and polysaccharides such as aminodextran (e.g., U.S. Pat. No.
4,699,784), and water. A carrier can also bear an agent by
noncovalent bonding or by encapsulation, such as within a liposome
vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers
specific for radionuclide agents include radiohalogenated small
molecules and chelating compounds. For example, U.S. Pat. No.
4,735,792 discloses representative radiohalogenated small molecules
and their synthesis. A radionuclide chelate can be formed from
chelating compounds that include those containing nitrogen and
sulfur atoms as the donor atoms for binding the metal, metal oxide,
radionuclide. For example, U.S. Pat. No. 4,673,562 discloses
representative chelating compounds and their synthesis.
[0321] Methods of preparing pharmaceutical formulations typically
include the step of bringing into association the active ingredient
with the carrier, which constitutes one or more accessory
ingredients. Formulations can be prepared by uniformly and
intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers, or both, and
then, if necessary, shaping the product into the desired
formulation.
[0322] Formulations suitable for intravenous, intramuscular,
subcutaneous, or intraperitoneal administration can comprise
sterile aqueous solutions of the active ingredient with solutions,
which can be isotonic with the blood of the recipient. Such
formulations can be prepared by dissolving solid active ingredient
in water containing physiologically compatible substances such as
sodium chloride (e.g., 0.1-2.0 M), glycine, and the like, and
having a buffered pH compatible with physiological conditions to
produce an aqueous solution, and rendering the solution sterile.
These may be present in unit or multi-dose containers, for example,
sealed ampoules or vials.
[0323] Exemplary formulations of the invention can incorporate a
stabilizer. Exemplary stabilizers include polyethylene glycol,
proteins, saccharides, amino acids, inorganic acids, detergents,
and organic acids, which can be used either alone or as admixtures.
These stabilizers can be incorporated in an amount of, for example,
0.11-10,000 parts by weight per part by weight of an agent. If two
or more stabilizers are to be used, their total amount can be
within the range specified above. These stabilizers can be used in
aqueous solutions at an appropriate concentration and pH. The
specific osmotic pressure of such aqueous solutions can be in the
range of 0.1-3.0 osmoles, preferably in the range of 0.8-1.2. The
pH of the aqueous solution can be adjusted to be within the range
of 5.0-9.0, preferably within the range of 6-8. In formulating an
antibody or antibody-drug conjugate, an anti-adsorption agent can
be used.
[0324] Additional pharmaceutical methods can be employed to control
duration of action. Controlled release can be achieved through the
use of polymer to complex or absorb the proteins or their
derivatives. Controlled delivery can be achieved by selecting
appropriate macromolecules (e.g., polyester, polyamino acids,
polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,
carboxymethylcellulose, or protamine sulfate) and the concentration
of macromolecules as well as the methods of incorporation in order
to control release. Another possible method to control the duration
of action by controlled-release preparations is to incorporate an
anti-LCM antibody into particles of a polymeric material such as
polyesters, polyamino acids, hydrogels, poly(lactic acid) or
ethylene vinylacetate copolymers. Alternatively, instead of
incorporating these agents into polymeric particles, it is possible
to entrap these materials in microcapsules prepared, for example,
by coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly(methylmethacylate) microcapsules, respectively, or in
colloidal drug delivery systems, for example, liposomes, albumin
microspheres, microemulsions, nanoparticles, and nanocapsules or in
macroemulsions.
[0325] When oral preparations are desired, the compositions can be
combined with typical carriers, such as lactose, sucrose, starch,
talc magnesium stearate, crystalline cellulose, methyl cellulose,
carboxymethyl cellulose, glycerin, sodium alginate or gum arabic,
among others.
[0326] Any of the therapeutic agents provided herein may be
administered in combination with other therapeutic agents.
Selection of agents for use in combination therapy can be made by
one of ordinary skill in the art according to conventional
pharmaceutical principles. A combination of therapeutic agents may
act synergistically to affect treatment of a particular disorder at
a lower dosage of each agent.
[0327] 7. Methods of Detection and Diagnosis Based on LCM
Proteins
[0328] LCM proteins are useful for diagnosing a disease,
particularly diseases in which the protein is over- or
under-expressed, especially cancer, and particularly lung cancer.
The diagnostic methods may be further suitable for monitoring
disease progression in patients undergoing treatment, or for
testing for reoccurrence of disease in patients who were previously
treated for a disease, for example. Accordingly, exemplary
embodiments of the invention provide methods for detecting the
presence of, or abundance levels of, a LCM protein in a biological
sample.
[0329] In vitro techniques for detection of proteins include, but
are not limited to, enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence using a
detection reagent, such as an antibody or protein binding agent.
Alternatively, a protein can be detected in vivo in a individual by
introducing into the individual a labeled antibody (or other types
of detection agent) specific for the protein marker. For example,
an antibody can be labeled with a radioactive marker whose presence
and location in a individual can be detected by standard imaging
techniques. Also useful are methods that detect variants of a
protein (e.g., allelic variants or mutations) and methods that
detect fragments of a protein in a sample.
[0330] Examples of immunoassays that can be used in accordance with
exemplary embodiments of the invention include, but are not limited
to, competitive and non-competitive assays using techniques such as
Western blots, radioimmunoassays, ELISA, "sandwich" immunoassays,
immunoprecipitation assays, precipitation reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays, and protein A immunoassays, as well as fluorescence
polarization immunoassay (FPIA), fluorescence immunoassay (FIA),
enzyme immunoassay (EIA), and nephelometric inhibition immunoassay
(NIA). Immunoassays such as these are well known in the art and are
described in, for example, Ausubel et al., Current Protocols in
Molecular Biology, 1992-2006.
[0331] For example, ELISA can be used to detect or quantify one or
more LCM. For example, ELISA (or other types of LCM assays) can be
used to detect LCM in, for example, a high-risk individual or
population, in an individual suspected of having lung cancer, or in
an individual with no suspicion of having lung cancer (e.g., an
individual undergoing routine screening for lung cancer).
[0332] In certain exemplary ELISA methods, an antibody that
specifically binds to an LCM antigen may be coated to the well of a
suitable container (e.g., a 96 well microtiter plate), a patient
sample (e.g., a serum sample) can be added to the well and
incubated for a period of time, and the presence of the LCM antigen
in the patient sample can be detected upon binding of the LCM
antigen in the patient sample to the antibody that is coated to the
well. In this instance, a second antibody conjugated to a
detectable moeity may optionally be added following the addition of
the patient sample to the coated well. ELISA methods such as these
may be modified or optimized as desired.
[0333] Further, instead of coating the well with an antibody to an
LCM antigen, the LCM antigen itself may be coated to the well.
Thus, in certain exemplary ELISA methods, an LCM antigen can be
coated to the well of a suitable container (e.g., a 96 well
microtiter plate), an antibody (which may optionally be conjugated
to a detectable moiety such as an enzymatic substrate like
horseradish peroxidase or alkaline phosphatase) to the LCM antigen
can be added to the well and incubated for a period of time, and
the presence of the LCM antigen can be detected. The antibody to
the LCM antigen does not have to be conjugated to a detectable
moiety; for example, a second antibody (which recognizes the
antibody to the LCM antigen) conjugated to a detectable moeity may
be added to the well. ELISA methods such as these may be modified
or optimized as desired.
[0334] Proteins can be isolated from a biological sample (such as
from a patient having a disease) and assayed for the presence of a
mutation. A mutation can include, for example, one or more amino
acid substitutions, deletions, insertions, rearrangements (such as
from aberrant splicing events), or inappropriate post-translational
modifications. Examples of analytic methods useful for detecting
mutations in a protein include, but are not limited to, altered
electrophoretic mobility, altered tryptic peptide digest, altered
protein activity in cell-based or cell-free assays, alteration in
substrate or antibody-binding patterns, altered isoelectric point,
and direct amino acid sequencing.
[0335] Information obtained by detecting a protein can be used, for
example, to determine prognosis and appropriate course of treatment
for a disease. For example, individuals with a particular LCM
expression level or stage of disease may respond differently to a
given treatment that individuals lacking LCM expression, or
individuals over- or under-expressing LCM. Information obtained
from diagnostic methods of the invention can provide for the
personalization of diagnosis and treatment.
[0336] In exemplary embodiments, the invention provides methods for
diagnosing disease (including, for example, monitoring treatment
response or recurrence of disease following treatment) in a
individual comprising: determining the abundance level of LCM
(e.g., LCM protein or nucleic acid, or protein or nucleic acid
fragments thereof) in a test sample from the individual; wherein a
difference in the abundance level of LCM relative to the abundance
level of LCM in a test sample from a healthy individual, or the
level established for a healthy individual, is indicative of
disease.
[0337] Exemplary embodiments of the invention provide methods for
diagnosing diseases having differential protein expression. For
example, normal, control, or standard values (e.g., that represent
typical expression levels of a protein in healthy individuals) can
be established, such as by combining body fluids, tissues, or cell
extracts taken from a normal healthy mammalian or human individual
with specific antibodies to a protein under conditions for complex
formation. Standard values for complex formation in normal and
disease tissues can be established by various methods, such as
photometric means. Complex formation, as it is expressed in a test
sample, can be compared with the standard values. Deviation from a
normal standard and toward a disease standard can provide
parameters for disease diagnosis or prognosis while deviation away
from a disease standard and toward a normal standard can be used to
evaluate treatment efficacy, for example.
[0338] Immunological methods for detecting and measuring complex
formation as a measure of protein expression using either specific
polyclonal or monoclonal antibodies are known in the art. Examples
of such techniques include ELISAs, radioimmunoassays (RIAs), flow
cytometry (also referred to as fluorescence-activated cell sorting,
or FACS), and antibody arrays. Such immunoassays typically involve
the measurement of complex formation between a protein and its
specific antibody. These assays and their quantitation against
purified, labeled standards are well known in the art (Ausubel,
supra, unit 10.1-10.6). For example, a two-site, monoclonal-based
immunoassay utilizing antibodies reactive to two non-interfering
epitopes can be utilized, and competitive binding assay can also be
utilized (Pound (1998) Immunochemical Protocols, Humana Press,
Totowa N.J.).
[0339] For diagnostic applications, an antibody can be labeled with
a detectable moiety (interchangeably referred to as a "label" or
"detectable substance"), such as to facilitate detection by various
imaging methods. Methods for detection of labels include, but are
not limited to, fluorescence, light, confocal, and electron
microscopy; magnetic resonance imaging and spectroscopy;
fluoroscopy, computed tomography and positron emission tomography.
Examples of suitable labels include, but are not limited to,
fluorescein, rhodamine, eosin and other fluorophores,
radioisotopes, gold, gadolinium and other lanthanides, paramagnetic
iron, fluorine-18 and other positron-emitting radionuclides.
Additionally, labels may be bi- or multi-functional and be
detectable by more than one of the methods listed. Antibodies may
be directly or indirectly labeled. Attachment of labels to
antibodies includes covalent attachment of a label, incorporation
of a label into an antibody, and covalent attachment of a chelating
compound for binding of a label, among others well known in the
art.
[0340] Numerous detectable moieties are available for labeling
antibodies, including, but not limited to, those in the following
categories:
[0341] (a) Radioisotopes, such as .sup.36S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. An antibody can be labeled with a
radioisotope using the techniques described in Current Protocols in
Immunology, vol 1-2, Coligen et al., Ed., Wiley-Interscience, New
York, Pubs. (1991-2006), for example, and radioactivity can be
measured using scintillation counting.
[0342] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. Fluorescent labels can be conjugated to an antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorometer.
[0343] (c) Various enzyme-substrate labels are available (e.g.,
U.S. Pat. Nos. 4,275,149 and 4,318,980). An enzyme generally
catalyzes a chemical alteration of a chromogenic substrate which
can be measured using various techniques. For example, an enzyme
may catalyze a color change in a substrate, which can be measured
spectrophotometrically. Alternatively, an enzyme may alter the
fluorescence or chemiluminescence of a substrate. Techniques for
quantifying a change in fluorescence are described herein and well
known in the art A chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light which can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay, in
Methods in Enzyme. (Ed. J. Langone & H. Van Vunakis), Academic
press, New York, 73: 147-166 (1981).
[0344] A label can be indirectly conjugated with an antibody. The
skilled artisan will be aware of various techniques for achieving
this. For example, an antibody can be conjugated with biotin and
any of the three broad categories of labels mentioned above can be
conjugated with avidin, or vice versa. Biotin binds selectively to
avidin and thus, the label can be conjugated with the antibody in
this indirect manner. Alternatively, to achieve indirect
conjugation of a label with an antibody, an antibody can be
conjugated with a small hapten (e.g., digoxin) and one of the
different types of labels mentioned above can be conjugated with an
anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect
conjugation of a label with an antibody can be achieved.
[0345] Antibodies can be used to isolate LCM proteins by standard
techniques, such as affinity chromatography or immunoprecipitation,
and antibodies can facilitate the purification of the natural
protein from cells and recombinantly-produced protein expressed in
host cells. Biological samples can be tested directly for the
presence of a LCM protein by assays (e.g., ELISA or
radioimmunoassay) and format (e.g., microwells, dipstick, etc., as
described in International Patent Publication WO 93/03367).
Alternatively, proteins in a sample can be size separated (e.g., by
polyacrylamide gel electrophoresis (PAGE)), in the presence or
absence of sodium dodecyl sulfate (SDS), and the presence of a LCM
detected by immunoblotting (e.g., Western blotting).
[0346] Antibody binding can also be detected by "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation
reactions, immunodiffusion assays, in situ immunoassays (e.g.,
using colloidal gold, enzyme or radioisotope labels, for example),
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays, etc.), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.
[0347] In certain exemplary embodiments, antibody binding can be
detected by detecting a label on the primary antibody. In other
exemplary embodiments, a primary antibody can be detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In further exemplary embodiments, the secondary antibody
is labeled. Numerous means are known in the art for detecting
binding in an immunoassay and are within the scope of the
invention. In some embodiments, an automated detection assay is
utilized. Methods for the automation of immunoassays are well known
in the art (e.g., U.S. Pat. Nos. 5,885,530: 4,981,785: 6,159,750:
and 5,358,691, each of which is herein incorporated by reference).
In some embodiments, the analysis and presentation of results are
also automated. For example, in some embodiments, software that
generates a prognosis based on the presence or absence of one or
more antigens can be implemented.
[0348] Competitive binding assays typically rely on the ability of
a labeled standard to compete with a test sample for binding with a
limited amount of antibody. The amount of antigen in the test
sample is inversely proportional to the amount of standard that
becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies generally are
insolubilized before or after the competition. As a result, the
standard and test sample that are bound to the antibodies can be
separated from the standard and test sample that remain
unbound.
[0349] Sandwich assays typically involve the use of two antibodies,
each capable of binding to a different immunogenic portion, or
epitope, of the protein to be detected. In typical sandwich assays,
the test sample to be analyzed is bound by a first antibody, which
is immobilized on a solid support, and thereafter a second antibody
binds to the test sample, thus forming an insoluble three-part
complex (e.g., U.S. Pat. No. 4,376,110). The second antibody can
itself be labeled with a detectable moiety (direct sandwich assays)
or can be measured using an anti-immunoglobulin antibody that is
labeled with a detectable moiety (indirect sandwich assay). For
example, one type of sandwich assay is an ELISA assay, in which
case the detectable moiety is an enzyme.
[0350] Antibodies can also be used for in vivo diagnostic assays.
Generally, an antibody can be labeled with a radionuclide (such as
.sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.3H, .sup.32P or
.sup.35S), so that disease cells or tissues can be localized using
immunoscintiography, for example. In certain embodiment, antibodies
or fragments thereof bind to the extracellular domains of two or
more LCM proteins and the affinity value (Kd) is less than
1.times.10.sup.8 M.
[0351] For immunohistochemistry, a disease tissue sample may be,
for example, fresh or frozen or may be embedded in paraffin and
fixed with a preservative such as formalin. A fixed or embedded
section can be contacted with a labeled primary antibody and
secondary antibody, wherein the antibody is used to detect LCM
protein expression in situ.
[0352] Antibodies can be used to detect a marker protein in situ,
in vitro, or in a cell lysate or supernatant in order to evaluate
the abundance and pattern of expression. Also, antibodies can be
used to assess abnormal tissue distribution or abnormal expression
during development or progression of a biological condition.
Antibodies against LCM proteins are useful for detecting the
presence of the proteins in cells or tissues to determine the
pattern of expression of the proteins among various tissues in an
organism and over the course of the organism's development.
[0353] Further, antibodies can be used to assess expression in
disease states such as in active stages of a disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by inappropriate
tissue distribution, developmental expression, or level of
expression of a protein, or expressed/processed form, for example,
an antibody can be prepared against the normal protein. If a
disorder is characterized by a specific mutation in a protein,
antibodies specific for the mutant protein can be used to assay for
the presence of the specific mutant protein and to target the
mutant protein for therapeutic purposes. Antibodies are also useful
as diagnostic tools, as immunological markers for aberrant protein
analyzed by electrophoretic mobility, isoelectric point, tryptic
peptide digest, and other physical assays known in the art.
[0354] Certain exemplary diagnostic methods of the invention can
also include monitoring a treatment modality. Accordingly, where
treatment is ultimately aimed at correcting, for example, the
function, activity, expression level, tissue distribution, or
developmental expression of a protein, antibodies directed against
the protein can be used to monitor therapeutic efficacy and to
modify a treatment regimen as necessary.
[0355] Additionally, antibodies to a marker protein are useful in
pharmacogenomic analysis. For example, antibodies prepared against
polymorphic proteins can be used to identify individuals that
require modified treatment modalities. Moreover, the marker
proteins and antibodies thereto can be used for clinical trials,
such as to identify individuals that should be included (e.g.,
individuals more likely to respond to a therapy) or excluded (e.g.,
individuals less likely to respond to a therapy, or individuals
more likely to experience harmful side effects from a therapy) from
a clinical trial.
[0356] The invention also encompasses kits for using antibodies to
detect the presence of a marker protein in a biological sample. An
exemplary kit can comprise antibodies such as a labeled or
labelable antibody and a compound or agent for detecting protein in
a biological sample; means for determining the amount of protein in
the sample; means for comparing the amount of protein in the sample
with a standard; and instructions for use. Such a kit can be
configured to detect a single marker protein or epitope or can be
configured to detect one of a multitude of epitopes, such as in an
antibody detection array.
[0357] LC/MS and ICAT
[0358] In certain exemplary embodiments, the invention provides
detection or diagnostic methods of a LCM by using LC/MS. Proteins
can be prepared from cells by methods known in the art (e.g., Zhang
et al., Nature Biotechnology 21(6):660-666 (2003)). The
differential expression of proteins in disease and healthy (or
drug-resistant and drug-sensitive, for example) samples can be
quantitated using mass spectrometry and ICAT (Isotope Coded
Affinity Tag) labeling, which is known in the art. ICAT is an
isotope label technique that allows for discrimination between two
populations of proteins, such as a healthy and a disease sample.
Over-expression or under-expression of a LCM protein, as measured
by ICAT, can indicate, for example, the likelihood of having or
developing a disease or an associated pathology.
[0359] LC/MS spectra can be collected for labeled samples and
processed as follows. The raw scans from the LC/MS instrument can
be individualed to peak detection and noise reduction software.
Filtered peak lists can then be used to detect `features`
corresponding to specific peptides from the original sample(s).
Features are characterized by their mass/charge ratio, charge,
retention time, isotope pattern, and/or intensity, for example.
[0360] The intensity of a peptide present in both healthy and
disease samples can be used to calculate the differential
expression, or relative abundance, of the peptide. The intensity of
a peptide found exclusively in one sample can be used to calculate
a theoretical expression ratio for that peptide (singleton).
Expression ratios can be calculated for each peptide in an assay or
experiment.
[0361] Statistical tests can be performed to assess the robustness
of the data and select statistically significant differentials. To
ensure the accuracy of data, the following steps can be taken: a)
ensure that similar features are detected in all replicates of an
experiment; b) assess the distribution of the log ratios of all
peptides (a Gaussian is expected); c) calculate the overall pair
wise correlations between ICAT LC/MS maps to ensure that the
expression ratios for peptides are reproducible across multiple
replicates; and d) aggregate multiple experiments in order to
compare the expression ratio of a peptide in multiple diseases or
disease samples.
[0362] 8. Methods of Treatment Based on LCM Proteins
[0363] a. Antibody Therapy
[0364] Antibodies of the invention can be used for therapeutic
purposes. It is contemplated that antibodies of the invention may
be used to treat a mammal, preferably a human, with a disease,
especially cancer, and particularly lung cancer. The antibodies can
be delivered alone, in a pharmaceutical composition (such as with a
carrier), or conjugated to one or more therapeutic agents, for
example.
[0365] Antibodies can be useful for modulating (e.g., agonizing or
antagonizing) protein function, such as for therapeutic purposes.
Antibodies can also be useful for inhibiting protein function by,
for example, blocking the binding of a LCM protein to a binding
partner such as a substrate, which can be useful therapeutically.
Antibodies can be prepared against, for example, specific portions
of a protein that contain domains required for protein function, or
against intact protein that is associated with a cell membrane.
[0366] Antibodies of the invention can also be used for enhancing
the immune response. The antibodies can be administered in amounts
similar to those used for other therapeutic administrations of
antibodies. For example, pooled gamma globulin can be administered
at a range of about 1 mg to about 100 mg per patient.
[0367] Antibodies reactive with LCM proteins can be administered
alone or in conjunction with other therapies, such as anti-cancer
therapies, to a mammal afflicted with cancer or other disease.
Examples of anti-cancer therapies include, but are not limited to,
chemotherapy, radiation therapy, and adoptive immunotherapy therapy
with TIL (tumor infiltrating lymphocytes).
[0368] The selection of an antibody subclass for therapy may depend
upon the nature of the antigen to be acted upon. For example, an
IgM may be preferred in situations where the antigen is highly
specific for the disease marker and rarely occurs on normal cells.
However, where the disease-associated antigen is also expressed in
normal tissues, although at lower levels, the IgG subclass may be
preferred. The IgG subclass may be preferred in these instances
because the binding of at least two IgG molecules in close
proximity is typically required to activate complement, and
therefore less complement-mediated damage may occur in normal
tissues that express smaller amounts of the antigen and thus bind
fewer IgG antibody molecules. Furthermore, IgG molecules, by being
smaller, may be more able than IgM molecules to localize to a
diseased tissue.
[0369] A mechanism for antibody therapy can be that a therapeutic
antibody recognizes a soluble or cell surface marker protein that
is expressed (preferably, over-expressed) in a disease cell. By NK
cell or complement activation, or conjugation of the antibody with
an immunotoxin or radiolabel, the interaction of the antibody with
the marker protein can abrogate ligand/receptor interaction or
activation of apoptosis, for example.
[0370] Potential mechanisms of antibody-mediated cytotoxicity of
diseased cells include phagocyte (antibody-dependent cellular
cytotoxicity (ADCC)), complement (complement-dependent cytotoxicity
(CDC)), naked antibody (receptor cross-linking apoptosis and growth
factor inhibition), or targeted payload labeled with a therapeutic
agent, such as a radionuclide, immunotoxin, or
immunochemotherapeutic or other therapeutic agent.
[0371] In certain exemplary embodiments, an antibody is
administered to a nonhuman mammal for the purposes of obtaining
preclinical data, for example. Exemplary nonhuman mammals to be
treated include nonhuman primates, dogs, cats, rodents, and other
mammals in which preclinical studies are performed. Such mammals
may be established animal models for a disease or may be used to
study toxicity of an antibody of interest, for example. Dose
escalation studies may be performed in the mammal, for example.
[0372] An antibody can be administered to an individual by any
suitable means, including parenteral, subcutaneous,
intraperitoneal, intrapulmonary, and intranasal, and, if desired
for local immunomodulatory treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. In
addition, an antibody can be administered by pulse infusion,
particularly with declining doses of the antibody. The dosing can
be given by injections, such as intravenous or subcutaneous
injections, which may depend in part on whether the administration
is brief or chronic.
[0373] For the prevention or treatment of a disease, the
appropriate dosage of an antibody may depend on the type of disease
to be treated, the severity and the course of the disease, whether
the antibody is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, and the discretion of the attending
physician.
[0374] Depending on the type and severity of disease, about 1
.mu.g/kg to 150 mg/kg (e.g., 0.1-20 mg/kg) of antibody can be an
initial candidate dosage for administration to a patient, whether,
for example, by one or more separate administrations, or by
continuous infusion. A typical daily dosage may range from about 1
.mu.g/kg to 100 mg/kg or more, depending on such factors as those
mentioned above. An antibody-drug conjugate can be administered
from about 1 .mu.g/kg to 50 mg/kg, typically from about 0.1-20
mg/kg, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
may range from about 0.1 mg/kg to 10 mg/kg, or from about 0.3 mg/kg
to about 7.5 mg/kg, depending on such factors as those mentioned
above. For repeated administrations over several days or longer,
depending on the condition, the treatment can be sustained until a
desired suppression of disease symptoms occurs. However, other
dosage regimens may be useful. Therapy progress can be monitored by
conventional techniques and assays.
[0375] Antibody composition can be formulated, dosed, and
administered in a manner consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners.
[0376] An antibody may optionally be formulated with, or
administered with, one or more therapeutic agents used to prevent
or treat the disorder in question. For example, an antibody can be
administered as a co-therapy with a standard of care therapeutic
for the specific disease being treated.
[0377] b. Other Immunotherapy
[0378] An "immunogenic peptide" is a peptide that comprises an
allele-specific motif such that the peptide typically will bind an
MHC allele (HLA in human) and be capable of inducing a CTL
(cytotoxic T-lymphocytes) response. Thus, immunogenic peptides
typically are capable of binding to an appropriate class I or II
MHC molecule and inducing a cytotoxic T cell or T helper cell
response against the antigen from which the immunogenic peptide is
derived.
[0379] Peptides derived from a LCM protein can be modified to
increase their immunogenicity, such as by enhancing the binding of
the peptide to the MHC molecules in which the peptide is presented.
The peptide or modified peptide can be conjugated to a carrier
molecule to enhance the antigenicity of the peptide. Examples of
carrier molecules, include, but are not limited to, human albumin,
bovine albumin, lipoprotein and keyhole limpet hemo-cyanin ("Basic
and Clinical Immunology" (1991) Stites and Terr (eds) Appleton and
Lange, Norwalk Conn., San Mateo, Calif.).
[0380] Further, amino acid sequence variants of a peptide can be
prepared, such as by altering the nucleic acid sequence of the DNA
which encodes the peptide, or by peptide synthesis. At the genetic
level, these variants can be prepared by, for example,
site-directed mutagenesis of nucleotides in the DNA encoding the
peptide, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. The variants can
exhibit the same qualitative biological activity as the nonvariant
peptide.
[0381] Exemplary embodiments of the invention provide peptides or
modified peptides derived from a LCM protein that are
differentially expressed in disease. Examples of peptide
modifications include, but are not limited to, substitutions,
deletions, or additions of one or more amino acids in a given
immunogenic peptide sequence, or mutation of existing amino acids
within a given immunogenic peptide sequence, or derivatization of
existing amino acids within a given immunogenic peptide sequence.
Any amino acid in an immunogenic peptide sequence may be modified.
In some embodiments, at least one amino acid can be substituted or
replaced within the given immunogenic peptide sequence. Any amino
acid may be used to substitute or replace a given amino acid within
the immunogenic peptide sequence. Modified peptides can include any
immunogenic peptide obtained from differentially expressed
proteins, which has been modified and exhibits enhanced binding to
the MHC molecule with which it associates when presented to a
T-cell. These modified peptides can be synthetically or
recombinantly produced by conventional methods, for example.
[0382] In certain exemplary embodiments of the invention, the
peptides comprise, or consist of, sequences of about 5-30 amino
acids in length which are immunogenic (i.e., capable of inducing an
immune response when injected into a individual).
[0383] In certain exemplary embodiments, the peptides may be used,
for example, to treat T cell-mediated pathologies. The term "T
cell-mediated pathologies" refers to any condition in which an
inappropriate T cell response is a component of the pathology. The
term is intended to encompass both T cell mediated diseases and
diseases resulting from unregulated clonal T cell replication.
[0384] Modified (e.g., recombinant) or natural LCM proteins, or
fragments thereof, can be used as a vaccine either prophylactically
or therapeutically. When provided prophylactically, a vaccine can
be provided in advance of any evidence of disease. The prophylactic
administration of a disease vaccine may serve to prevent or
attenuate a disease in a mammal such as a human.
[0385] An exemplary vaccine formulation can comprise an immunogen
that induces an immune response directed against a
disease-associated antigen such as a LCM protein. For example, a
substantially or partially purified LCM protein or fragments
thereof can be administered as a vaccine in a pharmaceutically
acceptable carrier. An immunogen can be administered in a pure or
substantially pure form, or can be administered as a pharmaceutical
composition, formulation, or preparation. Exemplary doses of
protein that can be administered are about 0.001 to about 100 mg
per patient, or about 0.01 to about 100 mg per patient.
Immunization can be repeated as necessary until a sufficient titer
of anti-immunogen antibody or immune cells has been obtained.
[0386] Vaccine can be prepared using, for example, recombinant
protein or expression vectors comprising a nucleic acid sequence
encoding all or part of a LCM protein. Examples of vectors that can
be used in vaccines include, but are not limited to, defective
retroviral vectors, adenoviral vectors vaccinia viral vectors, fowl
pox viral vectors, or other viral vectors (Mulligan, R. C., (1993)
Science 260:926-932). The vectors can be introduced into a mammal
(e.g., a human) either prior to any evidence of a disease or to
mediate regression of a disease in a mammal afflicted with the
disease. Examples of methods for administering a viral vector into
mammals include, but are not limited to, exposure of cells to the
virus ex vivo, or injection of the retrovirus or a producer cell
line of the virus into the affected tissue, or intravenous
administration of the virus. Alternatively, the vector can be
administered locally by direct injection into a disease lesion or
topical application in a pharmaceutically acceptable carrier. The
quantity of viral vector to be administered can be based on the
titer of virus particles. An exemplary range can be about 10.sup.6
to about 10.sup.11 virus particles per mammal.
[0387] After immunization, the efficacy of the vaccine can be
assessed by, for example, the production of antibodies or immune
cells that recognize the antigen, as assessed by specific lytic
activity, specific cytokine production, or disease regression,
which can be measured using conventional methods. If the mammal to
be immunized is already afflicted with a disease, the vaccine can
be administered in conjunction with other therapeutic treatments.
Examples of other therapeutic treatments include, but are not
limited to, adoptive T cell immunotherapy and coadministration of
cytokines or other therapeutic drugs.
[0388] In certain embodiments, mammals, preferably humans, at high
risk for disease, especially cancer, are prophylactically treated
with vaccines of the invention. Examples include, but are not
limited to, individuals with a family history of a disease,
individuals who themselves have a history of disease (e.g., cancer
that has been previously resected and at risk for reoccurrence), or
individuals already afflicted with a disease. When provided
therapeutically, a vaccine can be provided to enhance the patient's
own immune response to a disease antigen. An exemplary vaccine,
which acts as an immunogen, can be a cell, cell lysate from cells
transfected with a recombinant expression vector, or a culture
supernatant containing the expressed protein, for example.
Alternatively, an immunogen can be, for example, a partially or
substantially purified recombinant protein, peptide, or analog
thereof, or a modified protein, peptide, or analog thereof. The
proteins or peptides can be, for example, conjugated with
lipoprotein or administered in liposomal form or with adjuvant.
[0389] Vaccination can be carried out using conventional methods.
For example, an immunogen can be used in a suitable diluent such as
saline or water, or complete or incomplete adjuvants. Further, an
immunogen may or may not be bound to a carrier, including carriers
to increase the immunogenicity of the immunogen. Examples of
carrier molecules include, but are not limited to, bovine serum
albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and
the like. An immunogen also may be coupled with lipoproteins or
administered in liposomal form or with adjuvants. An immunogen can
be administered by any route appropriate for antibody production
such as intravenous, intraperitoneal, intramuscular, subcutaneous,
and the like. An immunogen can be administered once or at periodic
intervals until a significant titer of anti-LCM immune cells or
anti-LCM antibody is produced. The presence of anti-LCM immune
cells can be assessed by measuring the frequency of precursor CTL
(cytotoxic T-lymphocytes) against LCM antigen prior to and after
immunization by a CTL precursor analysis assay (Coulie et al.,
1992, International Journal Of Cancer 50:289-297). An immunoassay
can be used to detect antibody in serum.
[0390] The safety of a vaccine can be determined by examining the
effect of immunization on the general health of an immunized animal
(e.g., weight change, fever, change in appetite or behavior, etc.)
and looking for pathological changes during autopsies. After
initial testing in animals, a vaccine can be tested in patients
having a disease of interest. Conventional methods can be used to
evaluate the immune response of a patient to determine the
efficiency of the vaccine.
[0391] In certain exemplary embodiments of the invention, a LCM
protein or fragments thereof, or a modified LCM protein, can be
exposed to dendritic cells cultured in vitro. The cultured
dendritic cells provide a means of producing T-cell dependent
antigens comprised of dendritic cell-modified antigen or dendritic
cells pulsed with antigen, in which the antigen is processed and
expressed on the antigen-activated dendritic cell. The
antigen-activated dendritic cells or processed dendritic cell
antigens can be used as immunogens for vaccines or for the
treatment of diseases. The dendritic cells can be exposed to the
antigen for sufficient time to allow the antigens to be
internalized and presented on the surface of dendritic cells. The
resulting dendritic cells or the dendritic cell-processed antigens
can then be administered to an individual in need of therapy. Such
methods are described in Steinman et al. (WO93/208185) and in
Banchereau et al. (EPO Application 0563485A1).
[0392] In certain exemplary embodiments of the invention, T-cells
isolated from individuals can be exposed to a LCM protein or
fragment thereof, or a modified LCM protein, in vitro and then
administered in a therapeutically effective amount to a patient in
need of such treatment. Examples of where T-lymphocytes can be
isolated include, but are not limited to, peripheral blood cells
lymphocytes (PBL), lymph nodes, or tumor infiltrating lymphocytes
(TIL). Such lymphocytes can be isolated from the individual to be
treated or from a donor by methods known in the art and cultured in
vitro (Kawakami et al., 1989, J. Immunol. 142: 2453-3461).
Lymphocytes can be cultured in media such as RPMI or RPMI 1640 or
AIM V for 1-10 weeks. Viability can be assessed by trypan blue dye
exclusion assay. Examples of how these sensitized T-cells can be
administered to a mammal include, but are not limited to,
intravenously, intraperitoneally, or intralesionally. Parameters
that can be assessed to determine the efficacy of these sensitized
T-lymphocytes include, but are not limited to, production of immune
cells in the mammal being treated or tumor regression. Conventional
methods can be used to assess these parameters. Such treatment can
be given in conjunction with cytokines or gene-modified cells, for
example (Rosenberg et al., 1992, Human Gene Therapy, 3: 75-90;
Rosenberg et al., 1992, Human Gene Therapy, 3: 57-73).
[0393] 9. Screening Methods Using LCM Proteins
[0394] Exemplary embodiments of the invention provide methods of
screening for agents (interchangeably referred to by such terms as
candidate agents, compounds, or candidate compounds) that modulate
LCM protein activity (interchangeably referred to as protein
function). Examples of candidate agents include, but are not
limited to, proteins, peptides, antibodies, nucleic acids (such as
antisense and RNAi nucleic acid molecules), and small molecules.
Exemplary embodiments of the invention further provide agents
identified by these screening methods, and methods of using these
agents, such as for treating diseases, especially cancer, and
particularly lung cancer.
[0395] Exemplary screening methods can typically comprise the steps
of (i) contacting a LCM protein with a candidate agent, and (ii)
assaying for LCM protein activity, wherein a change in protein
activity in the presence of the agent relative to protein activity
in the absence of the agent indicates that the agent modulates LCM
protein activity.
[0396] Other exemplary screening methods can determine a candidate
agent's ability to modulate LCM expression. Exemplary methods can
typically comprise the steps of (i) contacting a candidate agent
with a system that is capable of expressing LCM protein or LCM
mRNA, and (ii) assaying for the level of LCM protein or LCM mRNA,
wherein a change in the level in the presence of the agent relative
to the level in the absence of the agent indicates that the agent
modulates LCM expression levels.
[0397] Exemplary embodiments of the invention further provide
methods to screen for agents that bind to LCM proteins. Exemplary
methods can typically comprise the steps of contacting a LCM
protein with a test agent and measuring the extent of binding of
the agent to the LCM protein.
[0398] LCM proteins can be used to identify agents that modulate
activity of a protein in its natural state or an altered form that
causes a specific disease or pathology. LCM proteins and
appropriate variants and fragments can be used in high-throughput
screens to assay candidate compounds for their ability to bind to
LCM. These compounds can be further screened against functional LCM
proteins to determine the effect of the compound on the protein's
activity. Further, these compounds can be tested in animal or
invertebrate systems to determine activity/effectiveness. Compounds
can be identified that activate (agonist) or inactivate
(antagonist) LCM proteins to a desired degree.
[0399] LCM proteins can be used to screen agents for their ability
to stimulate or inhibit interaction between a LCM protein and a
target molecule that normally interacts with the LCM protein (e.g.,
a substrate, an extracellular binding ligand, or a component of a
signal pathway that a LCM protein normally interacts with such as a
cytosolic signal protein). Exemplary assays can include the steps
of combining a LCM protein or fragment thereof with a candidate
compound under conditions that allow the LCM protein (or fragment
thereof) to interact with a target molecule, and detecting the
formation of a complex between the LCM protein and the target
molecule or detecting the biochemical consequence of the
interaction between the LCM protein and the target molecule, such
as any of the associated effects of signal transduction (e.g.,
protein phosphorylation, cAMP turnover, adenylate cyclase
activation, etc.). Any of the biological or biochemical functions
mediated by a LCM protein can be used as an endpoint assay to
identify an agent that modulates LCM activity.
[0400] Candidate compounds or agents include, but are not limited
to, 1) peptides such as soluble peptides, including Ig-tailed
fusion peptides and members of random peptide libraries (see, e.g.,
Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature
354:84-86 (1991)) and combinatorial chemistry-derived molecular
libraries made of D- and/or L-configuration amino acids; 2)
phosphopeptides (e.g., members of random and partially degenerate,
directed phosphopeptide libraries, see, e.g., Songyang et al., Cell
72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric, and single chain antibodies as
well as Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0401] An exemplary candidate compound or agent is a soluble
fragment of a LCM that competes for substrate binding. Other
exemplary candidate compounds include mutant LCM proteins or
appropriate fragments containing mutations that affect LCM function
and thus compete for substrate. Accordingly, a fragment that
competes for substrate, for example with a higher affinity, or a
fragment that binds substrate but does not allow release, is
encompassed by the invention.
[0402] Compounds can also be screened by using chimeric proteins in
which any portion of a protein such as an amino terminal
extracellular domain, a transmembrane domain (e.g., transmembrane
segments or intracellular or extracellular loops), or a carboxy
terminal intracellular domain can be replaced in whole or part by
heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate than the substrate that is recognized by a
native marker protein. Accordingly, a different set of signal
transduction components can be available as an end-point assay for
activation, thereby allowing assays to be performed in other than
the specific host cell from which a marker is derived.
[0403] Competition binding assays can also be used to screen for
compounds that interact with a marker protein (e.g., binding
partners and/or ligands). For example, a test compound can be
exposed to a marker protein under conditions that allow the test
compound to bind or otherwise interact with the marker protein.
Soluble marker protein can also be added to the mixture. If the
test compound interacts with the soluble marker protein, it can
decrease the amount of complex formed or activity of the marker
protein. This type of assay is particularly useful in instances in
which compounds are sought that interact with specific regions of a
marker protein. Thus, the soluble marker protein that competes with
the marker protein can contain peptide sequences corresponding to
the marker region of interest.
[0404] To perform cell-free drug screening assays, it may be
desirable to immobilize either a LCM protein (or fragment thereof)
or a molecule that binds the LCM protein (referred to herein as a
"binding partner") to facilitate separation of complexes from
uncomplexed forms, as well as to facilitate automation of the
assays.
[0405] Techniques for immobilizing proteins on matrices can be
utilized in exemplary drug screening assays. In exemplary
embodiments, a fusion protein can be provided which adds a domain
that allows a protein to be bound to a matrix. For example,
glutathione-S-transferase fusion proteins can be adsorbed onto
glutathione SEPHAROSE beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtitre plates, which are then combined
with cell lysates (e.g., .sup.35S-labeled) and a candidate
compound, and the mixture incubated under conditions conducive to
complex formation (e.g., at physiological conditions for salt and
pH). Following incubation, the beads can be washed to remove any
unbound label, and the matrix immobilized and radiolabel determined
directly, or in the supernatant after the complexes are
dissociated. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of a binding
partner found in the bead fraction quantitated from the gel using
standard electrophoretic techniques. For example, either a marker
protein or a binding partner can be immobilized by conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies that are reactive with a marker protein
but do not interfere with binding of the marker protein to its
binding partner can be derivatized to the wells of a plate, and the
marker protein trapped in the wells by antibody conjugation.
Preparations of a binding partner and a candidate compound can be
incubated in marker protein-presenting wells and the amount of
complex trapped in the well can be quantitated. Methods for
detecting such complexes, in addition to those described for
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with a binding partner, or which are
reactive with a marker protein and compete with the binding
partner, as well as marker protein-linked assays which rely on
detecting an enzymatic activity associated with a binding
partner.
[0406] In exemplary embodiments of the invention, a LCM protein can
be used as a "bait protein" in a two-hybrid assay or three-hybrid
assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993)
Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with a LCM
protein and are involved in the protein's activity. The two-hybrid
system is based on the modular nature of most transcription
factors, which typically consist of separable DNA-binding and
activation domains. In exemplary embodiments, the two-hybrid assay
can utilize two different DNA constructs. In one construct, a gene
that encodes a LCM protein can be fused to a gene encoding the DNA
binding domain of a known transcription factor (e.g., GAL-4). In
the other construct, a DNA sequence from a library of DNA sequences
that encode an unidentified protein ("prey" or "sample") can be
fused to a gene that encodes the activation domain of the known
transcription factor. If the "bait" and the "prey" proteins are
able to interact in vivo, forming a LCM-dependent complex, the
DNA-binding and activation domains of the transcription factor are
brought into close proximity. This proximity allows transcription
of a reporter gene (e.g., LacZ), which can be operably linked to a
transcriptional regulatory site responsive to the transcription
factor. Expression of the reporter gene can be detected and cell
colonies containing the functional transcription factor can be
isolated and used to obtain the cloned gene that encodes the
protein that interacts with the LCM protein.
[0407] Agents that modulate a LCM protein can be identified using
one or more of the above assays, alone or in combination. For
example, a cell-based or cell free system can be used for initial
identification of agents, and then activity of the agents can be
confirmed in an animal or other model system. Such model systems
are well known in the art and can readily be employed in this
context.
[0408] 10. Diagnosis, Treatment, and Screening Methods Using LCM
Nucleic Acid Molecules
[0409] The nucleic acid molecules of the invention are useful, for
example, as probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as
hybridization probes for messenger RNA, transcript/cDNA, and
genomic DNA to detect or isolate full-length cDNA and genomic
clones encoding a LCM protein, or variants thereof. The nucleic
acid molecules are also useful as primers for PCR to amplify any
given region of a nucleic acid molecule and are useful to
synthesize antisense molecules of desired length and sequence. The
nucleic acid molecules are also useful for producing ribozymes
corresponding to all, or a part, of the mRNA produced from the
nucleic acid molecules described herein.
[0410] The nucleic acid molecules are also useful for constructing
recombinant vectors. Exemplary vectors include expression vectors
that express a portion of, or all of, a LCM protein. The nucleic
acid molecules are also useful for expressing antigenic portions of
the proteins. The nucleic acid molecules are also useful for
constructing host cells expressing a part, or all, of the proteins.
The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the proteins.
[0411] A primer or probe can correspond to any sequence along the
entire length of a LCM-encoding nucleic acid molecule. Accordingly,
a primer or probe can be derived from 5' noncoding regions, coding
regions, or 3' noncoding regions, for example.
[0412] Exemplary in vitro techniques for detection of mRNA include
Northern hybridizations and in situ hybridizations. Exemplary in
vitro techniques for detecting DNA include Southern hybridizations
and in situ hybridization. Reverse transcriptase PCR amplification
(RT-PCR) and the like can also be used for detecting RNA
expression. A specific exemplary method of detection comprises
using TaqMan technology (Applied Biosystems, Foster City,
Calif.).
[0413] a. Methods of Diagnosis Using Nucleic Acids
[0414] Nucleic acid molecules of the invention are useful, for
example, as hybridization probes for determining the presence,
level, form, and/or distribution of nucleic acid expression.
Exemplary probes can be used to detect the presence of, or to
determine levels of, a specific nucleic acid molecule in cells,
tissues, and in organisms. Accordingly, probes corresponding to a
LCM described herein can be used to assess expression and/or gene
copy number in a given cell, tissue, or organism, which can be
applied to, for example, diagnosis of disorders involving an
increase or decrease in LCM protein expression relative to normal
LCM protein expression levels.
[0415] Probes can be used as part of a diagnostic test kit for
identifying cells or tissues that express LCM protein
differentially, such as by measuring a level of a LCM-encoding
nucleic acid (e.g., mRNA or genomic DNA) in a sample of cells from
a individual, or determining if a LCM-encoding nucleic acid is
mutated.
[0416] Exemplary embodiments of the invention encompass kits for
detecting the presence of LCM-encoding nucleic acid (e.g., mRNA or
genomic DNA) in a biological sample. For example, an exemplary kit
can comprise reagents such as a labeled or labelable nucleic acid
or agent capable of detecting LCM nucleic acid in a biological
sample; means for determining the amount of LCM nucleic acid in the
sample; and means for comparing the amount of LCM nucleic acid in
the sample with a standard. The compound or agent can be packaged
in a suitable container. The kit can further comprise instructions
for using the kit to detect LCM nucleic acid.
[0417] The nucleic acid molecules are useful in diagnostic assays
for qualitative changes in LCM nucleic acid expression, and
particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in LCM genes
and gene expression products such as mRNA. The nucleic acid
molecules can be used as hybridization probes to detect naturally
occurring genetic mutations in a LCM gene and to determine whether
a individual with the mutation is at risk for a disorder caused by
the mutation. Examples of mutations include deletions, additions,
or substitutions of one or more nucleotides in a gene, chromosomal
rearrangements (such as inversions or transpositions), and
modification of genomic DNA such as aberrant methylation patterns
or changes in gene copy number (such as amplification). Detection
of a mutated form of a LCM gene associated with a dysfunction can
provide a diagnostic tool for an active disease or susceptibility
to disease in instances in which the disease results from
overexpression, underexpression, or altered expression of a LCM
protein, for example.
[0418] Mutations in a LCM gene can be detected at the nucleic acid
level by a variety of techniques. For example, genomic DNA, RNA, or
cDNA can be analyzed directly or can be amplified (e.g., using PCR)
prior to analysis. In certain exemplary embodiments, detection of a
mutation involves the use of a probe/primer in a PCR reaction (see,
e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or
RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran et al., Science 241:1077-1080 (1988) and
Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can
be particularly useful for detecting point mutations in a gene (see
Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). Exemplary
methods such as these can include the steps of collecting a sample
of cells from a patient, isolating nucleic acid (e.g., genomic,
mRNA, or both) from the cells of the sample, contacting the nucleic
acid with one or more primers which specifically hybridize to a
marker nucleic acid under conditions such that hybridization and
amplification of the marker nucleic acid (if present) occurs, and
detecting the presence or absence of an amplification product, or
detecting the size of the amplification product and comparing the
length to a control sample. Deletions and insertions can be
detected by a change in size of the amplified product compared to a
normal genotype. Point mutations can be identified by hybridizing
amplified DNA to normal RNA or antisense DNA sequences, for
example.
[0419] Alternatively, mutations in a LCM gene can be identified,
for example, by alterations in restriction enzyme digestion
patterns as determined by gel electrophoresis. Further,
sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used
to identify the presence of specific mutations by development or
loss of a ribozyme cleavage site. Perfectly matched sequences can
be distinguished from mismatched sequences by nuclease cleavage
digestion assays or by differences in melting temperature.
[0420] Sequence changes at specific locations can be assessed by
nuclease protection assays such as RNase and S1 protection, or
chemical cleavage methods. Furthermore, sequence differences
between a mutant LCM gene and a corresponding wild-type gene can be
determined by direct DNA sequencing. A variety of automated
sequencing procedures can be utilized when performing diagnostic
assays (Naeve, C. W., (1995) Biotechniques 19:448), including
sequencing by mass spectrometry (e.g., PCT International
Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr.
36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol.
38:147-159 (1993)).
[0421] Other methods for detecting mutations in a nucleic acid
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et
al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (DGGE) (Myers et al.,
Nature 313:495 (1985)). Examples of other techniques for detecting
point mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0422] B. Methods of Monitoring Treatment and Pharmacogenomic
Methods Using Nucleic Acids
[0423] Nucleic acid molecules of the invention are also useful for
monitoring the effectiveness of modulating agents on the expression
or activity of a LCM gene, such as in clinical trials or in a
treatment regimen. For example, the gene expression pattern of a
LCM gene can serve as a barometer for the continuing effectiveness
of treatment with a compound, particularly with compounds to which
a patient can develop resistance. The gene expression pattern can
also serve as a marker indicative of a physiological response of
the affected cells to the compound. For example, based on
monitoring nucleic acid expression, the administration of a
compound can be increased or alternative compounds to which the
patient has not become resistant can be administered instead.
Similarly, if the level of nucleic acid expression falls below a
desirable level, administration of the compound can be
commensurately decreased.
[0424] The nucleic acid molecules are also useful for testing an
individual for a genotype that, while not necessarily causing a
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules provided herein can be used
to assess the mutation content of a marker gene in an individual in
order to select an appropriate compound or dosage regimen for
treatment. For example, marker nucleic acid molecules having
genetic variations that affect treatment can provide diagnostic
markers that can be used to tailor treatment to an individual.
Accordingly, the production of recombinant cells and animals having
these genetic variations allows effective clinical design of
treatment compounds and dosage regimens, for example.
[0425] c. Methods of Treatment Using Nucleic Acids
[0426] Nucleic acid molecules of the invention are useful to design
antisense constructs to control LCM gene expression in cells,
tissues, and organisms. An antisense nucleic acid molecule
typically blocks translation of mRNA into LCM protein by
hybridizing to marker mRNA in a sequence-specific manner. Nucleic
acid molecules of the invention can also be used to specifically
suppress gene expression by methods such as RNA interference
(RNAi). RNAi and antisense-based gene suppression are well known in
the art (e.g., Science 288:1370-1372, 2000). RNAi typically
operates on a post-transcriptional level and is sequence specific.
RNAi and antisense nucleic acid molecules are useful for treating
diseases, especially cancer. RNAi fragments, particularly
double-stranded (ds) RNAi, as well as antisense nucleic acid
molecules can also be used to generate loss-of-function phenotypes
by suppressing gene expression. Accordingly, exemplary embodiments
of the invention provide RNAi and antisense nucleic acid molecules,
and methods of using these RNAi and antisense nucleic acid
molecules, such as for therapy or for modulating cell function.
Nucleic acid molecules may also be produced that are complementary
to a region of a gene involved in transcription, such as to
hybridize to the gene to prevent transcription.
[0427] Exemplary embodiments of the invention relate to isolated
RNA molecules (double-stranded; single-stranded) that are about 17
to about 29 nucleotides (nt) in length, and more particularly about
21 to about 25 nt in length, which mediate RNAi (e.g., degradation
of mRNA, and such mRNA may be referred to herein as mRNA to be
degraded). With respect to RNAi, the terms RNA, RNA molecule(s),
RNA segment(s), and RNA fragment(s) are used interchangeably to
refer to RNA that mediates RNAi. These terms include
double-stranded RNA, single-stranded RNA, isolated RNA (e.g.,
partially purified RNA, essentially pure RNA, synthetic RNA,
recombinantly produced RNA), as well as altered RNA that differs
from naturally occurring RNA by the addition, deletion,
substitution, and/or alteration of one or more nucleotides. Such
alterations can include, for example, addition of non-nucleotide
material, such as to the end(s) of a 21-25 nt RNA or internally (at
one or more nucleotides of the RNA). Nucleotides in exemplary RNA
molecules of the invention can also comprise non-standard
nucleotides, including non-naturally occurring nucleotides or
deoxyribonucleotides. Collectively, all such altered RNAs are
referred to as analogs or analogs of naturally-occurring RNA. RNA
of 21-25 nt typically need only be sufficiently similar to natural
RNA that it has the ability to mediate RNAi. As used herein, the
phrase "mediates RNAi" refers to the ability to distinguish which
RNAs are to be degraded by RNAi processes. RNA that mediates RNAi
directs degradation of particular mRNAs by RNAi processes. Such RNA
may include RNAs of various structures, including short hairpin
RNA.
[0428] In certain exemplary embodiments, the invention relates to
RNA molecules of about 21 to about 25 nt that direct cleavage of
specific mRNA to which their sequence corresponds. It is not
necessary that there be a perfect correspondence (i.e., match) of
the sequences, but the correspondence must be sufficient to enable
the RNA to direct RNAi cleavage of the marker mRNA (Holen et al.,
Nucleic Acids Res. 33:4704-4710 (2005)). In an exemplary
embodiment, the 21-25 nt RNA molecules of the invention comprise a
3' hydroxyl group.
[0429] Certain exemplary embodiments of the invention relate to
21-25 nt RNAs of specific genes, produced by chemical synthesis or
recombinant DNA techniques, that mediate RNAi. As used herein, the
term "isolated RNA" includes RNA obtained by any means, including
processing or cleavage of dsRNA, production by chemical synthetic
methods, and production by recombinant DNA techniques, for example.
Exemplary embodiments of the invention further relate to uses of
the 21-25 nt RNAs, such as for therapeutic or prophylactic
treatment and compositions comprising 21-25 nt RNAs that mediate
RNAi, such as pharmaceutical compositions comprising 21-25 nt RNAs
and an appropriate carrier.
[0430] Further exemplary embodiments of the invention relate to
methods of mediating RNAi of genes of a patient. For example, RNA
of about 21 to about 25 nt which targets a specific mRNA to be
degraded can be introduced into a patient's cells. The cells can be
maintained under conditions allowing degradation of the mRNA,
resulting in RNA-mediated interference of the mRNA of the gene in
the cells of the patient. Treatment of cancer patients, for
example, with RNAi may inhibit the growth and spread of the cancer
and reduce tumor size. Treatment of patients using RNAi can also be
in combination with other therapies. For example, RNAi can be used
in combination with other treatment modalities, such as
chemotherapy, radiation therapy, and other treatments. In an
exemplary embodiment, a chemotherapy agent is used in combination
with RNAi. In a further exemplary embodiment, GEMZAR (gemcitabine
HCl) chemotherapy is used with RNAi.
[0431] Treatment of certain diseases by RNAi may require
introduction of the RNA into the disease cells. RNA can be directly
introduced into a cell, or introduced extracellularly into a
cavity, interstitial space, into the circulation of a patient, or
introduced orally, for example. Physical methods of introducing
nucleic acids, such as injection directly into a cell or
extracellular injection into a patient, may also be used. RNA may
be introduced into vascular or extravascular circulation, the blood
or lymph system, or the cerebrospinal fluid, for example. RNA may
be introduced into an embryonic stem cell or another multipotent
cell, which may be derived from a patient. Physical methods of
introducing nucleic acids include injection of a solution
containing the RNA, bombardment by particles covered by the RNA,
soaking cells or tissue in a solution of the RNA, or
electroporation of cell membranes in the presence of the RNA. A
viral construct packaged into a viral particle may be used to
introduce an expression construct into a cell, with the construct
expressing the RNA. Other methods known in the art for introducing
nucleic acids to cells may be used, such as lipid-mediated carrier
transport, chemical-mediated transport, and the like. The RNA may
be introduced along with components that perform one or more of the
following activities: enhance RNA uptake by the cell, promote
annealing of the duplex strands, stabilize the annealed strands, or
otherwise increase inhibition of the marker gene.
[0432] Exemplary RNA of the invention can be used alone or as a
component of a kit having at least one reagent for carrying out in
vitro or in vivo introduction of the RNA to a cell, tissue/fluid,
or patient. Exemplary components of a kit include dsRNA and a
vehicle that promotes introduction of the dsRNA. A kit may also
include instructions for using the kit.
[0433] Certain exemplary embodiments of the invention provide
compositions and methods for cleavage of mRNA by ribozymes having
nucleotide sequences complementary to one or more regions in the
mRNA, thereby attenuating the translation of the mRNA. Examples of
regions in mRNA that can be targeted by ribozymes include coding
regions, particularly coding regions corresponding to catalytic or
other functional activities of a marker protein, such as substrate
binding. These compositions and methods may be used to treat a
disorder characterized by abnormal or undesired marker nucleic acid
expression.
[0434] In certain exemplary embodiments, nucleic acid molecules of
the invention may be used for gene therapy in individuals having
cells that are aberrant in gene expression of a marker. For
example, recombinant cells that have been engineered ex vivo (which
can include an individual's own cells) can be introduced into an
individual where the cells produce the desired marker protein to
thereby treat the individual.
[0435] d. Methods of Screening Using Nucleic Acids
[0436] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate LCM nucleic acid
expression.
[0437] Exemplary embodiments of the invention thus provide methods
for identifying a compound that can be used to treat a disease
associated with differential expression of a LCM gene, especially
cancer. Exemplary methods can typically include assaying the
ability of a compound to modulate the expression of a marker
nucleic acid to thereby identify a compound that can be used to
treat a disorder characterized by undesired marker nucleic acid
expression. The assays can be performed in cell-based or cell-free
systems. Examples of cell-based assays include cells naturally
expressing marker nucleic acid or recombinant cells genetically
engineered to express specific marker nucleic acid sequences.
[0438] Assays for marker nucleic acid expression can involve direct
assay of marker nucleic acid levels, such as mRNA levels, or on
collateral compounds involved in a signal pathway. Further, the
expression of genes that are up- or down-regulated in response to a
signal pathway can also be assayed. In these embodiments, the
regulatory regions of these genes can be operably linked to a
reporter gene such as luciferase.
[0439] Thus, in exemplary embodiments, modulators of gene
expression of a marker can be identified in methods wherein a cell
is contacted with a candidate agent and the expression of marker
mRNA determined. The level of expression of marker mRNA in the
presence of the candidate agent is compared to the level of
expression of marker mRNA in the absence of the candidate agent.
The candidate agent can then be identified as a modulator of marker
nucleic acid expression based on this comparison and may be used,
for example, to treat a disorder characterized by aberrant marker
nucleic acid expression. When expression of marker mRNA is
statistically significantly greater in the presence of the
candidate agent than in its absence, the candidate agent is
identified as a stimulator (agonist) of nucleic acid expression.
When nucleic acid expression is statistically significantly less in
the presence of the candidate agent than in its absence, the
candidate compound is identified as an inhibitor (antagonist) of
nucleic acid expression.
[0440] 11. Arrays and Expression Analysis
[0441] "Array" (interchangeably referred to as "microarray")
typically refers to an arrangement of at least one, but more
typically at least two, nucleic acid molecules, proteins, or
antibodies on a substrate. In certain exemplary arrangements, at
least one of the nucleic acid molecules, proteins, or antibodies
typically represents a control or standard, and other nucleic acid
molecules, proteins, or antibodies are of diagnostic or therapeutic
interest. In exemplary embodiments, the arrangement of nucleic acid
molecules, proteins, or antibodies on the substrate is such that
the size and signal intensity of each labeled complex (e.g., formed
between each nucleic acid molecule and a complementary nucleic
acid, or between each protein and a ligand or antibody, or between
each antibody and a protein to which the antibody specifically
binds) is individually distinguishable.
[0442] An "expression profile" is a representation of marker
expression in a sample. A nucleic acid expression profile can be
produced using, for example, arrays, sequencing, hybridization, or
amplification technologies for nucleic acids from a sample. A
protein expression profile can be produced using, for example,
arrays, gel electrophoresis, mass spectrometry, or antibodies (and,
optionally, labeling moieties) which specifically bind proteins.
Nucleic acids, proteins, or antibodies can be attached to a
substrate or provided in solution, and their detection can be based
on methods well known in the art.
[0443] A substrate includes, but is not limited to, glass, paper,
nylon or other type of membrane, filter, chip, metal, or any other
suitable solid or semi-solid (e.g., gel) support.
[0444] Exemplary arrays can be prepared and used according to the
methods described in U.S. Pat. No. 5,837,832; PCT application
WO95/11995; Lockhart et al., 1996, Nat. Biotech. 14: 1675-1680;
Schena et al., 1996; Proc. Natl. Acad. Sci. 93: 10614-10619; and
U.S. Pat. No. 5,807,522. Exemplary embodiments of the invention
also provide antibody arrays (see, e.g., de Wildt et al. (2000)
Nat. Biotechnol. 18:989-94).
[0445] Certain exemplary embodiments of the invention provide a
nucleic acid array for assaying marker expression, which can be
composed of single-stranded nucleic acid molecules, usually either
synthetic antisense oligonucleotides or fragments of cDNAs, fixed
to a solid support. The oligonucleotides can be, for example, about
6-60 nucleotides in length, about 15-30 nucleotides in length, or
about 20-25 nucleotides in length.
[0446] To produce oligonucleotides to a marker nucleic acid
molecule for an array, the marker nucleic acid molecule of interest
is typically examined using a computer algorithm to identify
oligonucleotides of defined length that are unique to the nucleic
acid molecule, have a GC content within a range suitable for
hybridization, and lack predicted secondary structure that may
interfere with hybridization. In certain instances, it may be
desirable to use pairs of oligonucleotides on an array. In
exemplary embodiments, the "pairs" can be identical, except for one
nucleotide (which can be located in the center of the sequence, for
example). The second oligonucleotide in the pair (mismatched by
one) serves as a control. Any number of oligonucleotide pairs may
be utilized.
[0447] Oligonucleotides can be synthesized on the surface of a
substrate, such as by using a light-directed chemical process or by
using a chemical coupling procedure and an ink jet application
apparatus (e.g., PCT application WO95/251116).
[0448] In some exemplary embodiments, an array can be used to
diagnose or monitor the progression of disease, for example, by
assaying marker expression.
[0449] For example, an oligonucleotide probe specific for a marker
can be labeled by standard methods and added to a biological sample
from a patient under conditions that allow for the formation of
hybridization complexes. After an incubation period, the sample can
be washed and the amount of label (or signal) associated with
hybridization complexes can be quantified and compared with a
standard value. If complex formation in the patient sample is
significantly altered (higher or lower) in comparison to a normal
(e.g., healthy) standard, or is similar to a disease standard, this
differential expression can be diagnostic of a disorder.
[0450] By analyzing changes in patterns of marker expression,
disease may be diagnosed at earlier stages before a patient is
symptomatic. In exemplary embodiments of the invention, arrays or
marker expression analysis methods can be used to formulate a
diagnosis or prognosis, to design a treatment regimen, and/or to
monitor the efficacy of treatment. For example, a treatment dosage
can be established that causes a change in marker expression
patterns indicative of successful treatment, and marker expression
patterns associated with the onset of undesirable side effects can
be avoided. In further exemplary embodiments, assays of marker
expression can be repeated on a regular basis to determine if the
level of marker expression in a patient begins to approximate that
which is observed in a normal individual. The results obtained from
successive assays may be used to show the efficacy of treatment
over a period ranging from several days to years, for example.
[0451] Exemplary arrays of the invention can also be used to screen
candidate agents, such as to identify agents that produce a marker
expression profile similar to that caused by known therapeutic
agents, with the expectation that agents that cause a similar
expression profile of a marker may have similar therapeutic effects
and/or modes of action on the marker.
EXAMPLES
[0452] Exemplary embodiments of the invention are further described
in the following examples, which do not limit the scope of the
invention.
[0453] 1. Tissue Samples and Cell Lines
[0454] Tissue Processing and Preparation of Single Cell Suspensions
from Tissue
[0455] Tissue samples (e.g., normal tissues or disease tissues such
as surgically resected neoplastic or metastatic lesions) can be
procured from clinical sites and transported in transport buffer.
Tissues can be collected as remnant tissues following surgical
resection of cancer (or other disease) tissues. Remnant tissues are
supplied following processing for pathological diagnosis according
to proper standards of patient care. Normal tissue specimens can be
normal tissue adjacent to tumors (or other disease tissue) that is
collected during tumor resection. Normal tissue from healthy
patients not having cancer (or other disease of interest) can also
be included, such as to reduce the contribution from pre-neoplastic
changes that may exist in normal adjacent tissue. Procurement of
tissue samples is carried out in an anonymous manner in compliance
with federally mandated ethical and legal guidelines (HIPAA) and in
accordance with clinical institution ethical review board and
internal institutional review board guidelines.
[0456] Tissue can be crudely minced and incubated for 20-30 minutes
with periodic agitation at 37.degree. C. in Enzyme Combination #1
(200 units collagenase, cat#C5894 Sigma; 126 .mu.g DNAse I,
cat#D4513 Sigma (in 10 mM Tris/HCl pH7.5); 50 mM NaCl; 10 mM MgCl2;
0.05% elastase, cat#E7885 Sigma) (additionally, hyaluronidase
enzyme may also be utilized). D-PBS is added at 3.times. the volume
of the enzyme combination, the tissue finely minced, and
disassociated cells passed through a 200 .mu.m filter. The cells
are washed twice with D-PBS. Red blood cells are lysed with
PharMLyse (BD Biosciences) when necessary. Cell number and
viability are determined by PI exclusion (GUAVA). Cells at a total
cell number greater than 20.times.10.sup.6 are sorted using a
high-speed sorter (MoFlo Cytomation) for epithelial cells (EpCAM
positive).
[0457] The remaining undigested tissue is incubated for 20-30
minutes with periodic agitation at 37.degree. C. in Enzyme
Combination #2 (1.times. Liberase Blendzyme 1, cat#988-417 Roche;
1.times. Liberase Blendzyme 3, cat#814-184 Roche; 0.05% elastase,
cat#E7885 Sigma). D-PBS is added at 3.times. the volume of the
enzyme combination, and the tissue finely minced until tissue is
completely disassociated. The cells are passed through a 200 .mu.m
filter, washed twice with D-PBS, and pooled with cells from the
Enzyme Combination #1 digestion.
[0458] Cells are passed through a 70 .mu.m filter for single cell
suspension, and cell number and viability are determined by PI
exclusion (GUAVA). When needed, red blood cells are lysed with
PharMLyse (BD Biosciences). Cells are incubated in 20 ml of
1.times. PharMLyse in D-PBS for 30 seconds with gentle agitation
and cells pelleted at 300.times.g for 5 minutes at 4.degree. C.
Cells are washed once in D-PBS and cell number and viability are
recalculated by PI exclusion using the GUAVA. Cells at a total cell
number greater than 20.times.10.sup.6 are sorted using a high-speed
sorter (MoFlo Cytomation) for epithelial cells (EpCAM
positive).
[0459] Single cell suspensions can also be prepared from tissue
samples as follows: specimens are washed in DTT for 15 min,
digested with Dispase (30-60 min), then filtered twice (380
.mu.m/74 .mu.m) before red blood cells are removed through addition
of ACK lysis buffer. Epithelial (EpCAM) and leukocyte (CD45)
content and cellular viability (PI exclusion) can be determined
through flow cytometry analysis (LSR I, BD Biosciences, San Jose,
Calif.).
[0460] The epithelial content of both disease and normal specimens
can be enriched through depletion of immune CD45-positive cells by
flow cytometry or purification of Epithelial Cell Surface Antigen
(ECSA/EpCam)-positive cells by bead capture.
[0461] Bead capture of epithelial cells can be performed using a
Dynal CELLection Epithelial Enrich kit (Invitrogen, Carlsbad,
Calif.) as follows. Dynal CELLection beads at a concentration of
2.times.10.sup.8 beads are incubated with 1.times.10.sup.8 cells in
HBSS with 10% fetal calf serum for 30 minutes at 4.degree. C. Cells
and beads are placed in a magnet system Dynal MPC for 2 minutes.
Bead/cell complexes are washed in RPMI 1640 media with 1% fetal
calf serum. Cells are released from the bead complex with 15 minute
incubation with DNase with agitation in RPMI with 1% fetal calf
serum.
[0462] DynalBead cell depletion of CD45 cells can be carried out as
follows. DynalBead M-450 CD45 beads and cells are incubated at a
concentration of 250 .mu.l beads per 2.times.10.sup.7 cells for 30
minutes at 4.degree. C. Bead/cell complexes are washed in DPBS
buffer with 2% fetal bovine serum. Cells and beads are placed in a
magnet system Dynal MPC for 2 minutes. The supernatant contains
EpCAM enriched cells.
[0463] Cell Line Culture
[0464] Cell lines can be obtained from the American Type Culture
Collection (ATCC, Manassas, Va.). For example, lung cancer cell
lines and normal control lung cell lines (e.g., Beas2B cells can be
used as a normal control lung cell line) can be used, such as to
determine the expression levels of markers (e.g., proteins or
encoding mRNA transcripts) in lung cancer cells compared with
normal lung cells. Cell lines can be grown in a culturing medium
that is supplemented as necessary with growth factors and serum, in
accordance with the ATCC guidelines for each particular cell line.
Cultures are established from frozen stocks in which the cells are
suspended in a freezing medium (cell culture medium with 10% DMSO
[v/v]) and flash frozen in liquid nitrogen. Frozen stocks prepared
in this way are stored in liquid nitrogen vapor. Cell cultures are
established by rapidly thawing frozen stocks at 37.degree. C.
Thawed stock cultures are slowly transferred to a culture vessel
containing a large volume of supplemented culture medium. For
maintenance of culture, cells are seeded at 1.times.10.sup.5
cells/per ml in medium and incubated at 37.degree. C. until
confluence of cells in the culture vessel exceeds 50% by area. At
this time, cells are harvested from the culture vessel using
enzymes or EDTA where necessary. The density of harvested, viable
cells is estimated by hemocytometry and the culture reseeded as
above. A passage of this nature is repeated no more than 25 times,
at which point the culture is destroyed and reestablished from
frozen stocks as described above.
[0465] Alternatively, for secreted protein analysis, cells can be
grown under routine tissue culture conditions in 490 cm.sup.2
roller bottles at an initial seeding density of approximately 15
million cells per roller bottle. When the cells reach .about.70-80%
confluence, the culturing media is removed, the cells are washed 3
times with D-PBS and once with CD293 protein-free media (Invitrogen
cat#11913-019), and the culturing media is replaced with CD293 for
generating conditioned media. Cells are incubated for 72 hours in
CD293 and the media is collected for analysis, such as mass
spectrometry analysis of secreted proteins (30-300 ml). Cell debris
is removed from the conditioned media by centrifugation at 300 g
for 5 minutes and filtering through a 0.2 micron filter prior to
analysis.
[0466] 2. Cloning and Expression of Marker Proteins
[0467] cDNA Retrieval
[0468] Peptide sequences can be searched using the BLAST algorithm
against relevant protein sequence databases to identify the
corresponding full-length protein (reference sequence). Each
full-length protein sequence can then be searched using the BLAST
algorithm against a human cDNA clone collection. For each sequence
of interest, clones can be pulled and streaked onto LB/Ampicillin
(100 .mu.g/ml) plates. Plasmid DNA is isolated using Qiagen spin
mini-prep kit and verified by restriction digest. Subsequently, the
isolated plasmid DNA is sequence verified against the reference
full-length protein sequence. Sequencing reactions are carried out
using Applied Biosystems BigDye Terminator kit followed by ethanol
precipitation. Sequence data is collected using the Applied
Biosystems 3700 Genetic Analyzer and analyzed by alignment to the
reference full-length protein sequence using the Clone Manager
alignment tool.
[0469] PCR
[0470] PCR primers are designed to amplify the region encoding the
full-length protein and/or any regions of the protein that are of
interest for expression (e.g., antigenic or hydrophilic regions as
determined by the Clone Manager sequence analysis tool). Primers
also contain 5' and 3' overhangs to facilitate cloning (see below).
PCR reactions contain 2.5 units Platinum Taq DNA Polymerase High
Fidelity (Invitrogen), 50 ng cDNA plasmid template, 1 .mu.M forward
and reverse primers, 800 .mu.M dNTP cocktail (Applied Biosystems),
and 2 mM MgSO.sub.4. After 20-30 cycles (94.degree. C. for 30
seconds, 55.degree. C. for 1 minute, and 73.degree. C. for 2
minutes), the resulting product is verified by sequence analysis
and quantitated by agarose gel electrophoresis.
[0471] Construction of Entry Clones
[0472] PCR products are cloned into an entry vector for use with
the Gateway recombination based cloning system (Invitrogen). These
vectors include pDonr221, pDonr201, pEntr/D-TOPO, or
pEntr/SD/D-TOPO and are used as described in the cloning methods
below.
[0473] TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO
[0474] For cloning using this method, the forward PCR primer
contains a 5' overhang containing the sequence "CACC". PCR products
are generated as described above and cloned into the entry vector
using the Invitrogen TOPO.RTM. cloning kit. Reactions are typically
carried out at room temperature for 10 minutes and subsequently
transformed into TOP10 chemically competent cells (Invitrogen, CA).
Candidate clones are picked, and plasmid DNA is prepared using a
Qiagen spin mini-prep kit and screened by restriction enzyme
digestion. Inserts are subsequently sequence-verified as described
above.
[0475] Gateway Cloning into pDonr201 or pDonr221
[0476] For cloning using this method, PCR primers contain forward
and reverse 5' overhangs. PCR products are generated as described
above. Protein-encoding nucleic acid molecules are recombined into
the entry vector using the Invitrogen Gateway BP Clonase enzyme
mix. Reactions are typically carried out at 25.degree. C. for 1
hour, treated with Proteinase K at 37.degree. C. for 10 minutes,
and transformed into Library Efficiency DH5.alpha.c chemically
competent cells (Invitrogen, CA). Candidate clones are picked,
plasmid DNA is prepared using a Qiagen spin mini-prep kit, and
screened by restriction enzyme digestion. Inserts are subsequently
sequence-verified as described above.
[0477] Construction of Expression Clones
[0478] Protein-encoding nucleic acid molecules are transferred from
the entry construct into a series of expression vectors using the
Gateway LR Clonase enzyme mix. Reactions are typically carried out
for 1 hour at 25.degree. C., treated with Proteinase K at
37.degree. C. for 10 minutes, and subsequently transformed into
Library Efficiency DH5a chemically competent cells (Invitrogen).
Candidate clones are picked, plasmid DNA is prepared using a Qiagen
spin mini-prep kit, and screened by restriction enzyme digestion.
Expression vectors include, but are not limited to, pDest14,
pDest15, pDest17, pDest8, pDest10 and pDest20. These vectors allow
expression in systems such as E. coli and recombinant baculovirus.
Other vectors not listed here allow expression in yeast, mammalian
cells, or in vitro.
[0479] Expression of Recombinant Proteins in E. coli
[0480] Constructs are transformed into one or more of the following
host strains: BL21 S1, BL21 AI, (Invitrogen), Origami B (DE3),
Origami B (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS,
Rosetta-Gami (DE3), Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B
(DE3) pLysS (Novagen). The transformants are grown in LB with or
without NaCl and with appropriate antibiotics, at temperatures in
the range of 20-37.degree. C., with aeration. Expression is induced
with the addition of IPTG (0.03-0.30 mM) or NaCl (75-300 mM) when
the cells are in mid-log growth. Growth is continued for one to 24
hours post-induction. Cells are harvested by centrifugation in a
Sorvall RC-3C centrifuge in a H6000A rotor for 10 minutes at 3000
rpm at 4.degree. C. Cell pellets are stored at -80.degree. C.
[0481] Expression of Recombinant Proteins Using Baculovirus
Recombinant proteins are expressed using baculovirus in Sf21 fall
army worm ovarian cells. Recombinant baculoviruses are prepared
using the Bac-to-Bac system (Invitrogen) per the manufacturer's
instructions. Proteins are expressed on the large scale in Sf900
.mu.l serum-free medium (Invitrogen) in a 10 L bioreactor tank
(27.degree. C., 130 rpm, 50% dissolved oxygen for 48 hours).
[0482] 3. Recombinant Protein Purification
[0483] Recombinant proteins can be purified from E. coli and/or
insect cells using a variety of standard chromatography methods.
Briefly, cells are lysed using sonication or detergents. The
insoluble material is pelleted by centrifugation at 10,000.times.g
for 15 minutes. The supernatant is applied to an appropriate
affinity column. For example, His-tagged proteins are separated
using a pre-packed chelating sepharose column (Pharmacia) or
GST-tagged proteins are separated using a glutathione sepharose
column (Pharmacia). After using the affinity column, proteins are
further separated using various techniques, such as ion exchange
chromatography (columns from Pharmacia) to separate on the basis of
electrical charge or size exclusion chromatography (columns from
Tosohaas) to separate on the basis of molecular weight, size, and
shape.
[0484] Expression and purification of the protein can also be
achieved using either a mammalian cell expression system or an
insect cell expression system. The pUB6/V5-His vector system
(Invitrogen, CA) can be used to express cDNA in CHO cells. The
vector contains the selectable bsd gene, multiple cloning sites,
the promoter/enhancer sequence from the human ubiquitin C gene, a
C-terminal V5 epitope for antibody detection with anti-V5
antibodies, and a C-terminal polyhistidine (6.times. His) sequence
for rapid purification on PROBOND resin (Invitrogen, CA).
Transformed cells are selected on media containing blasticidin.
[0485] Spodoptera frugiperda (Sf9) insect cells are infected with
recombinant Autographica californica nuclear polyhedrosis virus
(baculovirus). The polyhedrin gene is replaced with the cDNA by
homologous recombination and the polyhedrin promoter drives cDNA
transcription. The protein is synthesized as a fusion protein with
6.times. His which enables purification as described above.
Purified proteins can be used to produce antibodies.
[0486] 4. Chemical Synthesis of Proteins
[0487] Proteins or portions thereof can be produced not only by
recombinant methods (such as described above), but also by using
chemical methods well known in the art. Solid phase peptide
synthesis can be carried out in a batchwise or continuous flow
process which sequentially adds .alpha.-amino- and side
chain-protected amino acid residues to an insoluble polymeric
support via a linker group. A linker group such as
methylamine-derivatized polyethylene glycol is attached to
poly(styrene-co-divinylbenzene) to form the support resin. The
amino acid residues are N-a-protected by acid labile Boc
(t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl) groups. The carboxyl group of the
protected amino acid is coupled to the amine of the linker group to
anchor the residue to the solid phase support resin.
Trifluoroacetic acid or piperidine are used to remove the
protecting group in the case of Boc or Fmoc, respectively. Each
additional amino acid is added to the anchored residue using a
coupling agent or pre-activated amino acid derivative, and the
resin is washed. The full-length peptide is synthesized by
sequential deprotection, coupling of derivitized amino acids, and
washing with dichloromethane and/or N,N-dimethylformamide. The
peptide is cleaved between the peptide carboxy terminus and the
linker group to yield a peptide acid or amide. (Novabiochem 1997/98
Catalog and Peptide Synthesis Handbook, San Diego Calif. pp.
S1-S20).
[0488] Automated synthesis can also be carried out on machines such
as the 431A peptide synthesizer (Applied Bio systems, Foster City,
Calif.). A protein or portion thereof can be purified by
preparative high performance liquid chromatography and its
composition confirmed by amino acid analysis or by sequencing
(Creighton, 1984, Proteins, Structures and Molecular Properties, W
H Freeman, New York N.Y.).
[0489] 5. Antibody Production
[0490] Polyclonal Antibodies
[0491] Polyclonal antibodies against recombinant proteins can be
raised in rabbits (Green Mountain Antibodies, Burlington, Vt.).
Briefly, two New Zealand rabbits are immunized with 0.1 mg of
antigen in complete Freund's adjuvant. Subsequent immunizations are
carried out using 0.05 mg of antigen in incomplete Freund's
adjuvant at days 14, 21, and 49. Bleeds are collected and screened
for recognition of the antigen by solid phase ELISA and Western
blot analysis. The IgG fraction is separated by centrifugation at
20,000.times.g for 20 minutes followed by a 50% ammonium sulfate
cut. The pelleted protein is resuspended in 5 mM Tris and separated
by ion exchange chromatography. Fractions are pooled based on IgG
content. Antigen-specific antibody is affinity purified using
Pierce AminoLink resin coupled to the appropriate antigen.
[0492] Isolation of Antibody Fragments Directed Against a Marker
Protein from a Library of scFvs
[0493] Naturally occurring V-genes isolated from human PBLs can be
constructed into a library of antibody fragments which contain
reactivities against a marker protein to which the donor may or may
not have been exposed (see, for example, U.S. Pat. No. 5,885,793,
incorporated herein by reference in its entirety).
[0494] Rescue of the library: A library of scFvs is constructed
from the RNA of human PBLs, as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 10.sup.9 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times. TY containing 1% glucose and 100
.mu.g/ml of ampicillin (2.times. TY-AMP-GLU) and grown to an O.D.
of 0.8 with shaking. Five ml of this culture is used to innoculate
50 ml of 2.times. TY-AMP-GLU, 2.times.10.sup.8 TU of delta gene 3
helper (M13 delta gene III, see PCT publication WO 92/01047) are
added and the culture incubated at 37.degree. C. for 45 minutes
without shaking and then at 37.degree. C. for 45 minutes with
shaking. The culture is centrifuged at 4000 rpm. for 10 min. and
the pellet resuspended in 2 liters of 2.times.TY containing 100
.mu.g/ml ampicillin and 50 .mu.g/ml kanamycin and grown overnight.
Phage are prepared as described in PCT publication WO 92/01047.
[0495] Preparation of M13 delta gene III: M13 delta gene III helper
phage does not encode gene III protein, hence the phage(mid)
displaying antibody fragments have a greater avidity of binding to
antigen. Infectious M13 delta gene III particles are made by
growing the helper phage in cells harboring a pUC19 derivative
supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 rpm for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phage particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 2001, Molecular Cloning: A
Laboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.), resuspended in 2 ml PBS and passed
through a 0.45 .mu.m filter (Minisart NML; Sartorius) to give a
final concentration of approximately 10.sup.13 transducing units/ml
(ampicillin-resistant clones).
[0496] Panning of the library: Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
a marker protein of interest. Tubes are blocked with 2% Marvel-PBS
for 2 hours at 37.degree. C. and then washed 3 times in PBS.
Approximately 10.sup.13 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an
over-and-under turntable and then left to stand for another 1.5
hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10
times with PBS. Phage are eluted by adding 1 ml of 100 mM
triethylamine and rotating 15 minutes on an under-and-over
turntable after which the solution is immediately neutralized with
0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phages are then used to infect 10
ml of mid-log E. coli TG1 by incubating eluted phage with bacteria
for 30 minutes at 37.degree. C. The E. coli are then plated on TYE
plates containing 1% glucose and 100 .mu.g/ml ampicillin. The
resulting bacterial library is then rescued with delta gene 3
helper phage as described above to prepare phage for a subsequent
round of selection. This process is then repeated for a total of 4
rounds of affinity purification with tube-washing increased to 20
times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3
and 4.
[0497] Characterization of binders: Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks et al., 1991, J. Mol. Biol. 222:
581-597) from single colonies for assay. ELISAs are performed with
microtitre plates coated with either 10 .mu.g/ml of the marker
protein of interest in 50 mM bicarbonate pH 9.6. Clones positive in
ELISA are further characterized by PCR fingerprinting (see, e.g.,
PCT publication WO 92/01047) and then by sequence analysis.
[0498] Monoclonal Antibodies
[0499] a) Materials:
[0500] 1. Complete Media No Sera (CMNS) for washing of the myeloma
and spleen cells; Hybridoma medium CM-HAT (Cell Mab (BD), 10% FBS
(or HS); 5% Origen HCF (hybridoma cloning factor) containing 4 mM
L-glutamine and antibiotics) to be used for plating hybridomas
after the fusion.
[0501] 2. Hybridoma medium CM-HT (no aminopterin) (Cell Mab (BD),
10% FBS 5% Origen HCF containing 4 mM L-glutamine and antibiotics)
to be used for fusion maintenance is stored in the refrigerator at
4-6.degree. C. The fusions are fed on days 4, 8, and 12, and
subsequent passages. Inactivated and pre-filtered commercial fetal
bovine serum (FBS) or horse serum (HS) are thawed and stored in the
refrigerator at 4.degree. C. and is pretested for myeloma growth
from single cells prior to use.
[0502] 3. The L-glutamine (200 mM, 100.times. solution), which is
stored at -20.degree. C., is thawed and warmed until completely in
solution. The L-glutamine is dispensed into media to supplement
growth. L-glutamine is added to 2 mM for myelomas and 4 mM for
hybridoma media.
[0503] Further, the penicillin, streptomycin, amphotericin
(antibacterial-antifungal stored at -20.degree. C.) is thawed and
added to Cell Mab Media to 1%.
[0504] 4. Myeloma growth media is Cell Mab Media (Cell Mab Media,
Quantum Yield, from BD, which is stored in the refrigerator at
4.degree. C. in the dark), to which is added L-glutamine to 2 mM
and antibiotic/antimycotic solution to 1% and is called CMNS.
[0505] 5. One bottle of PEG 1500 in Hepes (Roche, N.J.) is
prepared.
[0506] 6. 8-Azaguanine is stored as the dried powder supplied by
SIGMA at -700.degree. C. until needed. One vial/500 ml of media is
reconstituted and the entire contents are added to 500 ml media
(e.g., 2 vials/liter).
[0507] 7. Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza
(1.times.) stored in the refrigerator at 4.degree. C.
[0508] 8. Clonal cell medium D (Stemcell, Vancouver) contains HAT
and methyl cellulose for semi-solid direct cloning from the fusion.
This comes in 90 ml bottles with a CoA and is melted at 37.degree.
C. in a waterbath in the morning of the day of the fusion. The cap
is loosened and the bottle is left in a CO.sub.2 incubator to
sufficiently gas the medium D and bring the pH down.
[0509] 9. Hybridoma supplements HT [hypoxanthine, thymidine] to be
used in medium for the section of hybridomas and maintenance of
hybridomas through the cloning stages, respectively.
[0510] 10. Origen HCF can be obtained directly from Igen and is a
cell supernatant produced from a macrophage-like cell-line. It can
be thawed and aliqouted to 15 ml tubes at 5 ml per tube and stored
frozen at -20.degree. C. Positive hybridomas are fed HCF through
the first subcloning and are gradually weaned (individual
hybridomas can continue to be supplemented, as needed). This and
other additives are typically more effective in promoting new
hybridoma growth than conventional feeder layers.
[0511] b) Procedure:
[0512] To generate monoclonal antibodies, mice are immunized with
5-50 .mu.g of antigen, either intra-peritoneally (i.p.) or by
intravenous injection in the tail vein (i.v.). The antigen used can
be a recombinant marker protein of interest, for example. The
primary immunization takes place two months prior to the harvesting
of splenocytes from the mouse, and the immunization is typically
boosted by i.v. injection of 5-50 .mu.g of antigen every two weeks.
At least one week prior to the expected fusion date, a fresh vial
of myeloma cells is thawed and cultured. Several flasks of
different densities can be maintained so that a culture at the
optimum density is ensured at the time of fusion. An optimum
density can be 3-6.times.10.sup.5 cells/ml, for example. 2-5 days
before the scheduled fusion, a final immunization of approximately
5 .mu.g of antigen in PBS is administered (either i.p. or i.v).
[0513] Myeloma cells are washed with 30 ml serum free media by
centrifugation at 500 g at 4.degree. C. for 5 minutes. Viable cell
density is determined in resuspended cells using hemocytometry and
vital stains. Cells resuspended in complete growth medium are
stored at 37.degree. C. during the preparation of splenocytes.
Meanwhile, to test aminopterin sensitivity, 1.times.10.sup.6
myeloma cells are transferred to a 15 ml conical tube and
centrifuged at 500 g at 4.degree. C. for 5 minutes. The resulting
pellet is resuspended in 15 ml of HAT media and cells plated at 2
drops/well on a 96-well plate.
[0514] To prepare splenocytes from immunized mice, the animals are
euthanised and submerged in 70% ethanol. Under sterile conditions,
the spleen is surgically removed and placed in 10 ml of RPMI medium
supplemented with 20% fetal calf serum in a petri dish. Cells are
extricated from the spleen by infusing the organ with medium >50
times using a 21 g syringe.
[0515] Cells are harvested and washed by centrifugation (at 500 g
at 4.degree. C. for 5 minutes) with 30 ml of medium. Cells are
resuspended in 10 ml of medium and the density of viable cells
determined by hemocytometry using vital stains. The splenocytes are
mixed with myeloma cells at a ratio of 5:1 (spleen cells: myeloma
cells). Both the myeloma and spleen cells are washed twice more
with 30 ml of RPMI-CMNS, and the cells are spun at 800 rpm for 12
minutes.
[0516] Supernatant is removed and cells are resuspended in 5 ml of
RPMI-CMNS and are pooled to fill volume to 30 ml and spun down as
before. Then, the pellet is broken up by gently tapping on the flow
hood surface and resuspending in 1 ml of BMB REG1500 (prewarmed to
37.degree. C.) dropwise with a 1 cc needle over 1 minute.
[0517] RPMI-CMNS to the PEG cells and RPMI-CMNS are added to slowly
dilute out the PEG. Cells are centrifuged and diluted in 5 ml of
Complete media and 95 ml of Clonacell Medium D (HAT) media (with 5
ml of HCF). The cells are plated out 10 ml per small petri
plate.
[0518] Myeloma/HAT control is prepared as follows: dilute about
1000 P3X63 Ag8.653 myeloma cells into 1 ml of medium D and transfer
into a single well of a 24-well plate. Plates are placed in an
incubator, with two plates inside of a large petri plate, with an
additional petri plate full of distilled water, for 10-18 days
under 5% CO.sub.2 overlay at 37.degree. C. Clones are picked from
semisolid agarose into 96-well plates containing 150-200 .mu.l of
CM-HT. Supernatants are screened 4 days later in ELISA, and
positive clones are moved up to 24-well plates. Heavy growth
requires changing of the media at day 8 (+/-150 ml). The HCF can be
further decreased to 0.5% (gradually--2%, then 1%, then 0.5%) in
the cloning plates.
[0519] 6. Liquid Chromatography and Mass Spectrometry (LC/MS)
[0520] For LC/MS analysis, proteins are reduced in 2.5 mM DTT for 1
hour at 37.degree. C., and alkylated with ICAT.TM. reagent
according to the procedures recommended by the manufacturer
(Applied Biosystems, Framingham, Mass.). The reaction is quenched
by adding excess DTT. Proteins are digested using sequencing grade
modified trypsin overnight at 37.degree. C. followed by desalting
using 3 cc Oasis HLB solid phase extraction columns (Waters,
Milford, Mass.) and vacuum drying. Cysteine-containing peptides are
purified by avidin column (Applied Biosystems, Framingham, Mass.).
The peptides are reconstituted in buffer A (0.1% formic acid in
water) and separated over a C18 monomeric column (150 mm, 150 .mu.m
i.d., Grace Vydac 238EV5, 5 .mu.m) at a flow rate of 1.5 .mu.l/min
with a trap column. Peptides are eluted from the column using a
gradient, 3%-30% buffer B (0.1% formic acid in 90% acetonitrile) in
215 min, 30%-90% buffer B in 30 min. Eluted peptides are analyzed
using an online QSTAR XL system (MDS/Sciex, Toronto, ON). Peptide
ion peaks from the map are automatically detected with RESPEC.TM.
(PPL Inc., UK).
[0521] The sequence-composition of peptides detected, for example,
at higher levels in disease samples (or drug-resistant samples)
relative to adjacent normal tissue (or drug-sensitive samples) can
be resolved through tandem mass spectrometry and database analysis.
For data analysis, peptide ion peaks of LC/MS maps from normal and
disease samples can be aligned based on mass to charge ratio (m/z),
retention time (Rt), and charge state (z). The list of aligned
peptide ions is loaded into Spotfire.TM. (Spotfire Inc. Somerville,
Mass.). Intensities can be normalized before further differential
analysis between disease and normal samples. Differentially
expressed ions are manually verified before LC-MS/MS-based peptide
sequencing and database searching for protein/protein
identification.
[0522] For intensity normalization and expression analysis, a heat
map can be constructed by sorting the rows by the ratio of the mean
intensity in the disease samples to the mean intensity of the
normal samples. Rows are included if there is at least one MS/MS
identification of an ion in the row. The display colors are
determined for each row separately by assigning black to the median
intensity in the row, green to the lowest intensity in the row, and
red to the highest intensity.
[0523] Using a mass spectrometry procedure such as this, a
comprehensive analysis of proteins differentially expressed by
disease cells (or drug resistant cells, for example) compared with
normal cells (or cells responsive/sensitive to a drug, for example)
can be carried out.
[0524] 7. mRNA Expression Analysis
[0525] Expression of marker mRNA can be quantitated by RT-PCR using
TaqMan.RTM. technology. The Taqman.RTM. system couples a 5'
fluorogenic nuclease assay with PCR for real-time quantitation. A
probe is used to monitor the formation of the amplification
product.
[0526] Total RNA can be isolated from disease model cell lines
using an RNEasy Kit.RTM. (Qiagen, Valencia, Calif.) with DNase
treatment (per the manufacturer's instructions). Normal human
tissue RNAs can be acquired from commercial vendors (e.g., Ambion,
Austin, Tex.; Stratagene, La Jolla, Calif.; BioChain Institute,
Newington, N.H.), as well as RNAs from matched disease/normal
tissues.
[0527] Marker transcript sequences can be identified for
differentially expressed peptides by database searching using a
search algorithm such as BLAST. TaqMan.RTM. assays (PCR
primer/probe sets) specific for those transcripts can be obtained
from Applied Biosystems (AB) as part of the Assays on Demand.TM.
product line or by custom design through the AB Assays by
Design.sup.SM service. If desired, the assays can be designed to
span exon-exon borders so as not to amplify genomic DNA.
[0528] RT-PCR can be accomplished using AmpliTaq Gold.RTM. and
MultiScribe.TM. reverse transcriptase in the One Step RT-PCR Master
Mix reagent kit (AB) (according to the manufacturer's
instructions). Probe and primer concentrations are 250 nM and 900
nM, respectively, in a 15 .mu.l reaction. For each experiment, a
master mix of the above components is made and aliquoted into each
optical reaction well. Eight nanograms of total RNA is used as
template. Quantitative RT-PCR can be performed using the ABI
Prism.RTM. 7900HT Sequence Detection System (SDS). The following
cycling parameters are used: 48.degree. C. for 30 min. for one
cycle; 95.degree. C. for 10 min for one cycle; and 95.degree. C.
for 15 sec, 60.degree. C. for 1 min. for 40 cycles.
[0529] SDS software can be utilized to calculate the threshold
cycle (C.sub.T) for each reaction, and C.sub.T values are used to
quantitate the relative amount of starting template in the
reaction. The C.sub.T values for each set of reactions can be
averaged for all subsequent calculations
[0530] Data can be analyzed to determine estimated copy number per
cell. Gene expression can be quantitated relative to 18S rRNA
expression and copy number estimated assuming 5.times.10.sup.6
copies of 18S rRNA per cell. Alternatively, data can be analyzed
for fold difference in expression using an endogenous control for
normalization and expressed relative to a normal tissue or normal
cell line reference. The choice of endogenous control can be
determined empirically by testing various candidates against the
cell line and tissue RNA panels and selecting the one with the
least variation in expression. Relative changes in expression can
be quantitated using the 2.sup.-.DELTA..DELTA.CT method (Livak et
al., 2001, Methods 25: 402-408; User bulletin #2: ABI Prism 7700
Sequence Detection System). Alternatively, total RNA can be
quantitated using a RiboGreen RNA Quantitation Kit according to
manufacturer's instructions and the percentage mRNA expression
calculated using total RNA for normalization. Percentage knockdown
can then be calculated relative to a no addition control.
[0531] 8. Flow Cytometry (FACS) Analysis
[0532] Flow cytometry is interchangeably referred to as
fluorescence-activated cell sorting (FACS). Quantitative flow
cytometry can be used to compare the level of expression of a
protein on disease cells to the level found on normal cells, for
example.
[0533] Expression levels of a marker protein on primary tissue
samples can be quantified using the Quantum Simply Cellular System
(Bangs Laboratories, Fishers, Ind.) and a marker-specific antibody.
Normal adjacent and disease tissues can be processed into single
cell suspensions, as described above, which can be stained for
various markers (e.g., the epithelial marker EpCam) and the
marker-specific antibody. At least 0.5.times.10.sup.6 cells are
typically used for each analysis. Cells are washed once with Flow
Staining Buffer (0.5% BSA, 0.05% NaN3 in D-PBS). To the cells, 20
.mu.l of each marker-specific antibody are added. An additional 5
.mu.l of anti-EpCam antibody conjugated to APC can be added when
unsorted cells are used. Cells are incubated with antibodies for 30
minutes at 4.degree. C. Cells are washed once with Flow Staining
Buffer and either analyzed immediately on an LSR flow cytometry
apparatus or fixed in 1% formaldehyde and stored at 4.degree. C.
until LSR analysis. Antibodies used to detect a marker can be
PE-conjugated. PE-conjugated mouse IgG1k can used as an isotype
control antibody. Cells are analyzed by flow cytometry and epitope
copy number and the percentage of viable epithelial cells positive
for marker expression can be measured. Cell numbers and viability
can be determined by PI exclusion (GUAVA) for cells isolated from
both normal and disease tissue. Standard curve and samples can be
analyzed on a LSR I (BDBiosciences, San Jose Calif.) flow
cytometer. Antibody binding capacity for each lineage population
can be calculated using geometric means and linear regression.
[0534] Expression levels of a marker protein can be quantified in
cell lines with QIFIKIT flow cytometric indirect immunofluorescence
assay (Dako A/S) using a primary antibody to the marker. Briefly,
cells are detached with versene or trypsin and washed once with
complete media and then PBS. 5.times.10.sup.5 cells/sample are
incubated with saturating concentration (10 .mu.g/ml) of primary
antibody for 60 minutes at 4.degree. C. After washes, a
FITC-conjugated secondary antibody (1:50 dilution) is added for 45
minutes at 4.degree. C. QIFIKIT standard beads are simultaneously
labeled with the secondary antibody. Binding of antibodies is
analyzed by flow cytometry and specific antigen density is
calculated by subtracting background antibody equivalent from
antibody-binding capacity based on a standard curve of log mean
fluorescence intensity versus log antigen binding capacity.
[0535] Cells can also be prepared for flow cytometry analysis (as
well as other types of analysis) as follows: cells are incubated
with 1:100 dilution of BrdU in culturing media for 2-4 hours (BrdU
Flow Kit, cat#559619 BD Biosciences). Cells are washed 3 times with
D-PBS and disassociated from the flask with versene. Cell numbers
and viability can be determined by PI exclusion (GUAVA). Cells are
washed once with Flow Staining Buffer (0.5% BSA, 0.05% NaN.sub.3 in
D-PBS). Cells are incubated with 400 .mu.l of Cytofix/Cytoperm
Buffer (BrdU Flow Kit, BD Biosciences) for 15-30 minutes at
4.degree. C. Cells are washed once with Flow Staining Buffer and
resuspended in 400 .mu.l Cytoperm Plus Buffer (BrdU Flow Kit BD
Biosciences). Cells are incubated for 10 minutes at 4.degree. C.
and washed once with 1.times. Perm/Wash Buffer (BrdU Flow Kit, BD
Biosciences). Cells are incubated for 1 hour at 37.degree. C.
protected from light in DNAse solution (BrdU Flow Kit, BD
Biosciences). Cells are washed once with 1.times. Perm/Wash Buffer
and incubated for 20 min at room temperature with anti-BrdU
FITC-conjugated antibody (BrdU Flow Kit, BD Biosciences),
PE-conjugated active caspase 3 (BD Biosciences cat#550821), and PE
mouse IgG2.beta. isotype control. Cells are washed once with
1.times. Perm/Wash Buffer and resuspended in DAPI for LSR flow
cytometry analysis.
[0536] 9. Immunohistochemistry (IHC)
[0537] IHC of Tissue Sections
[0538] Paraffin embedded, fixed tissue sections (e.g., from disease
tissue samples such as solid tumors or other cancer tissues) can be
obtained from a panel of normal tissues as well as tumor (or other
disease) samples with matched normal adjacent tissues, along with
replicate sections (if desired). For example, for an initial survey
of marker expression, a panel of common cancer formalin-fixed
paraffin-embedded (FFPE) tissue microarrays (TMAs) can be used for
analysis, and such TMAs can be obtained from commercial sources
(TriStar, Rockville, Md.; USBiomax, Rockville, Md.; Imgenex, San
Diego, Calif.; Petagen/Abxis, Seoul, Korea). Sections can be
stained with hemotoxylin and eosin and histologically examined to
ensure adequate representation of cell types in each tissue
section.
[0539] An identical set of tissues can be obtained from frozen
sections for use in those instances where it is not possible to
generate antibodies that are suitable for fixed sections. Frozen
tissues do not require an antigen retrieval step.
[0540] Paraffin Fixed Tissue Sections
[0541] An exemplary protocol for hemotoxylin and eosin staining of
paraffin embedded, fixed tissue sections is as follows. Sections
are deparaffinized in three changes of xylene or xylene substitute
for 2-5 minutes each. Sections are rinsed in two changes of
absolute alcohol for 1-2 minutes each, in 95% alcohol for 1 minute,
followed by 80% alcohol for 1 minute. Slides are washed in running
water and stained in Gill solution 3 hemotoxylin for 3-5 minutes.
Following a vigorous wash in running water for 1 minute, sections
are stained in Scott's solution for 2 minutes. Sections are washed
for 1 minute in running water and then counterstained in eosin
solution for 2-3 minutes, depending upon the desired staining
intensity. Following a brief wash in 95% alcohol, sections are
dehydrated in three changes of absolute alcohol for 1 minute each
and three changes of xylene or xylene substitute for 1-2 minutes
each. Slides are coverslipped and stored for analysis.
[0542] Optimization of Antibody Staining
[0543] For each antibody, a positive and negative control sample
can be generated using data from ICAT analysis of disease cell
lines or tissues. Cells can be selected that are known to express
low levels of a particular marker as determined from the ICAT data,
and this cell line can be used as a reference normal control.
Similarly, a disease cell line that is determined to over-express
the marker can also be selected.
[0544] Antigen Retrieval
[0545] Sections are deparaffinized and rehydrated by washing 3
times for 5 minutes in xylene, two times for 5 minutes in 100%
ethanol, two times for 5 minutes in 95% ethanol, and once for 5
minutes in 80% ethanol. Sections are then placed in endogenous
blocking solution (methanol+2% hydrogen peroxide) and incubated for
20 minutes at room temperature. Sections are rinsed twice for 5
minutes each in deionized water and twice for 5 minutes in
phosphate buffered saline (PBS), pH 7.4.
[0546] Alternatively, where necessary, sections are de-parrafinized
by High Energy Antigen Retrieval as follows: sections are washed
three times for 5 minutes in xylene, two times for 5 minutes in
100% ethanol, two times for 5 minutes in 95% ethanol, and once for
5 minutes in 80% ethanol. Sections are placed in a Coplin jar with
dilute antigen retrieval solution (10 mM citrate acid, pH 6). The
Coplin jar containing slides is placed in a vessel filled with
water and microwaved on high for 2-3 minutes (700 watt oven).
Following cooling for 2-3 minutes, steps 3 and 4 are repeated four
times (depending on the tissue), followed by cooling for 20 minutes
at room temperature. Sections are then rinsed in deionized water
(two times for 5 minutes), placed in modified endogenous oxidation
blocking solution (PBS+2% hydrogen peroxide), and rinsed for 5
minutes in PBS.
[0547] Alternatively, formalin fixed paraffin embedded tissues can
be deparaffinized and processed for antigen retrieval using the
EZ-retriever system (BioGenex, San Ramon, Calif.). EZ-antigen
Retrieval common solution is used for deparaffinization and
EZ-retrieval citrate-based buffer used for antigen retrieval.
Samples are pre-blocked with non-serum protein block (Dako A/S,
Glostrup, Denmark) for 15 min. Primary antibodies (at 2.5-5.0
.mu.g/ml, for example) are incubated overnight at room temperature.
Envision Plus system HRP (Dako A/S) is used for detection with
diaminobenzidine (DAB) as substrate for horseradish peroxidase.
[0548] Blocking and Staining
[0549] Sections are blocked with PBS/1% bovine serum albumin (PBA)
for 1 hour at room temperature followed by incubation in normal
serum diluted in PBA (2%) for 30 minutes at room temperature to
reduce non-specific binding of antibody. Incubations are performed
in a sealed humidity chamber to prevent air-drying of the tissue
sections. The choice of blocking serum is typically the same as the
species of the biotinylated secondary antibody. Excess antibody is
gently removed by shaking and sections covered with primary
antibody diluted in PBA and incubated either at room temperature
for 1 hour or overnight at 4.degree. C. (care is taken that the
sections do not touch during incubation). Sections are rinsed twice
for 5 minutes in PBS, shaking gently. Excess PBS is removed by
gently shaking. The sections are covered with diluted biotinylated
secondary antibody in PBA and incubated for 30 minutes to 1 hour at
room temperature in the humidity chamber. If using a monoclonal
primary antibody, addition of 2% rat serum can be used to decrease
the background on rat tissue sections. Following incubation,
sections are rinsed twice for 5 minutes in PBS, shaking gently.
Excess PBS is removed and sections incubated for 1 hour at room
temperature in Vectastain ABC reagent (as per kit instructions).
The lid of the humidity chamber is secured during all incubations
to ensure a moist environment. Sections are rinsed twice for 5
minutes in PBS, shaking gently.
[0550] Developing and Counterstaining
[0551] Sections are incubated for 2 minutes in peroxidase substrate
solution that is made up immediately prior to use as follows: 10 mg
diaminobenzidine (DAB) dissolved in 10 ml of 50 mM sodium phosphate
buffer, pH 7.4; 12.5 microliters 3% CoCl.sub.2/NiCl.sub.2 in
deionized water; and 1.25 microliters hydrogen peroxide.
[0552] Slides are rinsed well three times for 10 minutes in
deionized water and counterstained with 0.01% Light Green acidified
with 0.01% acetic acid for 1-2 minutes, depending on the desired
intensity of counterstain.
[0553] Slides are rinsed three times for 5 minutes with deionized
water and dehydrated two times for 2 minutes in 95% ethanol; two
times for 2 minutes in 100% ethanol; and two times for 2 minutes in
xylene. Stained slides are mounted for visualization by
microscopy.
[0554] Slides are scored manually using a microscope such as the
Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging, Thornwood,
N.Y.). Representative images are acquired using 40.times. objective
(400.times. magnification).
[0555] IHC Staining of Frozen Tissue Sections
[0556] For IHC staining of frozen tissue sections, fresh tissues
are embedded in OCT in plastic mold, without trapping air bubbles
surrounding the tissue. Tissues are frozen by setting the mold on
top of liquid nitrogen until 70-80% of the block turns white at
which point the mold is placed on dry ice. The frozen blocks are
stored at -80.degree. C. Blocks are sectioned with a cryostat with
care taken to avoid warming to greater than -10.degree. C.
Initially, the block is equilibrated in the cryostat for about 5
minutes and 6-10 mm sections are cut sequentially. Sections are
allowed to dry for at least 30 minutes at room temperature.
Following drying, tissues are stored at 4.degree. C. for short term
and -80.degree. C. for long term storage.
[0557] Sections are fixed by immersing in an acetone jar for 1-2
minutes at room temperature, followed by drying at room
temperature. Primary antibody is added (diluted in 0.05 M
Tris-saline [0.05 M Tris, 0.15 M NaCl, pH 7.4], 2.5% serum)
directly to the sections by covering the section dropwise to cover
the tissue entirely. Binding is carried out by incubation in a
chamber for 1 hour at room temperature. Without letting the
sections dry out, the secondary antibody (diluted in
Tris-saline/2.5% serum) is added in a similar manner to the primary
antibody and incubated as before (at least 45 minutes).
[0558] Following incubation, the sections are washed gently in
Tris-saline for 3-5 minutes and then in Tris-saline/2.5% serum for
another 3-5 minutes. If a biotinylated primary antibody is used, in
place of the secondary antibody incubation, slides are covered with
100 .mu.l of diluted alkaline phosphatase conjugated streptavidin,
incubated for 30 minutes at room temperature and washed as above.
Sections are incubated with alkaline phosphatase substrate (1 mg/ml
Fast Violet; 0.2 mg/ml Napthol AS-MX phosphate in Tris-Saline pH
8.5) for 10-20 minutes until the desired positive staining is
achieved at which point the reaction is stopped by washing twice
with Tris-saline. Slides are counter-stained with Mayer's
hematoxylin for 30 seconds and washed with tap water for 2-5
minutes. Sections are mounted with Mount coverslips and mounting
media.
[0559] 10. RNAi Assays in Cell Lines
[0560] RNAi Transfections
[0561] Expression of a marker can be knocked down by transfection
with small interfering RNA (siRNA) to that marker. Synthetic siRNA
oligonucleotides can be obtained from Dharmacon (Lafayette, Colo.)
or Qiagen (Valencia, Calif.). For siRNA transfection, cells (e.g.,
disease cells) can be seeded into 96 well tissue culture plates at
a density of 2,500 cells per well 24 hours before transfection.
Culture medium is removed and 50 .mu.l of reaction mix containing
siRNA (final concentration 1 to 100 nM) and 0.4 .mu.l of
DharmaFECT4 (Dharmacon, Lafayette, Colo.) diluted in Opti-MEM is
added to each well. An equal volume of complete medium follows and
the cells are then incubated at 5% CO.sub.2 at 37.degree. C. for 1
to 4 days.
[0562] Alternatively, in the initial screening phase, RNAi can be
performed using 100 nM (final) of Smartpools (Dharmacon, Lafayette,
Colo.), pool of 4- for Silencing siRNA duplexes (Qiagen, Valencia,
Calif.), or non-targeting negative control siRNA (Dharmacon or
Qiagen). In the breakout phase, each individual duplex is used at
100 nM (final). In the titration phase, individual duplex is used
at 0.1-100 nM (final). Transient transfections are carried out
using either Lipofectamine 2000 from Invitrogen (Carlsbad, Calif.)
or GeneSilencer from Gene Therapy Systems (San Diego, Calif.) (see
below). One day after transfections, total RNA is isolated using
the RNeasy 96 Kit (Qiagen) according to manufacturer's instructions
and expression of mRNA is quantitated using TaqMan technology.
Apoptosis and cell proliferation assays can be performed daily
using Apop-one homogeneous caspase-3/7 kit and Alamar Blue or
CellTiter 96 AQueous One Solution Cell Proliferation Assays (see
below).
[0563] RNAi Transfections--Lipofectamine 2000 and GeneSilencer
[0564] Transient RNAi transfections can be carried out using
Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) or GeneSilencer
(Gene Therapy Systems, San Diego, Calif.), such as on sub-confluent
disease cell lines, as described elsewhere (Elbashir et al., 2001,
Nature 411: 494-498; Caplen et al., 2001, Proc Natl Acad Sci USA
98: 9742-9747; Sharp, 2001, Genes and Development 15: 485-490).
Synthetic RNA to a gene of interest or non-targeting negative
control siRNA are transfected using Lipofectamine 2000 or
GeneSilencer according to manufacturer's instructions. Cells are
plated in 96-well plates in antibiotic-free medium. The next day,
the transfection reagent and siRNA are prepared for transfections
as follows.
[0565] 0.1-100 nM siRNA is resuspended in 20-25 .mu.l serum-free
media in each well (with Plus for Lipofectamine 2000) and incubated
at room temperature for 15 minutes. 0.1-1 .mu.l of Lipofectamine
2000 or 1-1.5 .mu.l of GeneSilencer is also resuspended in
serum-free medium to a final volume of 20-25 .mu.l per well. After
incubation, the diluted siRNA and either the Lipofectamine 2000 or
the GeneSilencer are combined and incubated for 15 minutes
(Lipofectamine 2000) or 5-20 minutes (GeneSilencer) at room
temperature. Media is then removed from the cells and the combined
siRNA-Lipofectamine 2000 reagent or siRNA-GeneSilencer reagent is
added to a final volume of 50 .mu.l per well. After further
incubation at 37.degree. C. for 4 hours, 50 .mu.l serum-containing
medium is added back to the cells. 1-4 days after transfection,
expression of mRNA can be quantitated by RT-PCR using TaqMan
technology, and protein expression levels can be measured by flow
cytometry. Apoptosis and proliferation assays can be performed
daily using Apop-one homogeneous caspase-3/7 kit and Alamar Blue or
CellTiter 96 AQueous One Solution Cell Proliferation Assays (see
below).
[0566] mRNA and Protein Knockdowns
[0567] Knockdown of marker mRNA levels can be monitored by Q-PCR
one day after siRNA transfection by using a TaqMan.RTM. assay
(Applied Biosystems, Foster City, Calif.). RT-PCR is accomplished
in a one-step reaction by using M-MLV reverse transcriptase
(Promega, Madison, Wis.) and AmpliTaq Gold.RTM. (ABI) and analyzed
on the ABI Prism.RTM. 7900HT Sequence Detection System (ABI).
Relative gene expression can be quantitated by the .DELTA..DELTA.Ct
method (User Bulletin #2, ABI) with 18S rRNA serving as the
endogenous control.
[0568] Protein knockdown can be monitored by FACS four days after
transfection by using an antibody to the marker. The samples can be
run on a LSR flow cytometer (BD Biosciences, San Jose, Calif.) and
live cells monitored by using PI exclusion (50 .mu.g/ml PI, 2.5
units/ml RNase A, 0.1% Triton X-100 in D-PBS). The data can be
analyzed using CellQuest software.
[0569] Cell Proliferation--Alamar Blue
[0570] Cell growth can be assessed four days after transfection by
adding a 1:10 dilution of Alamar blue reagent (Invitrogen,
Carlsbad, Calif. or Biosource, Camarillo, Calif.) and incubated for
2 hours at 37.degree. C. Analysis can be performed on a
Spectrafluor Plus (Tecan, Durham, N.C.) set at excitation
wavelength of 530 nm and emission wavelength of 595 nm.
[0571] Cell Proliferation--MTS
[0572] Alternatively, cell proliferation assays can be performed
using a CellTiter 96 AQueous One Solution Cell Proliferation Assay
kit (Promega, Madison, Wis.). 200 of CellTiter 96 AQueous One
Solution is added to 1000 of culture medium. The plates are then
incubated for 1-4 hours at 37.degree. C. in a humidified 5%
CO.sub.2 incubator. After incubation, the change in absorbance is
read at 490 nm.
[0573] Apoptosis
[0574] Apoptosis assays can be performed using the Apop-one
homogeneous caspase-3/7 kit (Promega, Madison, Wis.). Briefly, the
caspase-3/7 substrate is thawed to room temperature and diluted
1:100 with buffer. The diluted substrate is then added 1:1 to
cells, control, or blank. The plates are then placed on a plate
shaker for 30 minutes to 18 hours at 300-500 rpm. The fluorescence
of each well is then measured using an excitation wavelength of
485+/-20 nm and an emission wavelength of 530+/-25 nm.
[0575] 11. Antibody Assays in Cell Lines
[0576] Cytotoxicity Assays
[0577] Cytotoxicity can be measured using a Resazurin (Sigma, Mo.)
dye reduction assay (McMillian et al., 2002, Cell Biol. Toxicol.
18:157-173). Briefly, cells are plated at 1,000-5,500 cells/well in
96 well plates, allowed to attach to the plates for 18 hours before
addition of fresh media with or without antibody. After 96-144
hours of exposure to antibody, resazurin is added to cells to a
final concentration of 50 .mu.M. Cells are incubated for 2-6 hours
depending on dye conversion of cell lines, and dye reduction is
measured on a Fusion HT fluorescent plate reader (Packard
Instruments, Meridien, Conn.) with excitation and emission
wavelengths of 530 nm and 590 nm, respectively. The IC.sub.50 value
is defined here as the drug concentration that results in 50%
reduction in growth or viability as compared with untreated control
cultures.
[0578] Assays for Antibody-Dependent Cellular Cytotoxicity
[0579] Antibody-dependent cellular cytotoxicity (ADCC) assays can
be carried out as follows. Cultured disease cells (e.g., tumor
cells) are labeled with 100 .mu.Ci .sup.51Cr for 1 hour (Livingston
et al., 1997, Cancer Immunol. Immunother. 43, 324-330). After being
washed three times with culture medium, cells are resuspended at
10.sup.5/ml, and 100 .mu.l/well are plated onto 96-well
round-bottom plates. A range of antibody concentrations are applied
to the wells, including an isotype control together with donor
peripheral blood mononuclear cells that are plated at a 100:1 and
50:1 ratio. After an 18 hour incubation at 37.degree. C.,
supernatant (30 .mu.l/well) is harvested and transferred onto
Lumaplate 96 (Packard), dried, and read in a Packard Top-Count NXT
.gamma. counter. Spontaneous release is determined by cpm of
disease cells incubated with medium and maximum release by cpm of
disease cells plus 1% Triton X-100 (Sigma). Specific lysis is
defined as: % specific lysis=[(experimental release-spontaneous
release)/(maximum release-spontaneous release)].times.100. The
percent ADCC is expressed as peak specific lysis postimmune
subtracted by preimmune percent specific lysis. A doubling of the
ADCC to >20% can typically be considered significant.
[0580] Assays for Complement Dependent Cytotoxicity
[0581] Chromium release assays to assess complement dependent
cytotoxicity (CDC) can be carried out as follows (Dickler et al.,
1999, Clin. Cancer Res. 5, 2773-2779). Cultured disease cells
(e.g., tumor cells) are washed in FCS-free media two times,
resuspended in 500 .mu.l of media, and incubated with 100 .mu.Ci
.sup.51Cr per 10 million cells for 2 hours at 37.degree. C. The
cells are then shaken every 15 min for 2 hours, washed 3 times in
media to achieve a concentration of approximately 20,000
cells/well, and then plated in round-bottom plates. The plates
contain either 50 .mu.l cells plus 50 .mu.l monoclonal antibody, 50
.mu.l cells plus serum (pre- and post-therapy), or 50 .mu.l cells
plus mouse serum as a control. The plates are incubated in a cold
room on a shaker for 45 min. Human complement of a 1:5 dilution
(resuspended in 1 ml of ice-cold water and diluted with 3% human
serum albumin) is added to each well at a volume of 100 .mu.l.
Control wells include those for maximum release of isotope in 10%
Triton X-100 (Sigma) and for spontaneous release in the absence of
complement with medium alone. The plates are incubated for 2 hours
at 37.degree. C., centrifuged for 3 min, and then 100 .mu.l of
supernatant is removed for radioactivity counting. The percentage
of specific lysis is calculated as follows: %
cytotoxicity=[(experimental release-spontaneous release)/(maximum
release-spontaneous release)].times.100. A doubling of the CDC to
>20% can typically be considered significant.
[0582] Cell Proliferation Assays
[0583] To measure cell proliferation, cells can be plated, grown
and treated as for the cytotoxicity assay (above) in 96 well
plates. After 96-144 hours of treatment, 0.5 .mu.Ci/well
.sup.3H-Thymidine (PerkinElmer, 6.7 Ci/mmol) is added to cells and
incubated for 4-6 hours at 37.degree. C., 5% CO.sub.2 in an
incubator. To lyse cells, plates are frozen overnight at
-20.degree. C. and then cell lysates are harvested using FilterMate
(Packard Instrument, Meridien, Conn.) into 96 well filter plates.
Radioactivity associated with cells is measured on a TopCount
(Packard) scintillation counter.
[0584] Other cell assays (e.g., proliferation assays such as Alamar
blue and MTS, and apoptosis assays) can be carried out using
antibodies, as described above for RNAi.
[0585] Testing of Function-Blocking Antibodies
[0586] For testing of function-blocking antibodies, sub-confluent
disease cell lines are serum-starved overnight. The next day,
serum-containing media is added back to the cells in the presence
of 5-50 ng/ml of function-blocking antibodies. After 2 or 5 days
incubation at 37.degree. C. 5% CO.sub.2, antibody binding is
examined by flow cytometry, and apoptosis and proliferation are
measured.
[0587] Cell Invasion
[0588] Cell invasion assays can be performed using a 96-well cell
invasion assay kit (Chemicon). After the cell invasion chamber
plates are adjusted to room temperature, 100 .mu.l serum-free media
is added to the interior of the inserts. 1-2 hours later, cell
suspensions of 1.times.10.sup.6 cells/ml are prepared. Media is
then carefully removed from the inserts and 100 .mu.l of prepared
cells are added into the insert +/-0 to 50 ng function blocking
antibodies. The cells are pre-incubated for 15 minutes at
37.degree. C. before 150 .mu.l of media containing 10% FBS is added
to the lower chamber. The cells are then incubated for 48 hours at
37.degree. C. After incubation, the cells from the top side of the
insert are discarded and the invasion chamber plates are then
placed on a new 96-well feeder tray containing 150 .mu.l of
pre-warmed cell detachment solution in the wells. The plates are
incubated for 30 minutes at 37.degree. C. and are periodically
shaken. Lysis buffer/dye solution (4 .mu.l CyQuant Dye/300 .mu.l
4.times. lysis buffer) is prepared and added to each well of
dissociation buffer/cells on feeder tray. The plates are incubated
for 15 minutes at room temperature before 150 .mu.l is transferred
to a new 96-well plate. Fluorescence of invading cells is then read
at 480 nm excitation and 520 nm emission.
[0589] Receptor Internalization
[0590] For quantification of receptor internalization, ELISA assays
can be performed essentially as described by Daunt et al. (Daunt et
al., 1997, Mol. Pharmacol. 51, 711-720). Cell lines are plated at
6.times.10.sup.5 cells per in a 24-well tissue culture dishes that
have previously been coated with 0.1 mg/ml poly-L-lysine. The next
day, the cells are washed once with PBS and incubated in DMEM at
37.degree. C. for several minutes. Agonist to the cell surface
marker of interest is then added to the wells at a pre-determined
concentration in prewarmed DMEM. The cells are then incubated for
various times at 37.degree. C. and reactions are stopped by
removing the media and fixing the cells in 3.7% formaldehyde/TBS
for 5 min at room temperature. The cells are then washed three
times with TBS and nonspecific binding blocked with TBS containing
1% BSA for 45 min at room temperature. The first antibody is added
at a pre-determined dilution in TBS/BSA for 1 hr at room
temperature. Three washes with TBS follow, and cells are briefly
reblocked for 15 min at room temperature. Incubation with goat
anti-mouse conjugated alkaline phosphatase (Bio-Rad) diluted 1:1000
in TBS/BSA is carried out for 1 hr at room temperature. The cells
are washed three times with TBS and a colorimetric alkaline
phosphatase substrate is added. When the adequate color change is
reached, 100 .mu.l samples are taken for colorimetric readings.
[0591] 12. Treatment with Antibodies
[0592] Treatment of Disease Cells with Monoclonal Antibodies.
[0593] Disease cells (e.g., cancer cells), or cells such as NIH 3T3
cells that express a marker of interest, are seeded at a density of
4.times.10.sup.4 cells per well in 96-well microtiter plates and
allowed to adhere for 2 hours. The cells are then treated with
different concentrations of monoclonal antibody (Mab) specific for
the marker protein of interest, or irrelevant isotype matched
(e.g., anti-rHuIFN-gamma) Mab, at 0.05, 0.5 or 5.0 .mu.g/ml. After
a 72 hour incubation, the cell monolayers are stained with crystal
violet dye for determination of relative percent viability (RPV)
compared to control (untreated) cells. Each treatment group can
have replicates. Cell growth inhibition is monitored.
[0594] In Vivo Treatment with Monoclonal Antibodies.
[0595] NIH 3T3 cells transfected with either an expression plasmid
that expresses the marker of interest or a neo-DHFR vector are
injected into nu/nu (athymic) mice subcutaneously at a dose of
10.sup.6 cells in 0.1 ml of phosphate-buffered saline. On days 0,
1, 5, and every 4 days thereafter, 100 .mu.g (0.1 ml in PBS) of a
Mab specific for the marker protein of interest, or an irrelevant
Mab, of the IgA2 subclass is injected intraperitoneally. Disease
progression (e.g., tumor occurrence and size) can be monitored for
a one month period of treatment, for example.
[0596] 13. Identification of LCM
[0597] A mass spectrometry (MS)-based proteomics platform was used
for the identification of secreted and shed proteins (secreted and
shed proteins are collectively referred to herein as soluble
proteins) and cell surface antigens that combines the discovery of
candidate biomarkers from human lung tumor specimens resected from
surgery and in a panel of lung cancer cell lines, followed by
validation of expression levels in patient serum (such as by using
ELISA). For example, proteomic analysis techniques such as
MALDI-TOF/TOF LC/MS-based protein expression analysis was used to
determine the expression levels of certain proteins in lung tumor
tissues and/or lung cancer cell lines (tissues and cell lines may
be collectively referred to herein as "samples") and in normal
tissues and/or normal cell lines, such that proteins that are
differentially expressed (e.g., over- or under-expressed) in lung
cancer samples compared with normal samples were identified.
[0598] Certain candidate markers were identified by mass
spectrometry-based methods that were differentially expressed on
the cell surface of lung tumors, lung cancer cell lines, or
secreted into the conditioned medium of cell lines. Certain of
these candidate markers that were identified as differentially
expressed by mass spectrometry, as well as certain other candidate
markers, were assayed by ELISA and scored in panels of lung cancer
patient sera and sera from individuals without lung cancer
(individuals without lung cancer are referred to herein as
"normal", "control", or "healthy" individuals). Individual markers
were scored "positive" for a given cancer sample if the value
exceeded a defined threshold (e.g., greater than or equal to two
standard deviations above the mean value for a group of "normal"
samples tested). From these candidate markers, lung cancer markers
("LCM") were identified that, particularly when used in
combination, distinguished lung cancer samples from healthy control
samples with various degrees of sensitivity and specificity.
[0599] Several methods and algorithms were applied to select
optimum panels/combinations of LCM including sum of the logs of the
ratios of the tumor concentration to the mean of the normal
concentration, defining a concentration cutoff manually for each
marker to optimize sensitivity and specificity, and use of Naive
Bayes to assign a probability that a sample is a tumor based on the
expression level of each marker. Additionally, ROC curves may be
constructed for each panel and their effectiveness may be evaluated
in several ways, such as maximizing the AUC of the ROC curve as
well as maximizing the sensitivity at a desired specificity or
maximizing the specificity at a desired sensitivity.
[0600] To further validate the specificity of certain panels,
co-morbidity studies were carried out to challenge certain panels
with other lung disease samples besides lung cancer, particularly
chronic obstructive pulmonary disease (COPD), asthma, bronchitis,
and other benign lung diseases (FIG. 20). Prevalence of COPD/asthma
is 10-25% in smokers. An initial panel of 30
bronchitis/asthma/benign lung disease samples was tested. Results
indicated that these co-morbidities may reduce specificity only
marginally if considered independent of false positives in 54
control samples (the specificity of the 9-member panel of Cyfra,
SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK in a 54
normal/53 lung tumor sample set was 98% on samples from smoking
controls).
[0601] 14. ELISA Immunoassays
[0602] Immunoassay kits, such as for performing ELISA assays, for
various LCM disclosed herein are commercially available. For
example, immunoassay kits can be obtained from a variety of
commercial sources, as follows: SLPI, MMP2, MIF, and OPN
immunoassay kits can be obtained from R&D Systems (Minneapolis,
Minn.); CYFRA 21-1 and SCC immunoassay kits can be obtained from
DRG-International (Mountainside, N.J.); DEFA1 immunoassay kits can
be obtained from Cell Sciences (Canton, Mass.); TIMP1 immunoassay
kits can be obtained from Siemens Healthcare Diagnostics
(Cambridge, Mass.); CEA and GRP immunoassay kits can be obtained
from IBL International (Toronto, Ontario); TFPI immunoassay kits
can be obtained from American Diagnostica (Stamford, Conn.); and
MDK immunoassay kits can be obtained from BioVendor (Candler, N.C.)
or R&D Systems (Minneapolis, Minn.). Assays can be performed
following manufacturers instructions. Plates can be read on a
Spectra Max M2 Microplate Reader (Molecular Devices, Sunnyvale,
Calif.) with the appropriate baseline correction for each
assay.
[0603] HNP1-3 (defensin, DEFA1) is employed as a representative
marker in the following exemplary ELISA protocol, which can be used
for the analysis of LCM. An HNP1-3 ELISA test kit can be used that
is a solid-phase enzyme-linked immunosorbent assay based on the
sandwich principle. Samples and standards are incubated in
microtiter wells coated with antibodies recognizing human HNP1-3.
During this incubation, human HNP1-3 is captured by solid bound
antibody. Unbound material present in the sample is removed by
washing. Biotinylated second antibody (tracer) to human HNP 1-3 is
then added to the wells. If HNP1-3 is present in the sample, the
tracer antibodies will bind to the captured HNP1-3. The excess
tracer is removed by washing. A streptavidin-peroxidase conjugate
is then applied to the wells, which reacts specifically with the
biotinylated tracer antibody bound onto the detected HNP1-3. The
excess streptavidin-peroxidase conjugate is removed by washing and
substrate tetramethylbenzidine (TMB) is added to the wells. Color
develops proportionally to the amount of human HNP1-3 present in
the sample. The enzyme reaction is stopped by the addition of
citric acid and the absorption at 450 nm is measured with a
spectrophotometer. A standard curve is obtained by plotting the
absorptions versus the corresponding concentrations of the known
standards. The concentration of human HNP1-3 in test samples, which
are run concurrently with the standards, can be determined from the
standard curve.
[0604] 15. Scoring of LCM Levels
[0605] This example describes an exemplary method of scoring LCM
levels using split-point analysis.
[0606] The term "split-point analysis" refers to a method adapted
from Mor et al., PNAS, (2005) 102, 7677-7682. In this exemplary
method, measurements for each marker are taken on all samples. A
cutoff value is determined for each marker. This cutoff value may
be set to, for example, maximize the accuracy of correct
classifications between the groups of interest (e.g., tumor and
control sample groups) or may be set to maximize the sensitivity or
specificity of one group. For each marker, a score is assigned to
that sample whenever the value of that marker is found to be on the
diseased side of the cutoff value (e.g., the side of the cutoff
corresponding to lung tumor samples). After all the measurements
have been taken on one sample, the scores are summed to produce a
total score for the panel of markers. All markers can be weighted
equally such that a panel of 9 markers may have a maximum score of
9 (each marker having a score of either 1 or 0) and a minimum score
of 0, for example. Alternatively, markers can be weighted
unequally, with a higher individual score for more significant
measures.
[0607] Other more sophisticated statistical modeling methods can
also be applied such as logistic regression (see, e.g., Planque et
al., Clin Cancer Res (2008), 14, 1355-1362) and decision tree
modeling (see, e.g., Patz et al., J Clin Oncol (2007), 25,
5578-5583).
[0608] An exemplary method of applying split-point analysis to an
LCM panel is described for illustrative purposes.
[0609] A patients sample can be tested to determine the patient's
likelihood of having lung cancer using a panel comprising the 9
biomarkers Cyfra, CEA, SLPI, OPN, MDK, TFPI, TIMP1, MMP2, and SCC
and the split and score method. The predetermined total score (or
threshold) for the panel can be set at 1 (or other value).
[0610] After obtaining a test sample from the patient, the amount
of each of the 9 biomarkers (Cyfra, CEA, SLPI, OPN, MDK, TFPI,
TIMP1, MMP2, SCC) in the patient's test sample is quantified. For
the purpose of this example, the amount of each of the 9 biomarkers
in the test sample is determined to be as follows (values are
expressed in ng/ml): Cyfra=0.891, CEA=4.087, SLPI=62.94,
OPN=21.514, MDK=0.174, TFPI=104.503, TIMP1=398.7, MMP2=194.41, and
SCC=1.35. The amount of each of these biomarkers is then compared
to the corresponding predetermined cutoff (or split point). For the
purpose of this example, the predetermined cutoffs for each of the
biomarkers are as follows: Cyfra=1.20, CEA=5.00, SLPI=52, OPN=32,
MDK=0.15, TFPI=150, TIMP1=385, MMP2=210, and SCC=2.2. For each
biomarker having an amount that is higher than its corresponding
predetermined cutoff (split point), a score of 1 can be assigned.
For each biomarker having an amount that is less than or equal to
its corresponding predetermined cutoff, a score of 0 can be
assigned. Thereupon, based on said comparison, each biomarker would
be assigned a score as follows: Cyfra=0, CEA=0, SLPI=1, OPN=0,
MDK=1, TFPI=0, TIMP1=1, MMP2=0, and SCC=0.
[0611] The score for each of the 9 biomarkers can then be combined
mathematically (e.g., by adding each of the scores of the
biomarkers together) to arrive at the total score for the patient.
In this particular example, the total score for the patient is 3
(the total score is calculated as follows: 0+0+1+0+1+0+1+0+0=3).
The total score for the patient is compared to the predetermined
total score, which is 1 in this particular example. A total score
greater than the predetermined total score of 1 would indicate a
positive result for the patient (i.e., in this particular example,
a total score of 2 or greater would indicate that the patient has
lung cancer). A total score equal to or less than 1 would indicate
a negative result for the patient. In this example, because the
patient's total score is greater than 1, the patient would be
considered to have a positive result (and thus may be referred for
further testing for an indication or suspicion of lung cancer). In
contrast, had the patient's total score been 1 or 0, the patient
would have been considered to have a negative result (and thus
would not be referred for any further testing).
[0612] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described methods and
compositions of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific exemplary embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
above-described modes for carrying out the invention, which are
obvious to those skilled in the field of molecular biology or
related fields, are intended to be within the scope of the
following claims.
TABLE-US-00001 Lengthy table referenced here
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TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150037820A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 1
1
651132PRTHomo sapiens 1Met Lys Ser Ser Gly Leu Phe Pro Phe Leu Val
Leu Leu Ala Leu Gly1 5 10 15 Thr Leu Ala Pro Trp Ala Val Glu Gly
Ser Gly Lys Ser Phe Lys Ala 20 25 30 Gly Val Cys Pro Pro Lys Lys
Ser Ala Gln Cys Leu Arg Tyr Lys Lys 35 40 45 Pro Glu Cys Gln Ser
Asp Trp Gln Cys Pro Gly Lys Lys Arg Cys Cys 50 55 60 Pro Asp Thr
Cys Gly Ile Lys Cys Leu Asp Pro Val Asp Thr Pro Asn65 70 75 80 Pro
Thr Arg Arg Lys Pro Gly Lys Cys Pro Val Thr Tyr Gly Gln Cys 85 90
95 Leu Met Leu Asn Pro Pro Asn Phe Cys Glu Met Asp Gly Gln Cys Lys
100 105 110 Arg Asp Leu Lys Cys Cys Met Gly Met Cys Gly Lys Ser Cys
Val Ser 115 120 125 Pro Val Lys Ala 130 2115PRTHomo sapiens 2Met
Pro Met Phe Ile Val Asn Thr Asn Val Pro Arg Ala Ser Val Pro1 5 10
15 Asp Gly Phe Leu Ser Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly
20 25 30 Lys Pro Pro Gln Tyr Ile Ala Val His Val Val Pro Asp Gln
Leu Met 35 40 45 Ala Phe Gly Gly Ser Ser Glu Pro Cys Ala Leu Cys
Ser Leu His Ser 50 55 60 Ile Gly Lys Ile Gly Gly Ala Gln Asn Arg
Ser Tyr Ser Lys Leu Leu65 70 75 80 Cys Gly Leu Leu Ala Glu Arg Leu
Arg Ile Ser Pro Asp Arg Val Tyr 85 90 95 Ile Asn Tyr Tyr Asp Met
Asn Ala Ala Asn Val Gly Trp Asn Asn Ser 100 105 110 Thr Phe Ala 115
3207PRTHomo sapiens 3Met Ala Pro Phe Glu Pro Leu Ala Ser Gly Ile
Leu Leu Leu Leu Trp1 5 10 15 Leu Ile Ala Pro Ser Arg Ala Cys Thr
Cys Val Pro Pro His Pro Gln 20 25 30 Thr Ala Phe Cys Asn Ser Asp
Leu Val Ile Arg Ala Lys Phe Val Gly 35 40 45 Thr Pro Glu Val Asn
Gln Thr Thr Leu Tyr Gln Arg Tyr Glu Ile Lys 50 55 60 Met Thr Lys
Met Tyr Lys Gly Phe Gln Ala Leu Gly Asp Ala Ala Asp65 70 75 80 Ile
Arg Phe Val Tyr Thr Pro Ala Met Glu Ser Val Cys Gly Tyr Phe 85 90
95 His Arg Ser His Asn Arg Ser Glu Glu Phe Leu Ile Ala Gly Lys Leu
100 105 110 Gln Asp Gly Leu Leu His Ile Thr Thr Cys Ser Phe Val Ala
Pro Trp 115 120 125 Asn Ser Leu Ser Leu Ala Gln Arg Arg Gly Phe Thr
Lys Thr Tyr Thr 130 135 140 Val Gly Cys Glu Glu Cys Thr Val Phe Pro
Cys Leu Ser Ile Pro Cys145 150 155 160 Lys Leu Gln Ser Gly Thr His
Cys Leu Trp Thr Asp Gln Leu Leu Gln 165 170 175 Gly Ser Glu Lys Gly
Phe Gln Ser Arg His Leu Ala Cys Leu Pro Arg 180 185 190 Glu Pro Gly
Leu Cys Thr Trp Gln Ser Leu Arg Ser Gln Ile Ala 195 200 205
4304PRTHomo sapiens 4Met Ile Tyr Thr Met Lys Lys Val His Ala Leu
Trp Ala Ser Val Cys1 5 10 15 Leu Leu Leu Asn Leu Ala Pro Ala Pro
Leu Asn Ala Asp Ser Glu Glu 20 25 30 Asp Glu Glu His Thr Ile Ile
Thr Asp Thr Glu Leu Pro Pro Leu Lys 35 40 45 Leu Met His Ser Phe
Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys 50 55 60 Ala Ile Met
Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu65 70 75 80 Glu
Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser 85 90
95 Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile
100 105 110 Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe
Leu Glu 115 120 125 Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg
Tyr Phe Tyr Asn 130 135 140 Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys
Tyr Gly Gly Cys Leu Gly145 150 155 160 Asn Met Asn Asn Phe Glu Thr
Leu Glu Glu Cys Lys Asn Ile Cys Glu 165 170 175 Asp Gly Pro Asn Gly
Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn 180 185 190 Ala Val Asn
Asn Ser Leu Thr Pro Gln Ser Thr Lys Val Pro Ser Leu 195 200 205 Phe
Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg Gly 210 215
220 Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val Ile
Gly225 230 235 240 Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly
Asn Glu Asn Asn 245 250 255 Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala
Cys Lys Lys Gly Phe Ile 260 265 270 Gln Arg Ile Ser Lys Gly Gly Leu
Ile Lys Thr Lys Arg Lys Arg Lys 275 280 285 Lys Gln Arg Val Lys Ile
Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 290 295 300 5434PRTHomo
sapiens 5Met Ser Ile Glu Lys Ile Trp Ala Arg Glu Ile Leu Asp Ser
Arg Gly1 5 10 15 Asn Pro Thr Val Glu Val Asp Leu Tyr Thr Ala Lys
Gly Leu Phe Arg 20 25 30 Ala Ala Val Pro Ser Gly Ala Ser Thr Gly
Ile Tyr Glu Ala Leu Glu 35 40 45 Leu Arg Asp Gly Asp Lys Gln Arg
Tyr Leu Gly Lys Gly Val Leu Lys 50 55 60 Ala Val Asp His Ile Asn
Ser Thr Ile Ala Pro Ala Leu Ile Ser Ser65 70 75 80 Gly Leu Ser Val
Val Glu Gln Glu Lys Leu Asp Asn Leu Met Leu Glu 85 90 95 Leu Asp
Gly Thr Glu Asn Lys Ser Lys Phe Gly Ala Asn Ala Ile Leu 100 105 110
Gly Val Ser Leu Ala Val Cys Lys Ala Gly Ala Ala Glu Arg Glu Leu 115
120 125 Pro Leu Tyr Arg His Ile Ala Gln Leu Ala Gly Asn Ser Asp Leu
Ile 130 135 140 Leu Pro Val Pro Ala Phe Asn Val Ile Asn Gly Gly Ser
His Ala Gly145 150 155 160 Asn Lys Leu Ala Met Gln Glu Phe Met Ile
Leu Pro Val Gly Ala Glu 165 170 175 Ser Phe Arg Asp Ala Met Arg Leu
Gly Ala Glu Val Tyr His Thr Leu 180 185 190 Lys Gly Val Ile Lys Asp
Lys Tyr Gly Lys Asp Ala Thr Asn Val Gly 195 200 205 Asp Glu Gly Gly
Phe Ala Pro Asn Ile Leu Glu Asn Ser Glu Ala Leu 210 215 220 Glu Leu
Val Lys Glu Ala Ile Asp Lys Ala Gly Tyr Thr Glu Lys Ile225 230 235
240 Val Ile Gly Met Asp Val Ala Ala Ser Glu Phe Tyr Arg Asp Gly Lys
245 250 255 Tyr Asp Leu Asp Phe Lys Ser Pro Thr Asp Pro Ser Arg Tyr
Ile Thr 260 265 270 Gly Asp Gln Leu Gly Ala Leu Tyr Gln Asp Phe Val
Arg Asp Tyr Pro 275 280 285 Val Val Ser Ile Glu Asp Pro Phe Asp Gln
Asp Asp Trp Ala Ala Trp 290 295 300 Ser Lys Phe Thr Ala Asn Val Gly
Ile Gln Ile Val Gly Asp Asp Leu305 310 315 320 Thr Val Thr Asn Pro
Lys Arg Ile Glu Arg Ala Val Glu Glu Lys Ala 325 330 335 Cys Asn Cys
Leu Leu Leu Lys Val Asn Gln Ile Gly Ser Val Thr Glu 340 345 350 Ala
Ile Gln Ala Cys Lys Leu Ala Gln Glu Asn Gly Trp Gly Val Met 355 360
365 Val Ser His Arg Ser Gly Glu Thr Glu Asp Thr Phe Ile Ala Asp Leu
370 375 380 Val Val Gly Leu Cys Thr Gly Gln Ile Lys Thr Gly Ala Pro
Cys Arg385 390 395 400 Ser Glu Arg Leu Ala Lys Tyr Asn Gln Leu Met
Arg Ile Glu Glu Glu 405 410 415 Leu Gly Asp Glu Ala Arg Phe Ala Gly
His Asn Phe Arg Asn Pro Ser 420 425 430 Val Leu6702PRTHomo sapiens
6Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln1 5
10 15 Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro
Thr 20 25 30 Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val
Ala Glu Gly 35 40 45 Lys Glu Val Leu Leu Leu Val His Asn Leu Pro
Gln His Leu Phe Gly 50 55 60 Tyr Ser Trp Tyr Lys Gly Glu Arg Val
Asp Gly Asn Arg Gln Ile Ile65 70 75 80 Gly Tyr Val Ile Gly Thr Gln
Gln Ala Thr Pro Gly Pro Ala Tyr Ser 85 90 95 Gly Arg Glu Ile Ile
Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile 100 105 110 Ile Gln Asn
Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp 115 120 125 Leu
Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu 130 135
140 Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp
Lys145 150 155 160 Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln
Asp Ala Thr Tyr 165 170 175 Leu Trp Trp Val Asn Asn Gln Ser Leu Pro
Val Ser Pro Arg Leu Gln 180 185 190 Leu Ser Asn Gly Asn Arg Thr Leu
Thr Leu Phe Asn Val Thr Arg Asn 195 200 205 Asp Thr Ala Ser Tyr Lys
Cys Glu Thr Gln Asn Pro Val Ser Ala Arg 210 215 220 Arg Ser Asp Ser
Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro225 230 235 240 Thr
Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn 245 250
255 Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe
260 265 270 Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile
Pro Asn 275 280 285 Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln
Ala His Asn Ser 290 295 300 Asp Thr Gly Leu Asn Arg Thr Thr Val Thr
Thr Ile Thr Val Tyr Ala305 310 315 320 Glu Pro Pro Lys Pro Phe Ile
Thr Ser Asn Asn Ser Asn Pro Val Glu 325 330 335 Asp Glu Asp Ala Val
Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr 340 345 350 Thr Tyr Leu
Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg 355 360 365 Leu
Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr 370 375
380 Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu
Ser385 390 395 400 Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu
Tyr Gly Pro Asp 405 410 415 Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr
Tyr Arg Pro Gly Val Asn 420 425 430 Leu Ser Leu Ser Cys His Ala Ala
Ser Asn Pro Pro Ala Gln Tyr Ser 435 440 445 Trp Leu Ile Asp Gly Asn
Ile Gln Gln His Thr Gln Glu Leu Phe Ile 450 455 460 Ser Asn Ile Thr
Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn465 470 475 480 Asn
Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val 485 490
495 Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro
500 505 510 Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu
Ala Gln 515 520 525 Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser
Leu Pro Val Ser 530 535 540 Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg
Thr Leu Thr Leu Phe Asn545 550 555 560 Val Thr Arg Asn Asp Ala Arg
Ala Tyr Val Cys Gly Ile Gln Asn Ser 565 570 575 Val Ser Ala Asn Arg
Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly 580 585 590 Pro Asp Thr
Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly 595 600 605 Ala
Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln 610 615
620 Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val
Leu625 630 635 640 Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr
Tyr Ala Cys Phe 645 650 655 Val Ser Asn Leu Ala Thr Gly Arg Asn Asn
Ser Ile Val Lys Ser Ile 660 665 670 Thr Val Ser Ala Ser Gly Thr Ser
Pro Gly Leu Ser Ala Gly Ala Thr 675 680 685 Val Gly Ile Met Ile Gly
Val Leu Val Gly Val Ala Leu Ile 690 695 700 7660PRTHomo sapiens
7Met Glu Ala Leu Met Ala Arg Gly Ala Leu Thr Gly Pro Leu Arg Ala1 5
10 15 Leu Cys Leu Leu Gly Cys Leu Leu Ser His Ala Ala Ala Ala Pro
Ser 20 25 30 Pro Ile Ile Lys Phe Pro Gly Asp Val Ala Pro Lys Thr
Asp Lys Glu 35 40 45 Leu Ala Val Gln Tyr Leu Asn Thr Phe Tyr Gly
Cys Pro Lys Glu Ser 50 55 60 Cys Asn Leu Phe Val Leu Lys Asp Thr
Leu Lys Lys Met Gln Lys Phe65 70 75 80 Phe Gly Leu Pro Gln Thr Gly
Asp Leu Asp Gln Asn Thr Ile Glu Thr 85 90 95 Met Arg Lys Pro Arg
Cys Gly Asn Pro Asp Val Ala Asn Tyr Asn Phe 100 105 110 Phe Pro Arg
Lys Pro Lys Trp Asp Lys Asn Gln Ile Thr Tyr Arg Ile 115 120 125 Ile
Gly Tyr Thr Pro Asp Leu Asp Pro Glu Thr Val Asp Asp Ala Phe 130 135
140 Ala Arg Ala Phe Gln Val Trp Ser Asp Val Thr Pro Leu Arg Phe
Ser145 150 155 160 Arg Ile His Asp Gly Glu Ala Asp Ile Met Ile Asn
Phe Gly Arg Trp 165 170 175 Glu His Gly Asp Gly Tyr Pro Phe Asp Gly
Lys Asp Gly Leu Leu Ala 180 185 190 His Ala Phe Ala Pro Gly Thr Gly
Val Gly Gly Asp Ser His Phe Asp 195 200 205 Asp Asp Glu Leu Trp Thr
Leu Gly Glu Gly Gln Val Val Arg Val Lys 210 215 220 Tyr Gly Asn Ala
Asp Gly Glu Tyr Cys Lys Phe Pro Phe Leu Phe Asn225 230 235 240 Gly
Lys Glu Tyr Asn Ser Cys Thr Asp Thr Gly Arg Ser Asp Gly Phe 245 250
255 Leu Trp Cys Ser Thr Thr Tyr Asn Phe Glu Lys Asp Gly Lys Tyr Gly
260 265 270 Phe Cys Pro His Glu Ala Leu Phe Thr Met Gly Gly Asn Ala
Glu Gly 275 280 285 Gln Pro Cys Lys Phe Pro Phe Arg Phe Gln Gly Thr
Ser Tyr Asp Ser 290 295 300 Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr
Arg Trp Cys Gly Thr Thr305 310 315 320 Glu Asp Tyr Asp Arg Asp Lys
Lys Tyr Gly Phe Cys Pro Glu Thr Ala 325 330 335 Met Ser Thr Val Gly
Gly Asn Ser Glu Gly Ala Pro Cys Val Phe Pro 340 345 350 Phe Thr Phe
Leu Gly Asn Lys Tyr Glu Ser Cys Thr Ser Ala Gly Arg 355 360 365 Ser
Asp Gly Lys Met Trp Cys Ala Thr Thr Ala Asn Tyr Asp Asp Asp 370 375
380 Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu
Val385 390 395 400 Ala Ala His Glu Phe
Gly His Ala Met Gly Leu Glu His Ser Gln Asp 405 410 415 Pro Gly Ala
Leu Met Ala Pro Ile Tyr Thr Tyr Thr Lys Asn Phe Arg 420 425 430 Leu
Ser Gln Asp Asp Ile Lys Gly Ile Gln Glu Leu Tyr Gly Ala Ser 435 440
445 Pro Asp Ile Asp Leu Gly Thr Gly Pro Thr Pro Thr Leu Gly Pro Val
450 455 460 Thr Pro Glu Ile Cys Lys Gln Asp Ile Val Phe Asp Gly Ile
Ala Gln465 470 475 480 Ile Arg Gly Glu Ile Phe Phe Phe Lys Asp Arg
Phe Ile Trp Arg Thr 485 490 495 Val Thr Pro Arg Asp Lys Pro Met Gly
Pro Leu Leu Val Ala Thr Phe 500 505 510 Trp Pro Glu Leu Pro Glu Lys
Ile Asp Ala Val Tyr Glu Ala Pro Gln 515 520 525 Glu Glu Lys Ala Val
Phe Phe Ala Gly Asn Glu Tyr Trp Ile Tyr Ser 530 535 540 Ala Ser Thr
Leu Glu Arg Gly Tyr Pro Lys Pro Leu Thr Ser Leu Gly545 550 555 560
Leu Pro Pro Asp Val Gln Arg Val Asp Ala Ala Phe Asn Trp Ser Lys 565
570 575 Asn Lys Lys Thr Tyr Ile Phe Ala Gly Asp Lys Phe Trp Arg Tyr
Asn 580 585 590 Glu Val Lys Lys Lys Met Asp Pro Gly Phe Pro Lys Leu
Ile Ala Asp 595 600 605 Ala Trp Asn Ala Ile Pro Asp Asn Leu Asp Ala
Val Val Asp Leu Gln 610 615 620 Gly Gly Gly His Ser Tyr Phe Phe Lys
Gly Ala Tyr Tyr Leu Lys Leu625 630 635 640 Glu Asn Gln Ser Leu Lys
Ser Val Lys Phe Gly Ser Ile Lys Ser Asp 645 650 655 Trp Leu Gly Cys
660 8352PRTHomo sapiens 8Met Arg Ser Leu Gly Ala Leu Leu Leu Leu
Leu Ser Ala Cys Leu Ala1 5 10 15 Val Ser Ala Gly Pro Val Pro Thr
Pro Pro Asp Asn Ile Gln Val Gln 20 25 30 Glu Asn Phe Asn Ile Ser
Arg Ile Tyr Gly Lys Trp Tyr Asn Leu Ala 35 40 45 Ile Gly Ser Thr
Cys Pro Trp Leu Lys Lys Ile Met Asp Arg Met Thr 50 55 60 Val Ser
Thr Leu Val Leu Gly Glu Gly Ala Thr Glu Ala Glu Ile Ser65 70 75 80
Met Thr Ser Thr Arg Trp Arg Lys Gly Val Cys Glu Glu Thr Ser Gly 85
90 95 Ala Tyr Glu Lys Thr Asp Thr Asp Gly Lys Phe Leu Tyr His Lys
Ser 100 105 110 Lys Trp Asn Ile Thr Met Glu Ser Tyr Val Val His Thr
Asn Tyr Asp 115 120 125 Glu Tyr Ala Ile Phe Leu Thr Lys Lys Phe Ser
Arg His His Gly Pro 130 135 140 Thr Ile Thr Ala Lys Leu Tyr Gly Arg
Ala Pro Gln Leu Arg Glu Thr145 150 155 160 Leu Leu Gln Asp Phe Arg
Val Val Ala Gln Gly Val Gly Ile Pro Glu 165 170 175 Asp Ser Ile Phe
Thr Met Ala Asp Arg Gly Glu Cys Val Pro Gly Glu 180 185 190 Gln Glu
Pro Glu Pro Ile Leu Ile Pro Arg Val Arg Arg Ala Val Leu 195 200 205
Pro Gln Glu Glu Glu Gly Ser Gly Gly Gly Gln Leu Val Thr Glu Val 210
215 220 Thr Lys Lys Glu Asp Ser Cys Gln Leu Gly Tyr Ser Ala Gly Pro
Cys225 230 235 240 Met Gly Met Thr Ser Arg Tyr Phe Tyr Asn Gly Thr
Ser Met Ala Cys 245 250 255 Glu Thr Phe Gln Tyr Gly Gly Cys Met Gly
Asn Gly Asn Asn Phe Val 260 265 270 Thr Glu Lys Glu Cys Leu Gln Thr
Cys Arg Thr Val Ala Ala Cys Asn 275 280 285 Leu Pro Ile Val Arg Gly
Pro Cys Arg Ala Phe Ile Gln Leu Trp Ala 290 295 300 Phe Asp Ala Val
Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys305 310 315 320 Gln
Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu Cys Arg Glu Tyr 325 330
335 Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg Phe Ser Asn
340 345 350 9400PRTHomo sapiens 9Met Thr Ser Tyr Ser Tyr Arg Gln
Ser Ser Ala Thr Ser Ser Phe Gly1 5 10 15 Gly Leu Gly Gly Gly Ser
Val Arg Phe Gly Pro Gly Val Ala Phe Arg 20 25 30 Ala Pro Ser Ile
His Gly Gly Ser Gly Gly Arg Gly Val Ser Val Ser 35 40 45 Ser Ala
Arg Phe Val Ser Ser Ser Ser Ser Gly Gly Tyr Gly Gly Gly 50 55 60
Tyr Gly Gly Val Leu Thr Ala Ser Asp Gly Leu Leu Ala Gly Asn Glu65
70 75 80 Lys Leu Thr Met Gln Asn Leu Asn Asp Arg Leu Ala Ser Tyr
Leu Asp 85 90 95 Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu
Glu Val Lys Ile 100 105 110 Arg Asp Trp Tyr Gln Lys Gln Gly Pro Gly
Pro Ser Arg Asp Tyr Ser 115 120 125 His Tyr Tyr Thr Thr Ile Gln Asp
Leu Arg Asp Lys Ile Leu Gly Ala 130 135 140 Thr Ile Glu Asn Ser Arg
Ile Val Leu Gln Ile Asp Asn Ala Arg Leu145 150 155 160 Ala Ala Asp
Asp Phe Arg Thr Lys Phe Glu Thr Glu Gln Ala Leu Arg 165 170 175 Met
Ser Val Glu Ala Asp Ile Asn Gly Leu Arg Arg Val Leu Asp Glu 180 185
190 Leu Thr Leu Ala Arg Thr Asp Leu Glu Met Gln Ile Glu Gly Leu Lys
195 200 205 Glu Glu Leu Ala Tyr Leu Lys Lys Asn His Glu Glu Glu Ile
Ser Thr 210 215 220 Leu Arg Gly Gln Val Gly Gly Gln Val Ser Val Glu
Val Asp Ser Ala225 230 235 240 Pro Gly Thr Asp Leu Ala Lys Ile Leu
Ser Asp Met Arg Ser Gln Tyr 245 250 255 Glu Val Met Ala Glu Gln Asn
Arg Lys Asp Ala Glu Ala Trp Phe Thr 260 265 270 Ser Arg Thr Glu Glu
Leu Asn Arg Glu Val Ala Gly His Thr Glu Gln 275 280 285 Leu Gln Met
Ser Arg Ser Glu Val Thr Asp Leu Arg Arg Thr Leu Gln 290 295 300 Gly
Leu Glu Ile Glu Leu Gln Ser Gln Leu Ser Met Lys Ala Ala Leu305 310
315 320 Glu Asp Thr Leu Ala Glu Thr Glu Ala Arg Phe Gly Ala Gln Leu
Ala 325 330 335 His Ile Gln Ala Leu Ile Ser Gly Ile Glu Ala Gln Leu
Gly Asp Val 340 345 350 Arg Ala Asp Ser Glu Arg Gln Asn Gln Glu Tyr
Gln Arg Leu Met Asp 355 360 365 Ile Lys Ser Arg Leu Glu Gln Glu Ile
Ala Thr Tyr Arg Ser Leu Leu 370 375 380 Glu Gly Gln Glu Asp His Tyr
Asn Asn Leu Ser Ala Ser Lys Val Leu385 390 395 400 10390PRTHomo
sapiens 10Met Asn Ser Leu Ser Glu Ala Asn Thr Lys Phe Met Phe Asp
Leu Phe1 5 10 15 Gln Gln Phe Arg Lys Ser Lys Glu Asn Asn Ile Phe
Tyr Ser Pro Ile 20 25 30 Ser Ile Thr Ser Ala Leu Gly Met Val Leu
Leu Gly Ala Lys Asp Asn 35 40 45 Thr Ala Gln Gln Ile Lys Lys Val
Leu His Phe Asp Gln Val Thr Glu 50 55 60 Asn Thr Thr Gly Lys Ala
Ala Thr Tyr His Val Asp Arg Ser Gly Asn65 70 75 80 Val His His Gln
Phe Gln Lys Leu Leu Thr Glu Phe Asn Lys Ser Thr 85 90 95 Asp Ala
Tyr Glu Leu Lys Ile Ala Asn Lys Leu Phe Gly Glu Lys Thr 100 105 110
Tyr Leu Phe Leu Gln Glu Tyr Leu Asp Ala Ile Lys Lys Phe Tyr Gln 115
120 125 Thr Ser Val Glu Ser Val Asp Phe Ala Asn Ala Pro Glu Glu Ser
Arg 130 135 140 Lys Lys Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Glu
Lys Ile Lys145 150 155 160 Asn Leu Ile Pro Glu Gly Asn Ile Gly Ser
Asn Thr Thr Leu Val Leu 165 170 175 Val Asn Ala Ile Tyr Phe Lys Gly
Gln Trp Glu Lys Lys Phe Asn Lys 180 185 190 Glu Asp Thr Lys Glu Glu
Lys Phe Trp Pro Asn Lys Asn Thr Tyr Lys 195 200 205 Ser Ile Gln Met
Met Arg Gln Tyr Thr Ser Phe His Phe Ala Ser Leu 210 215 220 Glu Asp
Val Gln Ala Lys Val Leu Glu Ile Pro Tyr Lys Gly Lys Asp225 230 235
240 Leu Ser Met Ile Val Leu Leu Pro Asn Glu Ile Asp Gly Leu Gln Lys
245 250 255 Leu Glu Glu Lys Leu Thr Ala Glu Lys Leu Met Glu Trp Thr
Ser Leu 260 265 270 Gln Asn Met Arg Glu Thr Arg Val Asp Leu His Leu
Pro Arg Phe Lys 275 280 285 Val Glu Glu Ser Tyr Asp Leu Lys Asp Thr
Leu Arg Thr Met Gly Met 290 295 300 Val Asp Ile Phe Asn Gly Asp Ala
Asp Leu Ser Gly Met Thr Gly Ser305 310 315 320 Arg Gly Leu Val Leu
Ser Gly Val Leu His Lys Ala Phe Val Glu Val 325 330 335 Thr Glu Glu
Gly Ala Glu Ala Ala Ala Ala Thr Ala Val Val Gly Phe 340 345 350 Gly
Ser Ser Pro Thr Ser Thr Asn Glu Glu Phe His Cys Asn His Pro 355 360
365 Phe Leu Phe Phe Ile Arg Gln Asn Lys Thr Asn Ser Ile Leu Phe Tyr
370 375 380 Gly Arg Phe Ser Ser Pro385 390 11314PRTHomo sapiens
11Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala1
5 10 15 Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln
Leu 20 25 30 Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn
Pro Asp Pro 35 40 45 Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn
Ala Val Ser Ser Glu 50 55 60 Glu Thr Asn Asp Phe Lys Gln Glu Thr
Leu Pro Ser Lys Ser Asn Glu65 70 75 80 Ser His Asp His Met Asp Asp
Met Asp Asp Glu Asp Asp Asp Asp His 85 90 95 Val Asp Ser Gln Asp
Ser Ile Asp Ser Asn Asp Ser Asp Asp Val Asp 100 105 110 Asp Thr Asp
Asp Ser His Gln Ser Asp Glu Ser His His Ser Asp Glu 115 120 125 Ser
Asp Glu Leu Val Thr Asp Phe Pro Thr Asp Leu Pro Ala Thr Glu 130 135
140 Val Phe Thr Pro Val Val Pro Thr Val Asp Thr Tyr Asp Gly Arg
Gly145 150 155 160 Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser Lys
Lys Phe Arg Arg 165 170 175 Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp
Glu Asp Ile Thr Ser His 180 185 190 Met Glu Ser Glu Glu Leu Asn Gly
Ala Tyr Lys Ala Ile Pro Val Ala 195 200 205 Gln Asp Leu Asn Ala Pro
Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser 210 215 220 Tyr Glu Thr Ser
Gln Leu Asp Asp Gln Ser Ala Glu Thr His Ser His225 230 235 240 Lys
Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn Glu 245 250
255 His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser Arg Glu
260 265 270 Phe His Ser His Glu Phe His Ser His Glu Asp Met Leu Val
Val Asp 275 280 285 Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe
Arg Ile Ser His 290 295 300 Glu Leu Asp Ser Ala Ser Ser Glu Val
Asn305 310 1294PRTHomo sapiens 12Met Arg Thr Leu Ala Ile Leu Ala
Ala Ile Leu Leu Val Ala Leu Gln1 5 10 15 Ala Gln Ala Glu Pro Leu
Gln Ala Arg Ala Asp Glu Val Ala Ala Ala 20 25 30 Pro Glu Gln Ile
Ala Ala Asp Ile Pro Glu Val Val Val Ser Leu Ala 35 40 45 Trp Asp
Glu Ser Leu Ala Pro Lys His Pro Gly Ser Arg Lys Asn Met 50 55 60
Ala Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr65
70 75 80 Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 85
90 13459PRTHomo sapiens 13Met Ala Pro Leu Cys Pro Ser Pro Trp Leu
Pro Leu Leu Ile Pro Ala1 5 10 15 Pro Ala Pro Gly Leu Thr Val Gln
Leu Leu Leu Ser Leu Leu Leu Leu 20 25 30 Val Pro Val His Pro Gln
Arg Leu Pro Arg Met Gln Glu Asp Ser Pro 35 40 45 Leu Gly Gly Gly
Ser Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp 50 55 60 Leu Pro
Ser Glu Glu Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu65 70 75 80
Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro 85
90 95 Glu Val Lys Pro Lys Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu
Asp 100 105 110 Leu Pro Thr Val Glu Ala Pro Gly Asp Pro Gln Glu Pro
Gln Asn Asn 115 120 125 Ala His Arg Asp Lys Glu Gly Asp Asp Gln Ser
His Trp Arg Tyr Gly 130 135 140 Gly Asp Pro Pro Trp Pro Arg Val Ser
Pro Ala Cys Ala Gly Arg Phe145 150 155 160 Gln Ser Pro Val Asp Ile
Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala 165 170 175 Leu Arg Pro Leu
Glu Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu 180 185 190 Leu Arg
Leu Arg Asn Asn Gly His Ser Val Gln Leu Thr Leu Pro Pro 195 200 205
Gly Leu Glu Met Ala Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln 210
215 220 Leu His Leu His Trp Gly Ala Ala Gly Arg Pro Gly Ser Glu His
Thr225 230 235 240 Val Glu Gly His Arg Phe Pro Ala Glu Ile His Val
Val His Leu Ser 245 250 255 Thr Ala Phe Ala Arg Val Asp Glu Ala Leu
Gly Arg Pro Gly Gly Leu 260 265 270 Ala Val Leu Ala Ala Phe Leu Glu
Glu Gly Pro Glu Glu Asn Ser Ala 275 280 285 Tyr Glu Gln Leu Leu Ser
Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser 290 295 300 Glu Thr Gln Val
Pro Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp305 310 315 320 Phe
Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys 325 330
335 Ala Gln Gly Val Ile Trp Thr Val Phe Asn Gln Thr Val Met Leu Ser
340 345 350 Ala Lys Gln Leu His Thr Leu Ser Asp Thr Leu Trp Gly Pro
Gly Asp 355 360 365 Ser Arg Leu Gln Leu Asn Phe Arg Ala Thr Gln Pro
Leu Asn Gly Arg 370 375 380 Val Ile Glu Ala Ser Phe Pro Ala Gly Val
Asp Ser Ser Pro Arg Ala385 390 395 400 Ala Glu Pro Val Gln Leu Asn
Ser Cys Leu Ala Ala Gly Asp Ile Leu 405 410 415 Ala Leu Val Phe Gly
Leu Leu Phe Ala Val Thr Ser Val Ala Phe Leu 420 425 430 Val Gln Met
Arg Arg Gln His Arg Arg Gly Thr Lys Gly Gly Val Ser 435 440 445 Tyr
Arg Pro Ala Glu Val Ala Glu Thr Gly Ala 450 455 14148PRTHomo
sapiens 14Met Arg Gly Ser Glu Leu Pro Leu Val Leu Leu Ala Leu Val
Leu Cys1 5 10 15 Leu Ala Pro Arg Gly Arg Ala Val Pro Leu Pro Ala
Gly Gly Gly Thr 20
25 30 Val Leu Thr Lys Met Tyr Pro Arg Gly Asn His Trp Ala Val Gly
His 35 40 45 Leu Met Gly Lys Lys Ser Thr Gly Glu Ser Ser Ser Val
Ser Glu Arg 50 55 60 Gly Ser Leu Lys Gln Gln Leu Arg Glu Tyr Ile
Arg Trp Glu Glu Ala65 70 75 80 Ala Arg Asn Leu Leu Gly Leu Ile Glu
Ala Lys Glu Asn Arg Asn His 85 90 95 Gln Pro Pro Gln Pro Lys Ala
Leu Gly Asn Gln Gln Pro Ser Trp Asp 100 105 110 Ser Glu Asp Ser Ser
Asn Phe Lys Asp Val Gly Ser Lys Gly Lys Val 115 120 125 Gly Arg Leu
Ser Ala Pro Gly Ser Gln Arg Glu Gly Arg Asn Pro Gln 130 135 140 Leu
Asn Gln Gln145 15430PRTHomo sapiens 15Met Ser Phe Thr Thr Arg Ser
Thr Phe Ser Thr Asn Tyr Arg Ser Leu1 5 10 15 Gly Ser Val Gln Ala
Pro Ser Tyr Gly Ala Arg Pro Val Ser Ser Ala 20 25 30 Ala Ser Val
Tyr Ala Gly Ala Gly Gly Ser Gly Ser Arg Ile Ser Val 35 40 45 Ser
Arg Ser Thr Ser Phe Arg Gly Gly Met Gly Ser Gly Gly Leu Ala 50 55
60 Thr Gly Ile Ala Gly Gly Leu Ala Gly Met Gly Gly Ile Gln Asn
Glu65 70 75 80 Lys Glu Thr Met Gln Ser Leu Asn Asp Arg Leu Ala Ser
Tyr Leu Asp 85 90 95 Arg Val Arg Ser Leu Glu Thr Glu Asn Arg Arg
Leu Glu Ser Lys Ile 100 105 110 Arg Glu His Leu Glu Lys Lys Gly Pro
Gln Val Arg Asp Trp Ser His 115 120 125 Tyr Phe Lys Ile Ile Glu Asp
Leu Arg Ala Gln Ile Phe Ala Asn Thr 130 135 140 Val Asp Asn Ala Arg
Ile Val Leu Gln Ile Asp Asn Ala Arg Leu Ala145 150 155 160 Ala Asp
Asp Phe Arg Val Lys Tyr Glu Thr Glu Leu Ala Met Arg Gln 165 170 175
Ser Val Glu Asn Asp Ile His Gly Leu Arg Lys Val Ile Asp Asp Thr 180
185 190 Asn Ile Thr Arg Leu Gln Leu Glu Thr Glu Ile Glu Ala Leu Lys
Glu 195 200 205 Glu Leu Leu Phe Met Lys Lys Asn His Glu Glu Glu Val
Lys Gly Leu 210 215 220 Gln Ala Gln Ile Ala Ser Ser Gly Leu Thr Val
Glu Val Asp Ala Pro225 230 235 240 Lys Ser Gln Asp Leu Ala Lys Ile
Met Ala Asp Ile Arg Ala Gln Tyr 245 250 255 Asp Glu Leu Ala Arg Lys
Asn Arg Glu Glu Leu Asp Lys Tyr Trp Ser 260 265 270 Gln Gln Ile Glu
Glu Ser Thr Thr Val Val Thr Thr Gln Ser Ala Glu 275 280 285 Val Gly
Ala Ala Glu Thr Thr Leu Thr Glu Leu Arg Arg Thr Val Gln 290 295 300
Ser Leu Glu Ile Asp Leu Asp Ser Met Arg Asn Leu Lys Ala Ser Leu305
310 315 320 Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu Gln
Met Glu 325 330 335 Gln Leu Asn Gly Ile Leu Leu His Leu Glu Ser Glu
Leu Ala Gln Thr 340 345 350 Arg Ala Glu Gly Gln Arg Gln Ala Gln Glu
Tyr Glu Ala Leu Leu Asn 355 360 365 Ile Lys Val Lys Leu Glu Ala Glu
Ile Ala Thr Tyr Arg Arg Leu Leu 370 375 380 Glu Asp Gly Glu Asp Phe
Asn Leu Gly Asp Ala Leu Asp Ser Ser Asn385 390 395 400 Ser Met Gln
Thr Ile Gln Lys Thr Thr Thr Arg Arg Ile Val Asp Gly 405 410 415 Lys
Val Val Ser Glu Thr Asn Asp Thr Lys Val Leu Arg His 420 425 430
16882PRTHomo sapiens 16Met Gly Pro Trp Ser Arg Ser Leu Ser Ala Leu
Leu Leu Leu Leu Gln1 5 10 15 Val Ser Ser Trp Leu Cys Gln Glu Pro
Glu Pro Cys His Pro Gly Phe 20 25 30 Asp Ala Glu Ser Tyr Thr Phe
Thr Val Pro Arg Arg His Leu Glu Arg 35 40 45 Gly Arg Val Leu Gly
Arg Val Asn Phe Glu Asp Cys Thr Gly Arg Gln 50 55 60 Arg Thr Ala
Tyr Phe Ser Leu Asp Thr Arg Phe Lys Val Gly Thr Asp65 70 75 80 Gly
Val Ile Thr Val Lys Arg Pro Leu Arg Phe His Asn Pro Gln Ile 85 90
95 His Phe Leu Val Tyr Ala Trp Asp Ser Thr Tyr Arg Lys Phe Ser Thr
100 105 110 Lys Val Thr Leu Asn Thr Val Gly His His His Arg Pro Pro
Pro His 115 120 125 Gln Ala Ser Val Ser Gly Ile Gln Ala Glu Leu Leu
Thr Phe Pro Asn 130 135 140 Ser Ser Pro Gly Leu Arg Arg Gln Lys Arg
Asp Trp Val Ile Pro Pro145 150 155 160 Ile Ser Cys Pro Glu Asn Glu
Lys Gly Pro Phe Pro Lys Asn Leu Val 165 170 175 Gln Ile Lys Ser Asn
Lys Asp Lys Glu Gly Lys Val Phe Tyr Ser Ile 180 185 190 Thr Gly Gln
Gly Ala Asp Thr Pro Pro Val Gly Val Phe Ile Ile Glu 195 200 205 Arg
Glu Thr Gly Trp Leu Lys Val Thr Glu Pro Leu Asp Arg Glu Arg 210 215
220 Ile Ala Thr Tyr Thr Leu Phe Ser His Ala Val Ser Ser Asn Gly
Asn225 230 235 240 Ala Val Glu Asp Pro Met Glu Ile Leu Ile Thr Val
Thr Asp Gln Asn 245 250 255 Asp Asn Lys Pro Glu Phe Thr Gln Glu Val
Phe Lys Gly Ser Val Met 260 265 270 Glu Gly Ala Leu Pro Gly Thr Ser
Val Met Glu Val Thr Ala Thr Asp 275 280 285 Ala Asp Asp Asp Val Asn
Thr Tyr Asn Ala Ala Ile Ala Tyr Thr Ile 290 295 300 Leu Ser Gln Asp
Pro Glu Leu Pro Asp Lys Asn Met Phe Thr Ile Asn305 310 315 320 Arg
Asn Thr Gly Val Ile Ser Val Val Thr Thr Gly Leu Asp Arg Glu 325 330
335 Ser Phe Pro Thr Tyr Thr Leu Val Val Gln Ala Ala Asp Leu Gln Gly
340 345 350 Glu Gly Leu Ser Thr Thr Ala Thr Ala Val Ile Thr Val Thr
Asp Thr 355 360 365 Asn Asp Asn Pro Pro Ile Phe Asn Pro Thr Thr Tyr
Lys Gly Gln Val 370 375 380 Pro Glu Asn Glu Ala Asn Val Val Ile Thr
Thr Leu Lys Val Thr Asp385 390 395 400 Ala Asp Ala Pro Asn Thr Pro
Ala Trp Glu Ala Val Tyr Thr Ile Leu 405 410 415 Asn Asp Asp Gly Gly
Gln Phe Val Val Thr Thr Asn Pro Val Asn Asn 420 425 430 Asp Gly Ile
Leu Lys Thr Ala Lys Gly Leu Asp Phe Glu Ala Lys Gln 435 440 445 Gln
Tyr Ile Leu His Val Ala Val Thr Asn Val Val Pro Phe Glu Val 450 455
460 Ser Leu Thr Thr Ser Thr Ala Thr Val Thr Val Asp Val Leu Asp
Val465 470 475 480 Asn Glu Ala Pro Ile Phe Val Pro Pro Glu Lys Arg
Val Glu Val Ser 485 490 495 Glu Asp Phe Gly Val Gly Gln Glu Ile Thr
Ser Tyr Thr Ala Gln Glu 500 505 510 Pro Asp Thr Phe Met Glu Gln Lys
Ile Thr Tyr Arg Ile Trp Arg Asp 515 520 525 Thr Ala Asn Trp Leu Glu
Ile Asn Pro Asp Thr Gly Ala Ile Ser Thr 530 535 540 Arg Ala Glu Leu
Asp Arg Glu Asp Phe Glu His Val Lys Asn Ser Thr545 550 555 560 Tyr
Thr Ala Leu Ile Ile Ala Thr Asp Asn Gly Ser Pro Val Ala Thr 565 570
575 Gly Thr Gly Thr Leu Leu Leu Ile Leu Ser Asp Val Asn Asp Asn Ala
580 585 590 Pro Ile Pro Glu Pro Arg Thr Ile Phe Phe Cys Glu Arg Asn
Pro Lys 595 600 605 Pro Gln Val Ile Asn Ile Ile Asp Ala Asp Leu Pro
Pro Asn Thr Ser 610 615 620 Pro Phe Thr Ala Glu Leu Thr His Gly Ala
Ser Ala Asn Trp Thr Ile625 630 635 640 Gln Tyr Asn Asp Pro Thr Gln
Glu Ser Ile Ile Leu Lys Pro Lys Met 645 650 655 Ala Leu Glu Val Gly
Asp Tyr Lys Ile Asn Leu Lys Leu Met Asp Asn 660 665 670 Gln Asn Lys
Asp Gln Val Thr Thr Leu Glu Val Ser Val Cys Asp Cys 675 680 685 Glu
Gly Ala Ala Gly Val Cys Arg Lys Ala Gln Pro Val Glu Ala Gly 690 695
700 Leu Gln Ile Pro Ala Ile Leu Gly Ile Leu Gly Gly Ile Leu Ala
Leu705 710 715 720 Leu Ile Leu Ile Leu Leu Leu Leu Leu Phe Leu Arg
Arg Arg Ala Val 725 730 735 Val Lys Glu Pro Leu Leu Pro Pro Glu Asp
Asp Thr Arg Asp Asn Val 740 745 750 Tyr Tyr Tyr Asp Glu Glu Gly Gly
Gly Glu Glu Asp Gln Asp Phe Asp 755 760 765 Leu Ser Gln Leu His Arg
Gly Leu Asp Ala Arg Pro Glu Val Thr Arg 770 775 780 Asn Asp Val Ala
Pro Thr Leu Met Ser Val Pro Arg Tyr Leu Pro Arg785 790 795 800 Pro
Ala Asn Pro Asp Glu Ile Gly Asn Phe Ile Asp Glu Asn Leu Lys 805 810
815 Ala Ala Asp Thr Asp Pro Thr Ala Pro Pro Tyr Asp Ser Leu Leu Val
820 825 830 Phe Asp Tyr Glu Gly Ser Gly Ser Glu Ala Ala Ser Leu Ser
Ser Leu 835 840 845 Asn Ser Ser Glu Ser Asp Lys Asp Gln Asp Tyr Asp
Tyr Leu Asn Glu 850 855 860 Trp Gly Asn Arg Phe Lys Lys Leu Ala Asp
Met Tyr Gly Gly Gly Glu865 870 875 880 Asp Asp17220PRTHomo sapiens
17Met Gly Ala Ala Ala Arg Thr Leu Arg Leu Ala Leu Gly Leu Leu Leu1
5 10 15 Leu Ala Thr Leu Leu Arg Pro Ala Asp Ala Cys Ser Cys Ser Pro
Val 20 25 30 His Pro Gln Gln Ala Phe Cys Asn Ala Asp Val Val Ile
Arg Ala Lys 35 40 45 Ala Val Ser Glu Lys Glu Val Asp Ser Gly Asn
Asp Ile Tyr Gly Asn 50 55 60 Pro Ile Lys Arg Ile Gln Tyr Glu Ile
Lys Gln Ile Lys Met Phe Lys65 70 75 80 Gly Pro Glu Lys Asp Ile Glu
Phe Ile Tyr Thr Ala Pro Ser Ser Ala 85 90 95 Val Cys Gly Val Ser
Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu Ile 100 105 110 Ala Gly Lys
Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys Asp 115 120 125 Phe
Ile Val Pro Trp Asp Thr Leu Ser Thr Thr Gln Lys Lys Ser Leu 130 135
140 Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys
Pro145 150 155 160 Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys
Leu Trp Met Asp 165 170 175 Trp Val Thr Glu Lys Asn Ile Asn Gly His
Gln Ala Lys Phe Phe Ala 180 185 190 Cys Ile Lys Arg Ser Asp Gly Ser
Cys Ala Trp Tyr Arg Gly Ala Ala 195 200 205 Pro Pro Lys Gln Glu Phe
Leu Asp Ile Glu Asp Pro 210 215 220 18742PRTHomo sapiens 18Met Asp
Lys Phe Trp Trp His Ala Ala Trp Gly Leu Cys Leu Val Pro1 5 10 15
Leu Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly 20
25 30 Val Phe His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr
Glu 35 40 45 Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro
Thr Met Ala 50 55 60 Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu
Thr Cys Arg Tyr Gly65 70 75 80 Phe Ile Glu Gly His Val Val Ile Pro
Arg Ile His Pro Asn Ser Ile 85 90 95 Cys Ala Ala Asn Asn Thr Gly
Val Tyr Ile Leu Thr Ser Asn Thr Ser 100 105 110 Gln Tyr Asp Thr Tyr
Cys Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp 115 120 125 Cys Thr Ser
Val Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro Ile Thr 130 135 140 Ile
Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly Glu145 150
155 160 Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp
Asp 165 170 175 Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr
Ser Gly Gly 180 185 190 Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro
Ile Pro Asp Glu Asp 195 200 205 Ser Pro Trp Ile Thr Asp Ser Thr Asp
Arg Ile Pro Ala Thr Thr Leu 210 215 220 Met Ser Thr Ser Ala Thr Ala
Thr Glu Thr Ala Thr Lys Arg Gln Glu225 230 235 240 Thr Trp Asp Trp
Phe Ser Trp Leu Phe Leu Pro Ser Glu Ser Lys Asn 245 250 255 His Leu
His Thr Thr Thr Gln Met Ala Gly Thr Ser Ser Asn Thr Ile 260 265 270
Ser Ala Gly Trp Glu Pro Asn Glu Glu Asn Glu Asp Glu Arg Asp Arg 275
280 285 His Leu Ser Phe Ser Gly Ser Gly Ile Asp Asp Asp Glu Asp Phe
Ile 290 295 300 Ser Ser Thr Ile Ser Thr Thr Pro Arg Ala Phe Asp His
Thr Lys Gln305 310 315 320 Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser
His Ser Asn Pro Glu Val 325 330 335 Leu Leu Gln Thr Thr Thr Arg Met
Thr Asp Val Asp Arg Asn Gly Thr 340 345 350 Thr Ala Tyr Glu Gly Asn
Trp Asn Pro Glu Ala His Pro Pro Leu Ile 355 360 365 His His Glu His
His Glu Glu Glu Glu Thr Pro His Ser Thr Ser Thr 370 375 380 Ile Gln
Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys385 390 395
400 Glu Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr Pro
405 410 415 Arg Glu Asp Ser His Ser Thr Thr Gly Thr Ala Ala Ala Ser
Ala His 420 425 430 Thr Ser His Pro Met Gln Gly Arg Thr Thr Pro Ser
Pro Glu Asp Ser 435 440 445 Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser
His Pro Met Gly Arg Gly 450 455 460 His Gln Ala Gly Arg Arg Met Asp
Met Asp Ser Ser His Ser Thr Thr465 470 475 480 Leu Gln Pro Thr Ala
Asn Pro Asn Thr Gly Leu Val Glu Asp Leu Asp 485 490 495 Arg Thr Gly
Pro Leu Ser Met Thr Thr Gln Gln Ser Asn Ser Gln Ser 500 505 510 Phe
Ser Thr Ser His Glu Gly Leu Glu Glu Asp Lys Asp His Pro Thr 515 520
525 Thr Ser Thr Leu Thr Ser Ser Asn Arg Asn Asp Val Thr Gly Gly Arg
530 535 540 Arg Asp Pro Asn His Ser Glu Gly Ser Thr Thr Leu Leu Glu
Gly Tyr545 550 555 560 Thr Ser His Tyr Pro His Thr Lys Glu Ser Arg
Thr Phe Ile Pro Val 565 570 575 Thr Ser Ala Lys Thr Gly Ser Phe Gly
Val Thr Ala Val Thr Val Gly 580 585 590 Asp Ser Asn Ser Asn Val Asn
Arg Ser Leu Ser Gly Asp Gln Asp Thr 595 600 605 Phe His Pro Ser Gly
Gly Ser His Thr Thr His Gly Ser Glu Ser Asp 610 615 620 Gly His Ser
His Gly Ser Gln Glu Gly Gly Ala Asn Thr Thr Ser Gly625 630 635 640
Pro Ile Arg Thr Pro Gln Ile Pro Glu Trp Leu Ile Ile Leu Ala Ser
645 650 655 Leu Leu Ala Leu Ala Leu Ile Leu Ala Val Cys Ile Ala Val
Asn Ser 660 665 670 Arg Arg Arg Cys Gly Gln Lys Lys Lys Leu Val Ile
Asn Ser Gly Asn 675 680 685 Gly Ala Val Glu Asp Arg Lys Pro Ser Gly
Leu Asn Gly Glu Ala Ser 690 695 700 Lys Ser Gln Glu Met Val His Leu
Val Asn Lys Glu Ser Ser Glu Thr705 710 715 720 Pro Asp Gln Phe Met
Thr Ala Asp Glu Thr Arg Asn Leu Gln Asn Val 725 730 735 Asp Met Lys
Ile Gly Val 740 19585PRTHomo sapiens 19Met Thr Pro Pro Arg Leu Phe
Trp Val Trp Leu Leu Val Ala Gly Thr1 5 10 15 Gln Gly Val Asn Asp
Gly Asp Met Arg Leu Ala Asp Gly Gly Ala Thr 20 25 30 Asn Gln Gly
Arg Val Glu Ile Phe Tyr Arg Gly Gln Trp Gly Thr Val 35 40 45 Cys
Asp Asn Leu Trp Asp Leu Thr Asp Ala Ser Val Val Cys Arg Ala 50 55
60 Leu Gly Phe Glu Asn Ala Thr Gln Ala Leu Gly Arg Ala Ala Phe
Gly65 70 75 80 Gln Gly Ser Gly Pro Ile Met Leu Asp Glu Val Gln Cys
Thr Gly Thr 85 90 95 Glu Ala Ser Leu Ala Asp Cys Lys Ser Leu Gly
Trp Leu Lys Ser Asn 100 105 110 Cys Arg His Glu Arg Asp Ala Gly Val
Val Cys Thr Asn Glu Thr Arg 115 120 125 Ser Thr His Thr Leu Asp Leu
Ser Arg Glu Leu Ser Glu Ala Leu Gly 130 135 140 Gln Ile Phe Asp Ser
Gln Arg Gly Cys Asp Leu Ser Ile Ser Val Asn145 150 155 160 Val Gln
Gly Glu Asp Ala Leu Gly Phe Cys Gly His Thr Val Ile Leu 165 170 175
Thr Ala Asn Leu Glu Ala Gln Ala Leu Trp Lys Glu Pro Gly Ser Asn 180
185 190 Val Thr Met Ser Val Asp Ala Glu Cys Val Pro Met Val Arg Asp
Leu 195 200 205 Leu Arg Tyr Phe Tyr Ser Arg Arg Ile Asp Ile Thr Leu
Ser Ser Val 210 215 220 Lys Cys Phe His Lys Leu Ala Ser Ala Tyr Gly
Ala Arg Gln Leu Gln225 230 235 240 Gly Tyr Cys Ala Ser Leu Phe Ala
Ile Leu Leu Pro Gln Asp Pro Ser 245 250 255 Phe Gln Met Pro Leu Asp
Leu Tyr Ala Tyr Ala Val Ala Thr Gly Asp 260 265 270 Ala Leu Leu Glu
Lys Leu Cys Leu Gln Phe Leu Ala Trp Asn Phe Glu 275 280 285 Ala Leu
Thr Gln Ala Glu Ala Trp Pro Ser Val Pro Thr Asp Leu Leu 290 295 300
Gln Leu Leu Leu Pro Arg Ser Asp Leu Ala Val Pro Ser Glu Leu Ala305
310 315 320 Leu Leu Lys Ala Val Asp Thr Trp Ser Trp Gly Glu Arg Ala
Ser His 325 330 335 Glu Glu Val Glu Gly Leu Val Glu Lys Ile Arg Phe
Pro Met Met Leu 340 345 350 Pro Glu Glu Leu Phe Glu Leu Gln Phe Asn
Leu Ser Leu Tyr Trp Ser 355 360 365 His Glu Ala Leu Phe Gln Lys Lys
Thr Leu Gln Ala Leu Glu Phe His 370 375 380 Thr Val Pro Phe Gln Leu
Leu Ala Arg Tyr Lys Gly Leu Asn Leu Thr385 390 395 400 Glu Asp Thr
Tyr Lys Pro Arg Ile Tyr Thr Ser Pro Thr Trp Ser Ala 405 410 415 Phe
Val Thr Asp Ser Ser Trp Ser Ala Arg Lys Ser Gln Leu Val Tyr 420 425
430 Gln Ser Arg Arg Gly Pro Leu Val Lys Tyr Ser Ser Asp Tyr Phe Gln
435 440 445 Ala Pro Ser Asp Tyr Arg Tyr Tyr Pro Tyr Gln Ser Phe Gln
Thr Pro 450 455 460 Gln His Pro Ser Phe Leu Phe Gln Asp Lys Arg Val
Ser Trp Ser Leu465 470 475 480 Val Tyr Leu Pro Thr Ile Gln Ser Cys
Trp Asn Tyr Gly Phe Ser Cys 485 490 495 Ser Ser Asp Glu Leu Pro Val
Leu Gly Leu Thr Lys Ser Gly Gly Ser 500 505 510 Asp Arg Thr Ile Ala
Tyr Glu Asn Lys Ala Leu Met Leu Cys Glu Gly 515 520 525 Leu Phe Val
Ala Asp Val Thr Asp Phe Glu Gly Trp Lys Ala Ala Ile 530 535 540 Pro
Ser Ala Leu Asp Thr Asn Ser Ser Lys Ser Thr Ser Ser Phe Pro545 550
555 560 Cys Pro Ala Gly His Phe Asn Gly Phe Arg Thr Val Ile Arg Pro
Phe 565 570 575 Tyr Leu Thr Asn Ser Ser Gly Val Asp 580 585
201255PRTHomo sapiens 20Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu
Leu Leu Ala Leu Leu1 5 10 15 Pro Pro Gly Ala Ala Ser Thr Gln Val
Cys Thr Gly Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro
Glu Thr His Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys
Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr
Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70 75 80 Gln
Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90
95 Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr
100 105 110 Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr
Thr Pro 115 120 125 Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu
Gln Leu Arg Ser 130 135 140 Leu Thr Glu Ile Leu Lys Gly Gly Val Leu
Ile Gln Arg Asn Pro Gln145 150 155 160 Leu Cys Tyr Gln Asp Thr Ile
Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175 Asn Gln Leu Ala Leu
Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190 His Pro Cys
Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205 Ser
Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215
220 Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln
Cys225 230 235 240 Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys
Leu Ala Cys Leu 245 250 255 His Phe Asn His Ser Gly Ile Cys Glu Leu
His Cys Pro Ala Leu Val 260 265 270 Thr Tyr Asn Thr Asp Thr Phe Glu
Ser Met Pro Asn Pro Glu Gly Arg 275 280 285 Tyr Thr Phe Gly Ala Ser
Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300 Ser Thr Asp Val
Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln305 310 315 320 Glu
Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330
335 Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu
340 345 350 Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly
Cys Lys 355 360 365 Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser
Phe Asp Gly Asp 370 375 380 Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro
Glu Gln Leu Gln Val Phe385 390 395 400 Glu Thr Leu Glu Glu Ile Thr
Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415 Asp Ser Leu Pro Asp
Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430 Gly Arg Ile
Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445 Gly
Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455
460 Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr
Val465 470 475 480 Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala
Leu Leu His Thr 485 490 495 Ala Asn Arg Pro Glu Asp Glu Cys Val Gly
Glu Gly Leu Ala Cys His 500 505 510 Gln Leu Cys Ala Arg Gly His Cys
Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525 Val Asn Cys Ser Gln Phe
Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540 Arg Val Leu Gln
Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys545 550 555 560 Leu
Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570
575 Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp
580 585 590 Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro
Asp Leu 595 600 605 Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu
Gly Ala Cys Gln 610 615 620 Pro Cys Pro Ile Asn Cys Thr His Ser Cys
Val Asp Leu Asp Asp Lys625 630 635 640 Gly Cys Pro Ala Glu Gln Arg
Ala Ser Pro Leu Thr Ser Ile Ile Ser 645 650 655 Ala Val Val Gly Ile
Leu Leu Val Val Val Leu Gly Val Val Phe Gly 660 665 670 Ile Leu Ile
Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685 Arg
Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695
700 Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu
Leu705 710 715 720 Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly
Thr Val Tyr Lys 725 730 735 Gly Ile Trp Ile Pro Asp Gly Glu Asn Val
Lys Ile Pro Val Ala Ile 740 745 750 Lys Val Leu Arg Glu Asn Thr Ser
Pro Lys Ala Asn Lys Glu Ile Leu 755 760 765 Asp Glu Ala Tyr Val Met
Ala Gly Val Gly Ser Pro Tyr Val Ser Arg 770 775 780 Leu Leu Gly Ile
Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu785 790 795 800 Met
Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810
815 Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly
820 825 830 Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu
Ala Ala 835 840 845 Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys
Ile Thr Asp Phe 850 855 860 Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu
Thr Glu Tyr His Ala Asp865 870 875 880 Gly Gly Lys Val Pro Ile Lys
Trp Met Ala Leu Glu Ser Ile Leu Arg 885 890 895 Arg Arg Phe Thr His
Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val 900 905 910 Trp Glu Leu
Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925 Arg
Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935
940 Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp
Met945 950 955 960 Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu
Val Ser Glu Phe 965 970 975 Ser Arg Met Ala Arg Asp Pro Gln Arg Phe
Val Val Ile Gln Asn Glu 980 985 990 Asp Leu Gly Pro Ala Ser Pro Leu
Asp Ser Thr Phe Tyr Arg Ser Leu 995 1000 1005 Leu Glu Asp Asp Asp
Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu 1010 1015 1020 Val Pro
Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly1025 1030
1035 1040Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg Ser
Gly Gly 1045 1050 1055 Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu
Glu Glu Ala Pro Arg 1060 1065 1070 Ser Pro Leu Ala Pro Ser Glu Gly
Ala Gly Ser Asp Val Phe Asp Gly 1075 1080 1085 Asp Leu Gly Met Gly
Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His 1090 1095 1100 Asp Pro
Ser Pro Leu Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu1105 1110
1115 1120Pro Ser Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser
Pro Gln 1125 1130 1135 Pro Glu Tyr Val Asn Gln Pro Asp Val Arg Pro
Gln Pro Pro Ser Pro 1140 1145 1150 Arg Glu Gly Pro Leu Pro Ala Ala
Arg Pro Ala Gly Ala Thr Leu Glu 1155 1160 1165 Arg Pro Lys Thr Leu
Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val 1170 1175 1180 Phe Ala
Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln1185 1190
1195 1200Gly Gly Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser
Pro Ala 1205 1210 1215 Phe Asp Asn Leu Tyr Tyr Trp Asp Gln Asp Pro
Pro Glu Arg Gly Ala 1220 1225 1230 Pro Pro Ser Thr Phe Lys Gly Thr
Pro Thr Ala Glu Asn Pro Glu Tyr 1235 1240 1245 Leu Gly Leu Asp Val
Pro Val 1250 125521431PRTHomo sapiens 21Met Arg Ala Leu Leu Ala Arg
Leu Leu Leu Cys Val Leu Val Val Ser1 5 10 15 Asp Ser Lys Gly Ser
Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp 20 25 30 Cys Leu Asn
Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile 35 40 45 His
Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile 50 55
60 Asp Lys Ser Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg
Gly65 70 75 80 Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro
Trp Asn Ser 85 90 95 Ala Thr Val Leu Gln Gln Thr Tyr His Ala His
Arg Ser Asp Ala Leu 100 105 110 Gln Leu Gly Leu Gly Lys His Asn Tyr
Cys Arg Asn Pro Asp Asn Arg 115 120 125 Arg Arg Pro Trp Cys Tyr Val
Gln Val Gly Leu Lys Pro Leu Val Gln 130 135 140 Glu Cys Met Val His
Asp Cys Ala Asp Gly Lys Lys Pro Ser Ser Pro145 150 155 160 Pro Glu
Glu Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg 165 170 175
Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp 180
185 190 Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr
Val 195 200 205 Cys Gly Gly Ser Leu Met Ser Pro Cys Trp Val Ile Ser
Ala Thr His 210 215 220 Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr
Ile Val Tyr Leu Gly225 230 235 240 Arg Ser Arg Leu Asn Ser Asn Thr
Gln Gly Glu Met Lys Phe Glu Val 245 250 255 Glu Asn Leu Ile Leu His
Lys Asp Tyr Ser Ala Asp Thr Leu Ala His 260 265 270 His Asn Asp Ile
Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys 275 280 285 Ala Gln
Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr 290 295 300
Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys305
310 315 320 Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met
Thr Val 325 330 335 Val Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro
His Tyr Tyr Gly 340 345 350 Ser Glu Val Thr Thr Lys Met Leu Cys Ala
Ala Asp Pro Gln Trp Lys 355 360 365 Thr Asp Ser Cys Gln Gly Asp
Ser
Gly Gly Pro Leu Val Cys Ser Leu 370 375 380 Gln Gly Arg Met Thr Leu
Thr Gly Ile Val Ser Trp Gly Arg Gly Cys385 390 395 400 Ala Leu Lys
Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu 405 410 415 Pro
Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 420 425 430
22266PRTHomo sapiens 22Met Met Ala Leu Gly Ala Ala Gly Ala Thr Arg
Val Phe Val Ala Met1 5 10 15 Val Ala Ala Ala Leu Gly Gly His Pro
Leu Leu Gly Val Ser Ala Thr 20 25 30 Leu Asn Ser Val Leu Asn Ser
Asn Ala Ile Lys Asn Leu Pro Pro Pro 35 40 45 Leu Gly Gly Ala Ala
Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro 50 55 60 Gly Ile Leu
Tyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn Tyr65 70 75 80 Gln
Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr Asp Glu Tyr 85 90
95 Cys Ala Ser Pro Thr Arg Gly Gly Asp Ala Gly Val Gln Ile Cys Leu
100 105 110 Ala Cys Arg Lys Arg Arg Lys Arg Cys Met Arg His Ala Met
Cys Cys 115 120 125 Pro Gly Asn Tyr Cys Lys Asn Gly Ile Cys Val Ser
Ser Asp Gln Asn 130 135 140 His Phe Arg Gly Glu Ile Glu Glu Thr Ile
Thr Glu Ser Phe Gly Asn145 150 155 160 Asp His Ser Thr Leu Asp Gly
Tyr Ser Arg Arg Thr Thr Leu Ser Ser 165 170 175 Lys Met Tyr His Thr
Lys Gly Gln Glu Gly Ser Val Cys Leu Arg Ser 180 185 190 Ser Asp Cys
Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser Lys 195 200 205 Ile
Cys Lys Pro Val Leu Lys Glu Gly Gln Val Cys Thr Lys His Arg 210 215
220 Arg Lys Gly Ser His Gly Leu Glu Ile Phe Gln Arg Cys Tyr Cys
Gly225 230 235 240 Glu Gly Leu Ser Cys Arg Ile Gln Lys Asp His His
Gln Ala Ser Asn 245 250 255 Ser Ser Arg Leu His Thr Cys Gln Arg His
260 265 23457PRTHomo sapiens 23Met Arg Ser Ala Ala Val Leu Ala Leu
Leu Leu Cys Ala Gly Gln Val1 5 10 15 Thr Ala Leu Pro Val Asn Ser
Pro Met Asn Lys Gly Asp Thr Glu Val 20 25 30 Met Lys Cys Ile Val
Glu Val Ile Ser Asp Thr Leu Ser Lys Pro Ser 35 40 45 Pro Met Pro
Val Ser Gln Glu Cys Phe Glu Thr Leu Arg Gly Asp Glu 50 55 60 Arg
Ile Leu Ser Ile Leu Arg His Gln Asn Leu Leu Lys Glu Leu Gln65 70 75
80 Asp Leu Ala Leu Gln Gly Ala Lys Glu Arg Ala His Gln Gln Lys Lys
85 90 95 His Ser Gly Phe Glu Asp Glu Leu Ser Glu Val Leu Glu Asn
Gln Ser 100 105 110 Ser Gln Ala Glu Leu Lys Glu Ala Val Glu Glu Pro
Ser Ser Lys Asp 115 120 125 Val Met Glu Lys Arg Glu Asp Ser Lys Glu
Ala Glu Lys Ser Gly Glu 130 135 140 Ala Thr Asp Gly Ala Arg Pro Gln
Ala Leu Pro Glu Pro Met Gln Glu145 150 155 160 Ser Lys Ala Glu Gly
Asn Asn Gln Ala Pro Gly Glu Glu Glu Glu Glu 165 170 175 Glu Glu Glu
Ala Thr Asn Thr His Pro Pro Ala Ser Leu Pro Ser Gln 180 185 190 Lys
Tyr Pro Gly Pro Gln Ala Glu Gly Asp Ser Glu Gly Leu Ser Gln 195 200
205 Gly Leu Val Asp Arg Glu Lys Gly Leu Ser Ala Glu Pro Gly Trp Gln
210 215 220 Ala Lys Arg Glu Glu Glu Glu Glu Glu Glu Glu Glu Ala Glu
Ala Gly225 230 235 240 Glu Glu Ala Val Pro Glu Glu Glu Gly Pro Thr
Val Val Leu Asn Pro 245 250 255 His Pro Ser Leu Gly Tyr Lys Glu Ile
Arg Lys Gly Glu Ser Arg Ser 260 265 270 Glu Ala Leu Ala Val Asp Gly
Ala Gly Lys Pro Gly Ala Glu Glu Ala 275 280 285 Gln Asp Pro Glu Gly
Lys Gly Glu Gln Glu His Ser Gln Gln Lys Glu 290 295 300 Glu Glu Glu
Glu Met Ala Val Val Pro Gln Gly Leu Phe Arg Gly Gly305 310 315 320
Lys Ser Gly Glu Leu Glu Gln Glu Glu Glu Arg Leu Ser Lys Glu Trp 325
330 335 Glu Asp Ser Lys Arg Trp Ser Lys Met Asp Gln Leu Ala Lys Glu
Leu 340 345 350 Thr Ala Glu Lys Arg Leu Glu Gly Gln Glu Glu Glu Glu
Asp Asn Arg 355 360 365 Asp Ser Ser Met Lys Leu Ser Phe Arg Ala Arg
Ala Tyr Gly Phe Arg 370 375 380 Gly Pro Gly Pro Gln Leu Arg Arg Gly
Trp Arg Pro Ser Arg Glu Glu385 390 395 400 Asp Ser Leu Glu Ala Gly
Leu Pro Leu Gln Val Arg Gly Tyr Pro Glu 405 410 415 Glu Lys Lys Glu
Glu Glu Gly Ser Ala Asn Arg Arg Pro Glu Asp Gln 420 425 430 Glu Leu
Glu Ser Leu Ser Ala Ile Glu Ala Glu Leu Glu Lys Val Ala 435 440 445
His Gln Leu Gln Ala Leu Arg Arg Gly 450 455 24232PRTHomo sapiens
24Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu1
5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu
Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp
Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val
Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe
Lys Pro Ser Cys Val Pro Leu65 70 75 80 Met Arg Cys Gly Gly Cys Cys
Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn
Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln
His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu
Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135
140 Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg
Tyr145 150 155 160 Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys
Leu Met Pro Trp 165 170 175 Ser Leu Pro Gly Pro His Pro Cys Gly Pro
Cys Ser Glu Arg Arg Lys 180 185 190 His Leu Phe Val Gln Asp Pro Gln
Thr Cys Lys Cys Ser Cys Lys Asn 195 200 205 Thr Asp Ser Arg Cys Lys
Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr 210 215 220 Cys Arg Cys Asp
Lys Pro Arg Arg225 230 25273PRTHomo sapiens 25Met Lys Lys Thr Gln
Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu1 5 10 15 Leu Leu Phe
Asn Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg 20 25 30 Val
Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 35 40
45 Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu
50 55 60 Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser
Asp Ser65 70 75 80 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser
Glu Gly Leu Ser 85 90 95 Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn
Ile Val Asp Asp Leu Val 100 105 110 Glu Cys Val Lys Glu Asn Ser Ser
Lys Asp Leu Lys Lys Ser Phe Lys 115 120 125 Ser Pro Glu Pro Arg Leu
Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe 130 135 140 Asn Arg Ser Ile
Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr145 150 155 160 Ser
Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg 165 170
175 Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser
180 185 190 Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala Lys Asn
Pro Pro 195 200 205 Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu
Pro Ala Leu Phe 210 215 220 Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala
Leu Tyr Trp Lys Lys Arg225 230 235 240 Gln Pro Ser Leu Thr Arg Ala
Val Glu Asn Ile Gln Ile Asn Glu Glu 245 250 255 Asp Asn Glu Ile Ser
Met Leu Gln Glu Lys Glu Arg Glu Phe Gln Glu 260 265 270
Val26491PRTHomo sapiens 26Met Asn Pro Ala Ala Glu Ala Glu Phe Asn
Ile Leu Leu Ala Thr Asp1 5 10 15 Ser Tyr Lys Val Thr His Tyr Lys
Gln Tyr Pro Pro Asn Thr Ser Lys 20 25 30 Val Tyr Ser Tyr Phe Glu
Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys 35 40 45 Leu Arg Lys Val
Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr 50 55 60 Ile Leu
Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe 85
90 95 Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His
Leu 100 105 110 Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile
Pro Arg Gly 115 120 125 Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro
Glu Cys Tyr Trp Leu 130 135 140 Thr Asn Trp Ile Glu Thr Ile Leu Val
Gln Ser Trp Tyr Pro Ile Thr145 150 155 160 Val Ala Thr Asn Ser Arg
Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu 165 170 175 Leu Glu Thr Ser
Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp 180 185 190 Phe Gly
Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala 195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu 210
215 220 Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly
Tyr225 230 235 240 Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala
Trp Gly Lys Asp 245 250 255 His Glu Lys Asp Ala Phe Glu His Ile Val
Thr Gln Phe Ser Ser Val 260 265 270 Pro Val Ser Val Val Ser Asp Ser
Tyr Asp Ile Tyr Asn Ala Cys Glu 275 280 285 Lys Ile Trp Gly Glu Asp
Leu Arg His Leu Ile Val Ser Arg Ser Thr 290 295 300 Gln Ala Pro Leu
Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr305 310 315 320 Val
Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu 325 330
335 Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350 Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu
Gly Met 355 360 365 Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe
Gly Ser Gly Gly 370 375 380 Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu
Leu Asn Cys Ser Phe Lys385 390 395 400 Cys Ser Tyr Val Val Thr Asn
Gly Leu Gly Ile Asn Val Phe Lys Asp 405 410 415 Pro Val Ala Asp Pro
Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu 420 425 430 His Arg Thr
Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly 435 440 445 Asp
Leu Glu Glu Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn 450 455
460 Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn
Ala465 470 475 480 Gln Leu Asn Ile Glu Leu Glu Ala Ala His His 485
490 27831PRTHomo sapiens 27Met Glu Arg Pro Trp Gly Ala Ala Asp Gly
Leu Ser Arg Trp Pro His1 5 10 15 Gly Leu Gly Leu Leu Leu Leu Leu
Gln Leu Leu Pro Pro Ser Thr Leu 20 25 30 Ser Gln Asp Arg Leu Asp
Ala Pro Pro Pro Pro Ala Ala Pro Leu Pro 35 40 45 Arg Trp Ser Gly
Pro Ile Gly Val Ser Trp Gly Leu Arg Ala Ala Ala 50 55 60 Ala Gly
Gly Ala Phe Pro Arg Gly Gly Arg Trp Arg Arg Ser Ala Pro65 70 75 80
Gly Glu Asp Glu Glu Cys Gly Arg Val Arg Asp Phe Val Ala Lys Leu 85
90 95 Ala Asn Asn Thr His Gln His Val Phe Asp Asp Leu Arg Gly Ser
Val 100 105 110 Ser Leu Ser Trp Val Gly Asp Ser Thr Gly Val Ile Leu
Val Leu Thr 115 120 125 Thr Phe His Val Pro Leu Val Ile Met Thr Phe
Gly Gln Ser Lys Leu 130 135 140 Tyr Arg Ser Glu Asp Tyr Gly Lys Asn
Phe Lys Asp Ile Thr Asp Leu145 150 155 160 Ile Asn Asn Thr Phe Ile
Arg Thr Glu Phe Gly Met Ala Ile Gly Pro 165 170 175 Glu Asn Ser Gly
Lys Val Val Leu Thr Ala Glu Val Ser Gly Gly Ser 180 185 190 Arg Gly
Gly Arg Ile Phe Arg Ser Ser Asp Phe Ala Lys Asn Phe Val 195 200 205
Gln Thr Asp Leu Pro Phe His Pro Leu Thr Gln Met Met Tyr Ser Pro 210
215 220 Gln Asn Ser Asp Tyr Leu Leu Ala Leu Ser Thr Glu Asn Gly Leu
Trp225 230 235 240 Val Ser Lys Asn Phe Gly Gly Lys Trp Glu Glu Ile
His Lys Ala Val 245 250 255 Cys Leu Ala Lys Trp Gly Ser Asp Asn Thr
Ile Phe Phe Thr Thr Tyr 260 265 270 Ala Asn Gly Ser Cys Lys Ala Asp
Leu Gly Ala Leu Glu Leu Trp Arg 275 280 285 Thr Ser Asp Leu Gly Lys
Ser Phe Lys Thr Ile Gly Val Lys Ile Tyr 290 295 300 Ser Phe Gly Leu
Gly Gly Arg Phe Leu Phe Ala Ser Val Met Ala Asp305 310 315 320 Lys
Asp Thr Thr Arg Arg Ile His Val Ser Thr Asp Gln Gly Asp Thr 325 330
335 Trp Ser Met Ala Gln Leu Pro Ser Val Gly Gln Glu Gln Phe Tyr Ser
340 345 350 Ile Leu Ala Ala Asn Asp Asp Met Val Phe Met His Val Asp
Glu Pro 355 360 365 Gly Asp Thr Gly Phe Gly Thr Ile Phe Thr Ser Asp
Asp Arg Gly Ile 370 375 380 Val Tyr Ser Lys Ser Leu Asp Arg His Leu
Tyr Thr Thr Thr Gly Gly385 390 395 400 Glu Thr Asp Phe Thr Asn Val
Thr Ser Leu Arg Gly Val Tyr Ile Thr 405 410 415 Ser Val Leu Ser Glu
Asp Asn Ser Ile Gln Thr Met Ile Thr Phe Asp 420 425 430 Gln Gly Gly
Arg Trp Thr His Leu Arg Lys Pro Glu Asn Ser Glu Cys 435 440 445 Asp
Ala Thr Ala Lys Asn Lys Asn Glu Cys Ser Leu His Ile His Ala 450 455
460 Ser Tyr Ser Ile Ser Gln Lys Leu Asn Val Pro Met Ala Pro Leu
Ser465 470 475 480 Glu Pro Asn Ala Val Gly Ile Val Ile Ala His Gly
Ser Val Gly Asp 485 490 495 Ala Ile Ser Val Met Val Pro Asp Val Tyr
Ile Ser Asp Asp Gly Gly 500 505 510 Tyr Ser Trp Thr Lys Met Leu Glu
Gly
Pro His Tyr Tyr Thr Ile Leu 515 520 525 Asp Ser Gly Gly Ile Ile Val
Ala Ile Glu His Ser Ser Arg Pro Ile 530 535 540 Asn Val Ile Lys Phe
Ser Thr Asp Glu Gly Gln Cys Trp Gln Thr Tyr545 550 555 560 Thr Phe
Thr Arg Asp Pro Ile Tyr Phe Thr Gly Leu Ala Ser Glu Pro 565 570 575
Gly Ala Arg Ser Met Asn Ile Ser Ile Trp Gly Phe Thr Glu Ser Phe 580
585 590 Leu Thr Ser Gln Trp Val Ser Tyr Thr Ile Asp Phe Lys Asp Ile
Leu 595 600 605 Glu Arg Asn Cys Glu Glu Lys Asp Tyr Thr Ile Trp Leu
Ala His Ser 610 615 620 Thr Asp Pro Glu Asp Tyr Glu Asp Gly Cys Ile
Leu Gly Tyr Lys Glu625 630 635 640 Gln Phe Leu Arg Leu Arg Lys Ser
Ser Val Cys Gln Asn Gly Arg Asp 645 650 655 Tyr Val Val Thr Lys Gln
Pro Ser Ile Cys Leu Cys Ser Leu Glu Asp 660 665 670 Phe Leu Cys Asp
Phe Gly Tyr Tyr Arg Pro Glu Asn Asp Ser Lys Cys 675 680 685 Val Glu
Gln Pro Glu Leu Lys Gly His Asp Leu Glu Phe Cys Leu Tyr 690 695 700
Gly Arg Glu Glu His Leu Thr Thr Asn Gly Tyr Arg Lys Ile Pro Gly705
710 715 720 Asp Lys Cys Gln Gly Gly Val Asn Pro Val Arg Glu Val Lys
Asp Leu 725 730 735 Lys Lys Lys Cys Thr Ser Asn Phe Leu Ser Pro Glu
Lys Gln Asn Ser 740 745 750 Lys Ser Asn Ser Val Pro Ile Ile Leu Ala
Ile Val Gly Leu Met Leu 755 760 765 Val Thr Val Val Ala Gly Val Leu
Ile Val Lys Lys Tyr Val Cys Gly 770 775 780 Gly Arg Phe Leu Val His
Arg Tyr Ser Val Leu Gln Gln His Ala Glu785 790 795 800 Ala Asn Gly
Val Asp Gly Val Asp Ala Leu Asp Thr Ala Ser His Thr 805 810 815 Asn
Lys Ser Gly Tyr His Asp Asp Ser Asp Glu Asp Leu Leu Glu 820 825 830
28143PRTHomo sapiens 28Met Gln His Arg Gly Phe Leu Leu Leu Thr Leu
Leu Ala Leu Leu Ala1 5 10 15 Leu Thr Ser Ala Val Ala Lys Lys Lys
Asp Lys Val Lys Lys Gly Gly 20 25 30 Pro Gly Ser Glu Cys Ala Glu
Trp Ala Trp Gly Pro Cys Thr Pro Ser 35 40 45 Ser Lys Asp Cys Gly
Val Gly Phe Arg Glu Gly Thr Cys Gly Ala Gln 50 55 60 Thr Gln Arg
Ile Arg Cys Arg Val Pro Cys Asn Trp Lys Lys Glu Phe65 70 75 80 Gly
Ala Asp Cys Lys Tyr Lys Phe Glu Asn Trp Gly Ala Cys Asp Gly 85 90
95 Gly Thr Gly Thr Lys Val Arg Gln Gly Thr Leu Lys Lys Ala Arg Tyr
100 105 110 Asn Ala Gln Cys Gln Glu Thr Ile Arg Val Thr Lys Pro Cys
Thr Pro 115 120 125 Lys Thr Lys Ala Lys Ala Lys Ala Lys Lys Gly Lys
Gly Lys Asp 130 135 140 29291PRTHomo sapiens 29Met Gln Arg Ala Arg
Pro Thr Leu Trp Ala Ala Ala Leu Thr Leu Leu1 5 10 15 Val Leu Leu
Arg Gly Pro Pro Val Ala Arg Ala Gly Ala Ser Ser Ala 20 25 30 Gly
Leu Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40
45 Ala Gln Cys Ala Pro Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu
50 55 60 Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly
Gln Pro65 70 75 80 Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu
Arg Cys Gln Pro 85 90 95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala
Leu Leu Asp Gly Arg Gly 100 105 110 Leu Cys Val Asn Ala Ser Ala Val
Ser Arg Leu Arg Ala Tyr Leu Leu 115 120 125 Pro Ala Pro Pro Ala Pro
Gly Asn Ala Ser Glu Ser Glu Glu Asp Arg 130 135 140 Ser Ala Gly Ser
Val Glu Ser Pro Ser Val Ser Ser Thr His Arg Val145 150 155 160 Ser
Asp Pro Lys Phe His Pro Leu His Ser Lys Ile Ile Ile Ile Lys 165 170
175 Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser
180 185 190 Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg
Glu Thr 195 200 205 Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr
Leu Asn His Leu 210 215 220 Lys Phe Leu Asn Val Leu Ser Pro Arg Gly
Val His Ile Pro Asn Cys225 230 235 240 Asp Lys Lys Gly Phe Tyr Lys
Lys Lys Gln Cys Arg Pro Ser Lys Gly 245 250 255 Arg Lys Arg Gly Phe
Cys Trp Cys Val Asp Lys Tyr Gly Gln Pro Leu 260 265 270 Pro Gly Tyr
Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met 275 280 285 Gln
Ser Lys 290 30258PRTHomo sapiens 30Met Leu Pro Leu Cys Leu Val Ala
Ala Leu Leu Leu Ala Ala Gly Pro1 5 10 15 Gly Pro Ser Leu Gly Asp
Glu Ala Ile His Cys Pro Pro Cys Ser Glu 20 25 30 Glu Lys Leu Ala
Arg Cys Arg Pro Pro Val Gly Cys Glu Glu Leu Val 35 40 45 Arg Glu
Pro Gly Cys Gly Cys Cys Ala Thr Cys Ala Leu Gly Leu Gly 50 55 60
Met Pro Cys Gly Val Tyr Thr Pro Arg Cys Gly Ser Gly Leu Arg Cys65
70 75 80 Tyr Pro Pro Arg Gly Val Glu Lys Pro Leu His Thr Leu Met
His Gly 85 90 95 Gln Gly Val Cys Met Glu Leu Ala Glu Ile Glu Ala
Ile Gln Glu Ser 100 105 110 Leu Gln Pro Ser Asp Lys Asp Glu Gly Asp
His Pro Asn Asn Ser Phe 115 120 125 Ser Pro Cys Ser Ala His Asp Arg
Arg Cys Leu Gln Lys His Phe Ala 130 135 140 Lys Ile Arg Asp Arg Ser
Thr Ser Gly Gly Lys Met Lys Val Asn Gly145 150 155 160 Ala Pro Arg
Glu Asp Ala Arg Pro Val Pro Gln Gly Ser Cys Gln Ser 165 170 175 Glu
Leu His Arg Ala Leu Glu Arg Leu Ala Ala Ser Gln Ser Arg Thr 180 185
190 His Glu Asp Leu Tyr Ile Ile Pro Ile Pro Asn Cys Asp Arg Asn Gly
195 200 205 Asn Phe His Pro Lys Gln Cys His Pro Ala Leu Asp Gly Gln
Arg Gly 210 215 220 Lys Cys Trp Cys Val Asp Arg Lys Thr Gly Val Lys
Leu Pro Gly Gly225 230 235 240 Leu Glu Pro Lys Gly Glu Leu Asp Cys
His Gln Leu Ala Asp Ser Phe 245 250 255 Arg Glu31463PRTHomo sapiens
31Met Gly Ala Gly Pro Ser Leu Leu Leu Ala Ala Leu Leu Leu Leu Leu1
5 10 15 Ser Gly Asp Gly Ala Val Arg Cys Asp Thr Pro Ala Asn Cys Thr
Tyr 20 25 30 Leu Asp Leu Leu Gly Thr Trp Val Phe Gln Val Gly Ser
Ser Gly Ser 35 40 45 Gln Arg Asp Val Asn Cys Ser Val Met Gly Pro
Gln Glu Lys Lys Val 50 55 60 Val Val Tyr Leu Gln Lys Leu Asp Thr
Ala Tyr Asp Asp Leu Gly Asn65 70 75 80 Ser Gly His Phe Thr Ile Ile
Tyr Asn Gln Gly Phe Glu Ile Val Leu 85 90 95 Asn Asp Tyr Lys Trp
Phe Ala Phe Phe Lys Tyr Lys Glu Glu Gly Ser 100 105 110 Lys Val Thr
Thr Tyr Cys Asn Glu Thr Met Thr Gly Trp Val His Asp 115 120 125 Val
Leu Gly Arg Asn Trp Ala Cys Phe Thr Gly Lys Lys Val Gly Thr 130 135
140 Ala Ser Glu Asn Val Tyr Val Asn Thr Ala His Leu Lys Asn Ser
Gln145 150 155 160 Glu Lys Tyr Ser Asn Arg Leu Tyr Lys Tyr Asp His
Asn Phe Val Lys 165 170 175 Ala Ile Asn Ala Ile Gln Lys Ser Trp Thr
Ala Thr Thr Tyr Met Glu 180 185 190 Tyr Glu Thr Leu Thr Leu Gly Asp
Met Ile Arg Arg Ser Gly Gly His 195 200 205 Ser Arg Lys Ile Pro Arg
Pro Lys Pro Ala Pro Leu Thr Ala Glu Ile 210 215 220 Gln Gln Lys Ile
Leu His Leu Pro Thr Ser Trp Asp Trp Arg Asn Val225 230 235 240 His
Gly Ile Asn Phe Val Ser Pro Val Arg Asn Gln Ala Ser Cys Gly 245 250
255 Ser Cys Tyr Ser Phe Ala Ser Met Gly Met Leu Glu Ala Arg Ile Arg
260 265 270 Ile Leu Thr Asn Asn Ser Gln Thr Pro Ile Leu Ser Pro Gln
Glu Val 275 280 285 Val Ser Cys Ser Gln Tyr Ala Gln Gly Cys Glu Gly
Gly Phe Pro Tyr 290 295 300 Leu Ile Ala Gly Lys Tyr Ala Gln Asp Phe
Gly Leu Val Glu Glu Ala305 310 315 320 Cys Phe Pro Tyr Thr Gly Thr
Asp Ser Pro Cys Lys Met Lys Glu Asp 325 330 335 Cys Phe Arg Tyr Tyr
Ser Ser Glu Tyr His Tyr Val Gly Gly Phe Tyr 340 345 350 Gly Gly Cys
Asn Glu Ala Leu Met Lys Leu Glu Leu Val His His Gly 355 360 365 Pro
Met Ala Val Ala Phe Glu Val Tyr Asp Asp Phe Leu His Tyr Lys 370 375
380 Lys Gly Ile Tyr His His Thr Gly Leu Arg Asp Pro Phe Asn Pro
Phe385 390 395 400 Glu Leu Thr Asn His Ala Val Leu Leu Val Gly Tyr
Gly Thr Asp Ser 405 410 415 Ala Ser Gly Met Asp Tyr Trp Ile Val Lys
Asn Ser Trp Gly Thr Gly 420 425 430 Trp Gly Glu Asn Gly Tyr Phe Arg
Ile Arg Arg Gly Thr Asp Glu Cys 435 440 445 Ala Ile Glu Ser Ile Ala
Val Ala Ala Thr Pro Ile Pro Lys Leu 450 455 460 32547PRTHomo
sapiens 32Met Ala Thr Met Val Pro Ser Val Leu Trp Pro Arg Ala Cys
Trp Thr1 5 10 15 Leu Leu Val Cys Cys Leu Leu Thr Pro Gly Val Gln
Gly Gln Glu Phe 20 25 30 Leu Leu Arg Val Glu Pro Gln Asn Pro Val
Leu Ser Ala Gly Gly Ser 35 40 45 Leu Phe Val Asn Cys Ser Thr Asp
Cys Pro Ser Ser Glu Lys Ile Ala 50 55 60 Leu Glu Thr Ser Leu Ser
Lys Glu Leu Val Ala Ser Gly Met Gly Trp65 70 75 80 Ala Ala Phe Asn
Leu Ser Asn Val Thr Gly Asn Ser Arg Ile Leu Cys 85 90 95 Ser Val
Tyr Cys Asn Gly Ser Gln Ile Thr Gly Ser Ser Asn Ile Thr 100 105 110
Val Tyr Gly Leu Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Pro Trp 115
120 125 Gln Pro Val Gly Gln Asn Phe Thr Leu Arg Cys Gln Val Glu Gly
Gly 130 135 140 Ser Pro Arg Thr Ser Leu Thr Val Val Leu Leu Arg Trp
Glu Glu Glu145 150 155 160 Leu Ser Arg Gln Pro Ala Val Glu Glu Pro
Ala Glu Val Thr Ala Thr 165 170 175 Val Leu Ala Ser Arg Asp Asp His
Gly Ala Pro Phe Ser Cys Arg Thr 180 185 190 Glu Leu Asp Met Gln Pro
Gln Gly Leu Gly Leu Phe Val Asn Thr Ser 195 200 205 Ala Pro Arg Gln
Leu Arg Thr Phe Val Leu Pro Val Thr Pro Pro Arg 210 215 220 Leu Val
Ala Pro Arg Phe Leu Glu Val Glu Thr Ser Trp Pro Val Asp225 230 235
240 Cys Thr Leu Asp Gly Leu Phe Pro Ala Ser Glu Ala Gln Val Tyr Leu
245 250 255 Ala Leu Gly Asp Gln Met Leu Asn Ala Thr Val Met Asn His
Gly Asp 260 265 270 Thr Leu Thr Ala Thr Ala Thr Ala Thr Ala Arg Ala
Asp Gln Glu Gly 275 280 285 Ala Arg Glu Ile Val Cys Asn Val Thr Leu
Gly Gly Glu Arg Arg Glu 290 295 300 Ala Arg Glu Asn Leu Thr Val Phe
Ser Phe Leu Gly Pro Ile Val Asn305 310 315 320 Leu Ser Glu Pro Thr
Ala His Glu Gly Ser Thr Val Thr Val Ser Cys 325 330 335 Met Ala Gly
Ala Arg Val Gln Val Thr Leu Asp Gly Val Pro Ala Ala 340 345 350 Ala
Pro Gly Gln Pro Ala Gln Leu Gln Leu Asn Ala Thr Glu Ser Asp 355 360
365 Asp Gly Arg Ser Phe Phe Cys Ser Ala Thr Leu Glu Val Asp Gly Glu
370 375 380 Phe Leu His Arg Asn Ser Ser Val Gln Leu Arg Val Leu Tyr
Gly Pro385 390 395 400 Lys Ile Asp Arg Ala Thr Cys Pro Gln His Leu
Lys Trp Lys Asp Lys 405 410 415 Thr Arg His Val Leu Gln Cys Gln Ala
Arg Gly Asn Pro Tyr Pro Glu 420 425 430 Leu Arg Cys Leu Lys Glu Gly
Ser Ser Arg Glu Val Pro Val Gly Ile 435 440 445 Pro Phe Phe Val Asn
Val Thr His Asn Gly Thr Tyr Gln Cys Gln Ala 450 455 460 Ser Ser Ser
Arg Gly Lys Tyr Thr Leu Val Val Val Met Asp Ile Glu465 470 475 480
Ala Gly Ser Ser His Phe Val Pro Val Phe Val Ala Val Leu Leu Thr 485
490 495 Leu Gly Val Val Thr Ile Val Leu Ala Leu Met Tyr Val Phe Arg
Glu 500 505 510 His Gln Arg Ser Gly Ser Tyr His Val Arg Glu Glu Ser
Thr Tyr Leu 515 520 525 Pro Leu Thr Ser Met Gln Pro Thr Glu Ala Met
Gly Glu Glu Pro Ser 530 535 540 Arg Ala Glu545 33349PRTHomo sapiens
33Met Thr Ala Ala Ser Met Gly Pro Val Arg Val Ala Phe Val Val Leu1
5 10 15 Leu Ala Leu Cys Ser Arg Pro Ala Val Gly Gln Asn Cys Ser Gly
Pro 20 25 30 Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg Cys Pro Ala
Gly Val Ser 35 40 45 Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val
Cys Ala Lys Gln Leu 50 55 60 Gly Glu Leu Cys Thr Glu Arg Asp Pro
Cys Asp Pro His Lys Gly Leu65 70 75 80 Phe Cys His Phe Gly Ser Pro
Ala Asn Arg Lys Ile Gly Val Cys Thr 85 90 95 Ala Lys Asp Gly Ala
Pro Cys Ile Phe Gly Gly Thr Val Tyr Arg Ser 100 105 110 Gly Glu Ser
Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp 115 120 125 Gly
Ala Val Gly Cys Met Pro Leu Cys Ser Met Asp Val Arg Leu Pro 130 135
140 Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly Lys
Cys145 150 155 160 Cys Glu Glu Trp Val Cys Asp Glu Pro Lys Asp Gln
Thr Val Val Gly 165 170 175 Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp
Thr Phe Gly Pro Asp Pro 180 185 190 Thr Met Ile Arg Ala Asn Cys Leu
Val Gln Thr Thr Glu Trp Ser Ala 195 200 205 Cys Ser Lys Thr Cys Gly
Met Gly Ile Ser Thr Arg Val Thr Asn Asp 210 215 220 Asn Ala Ser Cys
Arg Leu Glu Lys Gln Ser Arg Leu Cys Met Val Arg225 230 235 240 Pro
Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys Gly Lys Lys Cys 245 250
255 Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys Phe Glu Leu Ser Gly
260 265 270 Cys Thr Ser Met Lys Thr Tyr Arg Ala
Lys Phe Cys Gly Val Cys Thr 275 280 285 Asp Gly Arg Cys Cys Thr Pro
His Arg Thr Thr Thr Leu Pro Val Glu 290 295 300 Phe Lys Cys Pro Asp
Gly Glu Val Met Lys Lys Asn Met Met Phe Ile305 310 315 320 Lys Thr
Cys Ala Cys His Tyr Asn Cys Pro Gly Asp Asn Asp Ile Phe 325 330 335
Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala 340 345
34198PRTHomo sapiens 34Met Pro Leu Gly Leu Leu Trp Leu Gly Leu Ala
Leu Leu Gly Ala Leu1 5 10 15 His Ala Gln Ala Gln Asp Ser Thr Ser
Asp Leu Ile Pro Ala Pro Pro 20 25 30 Leu Ser Lys Val Pro Leu Gln
Gln Asn Phe Gln Asp Asn Gln Phe Gln 35 40 45 Gly Lys Trp Tyr Val
Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu 50 55 60 Asp Lys Asp
Pro Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu65 70 75 80 Asp
Lys Ser Tyr Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys 85 90
95 Asp Tyr Trp Ile Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe
100 105 110 Thr Leu Gly Asn Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr
Leu Val 115 120 125 Arg Val Val Ser Thr Asn Tyr Asn Gln His Ala Met
Val Phe Phe Lys 130 135 140 Lys Val Ser Gln Asn Arg Glu Tyr Phe Lys
Ile Thr Leu Tyr Gly Arg145 150 155 160 Thr Lys Glu Leu Thr Ser Glu
Leu Lys Glu Asn Phe Ile Arg Phe Ser 165 170 175 Lys Ser Leu Gly Leu
Pro Glu Asn His Ile Val Phe Pro Val Pro Ile 180 185 190 Asp Gln Cys
Ile Asp Gly 195 351210PRTHomo sapiens 35Met Arg Pro Ser Gly Thr Ala
Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15 Ala Leu Cys Pro Ala
Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser
Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu
Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55
60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu
Lys65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu
Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile
Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu Ala Val
Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys Glu Leu
Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala Val Arg
Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155 160 Ser Ile
Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180
185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys
Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly
Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln
Cys Ala Ala Gly Cys225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys
Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp
Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln
Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr
Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305
310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg
Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser
Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys
Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val Ala Phe
Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp Pro Gln
Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385 390 395 400 Ile Thr Gly
Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu
His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425
430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile
Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn
Trp Lys Lys Leu465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys
Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly
Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly
Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 Val Ser Arg
Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 Glu
Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550
555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly
Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn
Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His Val Cys
His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr Gly Cys Thr Gly Pro
Gly Leu Glu Gly Cys Pro Thr Asn Gly625 630 635 640 Pro Lys Ile Pro
Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655 Leu Leu
Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670
Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675
680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu
Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val
Leu Gly Ser705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu
Trp Ile Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val Ala Ile
Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn Lys Glu
Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp Asn Pro
His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780 Thr Val
Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp785 790 795
800 Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn
805 810 815 Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp
Arg Arg 820 825 830 Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu
Val Lys Thr Pro 835 840 845 Gln His Val Lys Ile Thr Asp Phe Gly Leu
Ala Lys Leu Leu Gly Ala 850 855 860 Glu Glu Lys Glu Tyr His Ala Glu
Gly Gly Lys Val Pro Ile Lys Trp865 870 875 880 Met Ala Leu Glu Ser
Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895 Val Trp Ser
Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910 Lys
Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920
925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr
930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg
Pro Lys945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met
Ala Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly Asp Glu
Arg Met His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe Tyr Arg
Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val Val Asp
Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe 1010 1015 1020 Ser
Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala1025
1030 1035 1040Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn
Gly Leu Gln 1045 1050 1055 Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu
Gln Arg Tyr Ser Ser Asp 1060 1065 1070 Pro Thr Gly Ala Leu Thr Glu
Asp Ser Ile Asp Asp Thr Phe Leu Pro 1075 1080 1085 Val Pro Glu Tyr
Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser 1090 1095 1100 Val
Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser1105
1110 1115 1120Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala Val
Gly Asn Pro 1125 1130 1135 Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys
Val Asn Ser Thr Phe Asp 1140 1145 1150 Ser Pro Ala His Trp Ala Gln
Lys Gly Ser His Gln Ile Ser Leu Asp 1155 1160 1165 Asn Pro Asp Tyr
Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn 1170 1175 1180 Gly
Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val1185
1190 1195 1200Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala 1205
121036368PRTHomo sapiens 36Met Trp Pro Leu Trp Arg Leu Val Ser Leu
Leu Ala Leu Ser Gln Ala1 5 10 15 Leu Pro Phe Glu Gln Arg Gly Phe
Trp Asp Phe Thr Leu Asp Asp Gly 20 25 30 Pro Phe Met Met Asn Asp
Glu Glu Ala Ser Gly Ala Asp Thr Ser Gly 35 40 45 Val Leu Asp Pro
Asp Ser Val Thr Pro Thr Tyr Ser Ala Met Cys Pro 50 55 60 Phe Gly
Cys His Cys His Leu Arg Val Val Gln Cys Ser Asp Leu Gly65 70 75 80
Leu Lys Ser Val Pro Lys Glu Ile Ser Pro Asp Thr Thr Leu Leu Asp 85
90 95 Leu Gln Asn Asn Asp Ile Ser Glu Leu Arg Lys Asp Asp Phe Lys
Gly 100 105 110 Leu Gln His Leu Tyr Ala Leu Val Leu Val Asn Asn Lys
Ile Ser Lys 115 120 125 Ile His Glu Lys Ala Phe Ser Pro Leu Arg Lys
Leu Gln Lys Leu Tyr 130 135 140 Ile Ser Lys Asn His Leu Val Glu Ile
Pro Pro Asn Leu Pro Ser Ser145 150 155 160 Leu Val Glu Leu Arg Ile
His Asp Asn Arg Ile Arg Lys Val Pro Lys 165 170 175 Gly Val Phe Ser
Gly Leu Arg Asn Met Asn Cys Ile Glu Met Gly Gly 180 185 190 Asn Pro
Leu Glu Asn Ser Gly Phe Glu Pro Gly Ala Phe Asp Gly Leu 195 200 205
Lys Leu Asn Tyr Leu Arg Ile Ser Glu Ala Lys Leu Thr Gly Ile Pro 210
215 220 Lys Asp Leu Pro Glu Thr Leu Asn Glu Leu His Leu Asp His Asn
Lys225 230 235 240 Ile Gln Ala Ile Glu Leu Glu Asp Leu Leu Arg Tyr
Ser Lys Leu Tyr 245 250 255 Arg Leu Gly Leu Gly His Asn Gln Ile Arg
Met Ile Glu Asn Gly Ser 260 265 270 Leu Ser Phe Leu Pro Thr Leu Arg
Glu Leu His Leu Asp Asn Asn Lys 275 280 285 Leu Ala Arg Val Pro Ser
Gly Leu Pro Asp Leu Lys Leu Leu Gln Val 290 295 300 Val Tyr Leu His
Ser Asn Asn Ile Thr Lys Val Gly Val Asn Asp Phe305 310 315 320 Cys
Pro Met Gly Phe Gly Val Lys Arg Ala Tyr Tyr Asn Gly Ile Ser 325 330
335 Leu Phe Asn Asn Pro Val Pro Tyr Trp Glu Val Gln Pro Ala Thr Phe
340 345 350 Arg Cys Val Thr Asp Arg Leu Ala Ile Gln Phe Gly Asn Tyr
Lys Lys 355 360 365 37211PRTHomo sapiens 37Met Thr Pro Trp Leu Gly
Leu Ile Val Leu Leu Gly Ser Trp Ser Leu1 5 10 15 Gly Asp Trp Gly
Ala Glu Ala Cys Thr Cys Ser Pro Ser His Pro Gln 20 25 30 Asp Ala
Phe Cys Asn Ser Asp Ile Val Ile Arg Ala Lys Val Val Gly 35 40 45
Lys Lys Leu Val Lys Glu Gly Pro Phe Gly Thr Leu Val Tyr Thr Ile 50
55 60 Lys Gln Met Lys Met Tyr Arg Gly Phe Thr Lys Met Pro His Val
Gln65 70 75 80 Tyr Ile His Thr Glu Ala Ser Glu Ser Leu Cys Gly Leu
Lys Leu Glu 85 90 95 Val Asn Lys Tyr Gln Tyr Leu Leu Thr Gly Arg
Val Tyr Asp Gly Lys 100 105 110 Met Tyr Thr Gly Leu Cys Asn Phe Val
Glu Arg Trp Asp Gln Leu Thr 115 120 125 Leu Ser Gln Arg Lys Gly Leu
Asn Tyr Arg Tyr His Leu Gly Cys Asn 130 135 140 Cys Lys Ile Lys Ser
Cys Tyr Tyr Leu Pro Cys Phe Val Thr Ser Lys145 150 155 160 Asn Glu
Cys Leu Trp Thr Asp Met Leu Ser Asn Phe Gly Tyr Pro Gly 165 170 175
Tyr Gln Ser Lys His Tyr Ala Cys Ile Arg Gln Lys Gly Gly Tyr Cys 180
185 190 Ser Trp Tyr Arg Gly Trp Ala Pro Pro Asp Lys Ser Ile Ile Asn
Ala 195 200 205 Thr Asp Pro 210 38728PRTHomo sapiens 38Met Trp Val
Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu1 5 10 15 Leu
His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25
30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys
Lys Val 50 55 60 Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg
Asn Lys Gly Leu65 70 75 80 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp
Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro Phe Asn Ser Met
Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu
Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile Ile Gly Lys
Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 Ser Gly
Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His145 150 155
160 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr
165 170 175 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe
Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro
Gln Cys Ser Glu 195 200 205 Val Glu Cys Met Thr Cys Asn Gly Glu Ser
Tyr Arg Gly Leu Met Asp 210 215 220 His Thr Glu Ser Gly Lys Ile Cys
Gln Arg Trp Asp His Gln Thr Pro225 230
235 240 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe
Asp 245 250 255 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro
Trp Cys Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys
Ala Ile Lys Thr Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr Asp
Val Pro Leu Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu
Gly Tyr Arg Gly Thr Val Asn Thr Ile305 310 315 320 Trp Asn Gly Ile
Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp
Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350
Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355
360 365 Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys
Asp 370 375 380 Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys
Asn Tyr Met385 390 395 400 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu
Thr Cys Ser Met Trp Asp 405 410 415 Lys Asn Met Glu Asp Leu His Arg
His Ile Phe Trp Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn
Tyr Cys Arg Asn Pro Asp Asp Asp Ala His 435 440 445 Gly Pro Trp Cys
Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 Pro Ile
Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu465 470 475
480 Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val
485 490 495 Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser
Leu Arg 500 505 510 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile
Lys Glu Ser Trp 515 520 525 Val Leu Thr Ala Arg Gln Cys Phe Pro Ser
Arg Asp Leu Lys Asp Tyr 530 535 540 Glu Ala Trp Leu Gly Ile His Asp
Val His Gly Arg Gly Asp Glu Lys545 550 555 560 Cys Lys Gln Val Leu
Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575 Ser Asp Leu
Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe
Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600
605 Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn
610 615 620 Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly
Asn Glu625 630 635 640 Lys Cys Ser Gln His His Arg Gly Lys Val Thr
Leu Asn Glu Ser Glu 645 650 655 Ile Cys Ala Gly Ala Glu Lys Ile Gly
Ser Gly Pro Cys Glu Gly Asp 660 665 670 Tyr Gly Gly Pro Leu Val Cys
Glu Gln His Lys Met Arg Met Val Leu 675 680 685 Gly Val Ile Val Pro
Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 700 Ile Phe Val
Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile705 710 715 720
Leu Thr Tyr Lys Val Pro Gln Ser 725 39393PRTHomo sapiens 39Met Glu
Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln1 5 10 15
Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20
25 30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro
Asp 35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp
Glu Ala Pro 50 55 60 Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro
Ala Pro Ala Ala Pro65 70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro
Ser Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr
Gln Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu His Ser Gly
Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn
Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135 140 Leu
Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met145 150
155 160 Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg
Cys 165 170 175 Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala
Pro Pro Gln 180 185 190 His Leu Ile Arg Val Glu Gly Asn Leu Arg Val
Glu Tyr Leu Asp Asp 195 200 205 Arg Asn Thr Phe Arg His Ser Val Val
Val Pro Tyr Glu Pro Pro Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr
Ile His Tyr Asn Tyr Met Cys Asn Ser225 230 235 240 Ser Cys Met Gly
Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255 Leu Glu
Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 260 265 270
Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275
280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser
Thr 290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln
Pro Lys Lys305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu
Gln Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu
Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu
Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365 Leu Lys Ser Lys
Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380 Phe Lys
Thr Glu Gly Pro Asp Ser Asp385 390 40638PRTHomo sapiens 40Met Ile
Leu Phe Lys Gln Ala Thr Tyr Phe Ile Ser Leu Phe Ala Thr1 5 10 15
Val Ser Cys Gly Cys Leu Thr Gln Leu Tyr Glu Asn Ala Phe Phe Arg 20
25 30 Gly Gly Asp Val Ala Ser Met Tyr Thr Pro Asn Ala Gln Tyr Cys
Gln 35 40 45 Met Arg Cys Thr Phe His Pro Arg Cys Leu Leu Phe Ser
Phe Leu Pro 50 55 60 Ala Ser Ser Ile Asn Asp Met Glu Lys Arg Phe
Gly Cys Phe Leu Lys65 70 75 80 Asp Ser Val Thr Gly Thr Leu Pro Lys
Val His Arg Thr Gly Ala Val 85 90 95 Ser Gly His Ser Leu Lys Gln
Cys Gly His Gln Ile Ser Ala Cys His 100 105 110 Arg Asp Ile Tyr Lys
Gly Val Asp Met Arg Gly Val Asn Phe Asn Val 115 120 125 Ser Lys Val
Ser Ser Val Glu Glu Cys Gln Lys Arg Cys Thr Asn Asn 130 135 140 Ile
Arg Cys Gln Phe Phe Ser Tyr Ala Thr Gln Thr Phe His Lys Ala145 150
155 160 Glu Tyr Arg Asn Asn Cys Leu Leu Lys Tyr Ser Pro Gly Gly Thr
Pro 165 170 175 Thr Ala Ile Lys Val Leu Ser Asn Val Glu Ser Gly Phe
Ser Leu Lys 180 185 190 Pro Cys Ala Leu Ser Glu Ile Gly Cys His Met
Asn Ile Phe Gln His 195 200 205 Leu Ala Phe Ser Asp Val Asp Val Ala
Arg Val Leu Thr Pro Asp Ala 210 215 220 Phe Val Cys Arg Thr Ile Cys
Thr Tyr His Pro Asn Cys Leu Phe Phe225 230 235 240 Thr Phe Tyr Thr
Asn Val Trp Lys Ile Glu Ser Gln Arg Asn Val Cys 245 250 255 Leu Leu
Lys Thr Ser Glu Ser Gly Thr Pro Ser Ser Ser Thr Pro Gln 260 265 270
Glu Asn Thr Ile Ser Gly Tyr Ser Leu Leu Thr Cys Lys Arg Thr Leu 275
280 285 Pro Glu Pro Cys His Ser Lys Ile Tyr Pro Gly Val Asp Phe Gly
Gly 290 295 300 Glu Glu Leu Asn Val Thr Phe Val Lys Gly Val Asn Val
Cys Gln Glu305 310 315 320 Thr Cys Thr Lys Met Ile Arg Cys Gln Phe
Phe Thr Tyr Ser Leu Leu 325 330 335 Pro Glu Asp Cys Lys Glu Glu Lys
Cys Lys Cys Phe Leu Arg Leu Ser 340 345 350 Met Asp Gly Ser Pro Thr
Arg Ile Ala Tyr Gly Thr Gln Gly Ser Ser 355 360 365 Gly Tyr Ser Leu
Arg Leu Cys Asn Thr Gly Asp Asn Ser Val Cys Thr 370 375 380 Thr Lys
Thr Ser Thr Arg Ile Val Gly Gly Thr Asn Ser Ser Trp Gly385 390 395
400 Glu Trp Pro Trp Gln Val Ser Leu Gln Val Lys Leu Thr Ala Gln Arg
405 410 415 His Leu Cys Gly Gly Ser Leu Ile Gly His Gln Trp Val Leu
Thr Ala 420 425 430 Ala His Cys Phe Asp Gly Leu Pro Leu Gln Asp Val
Trp Arg Ile Tyr 435 440 445 Ser Gly Ile Leu Asn Leu Ser Asp Ile Thr
Lys Asp Thr Pro Phe Ser 450 455 460 Gln Ile Lys Glu Ile Ile Ile His
Gln Asn Tyr Lys Val Ser Glu Gly465 470 475 480 Asn His Asp Ile Ala
Leu Ile Lys Leu Gln Ala Pro Leu Asn Tyr Thr 485 490 495 Glu Phe Gln
Lys Pro Ile Cys Leu Pro Ser Lys Gly Asp Thr Ser Thr 500 505 510 Ile
Tyr Thr Asn Cys Trp Val Thr Gly Trp Gly Phe Ser Lys Glu Lys 515 520
525 Gly Glu Ile Gln Asn Ile Leu Gln Lys Val Asn Ile Pro Leu Val Thr
530 535 540 Asn Glu Glu Cys Gln Lys Arg Tyr Gln Asp Tyr Lys Ile Thr
Gln Arg545 550 555 560 Met Val Cys Ala Gly Tyr Lys Glu Gly Gly Lys
Asp Ala Cys Lys Gly 565 570 575 Asp Ser Gly Gly Pro Leu Val Cys Lys
His Asn Gly Met Trp Arg Leu 580 585 590 Val Gly Ile Thr Ser Trp Gly
Glu Gly Cys Ala Arg Arg Glu Gln Pro 595 600 605 Gly Val Tyr Thr Lys
Val Ala Glu Tyr Met Asp Trp Ile Leu Glu Lys 610 615 620 Thr Gln Ser
Ser Asp Gly Lys Ala Gln Met Gln Ser Pro Ala625 630 635 41166PRTHomo
sapiens 41Met Ala Ser Gly Val Ala Val Ser Asp Gly Val Ile Lys Val
Phe Asn1 5 10 15 Asp Met Lys Val Arg Lys Ser Ser Thr Pro Glu Glu
Val Lys Lys Arg 20 25 30 Lys Lys Ala Val Leu Phe Cys Leu Ser Glu
Asp Lys Lys Asn Ile Ile 35 40 45 Leu Glu Glu Gly Lys Glu Ile Leu
Val Gly Asp Val Gly Gln Thr Val 50 55 60 Asp Asp Pro Tyr Ala Thr
Phe Val Lys Met Leu Pro Asp Lys Asp Cys65 70 75 80 Arg Tyr Ala Leu
Tyr Asp Ala Thr Tyr Glu Thr Lys Glu Ser Lys Lys 85 90 95 Glu Asp
Leu Val Phe Ile Phe Trp Ala Pro Glu Ser Ala Pro Leu Lys 100 105 110
Ser Lys Met Ile Tyr Ala Ser Ser Lys Asp Ala Ile Lys Lys Lys Leu 115
120 125 Thr Gly Ile Lys His Glu Leu Gln Ala Asn Cys Tyr Glu Glu Val
Lys 130 135 140 Asp Arg Cys Thr Leu Ala Glu Lys Leu Gly Gly Ser Ala
Val Ile Ser145 150 155 160 Leu Glu Gly Lys Pro Leu 165 42437PRTHomo
sapiens 42Met Ala Ala Gly Thr Leu Tyr Thr Tyr Pro Glu Asn Trp Arg
Ala Phe1 5 10 15 Lys Ala Leu Ile Ala Ala Gln Tyr Ser Gly Ala Gln
Val Arg Val Leu 20 25 30 Ser Ala Pro Pro His Phe His Phe Gly Gln
Thr Asn Arg Thr Pro Glu 35 40 45 Phe Leu Arg Lys Phe Pro Ala Gly
Lys Val Pro Ala Phe Glu Gly Asp 50 55 60 Asp Gly Phe Cys Val Phe
Glu Ser Asn Ala Ile Ala Tyr Tyr Val Ser65 70 75 80 Asn Glu Glu Leu
Arg Gly Ser Thr Pro Glu Ala Ala Ala Gln Val Val 85 90 95 Gln Trp
Val Ser Phe Ala Asp Ser Asp Ile Val Pro Pro Ala Ser Thr 100 105 110
Trp Val Phe Pro Thr Leu Gly Ile Met His His Asn Lys Gln Ala Thr 115
120 125 Glu Asn Ala Lys Glu Glu Val Arg Arg Ile Leu Gly Leu Leu Asp
Ala 130 135 140 Tyr Leu Lys Thr Arg Thr Phe Leu Val Gly Glu Arg Val
Thr Leu Ala145 150 155 160 Asp Ile Thr Val Val Cys Thr Leu Leu Trp
Leu Tyr Lys Gln Val Leu 165 170 175 Glu Pro Ser Phe Arg Gln Ala Phe
Pro Asn Thr Asn Arg Trp Phe Leu 180 185 190 Thr Cys Ile Asn Gln Pro
Gln Phe Arg Ala Val Leu Gly Glu Val Lys 195 200 205 Leu Cys Glu Lys
Met Ala Gln Phe Asp Ala Lys Lys Phe Ala Glu Thr 210 215 220 Gln Pro
Lys Lys Asp Thr Pro Arg Lys Glu Lys Gly Ser Arg Glu Glu225 230 235
240 Lys Gln Lys Pro Gln Ala Glu Arg Lys Glu Glu Lys Lys Ala Ala Ala
245 250 255 Pro Ala Pro Glu Glu Glu Met Asp Glu Cys Glu Gln Ala Leu
Ala Ala 260 265 270 Glu Pro Lys Ala Lys Asp Pro Phe Ala His Leu Pro
Lys Ser Thr Phe 275 280 285 Val Leu Asp Glu Phe Lys Arg Lys Tyr Ser
Asn Glu Asp Thr Leu Ser 290 295 300 Val Ala Leu Pro Tyr Phe Trp Glu
His Phe Asp Lys Asp Gly Trp Ser305 310 315 320 Leu Trp Tyr Ser Glu
Tyr Arg Phe Pro Glu Glu Leu Thr Gln Thr Phe 325 330 335 Met Ser Cys
Asn Leu Ile Thr Gly Met Phe Gln Arg Leu Asp Lys Leu 340 345 350 Arg
Lys Asn Ala Phe Ala Ser Val Ile Leu Phe Gly Thr Asn Asn Ser 355 360
365 Ser Ser Ile Ser Gly Val Trp Val Phe Arg Gly Gln Glu Leu Ala Phe
370 375 380 Pro Leu Ser Pro Asp Trp Gln Val Asp Tyr Glu Ser Tyr Thr
Trp Arg385 390 395 400 Lys Leu Asp Pro Gly Ser Glu Glu Thr Gln Thr
Leu Val Arg Glu Tyr 405 410 415 Phe Ser Trp Glu Gly Ala Phe Gln His
Val Gly Lys Ala Phe Asn Gln 420 425 430 Gly Lys Ile Phe Lys 435
43854PRTHomo sapiens 43Met Pro Pro Cys Ser Gly Gly Asp Gly Ser Thr
Pro Pro Gly Pro Ser1 5 10 15 Leu Arg Asp Arg Asp Cys Pro Ala Gln
Ser Ala Glu Tyr Pro Arg Asp 20 25 30 Arg Leu Asp Pro Arg Pro Gly
Ser Pro Ser Glu Ala Ser Ser Pro Pro 35 40 45 Phe Leu Arg Ser Arg
Ala Pro Val Asn Trp Tyr Gln Glu Lys Ala Gln 50 55 60 Val Phe Leu
Trp His Leu Met Val Ser Gly Ser Thr Thr Leu Leu Cys65 70 75 80 Leu
Trp Lys Gln Pro Phe His Val Ser Ala Phe Pro Val Thr Ala Ser 85 90
95 Leu Ala Phe Arg Gln Ser Gln Gly Ala Gly Gln His Leu Tyr Lys Asp
100 105 110 Leu Gln Pro Phe Ile Leu Leu Arg Leu Leu Met Pro Glu Glu
Thr Gln 115 120 125 Thr Gln Asp Gln Pro Met Glu Glu Glu Glu Val Glu
Thr Phe Ala Phe 130 135 140 Gln Ala Glu Ile Ala Gln Leu Met Ser Leu
Ile Ile Asn Thr Phe Tyr145 150 155 160 Ser Asn Lys Glu Ile Phe Leu
Arg Glu Leu Ile Ser Asn Ser Ser Asp 165
170 175 Ala Leu Asp Lys Ile Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys
Leu 180 185 190 Asp Ser Gly Lys Glu Leu His Ile Asn Leu Ile Pro Asn
Lys Gln Asp 195 200 205 Arg Thr Leu Thr Ile Val Asp Thr Gly Ile Gly
Met Thr Lys Ala Asp 210 215 220 Leu Ile Asn Asn Leu Gly Thr Ile Ala
Lys Ser Gly Thr Lys Ala Phe225 230 235 240 Met Glu Ala Leu Gln Ala
Gly Ala Asp Ile Ser Met Ile Gly Gln Phe 245 250 255 Gly Val Gly Phe
Tyr Ser Ala Tyr Leu Val Ala Glu Lys Val Thr Val 260 265 270 Ile Thr
Lys His Asn Asp Asp Glu Gln Tyr Ala Trp Glu Ser Ser Ala 275 280 285
Gly Gly Ser Phe Thr Val Arg Thr Asp Thr Gly Glu Pro Met Gly Arg 290
295 300 Gly Thr Lys Val Ile Leu His Leu Lys Glu Asp Gln Thr Glu Tyr
Leu305 310 315 320 Glu Glu Arg Arg Ile Lys Glu Ile Val Lys Lys His
Ser Gln Phe Ile 325 330 335 Gly Tyr Pro Ile Thr Leu Phe Val Glu Lys
Glu Arg Asp Lys Glu Val 340 345 350 Ser Asp Asp Glu Ala Glu Glu Lys
Glu Asp Lys Glu Glu Glu Lys Glu 355 360 365 Lys Glu Glu Lys Glu Ser
Glu Asp Lys Pro Glu Ile Glu Asp Val Gly 370 375 380 Ser Asp Glu Glu
Glu Glu Lys Lys Asp Gly Asp Lys Lys Lys Lys Lys385 390 395 400 Lys
Ile Lys Glu Lys Tyr Ile Asp Gln Glu Glu Leu Asn Lys Thr Lys 405 410
415 Pro Ile Trp Thr Arg Asn Pro Asp Asp Ile Thr Asn Glu Glu Tyr Gly
420 425 430 Glu Phe Tyr Lys Ser Leu Thr Asn Asp Trp Glu Asp His Leu
Ala Val 435 440 445 Lys His Phe Ser Val Glu Gly Gln Leu Glu Phe Arg
Ala Leu Leu Phe 450 455 460 Val Pro Arg Arg Ala Pro Phe Asp Leu Phe
Glu Asn Arg Lys Lys Lys465 470 475 480 Asn Asn Ile Lys Leu Tyr Val
Arg Arg Val Phe Ile Met Asp Asn Cys 485 490 495 Glu Glu Leu Ile Pro
Glu Tyr Leu Asn Phe Ile Arg Gly Val Val Asp 500 505 510 Ser Glu Asp
Leu Pro Leu Asn Ile Ser Arg Glu Met Leu Gln Gln Ser 515 520 525 Lys
Ile Leu Lys Val Ile Arg Lys Asn Leu Val Lys Lys Cys Leu Glu 530 535
540 Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu Asn Tyr Lys Lys Phe
Tyr545 550 555 560 Glu Gln Phe Ser Lys Asn Ile Lys Leu Gly Ile His
Glu Asp Ser Gln 565 570 575 Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg
Tyr Tyr Thr Ser Ala Ser 580 585 590 Gly Asp Glu Met Val Ser Leu Lys
Asp Tyr Cys Thr Arg Met Lys Glu 595 600 605 Asn Gln Lys His Ile Tyr
Tyr Ile Thr Gly Glu Thr Lys Asp Gln Val 610 615 620 Ala Asn Ser Ala
Phe Val Glu Arg Leu Arg Lys His Gly Leu Glu Val625 630 635 640 Ile
Tyr Met Ile Glu Pro Ile Asp Glu Tyr Cys Val Gln Gln Leu Lys 645 650
655 Glu Phe Glu Gly Lys Thr Leu Val Ser Val Thr Lys Glu Gly Leu Glu
660 665 670 Leu Pro Glu Asp Glu Glu Glu Lys Lys Lys Gln Glu Glu Lys
Lys Thr 675 680 685 Lys Phe Glu Asn Leu Cys Lys Ile Met Lys Asp Ile
Leu Glu Lys Lys 690 695 700 Val Glu Lys Val Val Val Ser Asn Arg Leu
Val Thr Ser Pro Cys Cys705 710 715 720 Ile Val Thr Ser Thr Tyr Gly
Trp Thr Ala Asn Met Glu Arg Ile Met 725 730 735 Lys Ala Gln Ala Leu
Arg Asp Asn Ser Thr Met Gly Tyr Met Ala Ala 740 745 750 Lys Lys His
Leu Glu Ile Asn Pro Asp His Ser Ile Ile Glu Thr Leu 755 760 765 Arg
Gln Lys Ala Glu Ala Asp Lys Asn Asp Lys Ser Val Lys Asp Leu 770 775
780 Val Ile Leu Leu Tyr Glu Thr Ala Leu Leu Ser Ser Gly Phe Ser
Leu785 790 795 800 Glu Asp Pro Gln Thr His Ala Asn Arg Ile Tyr Arg
Met Ile Lys Leu 805 810 815 Gly Leu Gly Ile Asp Glu Asp Asp Pro Thr
Ala Asp Asp Thr Ser Ala 820 825 830 Ala Val Thr Glu Glu Met Pro Pro
Leu Glu Gly Asp Asp Asp Thr Ser 835 840 845 Arg Met Glu Glu Val Asp
850 441192PRTHomo sapiens 44Met Glu Asp Leu Asp Gln Ser Pro Leu Val
Ser Ser Ser Asp Ser Pro1 5 10 15 Pro Arg Pro Gln Pro Ala Phe Lys
Tyr Gln Phe Val Arg Glu Pro Glu 20 25 30 Asp Glu Glu Glu Glu Glu
Glu Glu Glu Glu Glu Asp Glu Asp Glu Asp 35 40 45 Leu Glu Glu Leu
Glu Val Leu Glu Arg Lys Pro Ala Ala Gly Leu Ser 50 55 60 Ala Ala
Pro Val Pro Thr Ala Pro Ala Ala Gly Ala Pro Leu Met Asp65 70 75 80
Phe Gly Asn Asp Phe Val Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala 85
90 95 Ala Pro Pro Val Ala Pro Glu Arg Gln Pro Ser Trp Asp Pro Ser
Pro 100 105 110 Val Ser Ser Thr Val Pro Ala Pro Ser Pro Leu Ser Ala
Ala Ala Val 115 120 125 Ser Pro Ser Lys Leu Pro Glu Asp Asp Glu Pro
Pro Ala Arg Pro Pro 130 135 140 Pro Pro Pro Pro Ala Ser Val Ser Pro
Gln Ala Glu Pro Val Trp Thr145 150 155 160 Pro Pro Ala Pro Ala Pro
Ala Ala Pro Pro Ser Thr Pro Ala Ala Pro 165 170 175 Lys Arg Arg Gly
Ser Ser Gly Ser Val Asp Glu Thr Leu Phe Ala Leu 180 185 190 Pro Ala
Ala Ser Glu Pro Val Ile Arg Ser Ser Ala Glu Asn Met Asp 195 200 205
Leu Lys Glu Gln Pro Gly Asn Thr Ile Ser Ala Gly Gln Glu Asp Phe 210
215 220 Pro Ser Val Leu Leu Glu Thr Ala Ala Ser Leu Pro Ser Leu Ser
Pro225 230 235 240 Leu Ser Ala Ala Ser Phe Lys Glu His Glu Tyr Leu
Gly Asn Leu Ser 245 250 255 Thr Val Leu Pro Thr Glu Gly Thr Leu Gln
Glu Asn Val Ser Glu Ala 260 265 270 Ser Lys Glu Val Ser Glu Lys Ala
Lys Thr Leu Leu Ile Asp Arg Asp 275 280 285 Leu Thr Glu Phe Ser Glu
Leu Glu Tyr Ser Glu Met Gly Ser Ser Phe 290 295 300 Ser Val Ser Pro
Lys Ala Glu Ser Ala Val Ile Val Ala Asn Pro Arg305 310 315 320 Glu
Glu Ile Ile Val Lys Asn Lys Asp Glu Glu Glu Lys Leu Val Ser 325 330
335 Asn Asn Ile Leu His Asn Gln Gln Glu Leu Pro Thr Ala Leu Thr Lys
340 345 350 Leu Val Lys Glu Asp Glu Val Val Ser Ser Glu Lys Ala Lys
Asp Ser 355 360 365 Phe Asn Glu Lys Arg Val Ala Val Glu Ala Pro Met
Arg Glu Glu Tyr 370 375 380 Ala Asp Phe Lys Pro Phe Glu Arg Val Trp
Glu Val Lys Asp Ser Lys385 390 395 400 Glu Asp Ser Asp Met Leu Ala
Ala Gly Gly Lys Ile Glu Ser Asn Leu 405 410 415 Glu Ser Lys Val Asp
Lys Lys Cys Phe Ala Asp Ser Leu Glu Gln Thr 420 425 430 Asn His Glu
Lys Asp Ser Glu Ser Ser Asn Asp Asp Thr Ser Phe Pro 435 440 445 Ser
Thr Pro Glu Gly Ile Lys Asp Arg Ser Gly Ala Tyr Ile Thr Cys 450 455
460 Ala Pro Phe Asn Pro Ala Ala Thr Glu Ser Ile Ala Thr Asn Ile
Phe465 470 475 480 Pro Leu Leu Gly Asp Pro Thr Ser Glu Asn Lys Thr
Asp Glu Lys Lys 485 490 495 Ile Glu Glu Lys Lys Ala Gln Ile Val Thr
Glu Lys Asn Thr Ser Thr 500 505 510 Lys Thr Ser Asn Pro Phe Leu Val
Ala Ala Gln Asp Ser Glu Thr Asp 515 520 525 Tyr Val Thr Thr Asp Asn
Leu Thr Lys Val Thr Glu Glu Val Val Ala 530 535 540 Asn Met Pro Glu
Gly Leu Thr Pro Asp Leu Val Gln Glu Ala Cys Glu545 550 555 560 Ser
Glu Leu Asn Glu Val Thr Gly Thr Lys Ile Ala Tyr Glu Thr Lys 565 570
575 Met Asp Leu Val Gln Thr Ser Glu Val Met Gln Glu Ser Leu Tyr Pro
580 585 590 Ala Ala Gln Leu Cys Pro Ser Phe Glu Glu Ser Glu Ala Thr
Pro Ser 595 600 605 Pro Val Leu Pro Asp Ile Val Met Glu Ala Pro Leu
Asn Ser Ala Val 610 615 620 Pro Ser Ala Gly Ala Ser Val Ile Gln Pro
Ser Ser Ser Pro Leu Glu625 630 635 640 Ala Ser Ser Val Asn Tyr Glu
Ser Ile Lys His Glu Pro Glu Asn Pro 645 650 655 Pro Pro Tyr Glu Glu
Ala Met Ser Val Ser Leu Lys Lys Val Ser Gly 660 665 670 Ile Lys Glu
Glu Ile Lys Glu Pro Glu Asn Ile Asn Ala Ala Leu Gln 675 680 685 Glu
Thr Glu Ala Pro Tyr Ile Ser Ile Ala Cys Asp Leu Ile Lys Glu 690 695
700 Thr Lys Leu Ser Ala Glu Pro Ala Pro Asp Phe Ser Asp Tyr Ser
Glu705 710 715 720 Met Ala Lys Val Glu Gln Pro Val Pro Asp His Ser
Glu Leu Val Glu 725 730 735 Asp Ser Ser Pro Asp Ser Glu Pro Val Asp
Leu Phe Ser Asp Asp Ser 740 745 750 Ile Pro Asp Val Pro Gln Lys Gln
Asp Glu Thr Val Met Leu Val Lys 755 760 765 Glu Ser Leu Thr Glu Thr
Ser Phe Glu Ser Met Ile Glu Tyr Glu Asn 770 775 780 Lys Glu Lys Leu
Ser Ala Leu Pro Pro Glu Gly Gly Lys Pro Tyr Leu785 790 795 800 Glu
Ser Phe Lys Leu Ser Leu Asp Asn Thr Lys Asp Thr Leu Leu Pro 805 810
815 Asp Glu Val Ser Thr Leu Ser Lys Lys Glu Lys Ile Pro Leu Gln Met
820 825 830 Glu Glu Leu Ser Thr Ala Val Tyr Ser Asn Asp Asp Leu Phe
Ile Ser 835 840 845 Lys Glu Ala Gln Ile Arg Glu Thr Glu Thr Phe Ser
Asp Ser Ser Pro 850 855 860 Ile Glu Ile Ile Asp Glu Phe Pro Thr Leu
Ile Ser Ser Lys Thr Asp865 870 875 880 Ser Phe Ser Lys Leu Ala Arg
Glu Tyr Thr Asp Leu Glu Val Ser His 885 890 895 Lys Ser Glu Ile Ala
Asn Ala Pro Asp Gly Ala Gly Ser Leu Pro Cys 900 905 910 Thr Glu Leu
Pro His Asp Leu Ser Leu Lys Asn Ile Gln Pro Lys Val 915 920 925 Glu
Glu Lys Ile Ser Phe Ser Asp Asp Phe Ser Lys Asn Gly Ser Ala 930 935
940 Thr Ser Lys Val Leu Leu Leu Pro Pro Asp Val Ser Ala Leu Ala
Thr945 950 955 960 Gln Ala Glu Ile Glu Ser Ile Val Lys Pro Lys Val
Leu Val Lys Glu 965 970 975 Ala Glu Lys Lys Leu Pro Ser Asp Thr Glu
Lys Glu Asp Arg Ser Pro 980 985 990 Ser Ala Ile Phe Ser Ala Glu Leu
Ser Lys Thr Ser Val Val Asp Leu 995 1000 1005 Leu Tyr Trp Arg Asp
Ile Lys Lys Thr Gly Val Val Phe Gly Ala Ser 1010 1015 1020 Leu Phe
Leu Leu Leu Ser Leu Thr Val Phe Ser Ile Val Ser Val Thr1025 1030
1035 1040Ala Tyr Ile Ala Leu Ala Leu Leu Ser Val Thr Ile Ser Phe
Arg Ile 1045 1050 1055 Tyr Lys Gly Val Ile Gln Ala Ile Gln Lys Ser
Asp Glu Gly His Pro 1060 1065 1070 Phe Arg Ala Tyr Leu Glu Ser Glu
Val Ala Ile Ser Glu Glu Leu Val 1075 1080 1085 Gln Lys Tyr Ser Asn
Ser Ala Leu Gly His Val Asn Cys Thr Ile Lys 1090 1095 1100 Glu Leu
Arg Arg Leu Phe Leu Val Asp Asp Leu Val Asp Ser Leu Lys1105 1110
1115 1120Phe Ala Val Leu Met Trp Val Phe Thr Tyr Val Gly Ala Leu
Phe Asn 1125 1130 1135 Gly Leu Thr Leu Leu Ile Leu Ala Leu Ile Ser
Leu Phe Ser Val Pro 1140 1145 1150 Val Ile Tyr Glu Arg His Gln Ala
Gln Ile Asp His Tyr Leu Gly Leu 1155 1160 1165 Ala Asn Lys Asn Val
Lys Asp Ala Met Ala Lys Ile Gln Ala Lys Ile 1170 1175 1180 Pro Gly
Leu Lys Arg Lys Ala Glu1185 1190 45364PRTHomo sapiens 45Met Pro Tyr
Gln Tyr Pro Ala Leu Thr Pro Glu Gln Lys Lys Glu Leu1 5 10 15 Ser
Asp Ile Ala His Arg Ile Val Ala Pro Gly Lys Gly Ile Leu Ala 20 25
30 Ala Asp Glu Ser Thr Gly Ser Ile Ala Lys Arg Leu Gln Ser Ile Gly
35 40 45 Thr Glu Asn Thr Glu Glu Asn Arg Arg Phe Tyr Arg Gln Leu
Leu Leu 50 55 60 Thr Ala Asp Asp Arg Val Asn Pro Cys Ile Gly Gly
Val Ile Leu Phe65 70 75 80 His Glu Thr Leu Tyr Gln Lys Ala Asp Asp
Gly Arg Pro Phe Pro Gln 85 90 95 Val Ile Lys Ser Lys Gly Gly Val
Val Gly Ile Lys Val Asp Lys Gly 100 105 110 Val Val Pro Leu Ala Gly
Thr Asn Gly Glu Thr Thr Thr Gln Gly Leu 115 120 125 Asp Gly Leu Ser
Glu Arg Cys Ala Gln Tyr Lys Lys Asp Gly Ala Asp 130 135 140 Phe Ala
Lys Trp Arg Cys Val Leu Lys Ile Gly Glu His Thr Pro Ser145 150 155
160 Ala Leu Ala Ile Met Glu Asn Ala Asn Val Leu Ala Arg Tyr Ala Ser
165 170 175 Ile Cys Gln Gln Asn Gly Ile Val Pro Ile Val Glu Pro Glu
Ile Leu 180 185 190 Pro Asp Gly Asp His Asp Leu Lys Arg Cys Gln Tyr
Val Thr Glu Lys 195 200 205 Val Leu Ala Ala Val Tyr Lys Ala Leu Ser
Asp His His Ile Tyr Leu 210 215 220 Glu Gly Thr Leu Leu Lys Pro Asn
Met Val Thr Pro Gly His Ala Cys225 230 235 240 Thr Gln Lys Phe Ser
His Glu Glu Ile Ala Met Ala Thr Val Thr Ala 245 250 255 Leu Arg Arg
Thr Val Pro Pro Ala Val Thr Gly Ile Thr Phe Leu Ser 260 265 270 Gly
Gly Gln Ser Glu Glu Glu Ala Ser Ile Asn Leu Asn Ala Ile Asn 275 280
285 Lys Cys Pro Leu Leu Lys Pro Trp Ala Leu Thr Phe Ser Tyr Gly Arg
290 295 300 Ala Leu Gln Ala Ser Ala Leu Lys Ala Trp Gly Gly Lys Lys
Glu Asn305 310 315 320 Leu Lys Ala Ala Gln Glu Glu Tyr Val Lys Arg
Ala Leu Ala Asn Ser 325 330 335 Leu Ala Cys Gln Gly Lys Tyr Thr Pro
Ser Gly Gln Ala Gly Ala Ala 340 345 350 Ala Ser Glu Ser Leu Phe Val
Ser Asn His Ala Tyr 355 360 461203PRTHomo sapiens 46Met Ala Ala Cys
Gly Arg Val Arg Arg Met Phe Arg Leu Ser Ala Ala1 5 10 15 Leu His
Leu Leu Leu Leu Phe Ala Ala Gly Ala Glu Lys Leu Pro Gly 20 25 30
Gln Gly Val His Ser Gln Gly Gln Gly Pro Gly Ala Asn Phe Val Ser 35
40 45 Phe Val Gly Gln Ala Gly Gly Gly Gly Pro Ala Gly Gln Gln Leu
Pro 50 55 60 Gln Leu Pro Gln Ser Ser Gln Leu Gln Gln Gln
Gln Gln Gln Gln Gln65 70 75 80 Gln Gln Gln Gln Pro Gln Pro Pro Gln
Pro Pro Phe Pro Ala Gly Gly 85 90 95 Pro Pro Ala Arg Arg Gly Gly
Ala Gly Ala Gly Gly Gly Trp Lys Leu 100 105 110 Ala Glu Glu Glu Ser
Cys Arg Glu Asp Val Thr Arg Val Cys Pro Lys 115 120 125 His Thr Trp
Ser Asn Asn Leu Ala Val Leu Glu Cys Leu Gln Asp Val 130 135 140 Arg
Glu Pro Glu Asn Glu Ile Ser Ser Asp Cys Asn His Leu Leu Trp145 150
155 160 Asn Tyr Lys Leu Asn Leu Thr Thr Asp Pro Lys Phe Glu Ser Val
Ala 165 170 175 Arg Glu Val Cys Lys Ser Thr Ile Thr Glu Ile Lys Glu
Cys Ala Asp 180 185 190 Glu Pro Val Gly Lys Gly Tyr Met Val Ser Cys
Leu Val Asp His Arg 195 200 205 Gly Asn Ile Thr Glu Tyr Gln Cys His
Gln Tyr Ile Thr Lys Met Thr 210 215 220 Ala Ile Ile Phe Ser Asp Tyr
Arg Leu Ile Cys Gly Phe Met Asp Asp225 230 235 240 Cys Lys Asn Asp
Ile Asn Ile Leu Lys Cys Gly Ser Ile Arg Leu Gly 245 250 255 Glu Lys
Asp Ala His Ser Gln Gly Glu Val Val Ser Cys Leu Glu Lys 260 265 270
Gly Leu Val Lys Glu Ala Glu Glu Arg Glu Pro Lys Ile Gln Val Ser 275
280 285 Glu Leu Cys Lys Lys Ala Ile Leu Arg Val Ala Glu Leu Ser Ser
Asp 290 295 300 Asp Phe His Leu Asp Arg His Leu Tyr Phe Ala Cys Arg
Asp Asp Arg305 310 315 320 Glu Arg Phe Cys Glu Asn Thr Gln Ala Gly
Glu Gly Arg Val Tyr Lys 325 330 335 Cys Leu Phe Asn His Lys Phe Glu
Glu Ser Met Ser Glu Lys Cys Arg 340 345 350 Glu Ala Leu Thr Thr Arg
Gln Lys Leu Ile Ala Gln Asp Tyr Lys Val 355 360 365 Ser Tyr Ser Leu
Ala Lys Ser Cys Lys Ser Asp Leu Lys Lys Tyr Arg 370 375 380 Cys Asn
Val Glu Asn Leu Pro Arg Ser Arg Glu Ala Arg Leu Ser Tyr385 390 395
400 Leu Leu Met Cys Leu Glu Ser Ala Val His Arg Gly Arg Gln Val Ser
405 410 415 Ser Glu Cys Gln Gly Glu Met Leu Asp Tyr Arg Arg Met Leu
Met Glu 420 425 430 Asp Phe Ser Leu Ser Pro Glu Ile Ile Leu Ser Cys
Arg Gly Glu Ile 435 440 445 Glu His His Cys Ser Gly Leu His Arg Lys
Gly Arg Thr Leu His Cys 450 455 460 Leu Met Lys Val Val Arg Gly Glu
Lys Gly Asn Leu Gly Met Asn Cys465 470 475 480 Gln Gln Ala Leu Gln
Thr Leu Ile Gln Glu Thr Asp Pro Gly Ala Asp 485 490 495 Tyr Arg Ile
Asp Arg Ala Leu Asn Glu Ala Cys Glu Ser Val Ile Gln 500 505 510 Thr
Ala Cys Lys His Ile Arg Ser Gly Asp Pro Met Ile Leu Ser Cys 515 520
525 Leu Met Glu His Leu Tyr Thr Glu Lys Met Val Glu Asp Cys Glu His
530 535 540 Arg Leu Leu Glu Leu Gln Tyr Phe Ile Ser Arg Asp Trp Lys
Leu Asp545 550 555 560 Pro Val Leu Tyr Arg Lys Cys Gln Gly Asp Ala
Ser Arg Leu Cys His 565 570 575 Thr His Gly Trp Asn Glu Thr Ser Glu
Phe Met Pro Gln Gly Ala Val 580 585 590 Phe Ser Cys Leu Tyr Arg His
Ala Tyr Arg Thr Glu Glu Gln Gly Arg 595 600 605 Arg Leu Ser Arg Glu
Cys Arg Ala Glu Val Gln Arg Ile Leu His Gln 610 615 620 Arg Ala Met
Asp Val Lys Leu Asp Pro Ala Leu Gln Asp Lys Cys Leu625 630 635 640
Ile Asp Leu Gly Lys Trp Cys Ser Glu Lys Thr Glu Thr Gly Gln Glu 645
650 655 Leu Glu Cys Leu Gln Asp His Leu Asp Asp Leu Val Val Glu Cys
Arg 660 665 670 Asp Ile Val Gly Asn Leu Thr Glu Leu Glu Ser Glu Asp
Ile Gln Ile 675 680 685 Glu Ala Leu Leu Met Arg Ala Cys Glu Pro Ile
Ile Gln Asn Phe Cys 690 695 700 His Asp Val Ala Asp Asn Gln Ile Asp
Ser Gly Asp Leu Met Glu Cys705 710 715 720 Leu Ile Gln Asn Lys His
Gln Lys Asp Met Asn Glu Lys Cys Ala Ile 725 730 735 Gly Val Thr His
Phe Gln Leu Val Gln Met Lys Asp Phe Arg Phe Ser 740 745 750 Tyr Lys
Phe Lys Met Ala Cys Lys Glu Asp Val Leu Lys Leu Cys Pro 755 760 765
Asn Ile Lys Lys Lys Val Asp Val Val Ile Cys Leu Ser Thr Thr Val 770
775 780 Arg Asn Asp Thr Leu Gln Glu Ala Lys Glu His Arg Val Ser Leu
Lys785 790 795 800 Cys Arg Arg Gln Leu Arg Val Glu Glu Leu Glu Met
Thr Glu Asp Ile 805 810 815 Arg Leu Glu Pro Asp Leu Tyr Glu Ala Cys
Lys Ser Asp Ile Lys Asn 820 825 830 Phe Cys Ser Ala Val Gln Tyr Gly
Asn Ala Gln Ile Ile Glu Cys Leu 835 840 845 Lys Glu Asn Lys Lys Gln
Leu Ser Thr Arg Cys His Gln Lys Val Phe 850 855 860 Lys Leu Gln Glu
Thr Glu Met Met Asp Pro Glu Leu Asp Tyr Thr Leu865 870 875 880 Met
Arg Val Cys Lys Gln Met Ile Lys Arg Phe Cys Pro Glu Ala Asp 885 890
895 Ser Lys Thr Met Leu Gln Cys Leu Lys Gln Asn Lys Asn Ser Glu Leu
900 905 910 Met Asp Pro Lys Cys Lys Gln Met Ile Thr Lys Arg Gln Ile
Thr Gln 915 920 925 Asn Thr Asp Tyr Arg Leu Asn Pro Met Leu Arg Lys
Ala Cys Lys Ala 930 935 940 Asp Ile Pro Lys Phe Cys His Gly Ile Leu
Thr Lys Ala Lys Asp Asp945 950 955 960 Ser Glu Leu Glu Gly Gln Val
Ile Ser Cys Leu Lys Leu Arg Tyr Ala 965 970 975 Asp Gln Arg Leu Ser
Ser Asp Cys Glu Asp Gln Ile Arg Ile Ile Ile 980 985 990 Gln Glu Ser
Ala Leu Asp Tyr Arg Leu Asp Pro Gln Leu Gln Leu His 995 1000 1005
Cys Ser Asp Glu Ile Ser Ser Leu Cys Ala Glu Glu Ala Ala Ala Gln
1010 1015 1020 Glu Gln Thr Gly Gln Val Glu Glu Cys Leu Lys Val Asn
Leu Leu Lys1025 1030 1035 1040Ile Lys Thr Glu Leu Cys Lys Lys Glu
Val Leu Asn Met Leu Lys Glu 1045 1050 1055 Ser Lys Ala Asp Ile Phe
Val Asp Pro Val Leu His Thr Ala Cys Ala 1060 1065 1070 Leu Asp Ile
Lys His His Cys Ala Ala Ile Thr Pro Gly Arg Gly Arg 1075 1080 1085
Gln Met Ser Cys Leu Met Glu Ala Leu Glu Asp Lys Arg Val Arg Leu
1090 1095 1100 Gln Pro Glu Cys Lys Lys Arg Leu Asn Asp Arg Ile Glu
Met Trp Ser1105 1110 1115 1120Tyr Ala Ala Lys Val Ala Pro Ala Asp
Gly Phe Ser Asp Leu Ala Met 1125 1130 1135 Gln Val Met Thr Ser Pro
Ser Lys Asn Tyr Ile Leu Ser Val Ile Ser 1140 1145 1150 Gly Ser Ile
Cys Ile Leu Phe Leu Ile Gly Leu Met Cys Gly Arg Ile 1155 1160 1165
Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg Leu Gln Tyr Arg Ser
1170 1175 1180 Glu Thr Met Ala Tyr Lys Gly Leu Val Trp Ser Gln Asp
Val Thr Gly1185 1190 1195 1200Ser Pro Ala471070PRTHomo sapiens
47Met Gly Ala Ala Arg Gly Ser Pro Ala Arg Pro Arg Arg Leu Pro Leu1
5 10 15 Leu Ser Val Leu Leu Leu Pro Leu Leu Gly Gly Thr Gln Thr Ala
Ile 20 25 30 Val Phe Ile Lys Gln Pro Ser Ser Gln Asp Ala Leu Gln
Gly Arg Arg 35 40 45 Ala Leu Leu Arg Cys Glu Val Glu Ala Pro Gly
Pro Val His Val Tyr 50 55 60 Trp Leu Leu Asp Gly Ala Pro Val Gln
Asp Thr Glu Arg Arg Phe Ala65 70 75 80 Gln Gly Ser Ser Leu Ser Phe
Ala Ala Val Asp Arg Leu Gln Asp Ser 85 90 95 Gly Thr Phe Gln Cys
Val Ala Arg Asp Asp Val Thr Gly Glu Glu Ala 100 105 110 Arg Ser Ala
Asn Ala Ser Phe Asn Ile Lys Trp Ile Glu Ala Gly Pro 115 120 125 Val
Val Leu Lys His Pro Ala Ser Glu Ala Glu Ile Gln Pro Gln Thr 130 135
140 Gln Val Thr Leu Arg Cys His Ile Asp Gly His Pro Arg Pro Thr
Tyr145 150 155 160 Gln Trp Phe Arg Asp Gly Thr Pro Leu Ser Asp Gly
Gln Ser Asn His 165 170 175 Thr Val Ser Ser Lys Glu Arg Asn Leu Thr
Leu Arg Pro Ala Gly Pro 180 185 190 Glu His Ser Gly Leu Tyr Ser Cys
Cys Ala His Ser Ala Phe Gly Gln 195 200 205 Ala Cys Ser Ser Gln Asn
Phe Thr Leu Ser Ile Ala Asp Glu Ser Phe 210 215 220 Ala Arg Val Val
Leu Ala Pro Gln Asp Val Val Val Ala Arg Tyr Glu225 230 235 240 Glu
Ala Met Phe His Cys Gln Phe Ser Ala Gln Pro Pro Pro Ser Leu 245 250
255 Gln Trp Leu Phe Glu Asp Glu Thr Pro Ile Thr Asn Arg Ser Arg Pro
260 265 270 Pro His Leu Arg Arg Ala Thr Val Phe Ala Asn Gly Ser Leu
Leu Leu 275 280 285 Thr Gln Val Arg Pro Arg Asn Ala Gly Ile Tyr Arg
Cys Ile Gly Gln 290 295 300 Gly Gln Arg Gly Pro Pro Ile Ile Leu Glu
Ala Thr Leu His Leu Ala305 310 315 320 Glu Ile Glu Asp Met Pro Leu
Phe Glu Pro Arg Val Phe Thr Ala Gly 325 330 335 Ser Glu Glu Arg Val
Thr Cys Leu Pro Pro Lys Gly Leu Pro Glu Pro 340 345 350 Ser Val Trp
Trp Glu His Ala Gly Val Arg Leu Pro Thr His Gly Arg 355 360 365 Val
Tyr Gln Lys Gly His Glu Leu Val Leu Ala Asn Ile Ala Glu Ser 370 375
380 Asp Ala Gly Val Tyr Thr Cys His Ala Ala Asn Leu Ala Gly Gln
Arg385 390 395 400 Arg Gln Asp Val Asn Ile Thr Val Ala Thr Val Pro
Ser Trp Leu Lys 405 410 415 Lys Pro Gln Asp Ser Gln Leu Glu Glu Gly
Lys Pro Gly Tyr Leu Asp 420 425 430 Cys Leu Thr Gln Ala Thr Pro Lys
Pro Thr Val Val Trp Tyr Arg Asn 435 440 445 Gln Met Leu Ile Ser Glu
Asp Ser Arg Phe Glu Val Phe Lys Asn Gly 450 455 460 Thr Leu Arg Ile
Asn Ser Val Glu Val Tyr Asp Gly Thr Trp Tyr Arg465 470 475 480 Cys
Met Ser Ser Thr Pro Ala Gly Ser Ile Glu Ala Gln Ala Arg Val 485 490
495 Gln Val Leu Glu Lys Leu Lys Phe Thr Pro Pro Pro Gln Pro Gln Gln
500 505 510 Cys Met Glu Phe Asp Lys Glu Ala Thr Val Pro Cys Ser Ala
Thr Gly 515 520 525 Arg Glu Lys Pro Thr Ile Lys Trp Glu Arg Ala Asp
Gly Ser Ser Leu 530 535 540 Pro Glu Trp Val Thr Asp Asn Ala Gly Thr
Leu His Phe Ala Arg Val545 550 555 560 Thr Arg Asp Asp Ala Gly Asn
Tyr Thr Cys Ile Ala Ser Asn Gly Pro 565 570 575 Gln Gly Gln Ile Arg
Ala His Val Gln Leu Thr Val Ala Val Phe Ile 580 585 590 Thr Phe Lys
Val Glu Pro Glu Arg Thr Thr Val Tyr Gln Gly His Thr 595 600 605 Ala
Leu Leu Gln Cys Glu Ala Gln Gly Asp Pro Lys Pro Leu Ile Gln 610 615
620 Trp Lys Gly Lys Asp Arg Ile Leu Asp Pro Thr Lys Leu Gly Pro
Arg625 630 635 640 Met His Ile Phe Gln Asn Gly Ser Leu Val Ile His
Asp Val Ala Pro 645 650 655 Glu Asp Ser Gly Arg Tyr Thr Cys Ile Ala
Gly Asn Ser Cys Asn Ile 660 665 670 Lys His Thr Glu Ala Pro Leu Tyr
Val Val Asp Lys Pro Val Pro Glu 675 680 685 Glu Ser Glu Gly Pro Gly
Ser Pro Pro Pro Tyr Lys Met Ile Gln Thr 690 695 700 Ile Gly Leu Ser
Val Gly Ala Ala Val Ala Tyr Ile Ile Ala Val Leu705 710 715 720 Gly
Leu Met Phe Tyr Cys Lys Lys Arg Cys Lys Ala Lys Arg Leu Gln 725 730
735 Lys Gln Pro Glu Gly Glu Glu Pro Glu Met Glu Cys Leu Asn Gly Gly
740 745 750 Pro Leu Gln Asn Gly Gln Pro Ser Ala Glu Ile Gln Glu Glu
Val Ala 755 760 765 Leu Thr Ser Leu Gly Ser Gly Pro Ala Ala Thr Asn
Lys Arg His Ser 770 775 780 Thr Ser Asp Lys Met His Phe Pro Arg Ser
Ser Leu Gln Pro Ile Thr785 790 795 800 Thr Leu Gly Lys Ser Glu Phe
Gly Glu Val Phe Leu Ala Lys Ala Gln 805 810 815 Gly Leu Glu Glu Gly
Val Ala Glu Thr Leu Val Leu Val Lys Ser Leu 820 825 830 Gln Ser Lys
Asp Glu Gln Gln Gln Leu Asp Phe Arg Arg Glu Leu Glu 835 840 845 Met
Phe Gly Lys Leu Asn His Ala Asn Val Val Arg Leu Leu Gly Leu 850 855
860 Cys Arg Glu Ala Glu Pro His Tyr Met Val Leu Glu Tyr Val Asp
Leu865 870 875 880 Gly Asp Leu Lys Gln Phe Leu Arg Ile Ser Lys Ser
Lys Asp Glu Lys 885 890 895 Leu Lys Ser Gln Pro Leu Ser Thr Lys Gln
Lys Val Ala Leu Cys Thr 900 905 910 Gln Val Ala Leu Gly Met Glu His
Leu Ser Asn Asn Arg Phe Val His 915 920 925 Lys Asp Leu Ala Ala Arg
Asn Cys Leu Val Ser Ala Gln Arg Gln Val 930 935 940 Lys Val Ser Ala
Leu Gly Leu Ser Lys Asp Val Tyr Asn Ser Glu Tyr945 950 955 960 Tyr
His Phe Arg Gln Ala Trp Val Pro Leu Arg Trp Met Ser Pro Glu 965 970
975 Ala Ile Leu Glu Gly Asp Phe Ser Thr Lys Ser Asp Val Trp Ala Phe
980 985 990 Gly Val Leu Met Trp Glu Val Phe Thr His Gly Glu Met Pro
His Gly 995 1000 1005 Gly Gln Ala Asp Asp Glu Val Leu Ala Asp Leu
Gln Ala Gly Lys Ala 1010 1015 1020 Arg Leu Pro Gln Pro Glu Gly Cys
Pro Ser Lys Leu Tyr Arg Leu Met1025 1030 1035 1040Gln Arg Cys Trp
Ala Leu Ser Pro Lys Asp Arg Pro Ser Phe Ser Glu 1045 1050 1055 Ile
Ala Ser Ala Leu Gly Asp Ser Thr Val Asp Ser Lys Pro 1060 1065
107048493PRTHomo sapiens 48Met Leu Lys Ala Leu Phe Leu Thr Met Leu
Thr Leu Ala Leu Val Lys1 5 10 15 Ser Gln Asp Thr Glu Glu Thr Ile
Thr Tyr Thr Gln Cys Thr Asp Gly 20 25 30 Tyr Glu Trp Asp Pro Val
Arg Gln Gln Cys Lys Asp Ile Asp Glu Cys 35 40 45 Asp Ile Val Pro
Asp Ala Cys Lys Gly Gly Met Lys Cys Val Asn His 50 55 60 Tyr Gly
Gly Tyr Leu Cys Leu Pro Lys Thr Ala Gln Ile Ile Val Asn65 70 75 80
Asn Glu Gln Pro Gln Gln Glu Thr Gln Pro Ala Glu Gly Thr Ser Gly 85
90 95 Ala Thr Thr Gly Val Val Ala Ala Ser Ser Met Ala Thr Ser Gly
Val 100
105 110 Leu Pro Gly Gly Gly Phe Val Ala Ser Ala Ala Ala Val Ala Gly
Pro 115 120 125 Glu Met Gln Thr Gly Arg Asn Asn Phe Val Ile Arg Arg
Asn Pro Ala 130 135 140 Asp Pro Gln Arg Ile Pro Ser Asn Pro Ser His
Arg Ile Gln Cys Ala145 150 155 160 Ala Gly Tyr Glu Gln Ser Glu His
Asn Val Cys Gln Asp Ile Asp Glu 165 170 175 Cys Thr Ala Gly Thr His
Asn Cys Arg Ala Asp Gln Val Cys Ile Asn 180 185 190 Leu Arg Gly Ser
Phe Ala Cys Gln Cys Pro Pro Gly Tyr Gln Lys Arg 195 200 205 Gly Glu
Gln Cys Val Asp Ile Asp Glu Cys Thr Ile Pro Pro Tyr Cys 210 215 220
His Gln Arg Cys Val Asn Thr Pro Gly Ser Phe Tyr Cys Gln Cys Ser225
230 235 240 Pro Gly Phe Gln Leu Ala Ala Asn Asn Tyr Thr Cys Val Asp
Ile Asn 245 250 255 Glu Cys Asp Ala Ser Asn Gln Cys Ala Gln Gln Cys
Tyr Asn Ile Leu 260 265 270 Gly Ser Phe Ile Cys Gln Cys Asn Gln Gly
Tyr Glu Leu Ser Ser Asp 275 280 285 Arg Leu Asn Cys Glu Asp Ile Asp
Glu Cys Arg Thr Ser Ser Tyr Leu 290 295 300 Cys Gln Tyr Gln Cys Val
Asn Glu Pro Gly Lys Phe Ser Cys Met Cys305 310 315 320 Pro Gln Gly
Tyr Gln Val Val Arg Ser Arg Thr Cys Gln Asp Ile Asn 325 330 335 Glu
Cys Glu Thr Thr Asn Glu Cys Arg Glu Asp Glu Met Cys Trp Asn 340 345
350 Tyr His Gly Gly Phe Arg Cys Tyr Pro Arg Asn Pro Cys Gln Asp Pro
355 360 365 Tyr Ile Leu Thr Pro Glu Asn Arg Cys Val Cys Pro Val Ser
Asn Ala 370 375 380 Met Cys Arg Glu Leu Pro Gln Ser Ile Val Tyr Lys
Tyr Met Ser Ile385 390 395 400 Arg Ser Asp Arg Ser Val Pro Ser Asp
Ile Phe Gln Ile Gln Ala Thr 405 410 415 Thr Ile Tyr Ala Asn Thr Ile
Asn Thr Phe Arg Ile Lys Ser Gly Asn 420 425 430 Glu Asn Gly Glu Phe
Tyr Leu Arg Gln Thr Ser Pro Val Ser Ala Met 435 440 445 Leu Val Leu
Val Lys Ser Leu Ser Gly Pro Arg Glu His Ile Val Asp 450 455 460 Leu
Glu Met Leu Thr Val Ser Ser Ile Gly Thr Phe Arg Thr Ser Ser465 470
475 480 Val Leu Arg Leu Thr Ile Ile Val Gly Pro Phe Ser Phe 485 490
49661PRTHomo sapiens 49Met Glu Leu Gln Pro Pro Glu Ala Ser Ile Ala
Val Val Ser Ile Pro1 5 10 15 Arg Gln Leu Pro Gly Ser His Ser Glu
Ala Gly Val Gln Gly Leu Ser 20 25 30 Ala Gly Asp Asp Ser Glu Leu
Gly Ser His Cys Val Ala Gln Thr Gly 35 40 45 Leu Glu Leu Leu Ala
Ser Gly Asp Pro Leu Pro Ser Ala Ser Gln Asn 50 55 60 Ala Glu Met
Ile Glu Thr Gly Ser Asp Cys Val Thr Gln Ala Gly Leu65 70 75 80 Gln
Leu Leu Ala Ser Ser Asp Pro Pro Ala Leu Ala Ser Lys Asn Ala 85 90
95 Glu Val Thr Glu Thr Gly Phe His His Val Ser Gln Ala Asp Ile Glu
100 105 110 Phe Leu Thr Ser Ile Asp Pro Thr Ala Ser Ala Ser Gly Ser
Ala Gly 115 120 125 Ile Thr Gly Thr Met Ser Gln Asp Thr Glu Val Asp
Met Lys Glu Val 130 135 140 Glu Leu Asn Glu Leu Glu Pro Glu Lys Gln
Pro Met Asn Ala Ala Ser145 150 155 160 Gly Ala Ala Met Ser Leu Ala
Gly Ala Glu Lys Asn Gly Leu Val Lys 165 170 175 Ile Lys Val Ala Glu
Asp Glu Ala Glu Ala Ala Ala Ala Ala Lys Phe 180 185 190 Thr Gly Leu
Ser Lys Glu Glu Leu Leu Lys Val Ala Gly Ser Pro Gly 195 200 205 Trp
Val Arg Thr Arg Trp Ala Leu Leu Leu Leu Phe Trp Leu Gly Trp 210 215
220 Leu Gly Met Leu Ala Gly Ala Val Val Ile Ile Val Arg Ala Pro
Arg225 230 235 240 Cys Arg Glu Leu Pro Ala Gln Lys Trp Trp His Thr
Gly Ala Leu Tyr 245 250 255 Arg Ile Gly Asp Leu Gln Ala Phe Gln Gly
His Gly Ala Gly Asn Leu 260 265 270 Ala Gly Leu Lys Gly Arg Leu Asp
Tyr Leu Ser Ser Leu Lys Val Lys 275 280 285 Gly Leu Val Leu Gly Pro
Ile His Lys Asn Gln Lys Asp Asp Val Ala 290 295 300 Gln Thr Asp Leu
Leu Gln Ile Asp Pro Asn Phe Gly Ser Lys Glu Asp305 310 315 320 Phe
Asp Ser Leu Leu Gln Ser Ala Lys Lys Lys Ser Ile Arg Val Ile 325 330
335 Leu Asp Leu Thr Pro Asn Tyr Arg Gly Glu Asn Ser Trp Phe Ser Thr
340 345 350 Gln Val Asp Thr Val Ala Thr Lys Val Lys Asp Ala Leu Glu
Phe Trp 355 360 365 Leu Gln Ala Gly Val Asp Gly Phe Gln Val Arg Asp
Ile Glu Asn Leu 370 375 380 Lys Asp Ala Ser Ser Phe Leu Ala Glu Trp
Gln Asn Ile Thr Lys Gly385 390 395 400 Phe Ser Glu Asp Arg Leu Leu
Ile Ala Gly Thr Asn Ser Ser Asp Leu 405 410 415 Gln Gln Ile Leu Ser
Leu Leu Glu Ser Asn Lys Asp Leu Leu Leu Thr 420 425 430 Ser Ser Tyr
Leu Ser Asp Ser Gly Ser Thr Gly Glu His Thr Lys Ser 435 440 445 Leu
Val Thr Gln Tyr Leu Asn Ala Thr Gly Asn Arg Trp Cys Ser Trp 450 455
460 Ser Leu Ser Gln Ala Arg Leu Leu Thr Ser Phe Leu Pro Ala Gln
Leu465 470 475 480 Leu Arg Leu Tyr Gln Leu Met Leu Phe Thr Leu Pro
Gly Thr Pro Val 485 490 495 Phe Ser Tyr Gly Asp Glu Ile Gly Leu Asp
Ala Ala Ala Leu Pro Gly 500 505 510 Gln Pro Met Glu Ala Pro Val Met
Leu Trp Asp Glu Ser Ser Phe Pro 515 520 525 Asp Ile Pro Gly Ala Val
Ser Ala Asn Met Thr Val Lys Gly Gln Ser 530 535 540 Glu Asp Pro Gly
Ser Leu Leu Ser Leu Phe Arg Arg Leu Ser Asp Gln545 550 555 560 Arg
Ser Lys Glu Arg Ser Leu Leu His Gly Asp Phe His Ala Phe Ser 565 570
575 Ala Gly Pro Gly Leu Phe Ser Tyr Ile Arg His Trp Asp Gln Asn Glu
580 585 590 Arg Phe Leu Val Val Leu Asn Phe Gly Asp Val Gly Leu Ser
Ala Gly 595 600 605 Leu Gln Ala Ser Asp Leu Pro Ala Ser Ala Ser Leu
Pro Ala Lys Ala 610 615 620 Asp Leu Leu Leu Ser Thr Gln Pro Gly Arg
Glu Glu Gly Ser Pro Leu625 630 635 640 Glu Leu Glu Arg Leu Lys Leu
Glu Pro His Glu Gly Leu Leu Leu Arg 645 650 655 Phe Pro Tyr Ala Ala
660 50677PRTHomo sapiens 50Met Gln Pro Thr Leu Leu Leu Ser Leu Leu
Gly Ala Val Gly Leu Ala1 5 10 15 Ala Val Asn Ser Met Pro Val Asp
Asn Arg Asn His Asn Glu Gly Met 20 25 30 Val Thr Arg Cys Ile Ile
Glu Val Leu Ser Asn Ala Leu Ser Lys Ser 35 40 45 Ser Ala Pro Pro
Ile Thr Pro Glu Cys Arg Gln Val Leu Lys Thr Ser 50 55 60 Arg Lys
Asp Val Lys Asp Lys Glu Thr Thr Glu Asn Glu Asn Thr Lys65 70 75 80
Phe Glu Val Arg Leu Leu Arg Asp Pro Ala Asp Ala Ser Glu Ala His 85
90 95 Glu Ser Ser Ser Arg Gly Glu Ala Gly Ala Pro Gly Glu Glu Asp
Ile 100 105 110 Gln Gly Pro Thr Lys Ala Asp Thr Glu Lys Trp Ala Glu
Gly Gly Gly 115 120 125 His Ser Arg Glu Arg Ala Asp Glu Pro Gln Trp
Ser Leu Tyr Pro Ser 130 135 140 Asp Ser Gln Val Ser Glu Glu Val Lys
Thr Arg His Ser Glu Lys Ser145 150 155 160 Gln Arg Glu Asp Glu Glu
Glu Glu Glu Gly Glu Asn Tyr Gln Lys Gly 165 170 175 Glu Arg Gly Glu
Asp Ser Ser Glu Glu Lys His Leu Glu Glu Pro Gly 180 185 190 Glu Thr
Gln Asn Ala Phe Leu Asn Glu Arg Lys Gln Ala Ser Ala Ile 195 200 205
Lys Lys Glu Glu Leu Val Ala Arg Ser Glu Thr His Ala Ala Gly His 210
215 220 Ser Gln Glu Lys Thr His Ser Arg Glu Lys Ser Ser Gln Glu Ser
Gly225 230 235 240 Glu Glu Ala Gly Ser Gln Glu Asn His Pro Gln Glu
Ser Lys Gly Gln 245 250 255 Pro Arg Ser Gln Glu Glu Ser Glu Glu Gly
Glu Glu Asp Ala Thr Ser 260 265 270 Glu Val Asp Lys Arg Arg Thr Arg
Pro Arg His His His Gly Arg Ser 275 280 285 Arg Pro Asp Arg Ser Ser
Gln Gly Gly Ser Leu Pro Ser Glu Glu Lys 290 295 300 Gly His Pro Gln
Glu Glu Ser Glu Glu Ser Asn Val Ser Met Ala Ser305 310 315 320 Leu
Gly Glu Lys Arg Asp His His Ser Thr His Tyr Arg Ala Ser Glu 325 330
335 Glu Glu Pro Glu Tyr Gly Glu Glu Ile Lys Gly Tyr Pro Gly Val Gln
340 345 350 Ala Pro Glu Asp Leu Glu Trp Glu Arg Tyr Arg Gly Arg Gly
Ser Glu 355 360 365 Glu Tyr Arg Ala Pro Arg Pro Gln Ser Glu Glu Ser
Trp Asp Glu Glu 370 375 380 Asp Lys Arg Asn Tyr Pro Ser Leu Glu Leu
Asp Lys Met Ala His Gly385 390 395 400 Tyr Gly Glu Glu Ser Glu Glu
Glu Arg Gly Leu Glu Pro Gly Lys Gly 405 410 415 Arg His His Arg Gly
Arg Gly Gly Glu Pro Arg Ala Tyr Phe Met Ser 420 425 430 Asp Thr Arg
Glu Glu Lys Arg Phe Leu Gly Glu Gly His His Arg Val 435 440 445 Gln
Glu Asn Gln Met Asp Lys Ala Arg Arg His Pro Gln Gly Ala Trp 450 455
460 Lys Glu Leu Asp Arg Asn Tyr Leu Asn Tyr Gly Glu Glu Gly Ala
Pro465 470 475 480 Gly Lys Trp Gln Gln Gln Gly Asp Leu Gln Asp Thr
Lys Glu Asn Arg 485 490 495 Glu Glu Ala Arg Phe Gln Asp Lys Gln Tyr
Ser Ser His His Thr Ala 500 505 510 Glu Lys Arg Lys Arg Leu Gly Glu
Leu Phe Asn Pro Tyr Tyr Asp Pro 515 520 525 Leu Gln Trp Lys Ser Ser
His Phe Glu Arg Arg Asp Asn Met Asn Asp 530 535 540 Asn Phe Leu Glu
Gly Glu Glu Glu Asn Glu Leu Thr Leu Asn Glu Lys545 550 555 560 Asn
Phe Phe Pro Glu Tyr Asn Tyr Asp Trp Trp Glu Lys Lys Pro Phe 565 570
575 Ser Glu Asp Val Asn Trp Gly Tyr Glu Lys Arg Asn Leu Ala Arg Val
580 585 590 Pro Lys Leu Asp Leu Lys Arg Gln Tyr Asp Arg Val Ala Gln
Leu Asp 595 600 605 Gln Leu Leu His Tyr Arg Lys Lys Ser Ala Glu Phe
Pro Asp Phe Tyr 610 615 620 Asp Ser Glu Glu Pro Val Ser Thr His Gln
Glu Ala Glu Asn Glu Lys625 630 635 640 Asp Arg Ala Asp Gln Thr Val
Leu Thr Glu Asp Glu Lys Lys Glu Leu 645 650 655 Glu Asn Leu Ala Ala
Met Asp Leu Glu Leu Gln Lys Ile Ala Glu Lys 660 665 670 Phe Ser Gln
Arg Gly 675 51526PRTHomo sapiens 51Met Gly His Leu Ser Ala Pro Leu
His Arg Val Arg Val Pro Trp Gln1 5 10 15 Gly Leu Leu Leu Thr Ala
Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr 20 25 30 Thr Ala Gln Leu
Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly 35 40 45 Lys Glu
Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly 50 55 60
Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val65
70 75 80 Gly Tyr Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala
Asn Ser 85 90 95 Gly Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu
Ile Gln Asn Val 100 105 110 Thr Gln Asn Asp Thr Gly Phe Tyr Thr Leu
Gln Val Ile Lys Ser Asp 115 120 125 Leu Val Asn Glu Glu Ala Thr Gly
Gln Phe His Val Tyr Pro Glu Leu 130 135 140 Pro Lys Pro Ser Ile Ser
Ser Asn Asn Ser Asn Pro Val Glu Asp Lys145 150 155 160 Asp Ala Val
Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr 165 170 175 Leu
Trp Trp Ile Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln 180 185
190 Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn
195 200 205 Asp Thr Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser
Ala Asn 210 215 220 Arg Ser Asp Pro Val Thr Leu Asn Val Thr Tyr Gly
Pro Asp Thr Pro225 230 235 240 Thr Ile Ser Pro Ser Asp Thr Tyr Tyr
Arg Pro Gly Ala Asn Leu Ser 245 250 255 Leu Ser Cys Tyr Ala Ala Ser
Asn Pro Pro Ala Gln Tyr Ser Trp Leu 260 265 270 Ile Asn Gly Thr Phe
Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn 275 280 285 Ile Thr Val
Asn Asn Ser Gly Ser Tyr Thr Cys His Ala Asn Asn Ser 290 295 300 Val
Thr Gly Cys Asn Arg Thr Thr Val Lys Thr Ile Ile Val Thr Glu305 310
315 320 Leu Ser Pro Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr
Thr 325 330 335 Val Thr Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser
Thr Asn Asp 340 345 350 Thr Gly Ile Ser Ile Arg Trp Phe Phe Lys Asn
Gln Ser Leu Pro Ser 355 360 365 Ser Glu Arg Met Lys Leu Ser Gln Gly
Asn Thr Thr Leu Ser Ile Asn 370 375 380 Pro Val Lys Arg Glu Asp Ala
Gly Thr Tyr Trp Cys Glu Val Phe Asn385 390 395 400 Pro Ile Ser Lys
Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr 405 410 415 Asn Ala
Leu Pro Gln Glu Asn Gly Leu Ser Pro Gly Ala Ile Ala Gly 420 425 430
Ile Val Ile Gly Val Val Ala Leu Val Ala Leu Ile Ala Val Ala Leu 435
440 445 Ala Cys Phe Leu His Phe Gly Lys Thr Gly Arg Ala Ser Asp Gln
Arg 450 455 460 Asp Leu Thr Glu His Lys Pro Ser Val Ser Asn His Thr
Gln Asp His465 470 475 480 Ser Asn Asp Pro Pro Asn Lys Met Asn Glu
Val Thr Tyr Ser Thr Leu 485 490 495 Asn Phe Glu Ala Gln Gln Pro Thr
Gln Pro Thr Ser Ala Ser Pro Ser 500 505 510 Leu Thr Ala Thr Glu Ile
Ile Tyr Ser Glu Val Lys Lys Gln 515 520 525 52583PRTHomo sapiens
52Met Glu Ser Lys Gly Ala Ser Ser Cys Arg Leu Leu Phe Cys Leu Leu1
5 10 15 Ile Ser Ala Thr Val Phe Arg Pro Gly Leu Gly Trp Tyr Thr Val
Asn 20 25 30 Ser Ala Tyr Gly Asp Thr Ile Ile Ile Pro Cys Arg Leu
Asp Val Pro 35 40 45 Gln Asn Leu Met Phe Gly Lys Trp Lys
Tyr Glu Lys Pro Asp Gly Ser 50 55 60 Pro Val Phe Ile Ala Phe Arg
Ser Ser Thr Lys Lys Ser Val Gln Tyr65 70 75 80 Asp Asp Val Pro Glu
Tyr Lys Asp Arg Leu Asn Leu Ser Glu Asn Tyr 85 90 95 Thr Leu Ser
Ile Ser Asn Ala Arg Ile Ser Asp Glu Lys Arg Phe Val 100 105 110 Cys
Met Leu Val Thr Glu Asp Asn Val Phe Glu Ala Pro Thr Ile Val 115 120
125 Lys Val Phe Lys Gln Pro Ser Lys Pro Glu Ile Val Ser Lys Ala Leu
130 135 140 Phe Leu Glu Thr Glu Gln Leu Lys Lys Leu Gly Asp Cys Ile
Ser Glu145 150 155 160 Asp Ser Tyr Pro Asp Gly Asn Ile Thr Trp Tyr
Arg Asn Gly Lys Val 165 170 175 Leu His Pro Leu Glu Gly Ala Val Val
Ile Ile Phe Lys Lys Glu Met 180 185 190 Asp Pro Val Thr Gln Leu Tyr
Thr Met Thr Ser Thr Leu Glu Tyr Lys 195 200 205 Thr Thr Lys Ala Asp
Ile Gln Met Pro Phe Thr Cys Ser Val Thr Tyr 210 215 220 Tyr Gly Pro
Ser Gly Gln Lys Thr Ile His Ser Glu Gln Ala Val Phe225 230 235 240
Asp Ile Tyr Tyr Pro Thr Glu Gln Val Thr Ile Gln Val Leu Pro Pro 245
250 255 Lys Asn Ala Ile Lys Glu Gly Asp Asn Ile Thr Leu Lys Cys Leu
Gly 260 265 270 Asn Gly Asn Pro Pro Pro Glu Glu Phe Leu Phe Tyr Leu
Pro Gly Gln 275 280 285 Pro Glu Gly Ile Arg Ser Ser Asn Thr Tyr Thr
Leu Thr Asp Val Arg 290 295 300 Arg Asn Ala Thr Gly Asp Tyr Lys Cys
Ser Leu Ile Asp Lys Lys Ser305 310 315 320 Met Ile Ala Ser Thr Ala
Ile Thr Val His Tyr Leu Asp Leu Ser Leu 325 330 335 Asn Pro Ser Gly
Glu Val Thr Arg Gln Ile Gly Asp Ala Leu Pro Val 340 345 350 Ser Cys
Thr Ile Ser Ala Ser Arg Asn Ala Thr Val Val Trp Met Lys 355 360 365
Asp Asn Ile Arg Leu Arg Ser Ser Pro Ser Phe Ser Ser Leu His Tyr 370
375 380 Gln Asp Ala Gly Asn Tyr Val Cys Glu Thr Ala Leu Gln Glu Val
Glu385 390 395 400 Gly Leu Lys Lys Arg Glu Ser Leu Thr Leu Ile Val
Glu Gly Lys Pro 405 410 415 Gln Ile Lys Met Thr Lys Lys Thr Asp Pro
Ser Gly Leu Ser Lys Thr 420 425 430 Ile Ile Cys His Val Glu Gly Phe
Pro Lys Pro Ala Ile Gln Trp Thr 435 440 445 Ile Thr Gly Ser Gly Ser
Val Ile Asn Gln Thr Glu Glu Ser Pro Tyr 450 455 460 Ile Asn Gly Arg
Tyr Tyr Ser Lys Ile Ile Ile Ser Pro Glu Glu Asn465 470 475 480 Val
Thr Leu Thr Cys Thr Ala Glu Asn Gln Leu Glu Arg Thr Val Asn 485 490
495 Ser Leu Asn Val Ser Ala Ile Ser Ile Pro Glu His Asp Glu Ala Asp
500 505 510 Glu Ile Ser Asp Glu Asn Arg Glu Lys Val Asn Asp Gln Ala
Lys Leu 515 520 525 Ile Val Gly Ile Val Val Gly Leu Leu Leu Ala Ala
Leu Val Ala Gly 530 535 540 Val Val Tyr Trp Leu Tyr Met Lys Lys Ser
Lys Thr Ala Ser Lys His545 550 555 560 Val Asn Lys Asp Leu Gly Asn
Met Glu Glu Asn Lys Lys Leu Glu Glu 565 570 575 Asn Asn His Lys Thr
Glu Ala 580 53205PRTHomo sapiens 53Met Thr Glu Arg Arg Val Pro Phe
Ser Leu Leu Arg Gly Pro Ser Trp1 5 10 15 Asp Pro Phe Arg Asp Trp
Tyr Pro His Ser Arg Leu Phe Asp Gln Ala 20 25 30 Phe Gly Leu Pro
Arg Leu Pro Glu Glu Trp Ser Gln Trp Leu Gly Gly 35 40 45 Ser Ser
Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala Ile Glu 50 55 60
Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gln65
70 75 80 Leu Ser Ser Gly Val Ser Glu Ile Arg His Thr Ala Asp Arg
Trp Arg 85 90 95 Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu
Leu Thr Val Lys 100 105 110 Thr Lys Asp Gly Val Val Glu Ile Thr Gly
Lys His Glu Glu Arg Gln 115 120 125 Asp Glu His Gly Tyr Ile Ser Arg
Cys Phe Thr Arg Lys Tyr Thr Leu 130 135 140 Pro Pro Gly Val Asp Pro
Thr Gln Val Ser Ser Ser Leu Ser Pro Glu145 150 155 160 Gly Thr Leu
Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gln Ser 165 170 175 Asn
Glu Ile Thr Ile Pro Val Thr Phe Glu Ser Arg Ala Gln Leu Gly 180 185
190 Gly Pro Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys 195 200 205
54135PRTHomo sapiens 54Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn
Leu Lys Pro Gly Glu1 5 10 15 Cys Leu Arg Val Arg Gly Glu Val Ala
Pro Asp Ala Lys Ser Phe Val 20 25 30 Leu Asn Leu Gly Lys Asp Ser
Asn Asn Leu Cys Leu His Phe Asn Pro 35 40 45 Arg Phe Asn Ala His
Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys 50 55 60 Asp Gly Gly
Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe65 70 75 80 Gln
Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn 85 90
95 Leu Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg
100 105 110 Leu Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp
Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe Asp 130 135
55534PRTHomo sapiens 55Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly
Val His Val Gly Ala1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys Leu
Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Pro Glu Asp Pro Val Val
Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Cys Cys Ser Phe Ser
Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile Trp
Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala65 70 75 80 Glu
Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90
95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val
100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile
Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala
Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp
Leu Arg Pro Gly Asp Thr145 150 155 160 Val Thr Ile Thr Cys Ser Ser
Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp Gly
Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met
Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu 195 200 205 Arg
Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215
220 Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro
Gln225 230 235 240 Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro
Glu Asp Pro Val 245 250 255 Val Ala Leu Val Gly Thr Asp Ala Thr Leu
Arg Cys Ser Phe Ser Pro 260 265 270 Glu Pro Gly Phe Ser Leu Ala Gln
Leu Asn Leu Ile Trp Gln Leu Thr 275 280 285 Asp Thr Lys Gln Leu Val
His Ser Phe Thr Glu Gly Arg Asp Gln Gly 290 295 300 Ser Ala Tyr Ala
Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln305 310 315 320 Gly
Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly 325 330
335 Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val
340 345 350 Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr
Leu Glu 355 360 365 Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr
Ile Thr Cys Ser 370 375 380 Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val
Phe Trp Gln Asp Gly Gln385 390 395 400 Gly Val Pro Leu Thr Gly Asn
Val Thr Thr Ser Gln Met Ala Asn Glu 405 410 415 Gln Gly Leu Phe Asp
Val His Ser Val Leu Arg Val Val Leu Gly Ala 420 425 430 Asn Gly Thr
Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp 435 440 445 Ala
His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro 450 455
460 Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala
Leu465 470 475 480 Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile
Lys Gln Ser Cys 485 490 495 Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln
Asp Gly Glu Gly Glu Gly 500 505 510 Ser Lys Thr Ala Leu Gln Pro Leu
Lys His Ser Asp Ser Lys Glu Asp 515 520 525 Asp Gly Gln Glu Ile Ala
530 5610064PRTHomo sapiensVARIANT(1)...(10064)Xaa = Any Amino Acid
56Met Leu Lys Pro Ser Gly Leu Pro Gly Ser Ser Ser Pro Thr Arg Ser1
5 10 15 Leu Met Thr Gly Ser Arg Ser Thr Lys Ala Thr Pro Glu Met Asp
Ser 20 25 30 Gly Leu Thr Gly Ala Thr Leu Ser Pro Lys Thr Ser Thr
Gly Ala Ile 35 40 45 Val Val Thr Glu His Thr Leu Pro Phe Thr Ser
Pro Asp Lys Thr Leu 50 55 60 Ala Ser Pro Thr Ser Ser Val Val Gly
Arg Thr Thr Gln Ser Leu Gly65 70 75 80 Val Met Ser Ser Ala Leu Pro
Glu Ser Thr Ser Arg Gly Met Thr His 85 90 95 Ser Glu Gln Arg Thr
Ser Pro Ser Leu Ser Pro Gln Val Asn Gly Thr 100 105 110 Pro Ser Arg
Asn Tyr Pro Ala Thr Ser Met Val Ser Gly Leu Ser Ser 115 120 125 Pro
Arg Thr Arg Thr Ser Ser Thr Glu Gly Asn Phe Thr Lys Glu Ala 130 135
140 Ser Thr Tyr Thr Leu Thr Val Glu Thr Thr Ser Gly Pro Val Thr
Glu145 150 155 160 Lys Tyr Thr Val Pro Thr Glu Thr Ser Thr Thr Glu
Gly Asp Ser Thr 165 170 175 Glu Thr Pro Trp Asp Thr Arg Tyr Ile Pro
Val Lys Ile Thr Ser Pro 180 185 190 Met Lys Thr Phe Ala Asp Ser Thr
Ala Ser Lys Glu Asn Ala Pro Val 195 200 205 Ser Met Thr Pro Ala Glu
Thr Thr Val Thr Asp Ser His Thr Pro Gly 210 215 220 Arg Thr Asn Pro
Ser Phe Gly Thr Leu Tyr Ser Ser Phe Leu Asp Leu225 230 235 240 Ser
Pro Lys Gly Thr Pro Asn Ser Arg Gly Glu Thr Ser Leu Glu Leu 245 250
255 Ile Leu Ser Thr Thr Gly Tyr Pro Phe Ser Ser Pro Glu Pro Gly Ser
260 265 270 Ala Gly His Ser Arg Ile Ser Thr Ser Ala Pro Leu Ser Ser
Ser Ala 275 280 285 Ser Val Leu Asp Asn Lys Ile Ser Glu Thr Ser Ile
Phe Ser Gly Gln 290 295 300 Ser Leu Thr Ser Pro Leu Ser Pro Gly Val
Pro Glu Ala Arg Ala Ser305 310 315 320 Thr Met Pro Asn Ser Ala Ile
Pro Phe Ser Met Thr Leu Ser Asn Ala 325 330 335 Glu Thr Ser Ala Glu
Arg Val Arg Ser Thr Ile Ser Ser Leu Gly Thr 340 345 350 Pro Ser Ile
Ser Thr Lys Gln Thr Ala Glu Thr Ile Leu Thr Phe His 355 360 365 Ala
Phe Ala Glu Thr Met Asp Ile Pro Ser Thr His Ile Ala Lys Thr 370 375
380 Leu Ala Ser Glu Trp Leu Gly Ser Pro Gly Thr Leu Gly Gly Thr
Ser385 390 395 400 Thr Ser Ala Leu Thr Thr Thr Ser Pro Ser Thr Thr
Leu Val Ser Glu 405 410 415 Glu Thr Asn Thr His His Ser Thr Ser Gly
Lys Glu Thr Glu Gly Thr 420 425 430 Leu Asn Thr Ser Met Thr Pro Leu
Glu Thr Ser Ala Pro Gly Glu Glu 435 440 445 Ser Glu Met Thr Ala Thr
Leu Val Pro Thr Leu Gly Phe Thr Thr Leu 450 455 460 Asp Ser Lys Ile
Arg Ser Pro Ser Gln Val Ser Ser Ser His Pro Thr465 470 475 480 Arg
Glu Leu Arg Thr Thr Gly Ser Thr Ser Gly Arg Gln Ser Ser Ser 485 490
495 Thr Ala Ala His Gly Ser Ser Asp Ile Leu Arg Ala Thr Thr Ser Ser
500 505 510 Thr Ser Lys Ala Ser Ser Trp Thr Ser Glu Ser Thr Ala Gln
Gln Phe 515 520 525 Ser Glu Pro Gln His Thr Gln Trp Val Glu Thr Ser
Pro Ser Met Lys 530 535 540 Thr Glu Arg Pro Pro Ala Ser Thr Ser Val
Ala Ala Pro Ile Thr Thr545 550 555 560 Ser Val Pro Ser Val Val Ser
Gly Phe Thr Thr Leu Lys Thr Ser Ser 565 570 575 Thr Lys Gly Ile Trp
Leu Glu Glu Thr Ser Ala Asp Thr Leu Ile Gly 580 585 590 Glu Ser Thr
Ala Gly Pro Thr Thr His Gln Phe Ala Val Pro Thr Gly 595 600 605 Ile
Ser Met Thr Gly Gly Ser Ser Thr Arg Gly Ser Gln Gly Thr Thr 610 615
620 His Leu Leu Thr Arg Ala Thr Ala Ser Ser Glu Thr Ser Ala Asp
Leu625 630 635 640 Thr Leu Ala Thr Asn Gly Val Pro Val Ser Val Ser
Pro Ala Val Ser 645 650 655 Lys Thr Ala Ala Gly Ser Ser Pro Pro Gly
Gly Thr Lys Pro Ser Tyr 660 665 670 Thr Met Val Ser Ser Val Ile Pro
Glu Thr Ser Ser Leu Gln Ser Ser 675 680 685 Ala Phe Arg Glu Gly Thr
Ser Leu Gly Leu Thr Pro Leu Asn Thr Arg 690 695 700 His Pro Phe Ser
Ser Pro Glu Pro Asp Ser Ala Gly His Thr Lys Ile705 710 715 720 Ser
Thr Ser Ile Pro Leu Leu Ser Ser Ala Ser Val Leu Glu Asp Lys 725 730
735 Val Ser Ala Thr Ser Thr Phe Ser His His Lys Ala Thr Ser Ser Ile
740 745 750 Thr Thr Gly Thr Pro Glu Ile Ser Thr Lys Thr Lys Pro Ser
Ser Ala 755 760 765 Val Leu Ser Ser Met Thr Leu Ser Asn Ala Ala Thr
Ser Pro Glu Arg 770 775 780 Val Arg Asn Ala Thr Ser Pro Leu Thr His
Pro Ser Pro Ser Gly Glu785 790 795 800 Glu Thr Ala Gly Ser Val Leu
Thr Leu Ser Thr Ser Ala Glu Thr Thr 805 810 815 Asp Ser Pro Asn Ile
His Pro Thr Gly Thr Leu Thr Ser Glu Ser Ser 820 825 830 Glu Ser Pro
Ser Thr Leu Ser Leu Pro Ser Val Ser Gly Val Lys Thr 835 840 845 Thr
Phe Ser Ser Ser Thr Pro Ser Thr His Leu Phe Thr Ser Gly Glu 850 855
860 Glu Thr Glu Glu Thr Ser Asn Pro Ser Val Ser Gln Pro Glu Thr
Ser865 870 875 880 Val Ser Arg Val Arg Thr Thr Leu Ala Ser Thr Ser
Val Pro Thr Pro 885 890
895 Val Phe Pro Thr Met Asp Thr Trp Pro Thr Arg Ser Ala Gln Phe Ser
900 905 910 Ser Ser His Leu Val Ser Glu Leu Arg Ala Thr Ser Ser Thr
Ser Val 915 920 925 Thr Asn Ser Thr Gly Ser Ala Leu Pro Lys Ile Ser
His Leu Thr Gly 930 935 940 Thr Ala Thr Met Ser Gln Thr Asn Arg Asp
Thr Phe Asn Asp Ser Ala945 950 955 960 Ala Pro Gln Ser Thr Thr Trp
Pro Glu Thr Ser Pro Arg Phe Lys Thr 965 970 975 Gly Leu Pro Ser Ala
Thr Thr Thr Val Ser Thr Ser Ala Thr Ser Leu 980 985 990 Ser Ala Thr
Val Met Val Ser Lys Phe Thr Ser Pro Ala Thr Ser Ser 995 1000 1005
Met Glu Ala Thr Ser Ile Arg Glu Pro Ser Thr Thr Ile Leu Thr Thr
1010 1015 1020 Glu Thr Thr Asn Gly Pro Gly Ser Met Ala Val Ala Ser
Thr Asn Ile1025 1030 1035 1040Pro Ile Gly Lys Gly Tyr Ile Thr Glu
Gly Arg Leu Asp Thr Ser His 1045 1050 1055 Leu Pro Ile Gly Thr Thr
Ala Ser Ser Glu Thr Ser Met Asp Phe Thr 1060 1065 1070 Met Ala Lys
Glu Ser Val Ser Met Ser Val Ser Pro Ser Gln Ser Met 1075 1080 1085
Asp Ala Ala Gly Ser Ser Thr Pro Gly Arg Thr Ser Gln Phe Val Asp
1090 1095 1100 Thr Phe Ser Asp Asp Val Tyr His Leu Thr Ser Arg Glu
Ile Thr Ile1105 1110 1115 1120Pro Arg Asp Gly Thr Ser Ser Ala Leu
Thr Pro Gln Met Thr Ala Thr 1125 1130 1135 His Pro Pro Ser Pro Asp
Pro Gly Ser Ala Arg Ser Thr Trp Leu Gly 1140 1145 1150 Ile Leu Ser
Ser Ser Pro Ser Ser Pro Thr Pro Lys Val Thr Met Ser 1155 1160 1165
Ser Thr Phe Ser Thr Gln Arg Val Thr Thr Ser Met Ile Met Asp Thr
1170 1175 1180 Val Glu Thr Ser Arg Trp Asn Met Pro Asn Leu Pro Ser
Thr Thr Ser1185 1190 1195 1200Leu Thr Pro Ser Asn Ile Pro Thr Ser
Gly Ala Ile Gly Lys Ser Thr 1205 1210 1215 Leu Val Pro Leu Asp Thr
Pro Ser Pro Ala Thr Ser Leu Glu Ala Ser 1220 1225 1230 Glu Gly Gly
Leu Pro Thr Leu Ser Thr Tyr Pro Glu Ser Thr Asn Thr 1235 1240 1245
Pro Ser Ile His Leu Gly Ala His Ala Ser Ser Glu Ser Pro Ser Thr
1250 1255 1260 Ile Lys Leu Thr Met Ala Ser Val Val Lys Pro Gly Ser
Tyr Thr Pro1265 1270 1275 1280Leu Thr Phe Pro Ser Ile Glu Thr His
Ile His Val Ser Thr Ala Arg 1285 1290 1295 Met Ala Tyr Ser Ser Gly
Ser Ser Pro Glu Met Thr Ala Pro Gly Glu 1300 1305 1310 Thr Asn Thr
Gly Ser Thr Trp Asp Pro Thr Thr Tyr Ile Thr Thr Thr 1315 1320 1325
Asp Pro Lys Asp Thr Ser Ser Ala Gln Val Ser Thr Pro His Ser Val
1330 1335 1340 Arg Thr Leu Arg Thr Thr Glu Asn His Pro Lys Thr Glu
Ser Ala Thr1345 1350 1355 1360Pro Ala Ala Tyr Ser Gly Ser Pro Lys
Ile Ser Ser Ser Pro Asn Leu 1365 1370 1375 Thr Ser Pro Ala Thr Lys
Ala Trp Thr Ile Thr Asp Thr Thr Glu His 1380 1385 1390 Ser Thr Gln
Leu His Tyr Thr Lys Leu Ala Glu Lys Ser Ser Gly Phe 1395 1400 1405
Glu Thr Gln Ser Ala Pro Gly Pro Val Ser Val Val Ile Pro Thr Ser
1410 1415 1420 Pro Thr Ile Gly Ser Ser Thr Leu Glu Leu Thr Ser Asp
Val Pro Gly1425 1430 1435 1440Glu Pro Leu Val Leu Ala Pro Ser Glu
Gln Thr Thr Ile Thr Leu Pro 1445 1450 1455 Met Ala Thr Trp Leu Ser
Thr Ser Leu Thr Glu Glu Met Ala Ser Thr 1460 1465 1470 Asp Leu Asp
Ile Ser Ser Pro Ser Ser Pro Met Ser Thr Phe Ala Ile 1475 1480 1485
Phe Pro Pro Met Ser Thr Pro Ser His Glu Leu Ser Lys Ser Glu Ala
1490 1495 1500 Asp Thr Ser Ala Ile Arg Asn Thr Asp Ser Thr Thr Leu
Asp Gln His1505 1510 1515 1520Leu Gly Ile Arg Ser Leu Gly Arg Thr
Gly Asp Leu Thr Thr Val Pro 1525 1530 1535 Ile Thr Pro Leu Thr Thr
Thr Trp Thr Ser Val Ile Glu His Ser Thr 1540 1545 1550 Gln Ala Gln
Asp Thr Leu Ser Ala Thr Met Ser Pro Thr His Val Thr 1555 1560 1565
Gln Ser Leu Lys Asp Gln Thr Ser Ile Pro Ala Ser Ala Ser Pro Ser
1570 1575 1580 His Leu Thr Glu Val Tyr Pro Glu Leu Gly Thr Gln Gly
Arg Ser Ser1585 1590 1595 1600Ser Glu Ala Thr Thr Phe Trp Lys Pro
Ser Thr Asp Thr Leu Ser Arg 1605 1610 1615 Glu Ile Glu Thr Gly Pro
Thr Asn Ile Gln Ser Thr Pro Pro Met Asp 1620 1625 1630 Asn Thr Thr
Thr Gly Ser Ser Ser Ser Gly Val Thr Leu Gly Ile Ala 1635 1640 1645
His Leu Pro Ile Gly Thr Ser Ser Pro Ala Glu Thr Ser Thr Asn Met
1650 1655 1660 Ala Leu Glu Arg Arg Ser Ser Thr Ala Thr Val Ser Met
Ala Gly Thr1665 1670 1675 1680Met Gly Leu Leu Val Thr Ser Ala Pro
Gly Arg Ser Ile Ser Gln Ser 1685 1690 1695 Leu Gly Arg Val Ser Ser
Val Leu Ser Glu Ser Thr Thr Glu Gly Val 1700 1705 1710 Thr Asp Ser
Ser Lys Gly Ser Ser Pro Arg Leu Asn Thr Gln Gly Asn 1715 1720 1725
Thr Ala Leu Ser Ser Ser Leu Glu Pro Ser Tyr Ala Glu Gly Ser Gln
1730 1735 1740 Met Ser Thr Ser Ile Pro Leu Thr Ser Ser Pro Thr Thr
Pro Asp Val1745 1750 1755 1760Glu Phe Ile Gly Gly Ser Thr Phe Trp
Thr Lys Glu Val Thr Thr Val 1765 1770 1775 Met Thr Ser Asp Ile Ser
Lys Ser Ser Ala Arg Thr Glu Ser Ser Ser 1780 1785 1790 Ala Thr Leu
Met Ser Thr Ala Leu Gly Ser Thr Glu Asn Thr Gly Lys 1795 1800 1805
Glu Lys Leu Arg Thr Ala Ser Met Asp Leu Pro Ser Pro Thr Pro Ser
1810 1815 1820 Met Glu Val Thr Pro Trp Ile Ser Leu Thr Leu Ser Asn
Ala Pro Asn1825 1830 1835 1840Thr Thr Asp Ser Leu Asp Leu Ser His
Gly Val His Thr Ser Ser Ala 1845 1850 1855 Gly Thr Leu Ala Thr Asp
Arg Ser Leu Asn Thr Gly Val Thr Arg Ala 1860 1865 1870 Ser Arg Leu
Glu Asn Gly Ser Asp Thr Ser Ser Lys Ser Leu Ser Met 1875 1880 1885
Gly Asn Ser Thr His Thr Ser Met Thr Asp Thr Glu Lys Ser Glu Val
1890 1895 1900 Ser Ser Ser Ile His Pro Arg Pro Glu Thr Ser Ala Pro
Gly Ala Glu1905 1910 1915 1920Thr Thr Leu Thr Ser Thr Pro Gly Asn
Arg Ala Ile Ser Leu Thr Leu 1925 1930 1935 Pro Phe Ser Ser Ile Pro
Val Glu Glu Val Ile Ser Thr Gly Ile Thr 1940 1945 1950 Ser Gly Pro
Asp Ile Asn Ser Ala Pro Met Thr His Ser Pro Ile Thr 1955 1960 1965
Pro Pro Thr Ile Val Trp Thr Ser Thr Gly Thr Ile Glu Gln Ser Thr
1970 1975 1980 Gln Pro Leu His Ala Val Ser Ser Glu Lys Val Ser Val
Gln Thr Gln1985 1990 1995 2000Ser Thr Pro Tyr Val Asn Ser Val Ala
Val Ser Ala Ser Pro Thr His 2005 2010 2015 Glu Asn Ser Val Ser Ser
Gly Ser Ser Thr Ser Ser Pro Tyr Ser Ser 2020 2025 2030 Ala Ser Leu
Glu Ser Leu Asp Ser Thr Ile Ser Arg Arg Asn Ala Ile 2035 2040 2045
Thr Ser Trp Leu Trp Asp Leu Thr Thr Ser Leu Pro Thr Thr Thr Trp
2050 2055 2060 Pro Ser Thr Ser Leu Ser Glu Ala Leu Ser Ser Gly His
Ser Gly Val2065 2070 2075 2080Ser Asn Pro Ser Ser Thr Thr Thr Glu
Phe Pro Leu Phe Ser Ala Ala 2085 2090 2095 Ser Thr Ser Ala Ala Lys
Gln Arg Asn Pro Glu Thr Glu Thr His Gly 2100 2105 2110 Pro Gln Asn
Thr Ala Ala Ser Thr Leu Asn Thr Asp Ala Ser Ser Val 2115 2120 2125
Thr Gly Leu Ser Glu Thr Pro Val Gly Ala Ser Ile Ser Ser Glu Val
2130 2135 2140 Pro Leu Pro Met Ala Ile Thr Ser Arg Ser Asp Val Ser
Gly Leu Thr2145 2150 2155 2160Ser Glu Ser Thr Ala Asn Pro Ser Leu
Gly Thr Ala Ser Ser Ala Gly 2165 2170 2175 Thr Lys Leu Thr Arg Thr
Ile Ser Leu Pro Thr Ser Glu Ser Leu Val 2180 2185 2190 Ser Phe Arg
Met Asn Lys Asp Pro Trp Thr Val Ser Ile Pro Leu Gly 2195 2200 2205
Ser His Pro Thr Thr Asn Thr Glu Thr Ser Ile Pro Val Asn Ser Ala
2210 2215 2220 Gly Pro Pro Gly Leu Ser Thr Val Ala Ser Asp Val Ile
Asp Thr Pro2225 2230 2235 2240Ser Asp Gly Ala Glu Ser Ile Pro Thr
Val Ser Phe Ser Pro Ser Pro 2245 2250 2255 Asp Thr Glu Val Thr Thr
Ile Ser His Phe Pro Glu Lys Thr Thr His 2260 2265 2270 Ser Phe Arg
Thr Ile Ser Ser Leu Thr His Glu Leu Thr Ser Arg Val 2275 2280 2285
Thr Pro Ile Pro Gly Asp Trp Met Ser Ser Ala Met Ser Thr Lys Pro
2290 2295 2300 Thr Gly Ala Ser Pro Ser Ile Thr Leu Gly Glu Arg Arg
Thr Ile Thr2305 2310 2315 2320Ser Ala Ala Pro Thr Thr Ser Pro Ile
Val Leu Thr Ala Ser Phe Thr 2325 2330 2335 Glu Thr Ser Thr Val Ser
Leu Asp Asn Glu Thr Thr Val Lys Thr Ser 2340 2345 2350 Asp Ile Leu
Asp Ala Arg Lys Thr Asn Glu Leu Pro Ser Asp Ser Ser 2355 2360 2365
Ser Ser Ser Asp Leu Ile Asn Thr Ser Ile Ala Ser Ser Thr Met Asp
2370 2375 2380 Val Thr Lys Thr Ala Ser Ile Ser Pro Thr Ser Ile Ser
Gly Met Thr2385 2390 2395 2400Ala Ser Ser Ser Pro Ser Leu Phe Ser
Ser Asp Arg Pro Gln Val Pro 2405 2410 2415 Thr Ser Thr Thr Glu Thr
Asn Thr Ala Thr Ser Pro Ser Val Ser Ser 2420 2425 2430 Asn Thr Tyr
Ser Leu Asp Gly Gly Ser Asn Val Gly Gly Thr Pro Ser 2435 2440 2445
Thr Leu Pro Pro Phe Thr Ile Thr His Pro Val Glu Thr Ser Ser Ala
2450 2455 2460 Leu Leu Ala Trp Ser Arg Pro Val Arg Thr Phe Ser Thr
Met Val Ser2465 2470 2475 2480Thr Asp Thr Ala Ser Gly Glu Asn Pro
Thr Ser Ser Asn Ser Val Val 2485 2490 2495 Thr Ser Val Pro Ala Pro
Gly Thr Trp Ala Ser Val Gly Ser Thr Thr 2500 2505 2510 Asp Leu Pro
Ala Met Gly Phe Leu Lys Thr Ser Pro Ala Gly Glu Ala 2515 2520 2525
His Ser Leu Leu Ala Ser Thr Ile Glu Pro Ala Thr Ala Phe Thr Pro
2530 2535 2540 His Leu Ser Ala Ala Val Val Thr Gly Ser Ser Ala Thr
Ser Glu Ala2545 2550 2555 2560Ser Leu Leu Thr Thr Ser Glu Ser Lys
Ala Ile His Ser Ser Pro Gln 2565 2570 2575 Thr Pro Thr Thr Pro Thr
Ser Gly Ala Asn Trp Glu Thr Ser Ala Thr 2580 2585 2590 Pro Glu Ser
Leu Leu Val Val Thr Glu Thr Ser Asp Thr Thr Leu Thr 2595 2600 2605
Ser Lys Ile Leu Val Thr Asp Thr Ile Leu Phe Ser Thr Val Ser Thr
2610 2615 2620 Pro Pro Ser Lys Phe Pro Ser Thr Gly Thr Leu Ser Gly
Ala Ser Phe2625 2630 2635 2640Pro Thr Leu Leu Pro Asp Thr Pro Ala
Ile Pro Leu Thr Ala Thr Glu 2645 2650 2655 Pro Thr Ser Ser Leu Ala
Thr Ser Phe Asp Ser Thr Pro Leu Val Thr 2660 2665 2670 Ile Ala Ser
Asp Ser Leu Gly Thr Val Pro Glu Thr Thr Leu Thr Met 2675 2680 2685
Ser Glu Thr Ser Asn Gly Asp Ala Leu Val Leu Lys Thr Val Ser Asn
2690 2695 2700 Pro Asp Arg Ser Ile Pro Gly Ile Thr Ile Gln Gly Val
Thr Glu Ser2705 2710 2715 2720Pro Leu His Pro Ser Ser Thr Ser Pro
Ser Lys Ile Val Ala Pro Arg 2725 2730 2735 Asn Thr Thr Tyr Glu Gly
Ser Ile Thr Val Ala Leu Ser Thr Leu Pro 2740 2745 2750 Ala Gly Thr
Thr Gly Ser Leu Val Phe Ser Gln Ser Ser Glu Asn Ser 2755 2760 2765
Glu Thr Thr Ala Leu Val Asp Ser Ser Ala Gly Leu Glu Arg Ala Ser
2770 2775 2780 Val Met Pro Leu Thr Thr Gly Ser Gln Gly Met Ala Ser
Ser Gly Gly2785 2790 2795 2800Ile Arg Ser Gly Ser Thr His Ser Thr
Gly Thr Lys Thr Phe Ser Ser 2805 2810 2815 Leu Pro Leu Thr Met Asn
Pro Gly Glu Val Thr Ala Met Ser Glu Ile 2820 2825 2830 Thr Thr Asn
Arg Leu Thr Ala Thr Gln Ser Thr Ala Pro Lys Gly Ile 2835 2840 2845
Pro Val Lys Pro Thr Ser Ala Glu Ser Gly Leu Leu Thr Pro Val Ser
2850 2855 2860 Ala Ser Ser Ser Pro Ser Lys Ala Phe Ala Ser Leu Thr
Thr Ala Pro2865 2870 2875 2880Pro Ser Thr Trp Gly Ile Pro Gln Ser
Thr Leu Thr Phe Glu Phe Ser 2885 2890 2895 Glu Val Pro Ser Leu Asp
Thr Lys Ser Ala Ser Leu Pro Thr Pro Gly 2900 2905 2910 Gln Ser Leu
Asn Thr Ile Pro Asp Ser Asp Ala Ser Thr Ala Ser Ser 2915 2920 2925
Ser Leu Ser Lys Ser Pro Glu Lys Asn Pro Arg Ala Arg Met Met Thr
2930 2935 2940 Ser Thr Lys Ala Ile Ser Ala Ser Ser Phe Gln Ser Thr
Gly Phe Thr2945 2950 2955 2960Glu Thr Pro Glu Gly Ser Ala Ser Pro
Ser Met Ala Gly His Glu Pro 2965 2970 2975 Arg Val Pro Thr Ser Gly
Thr Gly Asp Pro Arg Tyr Ala Ser Glu Ser 2980 2985 2990 Met Ser Tyr
Pro Asp Pro Ser Lys Ala Ser Ser Ala Met Thr Ser Thr 2995 3000 3005
Ser Leu Ala Ser Lys Leu Thr Thr Leu Phe Ser Thr Gly Gln Ala Ala
3010 3015 3020 Arg Ser Gly Ser Ser Ser Ser Pro Ile Ser Leu Ser Thr
Glu Lys Glu3025 3030 3035 3040Thr Ser Phe Leu Ser Pro Thr Ala Ser
Thr Ser Arg Lys Thr Ser Leu 3045 3050 3055 Phe Leu Gly Pro Ser Met
Ala Arg Gln Pro Asn Ile Leu Val His Leu 3060 3065 3070 Gln Thr Ser
Ala Leu Thr Leu Ser Pro Thr Ser Thr Leu Asn Met Ser 3075 3080 3085
Gln Glu Glu Pro Pro Glu Leu Thr Ser Ser Gln Thr Ile Ala Glu Glu
3090 3095 3100 Glu Gly Thr Thr Ala Glu Thr Gln Thr Leu Thr Phe Thr
Pro Ser Glu3105 3110 3115 3120Thr Pro Thr Ser Leu Leu Pro Val Ser
Ser Pro Thr Glu Pro Thr Ala 3125 3130 3135 Arg Arg Lys Ser Ser Pro
Glu Thr Trp Ala Ser Ser Ile Ser Val Pro 3140 3145 3150 Ala Lys Thr
Ser Leu Val Glu Thr Thr Asp Gly Thr Leu Val Thr Thr 3155 3160 3165
Ile Lys Met Ser Ser Gln Ala Ala Gln Gly Asn Ser Thr Trp Pro Ala
3170 3175 3180 Pro Ala Glu Glu Thr Gly Thr Ser Pro Ala Gly Thr Ser
Pro Gly Ser3185 3190 3195 3200Pro Glu Val Ser Thr Thr Leu Lys Ile
Met Ser Ser Lys Glu Pro Ser 3205
3210 3215 Ile Ser Pro Glu Ile Arg Ser Thr Val Arg Asn Ser Pro Trp
Lys Thr 3220 3225 3230 Pro Glu Thr Thr Val Pro Met Glu Thr Thr Val
Glu Pro Val Thr Leu 3235 3240 3245 Gln Ser Thr Ala Leu Gly Ser Gly
Ser Thr Ser Ile Ser His Leu Pro 3250 3255 3260 Thr Gly Thr Thr Ser
Pro Thr Lys Ser Pro Thr Glu Asn Met Leu Ala3265 3270 3275 3280Thr
Glu Arg Val Ser Leu Ser Pro Ser Pro Pro Glu Ala Trp Thr Asn 3285
3290 3295 Leu Tyr Ser Gly Thr Pro Gly Gly Thr Arg Gln Ser Leu Ala
Thr Met 3300 3305 3310 Ser Ser Val Ser Leu Glu Ser Pro Thr Ala Arg
Ser Ile Thr Gly Thr 3315 3320 3325 Gly Gln Gln Ser Ser Pro Glu Leu
Val Ser Lys Thr Thr Gly Met Glu 3330 3335 3340 Phe Ser Met Trp His
Gly Ser Thr Gly Gly Thr Thr Gly Asp Thr His3345 3350 3355 3360Val
Ser Leu Ser Thr Ser Ser Asn Ile Leu Glu Asp Pro Val Thr Ser 3365
3370 3375 Pro Asn Ser Val Ser Ser Leu Thr Asp Lys Ser Lys His Lys
Thr Glu 3380 3385 3390 Thr Trp Val Ser Thr Thr Ala Ile Pro Ser Thr
Val Leu Asn Asn Lys 3395 3400 3405 Ile Met Ala Ala Glu Gln Gln Thr
Ser Arg Ser Val Asp Glu Ala Tyr 3410 3415 3420 Ser Ser Thr Ser Ser
Trp Ser Asp Gln Thr Ser Gly Ser Asp Ile Thr3425 3430 3435 3440Leu
Gly Ala Ser Pro Asp Val Thr Asn Thr Leu Tyr Ile Thr Ser Thr 3445
3450 3455 Ala Gln Thr Thr Ser Leu Val Ser Leu Pro Ser Gly Asp Gln
Gly Ile 3460 3465 3470 Thr Ser Leu Thr Asn Pro Ser Gly Gly Lys Thr
Ser Ser Ala Ser Ser 3475 3480 3485 Val Thr Ser Pro Ser Ile Gly Leu
Glu Thr Leu Arg Ala Asn Val Ser 3490 3495 3500 Ala Val Lys Ser Asp
Ile Ala Pro Thr Ala Gly His Leu Ser Gln Thr3505 3510 3515 3520Ser
Ser Pro Ala Glu Val Ser Ile Leu Asp Val Thr Thr Ala Pro Thr 3525
3530 3535 Pro Gly Ile Ser Thr Thr Ile Thr Thr Met Gly Thr Asn Ser
Ile Ser 3540 3545 3550 Thr Thr Thr Pro Asn Pro Glu Val Gly Met Ser
Thr Met Asp Ser Thr 3555 3560 3565 Pro Ala Thr Glu Arg Arg Thr Thr
Ser Thr Glu His Pro Ser Thr Trp 3570 3575 3580 Ser Ser Thr Ala Ala
Ser Asp Ser Trp Thr Val Thr Asp Met Thr Ser3585 3590 3595 3600Asn
Leu Lys Val Ala Arg Ser Pro Gly Thr Ile Ser Thr Met His Thr 3605
3610 3615 Thr Ser Phe Leu Ala Ser Ser Thr Glu Leu Asp Ser Met Ser
Thr Pro 3620 3625 3630 His Gly Arg Ile Thr Val Ile Gly Thr Ser Leu
Val Thr Pro Ser Ser 3635 3640 3645 Asp Ala Ser Ala Val Lys Thr Glu
Thr Ser Thr Ser Glu Arg Thr Leu 3650 3655 3660 Ser Pro Ser Asp Thr
Thr Ala Ser Thr Pro Ile Ser Thr Phe Ser Arg3665 3670 3675 3680Val
Gln Arg Met Ser Ile Ser Val Pro Asp Ile Leu Ser Thr Ser Trp 3685
3690 3695 Thr Pro Ser Ser Thr Glu Ala Glu Asp Val Pro Val Ser Met
Val Ser 3700 3705 3710 Thr Asp His Ala Ser Thr Lys Thr Asp Pro Asn
Thr Pro Leu Ser Thr 3715 3720 3725 Phe Leu Phe Asp Ser Leu Ser Thr
Leu Asp Trp Asp Thr Gly Arg Ser 3730 3735 3740 Leu Ser Ser Ala Thr
Ala Thr Thr Ser Ala Pro Gln Gly Ala Thr Thr3745 3750 3755 3760Pro
Gln Glu Leu Thr Leu Glu Thr Met Ile Ser Pro Ala Thr Ser Gln 3765
3770 3775 Leu Pro Phe Ser Ile Gly His Ile Thr Ser Ala Val Thr Pro
Ala Ala 3780 3785 3790 Met Ala Arg Ser Ser Gly Val Thr Phe Ser Arg
Pro Asp Pro Thr Ser 3795 3800 3805 Lys Lys Ala Glu Gln Thr Ser Thr
Gln Leu Pro Thr Thr Thr Ser Ala 3810 3815 3820 His Pro Gly Gln Val
Pro Arg Ser Ala Ala Thr Thr Leu Asp Val Ile3825 3830 3835 3840Pro
His Thr Ala Lys Thr Pro Asp Ala Thr Phe Gln Arg Gln Gly Gln 3845
3850 3855 Thr Ala Leu Thr Thr Glu Ala Arg Ala Thr Ser Asp Ser Trp
Asn Glu 3860 3865 3870 Lys Glu Lys Ser Thr Pro Ser Ala Pro Trp Ile
Thr Glu Met Met Asn 3875 3880 3885 Ser Val Ser Glu Asp Thr Ile Lys
Glu Val Thr Ser Ser Ser Ser Val 3890 3895 3900 Leu Lys Asp Pro Glu
Tyr Ala Gly His Lys Leu Gly Ile Trp Asp Asp3905 3910 3915 3920Phe
Ile Pro Lys Phe Gly Lys Ala Ala His Met Arg Glu Leu Pro Leu 3925
3930 3935 Leu Ser Pro Pro Gln Asp Lys Glu Ala Ile His Pro Ser Thr
Asn Thr 3940 3945 3950 Val Glu Thr Thr Gly Trp Val Thr Ser Ser Glu
His Ala Ser His Ser 3955 3960 3965 Thr Ile Pro Ala His Ser Ala Ser
Ser Lys Leu Thr Ser Pro Val Val 3970 3975 3980 Thr Thr Ser Thr Arg
Glu Gln Ala Ile Val Ser Met Ser Thr Thr Thr3985 3990 3995 4000Trp
Pro Glu Ser Thr Arg Ala Arg Thr Glu Pro Asn Ser Phe Leu Thr 4005
4010 4015 Ile Glu Leu Arg Asp Val Ser Pro Tyr Met Asp Thr Ser Ser
Thr Thr 4020 4025 4030 Gln Thr Ser Ile Ile Ser Ser Pro Gly Ser Thr
Ala Ile Thr Lys Gly 4035 4040 4045 Pro Arg Thr Glu Ile Thr Ser Ser
Lys Arg Ile Ser Ser Ser Phe Leu 4050 4055 4060 Ala Gln Ser Met Arg
Ser Ser Asp Ser Pro Ser Glu Ala Ile Thr Arg4065 4070 4075 4080Leu
Ser Asn Phe Pro Ala Met Thr Glu Ser Gly Gly Met Ile Leu Ala 4085
4090 4095 Met Gln Thr Ser Pro Pro Gly Ala Thr Ser Leu Ser Ala Pro
Thr Leu 4100 4105 4110 Asp Thr Ser Ala Thr Ala Ser Trp Thr Gly Thr
Pro Leu Ala Thr Thr 4115 4120 4125 Gln Arg Phe Thr Tyr Ser Glu Lys
Thr Thr Leu Phe Ser Lys Gly Pro 4130 4135 4140 Glu Asp Thr Ser Gln
Pro Ser Pro Pro Ser Val Glu Glu Thr Ser Ser4145 4150 4155 4160Ser
Ser Ser Leu Val Pro Ile His Ala Thr Thr Ser Pro Ser Asn Ile 4165
4170 4175 Leu Leu Thr Ser Gln Gly His Ser Pro Ser Ser Thr Pro Pro
Val Thr 4180 4185 4190 Ser Val Phe Leu Ser Glu Thr Ser Gly Leu Gly
Lys Thr Thr Asp Met 4195 4200 4205 Ser Arg Ile Ser Leu Glu Pro Gly
Thr Ser Leu Pro Pro Asn Leu Ser 4210 4215 4220 Ser Thr Ala Gly Glu
Ala Leu Ser Thr Tyr Glu Ala Ser Arg Asp Thr4225 4230 4235 4240Lys
Ala Ile His His Ser Ala Asp Thr Ala Val Thr Asn Met Glu Ala 4245
4250 4255 Thr Ser Ser Glu Tyr Ser Pro Ile Pro Gly His Thr Lys Pro
Ser Lys 4260 4265 4270 Ala Thr Ser Pro Leu Val Thr Ser His Ile Met
Gly Asp Ile Thr Ser 4275 4280 4285 Ser Thr Ser Val Phe Gly Ser Ser
Glu Thr Thr Glu Ile Glu Thr Val 4290 4295 4300 Ser Ser Val Asn Gln
Gly Leu Gln Glu Arg Ser Thr Ser Gln Val Ala4305 4310 4315 4320Ser
Ser Ala Thr Glu Thr Ser Thr Val Ile Thr His Val Ser Ser Gly 4325
4330 4335 Asp Ala Thr Thr His Val Thr Lys Thr Gln Ala Thr Phe Ser
Ser Gly 4340 4345 4350 Thr Ser Ile Ser Ser Pro His Gln Phe Ile Thr
Ser Thr Asn Thr Phe 4355 4360 4365 Thr Asp Val Ser Thr Asn Pro Ser
Thr Ser Leu Ile Met Thr Glu Ser 4370 4375 4380 Ser Gly Val Thr Ile
Thr Thr Gln Thr Gly Pro Thr Gly Ala Ala Thr4385 4390 4395 4400Gln
Gly Pro Tyr Leu Leu Asp Thr Ser Thr Met Pro Tyr Leu Thr Glu 4405
4410 4415 Thr Pro Leu Ala Val Thr Pro Asp Phe Met Gln Ser Glu Lys
Thr Thr 4420 4425 4430 Leu Ile Ser Lys Gly Pro Lys Asp Val Thr Trp
Thr Ser Pro Pro Ser 4435 4440 4445 Val Ala Glu Thr Ser Tyr Pro Ser
Ser Leu Thr Pro Phe Leu Val Thr 4450 4455 4460 Thr Ile Pro Pro Ala
Thr Ser Thr Leu Gln Gly Gln His Thr Ser Ser4465 4470 4475 4480Pro
Val Ser Ala Thr Ser Val Leu Thr Ser Gly Leu Val Lys Thr Thr 4485
4490 4495 Asp Met Leu Asn Thr Ser Met Glu Pro Val Thr Asn Ser Pro
Gln Asn 4500 4505 4510 Leu Asn Asn Pro Ser Asn Glu Ile Leu Ala Thr
Leu Ala Ala Thr Thr 4515 4520 4525 Asp Ile Glu Thr Ile His Pro Ser
Ile Asn Lys Ala Val Thr Asn Met 4530 4535 4540 Gly Thr Ala Ser Ser
Ala His Val Leu His Ser Thr Leu Pro Val Ser4545 4550 4555 4560Ser
Glu Pro Ser Thr Ala Thr Ser Pro Met Val Pro Ala Ser Ser Met 4565
4570 4575 Gly Asp Ala Leu Ala Ser Ile Ser Ile Pro Gly Ser Glu Thr
Thr Asp 4580 4585 4590 Ile Glu Gly Glu Pro Thr Ser Ser Leu Thr Ala
Gly Arg Lys Glu Asn 4595 4600 4605 Ser Thr Leu Gln Glu Met Asn Ser
Thr Thr Glu Ser Asn Ile Ile Leu 4610 4615 4620 Ser Asn Val Ser Val
Gly Ala Ile Thr Glu Ala Thr Lys Met Glu Val4625 4630 4635 4640Pro
Ser Phe Asp Ala Thr Phe Ile Pro Thr Pro Ala Gln Ser Thr Lys 4645
4650 4655 Phe Pro Asp Ile Phe Ser Val Ala Ser Ser Arg Leu Ser Asn
Ser Pro 4660 4665 4670 Pro Met Thr Ile Ser Thr His Met Thr Thr Thr
Gln Thr Gly Ser Ser 4675 4680 4685 Gly Ala Thr Ser Lys Ile Pro Leu
Ala Leu Asp Thr Ser Thr Leu Glu 4690 4695 4700 Thr Ser Ala Gly Thr
Pro Ser Val Val Thr Glu Gly Phe Ala His Ser4705 4710 4715 4720Lys
Ile Thr Thr Ala Met Asn Asn Asp Val Lys Asp Val Ser Gln Thr 4725
4730 4735 Asn Pro Pro Phe Gln Asp Glu Ala Ser Ser Pro Ser Ser Gln
Ala Pro 4740 4745 4750 Val Leu Val Thr Thr Leu Pro Ser Ser Val Ala
Phe Thr Pro Gln Trp 4755 4760 4765 His Ser Thr Ser Ser Pro Val Ser
Met Ser Ser Val Leu Thr Ser Ser 4770 4775 4780 Leu Val Lys Thr Ala
Gly Lys Val Asp Thr Ser Leu Glu Thr Val Thr4785 4790 4795 4800Ser
Ser Pro Gln Ser Met Ser Asn Thr Leu Asp Asp Ile Ser Val Thr 4805
4810 4815 Ser Ala Ala Thr Thr Asp Ile Glu Thr Thr His Pro Ser Ile
Asn Thr 4820 4825 4830 Val Val Thr Asn Val Gly Thr Thr Gly Ser Ala
Phe Glu Ser His Ser 4835 4840 4845 Thr Val Ser Ala Tyr Pro Glu Pro
Ser Lys Val Thr Ser Pro Asn Val 4850 4855 4860 Thr Thr Ser Thr Met
Glu Asp Thr Thr Ile Ser Arg Ser Ile Pro Lys4865 4870 4875 4880Ser
Ser Lys Thr Thr Arg Thr Glu Thr Glu Thr Thr Ser Ser Leu Thr 4885
4890 4895 Pro Lys Leu Arg Glu Thr Ser Ile Ser Gln Glu Ile Thr Ser
Ser Thr 4900 4905 4910 Glu Thr Ser Thr Val Pro Tyr Lys Glu Leu Thr
Gly Ala Thr Thr Glu 4915 4920 4925 Val Ser Arg Thr Asp Val Thr Ser
Ser Ser Ser Thr Ser Phe Pro Gly 4930 4935 4940 Pro Asp Gln Ser Thr
Val Ser Leu Asp Ile Ser Thr Glu Thr Asn Thr4945 4950 4955 4960Arg
Leu Ser Thr Ser Pro Ile Met Thr Glu Ser Ala Glu Ile Thr Ile 4965
4970 4975 Thr Thr Gln Thr Gly Pro His Gly Ala Thr Ser Gln Asp Thr
Phe Thr 4980 4985 4990 Met Asp Pro Ser Asn Thr Thr Pro Gln Ala Gly
Ile His Ser Ala Met 4995 5000 5005 Thr His Gly Phe Ser Gln Leu Asp
Val Thr Thr Leu Met Ser Arg Ile 5010 5015 5020 Pro Gln Asp Val Ser
Trp Thr Ser Pro Pro Ser Val Asp Lys Thr Ser5025 5030 5035 5040Ser
Pro Ser Ser Phe Leu Ser Ser Pro Ala Met Thr Thr Pro Ser Leu 5045
5050 5055 Ile Ser Ser Thr Leu Pro Glu Asp Lys Leu Ser Ser Pro Met
Thr Ser 5060 5065 5070 Leu Leu Thr Ser Gly Leu Val Lys Ile Thr Asp
Ile Leu Arg Thr Arg 5075 5080 5085 Leu Glu Pro Val Thr Ser Ser Leu
Pro Asn Phe Ser Ser Thr Ser Asp 5090 5095 5100 Lys Ile Leu Ala Thr
Ser Lys Asp Ser Lys Asp Thr Lys Glu Ile Phe5105 5110 5115 5120Pro
Ser Ile Asn Thr Glu Glu Thr Asn Val Lys Ala Asn Asn Ser Gly 5125
5130 5135 His Glu Ser His Ser Pro Ala Leu Ala Asp Ser Glu Thr Pro
Lys Ala 5140 5145 5150 Thr Thr Gln Met Val Ile Thr Thr Thr Val Gly
Asp Pro Ala Pro Ser 5155 5160 5165 Thr Ser Met Pro Val His Gly Ser
Ser Glu Thr Thr Asn Ile Lys Arg 5170 5175 5180 Glu Pro Thr Tyr Phe
Leu Thr Pro Arg Leu Arg Glu Thr Ser Thr Ser5185 5190 5195 5200Gln
Glu Ser Ser Phe Pro Thr Asp Thr Ser Phe Leu Leu Ser Lys Val 5205
5210 5215 Pro Thr Gly Thr Ile Thr Glu Val Ser Ser Thr Gly Val Asn
Ser Ser 5220 5225 5230 Ser Lys Ile Ser Thr Pro Asp His Asp Lys Ser
Thr Val Pro Pro Asp 5235 5240 5245 Thr Phe Thr Gly Glu Ile Pro Arg
Val Phe Thr Ser Ser Ile Lys Thr 5250 5255 5260 Lys Ser Ala Glu Met
Thr Ile Thr Thr Gln Ala Ser Pro Pro Glu Ser5265 5270 5275 5280Ala
Ser His Ser Thr Leu Pro Leu Asp Thr Ser Thr Thr Leu Ser Gln 5285
5290 5295 Gly Gly Thr His Ser Thr Val Thr Gln Gly Phe Pro Tyr Ser
Glu Val 5300 5305 5310 Thr Thr Leu Met Gly Met Gly Pro Gly Asn Val
Ser Trp Met Thr Thr 5315 5320 5325 Pro Pro Val Glu Glu Thr Ser Ser
Val Ser Ser Leu Met Ser Ser Pro 5330 5335 5340 Ala Met Thr Ser Pro
Ser Pro Val Ser Ser Thr Ser Pro Gln Ser Ile5345 5350 5355 5360Pro
Ser Ser Pro Leu Pro Val Thr Ala Leu Pro Thr Ser Val Leu Val 5365
5370 5375 Thr Thr Thr Asp Val Leu Gly Thr Thr Ser Pro Glu Ser Val
Thr Ser 5380 5385 5390 Ser Pro Pro Asn Leu Ser Ser Ile Thr His Glu
Arg Pro Ala Thr Tyr 5395 5400 5405 Lys Asp Thr Ala His Thr Glu Ala
Ala Met His His Ser Thr Asn Thr 5410 5415 5420 Ala Val Thr Asn Val
Gly Thr Ser Gly Ser Gly His Lys Ser Gln Ser5425 5430 5435 5440Ser
Val Leu Ala Asp Ser Glu Thr Ser Lys Ala Thr Pro Leu Met Ser 5445
5450 5455 Thr Thr Ser Thr Leu Gly Asp Thr Ser Val Ser Thr Ser Thr
Pro Asn 5460 5465 5470 Ile Ser Gln Thr Asn Gln Ile Gln Thr Glu Pro
Thr Ala Ser Leu Ser 5475 5480 5485 Pro Arg Leu Arg Glu Ser Ser Thr
Ser Glu Lys Thr Ser Ser Thr Thr 5490 5495 5500 Glu Thr Asn Thr Ala
Phe Ser Tyr Val Pro Thr Gly Ala Ile Thr Gln5505 5510 5515 5520Ala
Ser Arg Thr Glu Ile Ser Ser Ser Arg Thr Ser Ile Ser Asp Leu
5525 5530 5535 Asp Arg Pro Thr Ile Ala Pro Asp Ile Ser Thr Gly Met
Ile Thr Arg 5540 5545 5550 Leu Phe Thr Ser Pro Ile Met Thr Lys Ser
Ala Glu Met Thr Val Thr 5555 5560 5565 Thr Gln Thr Thr Thr Pro Gly
Ala Thr Ser Gln Gly Ile Leu Pro Trp 5570 5575 5580 Asp Thr Ser Thr
Thr Leu Phe Gln Gly Gly Thr His Ser Thr Val Ser5585 5590 5595
5600Gln Gly Phe Pro His Ser Glu Ile Thr Thr Leu Arg Ser Arg Thr Pro
5605 5610 5615 Gly Asp Val Ser Trp Met Thr Thr Pro Pro Val Glu Glu
Thr Ser Ser 5620 5625 5630 Gly Phe Ser Leu Met Ser Pro Ser Met Thr
Ser Pro Ser Pro Val Ser 5635 5640 5645 Ser Thr Ser Pro Glu Ser Ile
Pro Ser Ser Pro Leu Pro Val Thr Ala 5650 5655 5660 Leu Leu Thr Ser
Val Leu Val Thr Thr Thr Asn Val Leu Gly Thr Thr5665 5670 5675
5680Ser Pro Glu Thr Val Thr Ser Ser Pro Pro Asn Leu Ser Ser Pro Thr
5685 5690 5695 Gln Glu Arg Leu Thr Thr Tyr Lys Asp Thr Ala His Thr
Glu Ala Met 5700 5705 5710 His Ala Ser Met His Thr Asn Thr Ala Val
Ala Asn Val Gly Thr Ser 5715 5720 5725 Ile Ser Gly His Glu Ser Gln
Ser Ser Val Pro Ala Asp Ser His Thr 5730 5735 5740 Ser Lys Ala Thr
Ser Pro Met Gly Ile Thr Phe Ala Met Gly Asp Thr5745 5750 5755
5760Ser Val Ser Thr Ser Thr Pro Ala Phe Phe Glu Thr Arg Ile Gln Thr
5765 5770 5775 Glu Ser Thr Ser Ser Leu Ile Pro Gly Leu Arg Asp Thr
Arg Thr Ser 5780 5785 5790 Glu Glu Ile Asn Thr Val Thr Glu Thr Ser
Thr Val Leu Ser Glu Val 5795 5800 5805 Pro Thr Thr Thr Thr Thr Glu
Val Ser Arg Thr Glu Val Ile Thr Ser 5810 5815 5820 Ser Arg Thr Thr
Ile Ser Gly Pro Asp His Ser Lys Met Ser Pro Tyr5825 5830 5835
5840Ile Ser Thr Glu Thr Ile Thr Arg Leu Ser Thr Phe Pro Phe Val Thr
5845 5850 5855 Gly Ser Thr Glu Met Ala Ile Thr Asn Gln Thr Gly Pro
Ile Gly Thr 5860 5865 5870 Ile Ser Gln Ala Thr Leu Thr Leu Asp Thr
Ser Ser Thr Ala Ser Trp 5875 5880 5885 Glu Gly Thr His Ser Pro Val
Thr Gln Arg Phe Pro His Ser Glu Glu 5890 5895 5900 Thr Thr Thr Met
Ser Arg Ser Thr Lys Gly Val Ser Trp Gln Ser Pro5905 5910 5915
5920Pro Ser Val Glu Glu Thr Ser Ser Pro Ser Ser Pro Val Pro Leu Pro
5925 5930 5935 Ala Ile Thr Ser His Ser Ser Leu Tyr Ser Ala Val Ser
Gly Ser Ser 5940 5945 5950 Pro Thr Ser Ala Leu Pro Val Thr Ser Leu
Leu Thr Ser Gly Arg Arg 5955 5960 5965 Lys Thr Ile Asp Met Leu Asp
Thr His Ser Glu Leu Val Thr Ser Ser 5970 5975 5980 Leu Pro Ser Ala
Ser Ser Phe Ser Gly Glu Ile Leu Thr Ser Glu Ala5985 5990 5995
6000Ser Thr Asn Thr Glu Thr Ile His Phe Ser Glu Asn Thr Ala Glu Thr
6005 6010 6015 Asn Met Gly Thr Thr Asn Ser Met His Lys Leu His Ser
Ser Val Ser 6020 6025 6030 Ile His Ser Gln Pro Ser Gly His Thr Pro
Pro Lys Val Thr Gly Ser 6035 6040 6045 Met Met Glu Asp Ala Ile Val
Ser Thr Ser Thr Pro Gly Ser Pro Glu 6050 6055 6060 Thr Lys Asn Val
Asp Arg Asp Ser Thr Ser Pro Leu Thr Pro Glu Leu6065 6070 6075
6080Lys Glu Asp Ser Thr Ala Leu Val Met Asn Ser Thr Thr Glu Ser Asn
6085 6090 6095 Thr Val Phe Ser Ser Val Ser Leu Asp Ala Ala Thr Glu
Val Ser Arg 6100 6105 6110 Ala Glu Val Thr Tyr Tyr Asp Pro Thr Phe
Met Pro Ala Ser Ala Gln 6115 6120 6125 Ser Thr Lys Ser Pro Asp Ile
Ser Pro Glu Ala Ser Ser Ser His Ser 6130 6135 6140 Asn Ser Pro Pro
Leu Thr Ile Ser Thr His Lys Thr Ile Ala Thr Gln6145 6150 6155
6160Thr Gly Pro Ser Gly Val Thr Ser Leu Gly Gln Leu Thr Leu Asp Thr
6165 6170 6175 Ser Thr Ile Ala Thr Ser Ala Gly Thr Pro Ser Ala Arg
Thr Gln Asp 6180 6185 6190 Phe Val Asp Ser Glu Thr Thr Ser Val Met
Asn Asn Asp Leu Asn Asp 6195 6200 6205 Val Leu Lys Thr Ser Pro Phe
Ser Ala Glu Glu Ala Asn Ser Leu Ser 6210 6215 6220 Ser Gln Ala Pro
Leu Leu Val Thr Thr Ser Pro Ser Pro Val Thr Ser6225 6230 6235
6240Thr Leu Gln Glu His Ser Thr Ser Ser Leu Val Ser Val Thr Ser Val
6245 6250 6255 Pro Thr Pro Thr Leu Ala Lys Ile Thr Asp Met Asp Thr
Asn Leu Glu 6260 6265 6270 Pro Val Thr Arg Ser Pro Gln Asn Leu Arg
Asn Thr Leu Ala Thr Ser 6275 6280 6285 Glu Ala Thr Thr Asp Thr His
Thr Met His Pro Ser Ile Asn Thr Ala 6290 6295 6300 Met Ala Asn Val
Gly Thr Thr Ser Ser Pro Asn Glu Phe Tyr Phe Thr6305 6310 6315
6320Val Ser Pro Asp Ser Asp Pro Tyr Lys Ala Thr Ser Ala Val Val Ile
6325 6330 6335 Thr Ser Thr Ser Gly Asp Ser Ile Val Ser Thr Ser Met
Pro Arg Ser 6340 6345 6350 Ser Ala Met Lys Lys Ile Glu Ser Glu Thr
Thr Phe Ser Leu Ile Phe 6355 6360 6365 Arg Leu Arg Glu Thr Ser Thr
Ser Gln Lys Ile Gly Ser Ser Ser Asp 6370 6375 6380 Thr Ser Thr Val
Phe Asp Lys Ala Phe Thr Ala Ala Thr Thr Glu Val6385 6390 6395
6400Ser Arg Thr Glu Leu Thr Ser Ser Ser Arg Thr Ser Ile Gln Gly Thr
6405 6410 6415 Glu Lys Pro Thr Met Ser Pro Asp Thr Ser Thr Arg Ser
Val Thr Met 6420 6425 6430 Leu Ser Thr Phe Ala Gly Leu Thr Lys Ser
Glu Glu Arg Thr Ile Ala 6435 6440 6445 Thr Gln Thr Gly Pro His Arg
Ala Thr Ser Gln Gly Thr Leu Thr Trp 6450 6455 6460 Asp Thr Ser Ile
Thr Thr Ser Gln Ala Gly Thr His Ser Ala Met Thr6465 6470 6475
6480His Gly Phe Ser Gln Leu Asp Leu Ser Thr Leu Thr Ser Arg Val Pro
6485 6490 6495 Glu Tyr Ile Ser Gly Thr Ser Pro Pro Ser Val Glu Lys
Thr Ser Ser 6500 6505 6510 Ser Ser Ser Leu Leu Ser Leu Pro Ala Ile
Thr Ser Pro Ser Pro Val 6515 6520 6525 Pro Thr Thr Leu Pro Glu Ser
Arg Pro Ser Ser Pro Val His Leu Thr 6530 6535 6540 Ser Leu Pro Thr
Ser Gly Leu Val Lys Thr Thr Asp Met Leu Ala Ser6545 6550 6555
6560Val Ala Ser Leu Pro Pro Asn Leu Gly Ser Thr Ser His Lys Ile Pro
6565 6570 6575 Thr Thr Ser Glu Asp Ile Lys Asp Thr Glu Lys Met Tyr
Pro Ser Thr 6580 6585 6590 Asn Ile Ala Val Thr Asn Val Gly Thr Thr
Thr Ser Glu Lys Glu Ser 6595 6600 6605 Tyr Ser Ser Val Pro Ala Tyr
Ser Glu Pro Pro Lys Val Thr Ser Pro 6610 6615 6620 Met Val Thr Ser
Phe Asn Ile Arg Asp Thr Ile Val Ser Thr Ser Met6625 6630 6635
6640Pro Gly Ser Ser Glu Ile Thr Arg Ile Glu Met Glu Ser Thr Phe Ser
6645 6650 6655 Val Ala His Gly Leu Lys Gly Thr Ser Thr Ser Gln Asp
Pro Ile Val 6660 6665 6670 Ser Thr Glu Lys Ser Ala Val Leu His Lys
Leu Thr Thr Gly Ala Thr 6675 6680 6685 Glu Thr Ser Arg Thr Glu Val
Ala Ser Ser Arg Arg Thr Ser Ile Pro 6690 6695 6700 Gly Pro Asp His
Ser Thr Glu Ser Pro Asp Ile Ser Thr Glu Val Ile6705 6710 6715
6720Pro Ser Leu Pro Ile Ser Leu Gly Ile Thr Glu Ser Ser Asn Met Thr
6725 6730 6735 Ile Ile Thr Arg Thr Gly Pro Pro Leu Gly Ser Thr Ser
Gln Gly Thr 6740 6745 6750 Phe Thr Leu Asp Thr Pro Thr Thr Ser Ser
Arg Ala Gly Thr His Ser 6755 6760 6765 Met Ala Thr Gln Glu Phe Pro
His Ser Glu Met Thr Thr Val Met Asn 6770 6775 6780 Lys Asp Pro Glu
Ile Leu Ser Trp Thr Ile Pro Pro Ser Ile Glu Lys6785 6790 6795
6800Thr Ser Phe Ser Ser Ser Leu Met Pro Ser Pro Ala Met Thr Ser Pro
6805 6810 6815 Pro Val Ser Ser Thr Leu Pro Lys Thr Ile His Thr Thr
Pro Ser Pro 6820 6825 6830 Met Thr Ser Leu Leu Thr Pro Ser Leu Val
Met Thr Thr Asp Thr Leu 6835 6840 6845 Gly Thr Ser Pro Glu Pro Thr
Thr Ser Ser Pro Pro Asn Leu Ser Ser 6850 6855 6860 Thr Ser His Val
Ile Leu Thr Thr Asp Glu Asp Thr Thr Ala Ile Glu6865 6870 6875
6880Ala Met His Pro Ser Thr Ser Thr Ala Ala Thr Asn Val Glu Thr Thr
6885 6890 6895 Cys Ser Gly His Gly Ser Gln Ser Ser Val Leu Thr Asp
Ser Glu Lys 6900 6905 6910 Thr Lys Ala Thr Ala Pro Met Asp Thr Thr
Ser Thr Met Gly His Thr 6915 6920 6925 Thr Val Ser Thr Ser Met Ser
Val Ser Ser Glu Thr Thr Lys Ile Lys 6930 6935 6940 Arg Glu Ser Thr
Tyr Ser Leu Thr Pro Gly Leu Arg Glu Thr Ser Ile6945 6950 6955
6960Ser Gln Asn Ala Ser Phe Ser Thr Asp Thr Ser Ile Val Leu Ser Glu
6965 6970 6975 Val Pro Thr Gly Thr Thr Ala Glu Val Ser Arg Thr Glu
Val Thr Ser 6980 6985 6990 Ser Gly Arg Thr Ser Ile Pro Gly Pro Ser
Gln Ser Thr Val Leu Pro 6995 7000 7005 Glu Ile Ser Thr Arg Thr Met
Thr Arg Leu Phe Ala Ser Pro Thr Met 7010 7015 7020 Thr Glu Ser Ala
Glu Met Thr Ile Pro Thr Gln Thr Gly Pro Ser Gly7025 7030 7035
7040Ser Thr Ser Gln Asp Thr Leu Thr Leu Asp Thr Ser Thr Thr Lys Ser
7045 7050 7055 Gln Ala Lys Thr His Ser Thr Leu Thr Gln Arg Phe Pro
His Ser Glu 7060 7065 7070 Met Thr Thr Leu Met Ser Arg Gly Pro Gly
Asp Met Ser Trp Gln Ser 7075 7080 7085 Ser Pro Ser Leu Glu Asn Pro
Ser Ser Leu Pro Ser Leu Leu Ser Leu 7090 7095 7100 Pro Ala Thr Thr
Ser Pro Pro Pro Ile Ser Ser Thr Leu Pro Val Thr7105 7110 7115
7120Ile Ser Ser Ser Pro Leu Pro Val Thr Ser Leu Leu Thr Ser Ser Pro
7125 7130 7135 Val Thr Thr Thr Asp Met Leu His Thr Ser Pro Glu Leu
Val Thr Ser 7140 7145 7150 Ser Pro Pro Lys Leu Ser His Thr Ser Asp
Glu Arg Leu Thr Thr Gly 7155 7160 7165 Lys Asp Thr Thr Asn Thr Glu
Ala Val His Pro Ser Thr Asn Thr Ala 7170 7175 7180 Ala Ser Asn Val
Glu Ile Pro Ser Phe Gly His Glu Ser Pro Ser Ser7185 7190 7195
7200Ala Leu Ala Asp Ser Glu Thr Ser Lys Ala Thr Ser Pro Met Phe Ile
7205 7210 7215 Thr Ser Thr Gln Glu Asp Thr Thr Val Ala Ile Ser Thr
Pro His Phe 7220 7225 7230 Leu Glu Thr Ser Arg Ile Gln Lys Glu Ser
Ile Ser Ser Leu Ser Pro 7235 7240 7245 Lys Leu Arg Glu Thr Gly Ser
Ser Val Glu Thr Ser Ser Ala Ile Glu 7250 7255 7260 Thr Ser Ala Val
Leu Ser Glu Val Ser Ile Gly Ala Thr Thr Glu Ile7265 7270 7275
7280Ser Arg Thr Glu Val Thr Ser Ser Ser Arg Thr Ser Ile Ser Gly Ser
7285 7290 7295 Ala Glu Ser Thr Met Leu Pro Glu Ile Ser Thr Thr Arg
Lys Ile Ile 7300 7305 7310 Lys Phe Pro Thr Ser Pro Ile Leu Ala Glu
Ser Ser Glu Met Thr Ile 7315 7320 7325 Lys Thr Gln Thr Ser Pro Pro
Gly Ser Thr Ser Glu Ser Thr Phe Thr 7330 7335 7340 Leu Asp Thr Ser
Thr Thr Pro Ser Leu Val Ile Thr His Ser Thr Met7345 7350 7355
7360Thr Gln Arg Leu Pro His Ser Glu Ile Thr Thr Leu Val Ser Arg Gly
7365 7370 7375 Ala Gly Asp Val Pro Arg Pro Ser Ser Leu Pro Val Glu
Glu Thr Ser 7380 7385 7390 Pro Pro Ser Ser Gln Leu Ser Leu Ser Ala
Met Ile Ser Pro Ser Pro 7395 7400 7405 Val Ser Ser Thr Leu Pro Ala
Ser Ser His Ser Ser Ser Ala Ser Val 7410 7415 7420 Thr Ser Pro Leu
Thr Pro Gly Gln Val Lys Thr Thr Glu Val Leu Asp7425 7430 7435
7440Ala Ser Ala Glu Pro Glu Thr Ser Ser Pro Pro Ser Leu Ser Ser Thr
7445 7450 7455 Ser Val Glu Ile Leu Ala Thr Ser Glu Val Thr Thr Asp
Thr Glu Lys 7460 7465 7470 Ile His Pro Phe Pro Asn Thr Ala Val Thr
Lys Val Gly Thr Ser Ser 7475 7480 7485 Ser Gly His Glu Ser Pro Ser
Ser Val Leu Pro Asp Ser Glu Thr Thr 7490 7495 7500 Lys Ala Thr Ser
Ala Met Gly Thr Ile Ser Ile Met Gly Asp Thr Ser7505 7510 7515
7520Val Ser Thr Leu Thr Pro Ala Leu Ser Asn Thr Arg Lys Ile Gln Ser
7525 7530 7535 Glu Pro Ala Ser Ser Leu Thr Thr Arg Leu Arg Glu Thr
Ser Thr Ser 7540 7545 7550 Glu Glu Thr Ser Leu Ala Thr Glu Ala Asn
Thr Val Leu Ser Lys Val 7555 7560 7565 Ser Thr Gly Ala Thr Thr Glu
Val Ser Arg Thr Glu Ala Ile Ser Phe 7570 7575 7580 Ser Arg Thr Ser
Met Ser Gly Pro Glu Gln Ser Thr Met Ser Gln Asp7585 7590 7595
7600Ile Ser Ile Gly Thr Ile Pro Arg Ile Ser Ala Ser Ser Val Leu Thr
7605 7610 7615 Glu Ser Ala Lys Met Thr Ile Thr Thr Gln Thr Gly Pro
Ser Glu Ser 7620 7625 7630 Thr Leu Glu Ser Thr Leu Asn Leu Asn Thr
Ala Thr Thr Pro Ser Trp 7635 7640 7645 Val Glu Thr His Ser Ile Val
Ile Gln Gly Phe Pro His Pro Glu Met 7650 7655 7660 Thr Thr Ser Met
Gly Arg Gly Pro Gly Gly Val Ser Trp Pro Ser Pro7665 7670 7675
7680Pro Phe Val Lys Glu Thr Ser Pro Pro Ser Ser Pro Leu Ser Leu Pro
7685 7690 7695 Ala Val Thr Ser Pro His Pro Val Ser Thr Thr Phe Leu
Ala His Ile 7700 7705 7710 Pro Pro Ser Pro Leu Pro Val Thr Ser Leu
Leu Thr Ser Gly Pro Ala 7715 7720 7725 Thr Thr Thr Asp Ile Leu Gly
Thr Ser Thr Glu Pro Gly Thr Ser Ser 7730 7735 7740 Ser Ser Ser Leu
Ser Thr Thr Ser His Glu Arg Leu Thr Thr Tyr Lys7745 7750 7755
7760Asp Thr Ala His Thr Glu Ala Val His Pro Ser Thr Asn Thr Gly Gly
7765 7770 7775 Thr Asn Val Ala Thr Thr Ser Ser Gly Tyr Lys Ser Gln
Ser Ser Val 7780 7785 7790 Leu Ala Asp Ser Ser Pro Met Cys Thr Thr
Ser Thr Met Gly Asp Thr 7795 7800 7805 Ser Val Leu Thr Ser Thr Pro
Ala Phe Leu Glu Thr Arg Arg Ile Gln 7810 7815 7820 Thr Glu Leu Ala
Ser Ser Leu Thr Pro Gly Leu Arg Glu Ser Ser Gly7825 7830 7835
7840Ser Glu Gly Thr Ser Ser Gly Thr Lys Met Ser
Thr Val Leu Ser Lys 7845 7850 7855 Val Pro Thr Gly Ala Thr Thr Glu
Ile Ser Lys Glu Asp Val Thr Ser 7860 7865 7870 Ile Pro Gly Pro Ala
Gln Ser Thr Ile Ser Pro Asp Ile Ser Thr Arg 7875 7880 7885 Thr Val
Ser Trp Phe Ser Thr Ser Pro Val Met Thr Glu Ser Ala Glu 7890 7895
7900 Ile Thr Met Asn Thr His Thr Ser Pro Leu Gly Ala Thr Thr Gln
Gly7905 7910 7915 7920Thr Ser Thr Leu Ala Thr Ser Ser Thr Thr Ser
Leu Thr Met Thr His 7925 7930 7935 Ser Thr Ile Ser Gln Gly Phe Ser
His Ser Gln Met Ser Thr Leu Met 7940 7945 7950 Arg Arg Gly Pro Glu
Asp Val Ser Trp Met Ser Pro Pro Leu Leu Glu 7955 7960 7965 Lys Thr
Arg Pro Ser Phe Ser Leu Met Ser Ser Pro Ala Thr Thr Ser 7970 7975
7980 Pro Ser Pro Val Ser Ser Thr Leu Pro Glu Ser Ile Ser Ser Ser
Pro7985 7990 7995 8000Leu Pro Val Thr Ser Leu Leu Thr Ser Gly Leu
Ala Lys Thr Thr Asp 8005 8010 8015 Met Leu His Lys Ser Ser Glu Pro
Val Thr Asn Ser Pro Ala Asn Leu 8020 8025 8030 Ser Ser Thr Ser Val
Glu Ile Leu Ala Thr Ser Glu Val Thr Thr Asp 8035 8040 8045 Thr Glu
Lys Thr His Pro Ser Ser Asn Arg Thr Val Thr Asp Val Gly 8050 8055
8060 Thr Ser Ser Ser Gly His Glu Ser Thr Ser Phe Val Leu Ala Asp
Ser8065 8070 8075 8080Gln Thr Ser Lys Val Thr Ser Pro Met Val Ile
Thr Ser Thr Met Glu 8085 8090 8095 Asp Thr Ser Val Ser Thr Ser Thr
Pro Gly Phe Phe Glu Thr Ser Arg 8100 8105 8110 Ile Gln Thr Glu Pro
Thr Ser Ser Leu Thr Leu Gly Leu Arg Lys Thr 8115 8120 8125 Ser Ser
Ser Glu Gly Thr Ser Leu Ala Thr Glu Met Ser Thr Val Leu 8130 8135
8140 Ser Gly Val Pro Thr Gly Ala Thr Ala Glu Val Ser Arg Thr Glu
Val8145 8150 8155 8160Thr Ser Ser Ser Arg Thr Ser Ile Ser Gly Phe
Ala Gln Leu Thr Val 8165 8170 8175 Ser Pro Glu Thr Ser Thr Glu Thr
Ile Thr Arg Leu Pro Thr Ser Ser 8180 8185 8190 Ile Met Thr Glu Ser
Ala Glu Met Met Ile Lys Thr Gln Thr Asp Pro 8195 8200 8205 Pro Gly
Ser Thr Pro Glu Ser Thr His Thr Val Asp Ile Ser Thr Thr 8210 8215
8220 Pro Asn Trp Val Glu Thr His Ser Thr Val Thr Gln Arg Phe Ser
His8225 8230 8235 8240Ser Glu Met Thr Thr Leu Val Ser Arg Ser Pro
Gly Asp Met Leu Trp 8245 8250 8255 Pro Ser Gln Ser Ser Val Glu Glu
Thr Ser Ser Ala Ser Ser Leu Leu 8260 8265 8270 Ser Leu Pro Ala Thr
Thr Ser Pro Ser Pro Val Ser Ser Thr Leu Val 8275 8280 8285 Glu Asp
Phe Pro Ser Ala Ser Leu Pro Val Thr Ser Leu Leu Thr Pro 8290 8295
8300 Gly Leu Val Ile Thr Thr Asp Arg Met Gly Ile Ser Arg Glu Pro
Gly8305 8310 8315 8320Thr Ser Ser Thr Ser Asn Leu Ser Ser Thr Ser
His Glu Arg Leu Thr 8325 8330 8335 Thr Leu Glu Asp Thr Val Asp Thr
Glu Asp Met Gln Pro Ser Thr His 8340 8345 8350 Thr Ala Val Thr Asn
Val Arg Thr Ser Ile Ser Gly His Glu Ser Gln 8355 8360 8365 Ser Ser
Val Leu Ser Asp Ser Glu Thr Pro Lys Ala Thr Ser Pro Met 8370 8375
8380 Gly Thr Thr Tyr Thr Met Gly Glu Thr Ser Val Ser Ile Ser Thr
Ser8385 8390 8395 8400Asp Phe Phe Glu Thr Ser Arg Ile Gln Ile Glu
Pro Thr Ser Ser Leu 8405 8410 8415 Thr Ser Gly Leu Arg Glu Thr Ser
Ser Ser Glu Arg Ile Ser Ser Ala 8420 8425 8430 Thr Glu Gly Ser Thr
Val Leu Ser Glu Val Pro Ser Gly Ala Thr Thr 8435 8440 8445 Glu Val
Ser Arg Thr Glu Val Ile Ser Ser Arg Gly Thr Ser Met Ser 8450 8455
8460 Gly Pro Asp Gln Phe Thr Ile Ser Pro Asp Ile Ser Thr Glu Ala
Ile8465 8470 8475 8480Thr Arg Leu Ser Thr Ser Pro Ile Met Thr Glu
Ser Ala Glu Ser Ala 8485 8490 8495 Ile Thr Ile Glu Thr Gly Ser Pro
Gly Ala Thr Ser Glu Gly Thr Leu 8500 8505 8510 Thr Leu Asp Thr Ser
Thr Thr Thr Phe Trp Ser Gly Thr His Ser Thr 8515 8520 8525 Ala Ser
Pro Gly Phe Ser His Ser Glu Met Thr Thr Leu Met Ser Arg 8530 8535
8540 Thr Pro Gly Asp Val Pro Trp Pro Ser Leu Pro Ser Val Glu Glu
Ala8545 8550 8555 8560Ser Ser Val Ser Ser Ser Leu Ser Ser Pro Ala
Met Thr Ser Thr Ser 8565 8570 8575 Phe Phe Ser Ala Leu Pro Glu Ser
Ile Ser Ser Ser Pro His Pro Val 8580 8585 8590 Thr Ala Leu Leu Thr
Leu Gly Pro Val Lys Thr Thr Asp Met Leu Arg 8595 8600 8605 Thr Ser
Ser Glu Pro Glu Thr Ser Ser Pro Pro Asn Leu Ser Ser Thr 8610 8615
8620 Ser Ala Glu Ile Leu Ala Thr Ser Glu Val Thr Lys Asp Arg Glu
Lys8625 8630 8635 8640Ile His Pro Ser Ser Asn Thr Pro Val Val Asn
Val Gly Thr Val Ile 8645 8650 8655 Tyr Lys His Leu Ser Pro Ser Ser
Val Leu Ala Asp Leu Val Thr Thr 8660 8665 8670 Lys Pro Thr Ser Pro
Met Ala Thr Thr Ser Thr Leu Gly Asn Thr Ser 8675 8680 8685 Val Ser
Thr Ser Thr Pro Ala Phe Pro Glu Thr Met Met Thr Gln Pro 8690 8695
8700 Thr Ser Ser Leu Thr Ser Gly Leu Arg Glu Ile Ser Thr Ser Gln
Glu8705 8710 8715 8720Thr Ser Ser Ala Thr Glu Arg Ser Ala Ser Leu
Ser Gly Met Pro Thr 8725 8730 8735 Gly Ala Thr Thr Lys Val Ser Arg
Thr Glu Ala Leu Ser Leu Gly Arg 8740 8745 8750 Thr Ser Thr Pro Gly
Pro Ala Gln Ser Thr Ile Ser Pro Glu Ile Ser 8755 8760 8765 Thr Glu
Thr Ile Thr Arg Ile Ser Thr Pro Leu Thr Thr Thr Gly Ser 8770 8775
8780 Ala Glu Met Thr Ile Thr Pro Lys Thr Gly His Ser Gly Ala Ser
Ser8785 8790 8795 8800Gln Gly Thr Phe Thr Leu Asp Thr Ser Ser Arg
Ala Ser Trp Pro Gly 8805 8810 8815 Thr His Ser Ala Ala Thr His Arg
Ser Pro His Ser Gly Met Thr Thr 8820 8825 8830 Pro Met Ser Arg Gly
Pro Glu Asp Val Ser Trp Pro Ser Arg Pro Ser 8835 8840 8845 Val Glu
Lys Thr Ser Pro Pro Ser Ser Leu Val Ser Leu Ser Ala Val 8850 8855
8860 Thr Ser Pro Ser Pro Leu Tyr Ser Thr Pro Ser Glu Ser Ser His
Ser8865 8870 8875 8880Ser Pro Leu Arg Val Thr Ser Leu Phe Thr Pro
Val Met Met Lys Thr 8885 8890 8895 Thr Asp Met Leu Asp Thr Ser Leu
Glu Pro Val Thr Thr Ser Pro Pro 8900 8905 8910 Ser Met Asn Ile Thr
Ser Asp Glu Ser Leu Ala Thr Ser Lys Ala Thr 8915 8920 8925 Met Glu
Thr Glu Ala Ile Gln Leu Ser Glu Asn Thr Ala Val Thr Gln 8930 8935
8940 Met Gly Thr Ile Ser Ala Arg Gln Glu Phe Tyr Ser Ser Tyr Pro
Gly8945 8950 8955 8960Leu Pro Glu Pro Ser Lys Val Thr Ser Pro Val
Val Thr Ser Ser Thr 8965 8970 8975 Ile Lys Asp Ile Val Ser Thr Thr
Ile Pro Ala Ser Ser Glu Ile Thr 8980 8985 8990 Arg Ile Glu Met Glu
Ser Thr Ser Thr Leu Thr Pro Thr Pro Arg Glu 8995 9000 9005 Thr Ser
Thr Ser Gln Glu Ile His Ser Ala Thr Lys Pro Ser Thr Val 9010 9015
9020 Pro Tyr Lys Ala Leu Thr Ser Ala Thr Ile Glu Asp Ser Met Thr
Gln9025 9030 9035 9040Val Met Ser Ser Ser Arg Gly Pro Ser Pro Asp
Gln Ser Thr Met Ser 9045 9050 9055 Gln Asp Ile Ser Ser Glu Val Ile
Thr Arg Leu Ser Thr Ser Pro Ile 9060 9065 9070 Lys Ala Glu Ser Thr
Glu Met Thr Ile Thr Thr Gln Thr Gly Ser Pro 9075 9080 9085 Gly Ala
Thr Ser Arg Gly Thr Leu Thr Leu Asp Thr Ser Thr Thr Phe 9090 9095
9100 Met Ser Gly Thr His Ser Thr Ala Ser Gln Gly Phe Ser His Ser
Gln9105 9110 9115 9120Met Thr Ala Leu Met Ser Arg Thr Pro Gly Asp
Val Pro Trp Leu Ser 9125 9130 9135 His Pro Ser Val Glu Glu Ala Ser
Ser Ala Ser Phe Ser Leu Ser Ser 9140 9145 9150 Pro Val Met Thr Ser
Ser Ser Pro Val Ser Ser Thr Leu Pro Asp Ser 9155 9160 9165 Ile His
Ser Ser Ser Leu Pro Val Thr Ser Leu Leu Thr Ser Gly Leu 9170 9175
9180 Val Lys Thr Thr Glu Leu Leu Gly Thr Ser Ser Glu Pro Glu Thr
Ser9185 9190 9195 9200Ser Pro Pro Asn Leu Ser Ser Thr Ser Ala Glu
Ile Leu Ala Thr Thr 9205 9210 9215 Glu Val Thr Thr Asp Thr Glu Lys
Leu Glu Met Thr Asn Val Val Thr 9220 9225 9230 Ser Gly Tyr Thr His
Glu Ser Pro Ser Ser Val Leu Ala Asp Ser Val 9235 9240 9245 Thr Thr
Lys Ala Thr Ser Ser Met Gly Ile Thr Tyr Pro Thr Gly Asp 9250 9255
9260 Thr Asn Val Leu Thr Ser Thr Pro Ala Phe Ser Asp Thr Ser Arg
Ile9265 9270 9275 9280Gln Thr Lys Ser Lys Leu Ser Leu Thr Pro Gly
Leu Met Glu Thr Ser 9285 9290 9295 Ile Ser Glu Glu Thr Ser Ser Ala
Thr Glu Lys Ser Thr Val Leu Ser 9300 9305 9310 Ser Val Pro Thr Gly
Ala Thr Thr Glu Val Ser Arg Thr Glu Ala Ile 9315 9320 9325 Ser Ser
Ser Arg Thr Ser Ile Pro Gly Pro Ala Gln Ser Thr Met Ser 9330 9335
9340 Ser Asp Thr Ser Met Glu Thr Ile Thr Arg Ile Ser Thr Pro Leu
Thr9345 9350 9355 9360Arg Lys Glu Ser Thr Asp Met Ala Ile Thr Pro
Lys Thr Gly Pro Ser 9365 9370 9375 Gly Ala Thr Ser Gln Gly Thr Phe
Thr Leu Asp Ser Ser Ser Thr Ala 9380 9385 9390 Ser Trp Pro Gly Thr
His Ser Ala Thr Thr Gln Arg Phe Pro Gln Ser 9395 9400 9405 Val Val
Thr Thr Pro Met Ser Arg Gly Pro Glu Asp Val Ser Trp Pro 9410 9415
9420 Ser Pro Leu Ser Val Glu Lys Asn Ser Pro Pro Ser Ser Leu Val
Ser9425 9430 9435 9440Ser Ser Ser Val Thr Ser Pro Ser Pro Leu Tyr
Ser Thr Pro Ser Gly 9445 9450 9455 Ser Ser His Ser Ser Pro Val Pro
Val Thr Ser Leu Phe Thr Ser Ile 9460 9465 9470 Met Met Lys Ala Thr
Asp Met Leu Asp Ala Ser Leu Glu Pro Glu Thr 9475 9480 9485 Thr Ser
Ala Pro Asn Met Asn Ile Thr Ser Asp Glu Ser Leu Ala Thr 9490 9495
9500 Ser Lys Ala Thr Thr Glu Thr Glu Ala Ile His Val Phe Glu Asn
Thr9505 9510 9515 9520Ala Ala Ser His Val Glu Thr Thr Ser Ala Thr
Glu Glu Leu Tyr Ser 9525 9530 9535 Ser Ser Pro Gly Phe Ser Glu Pro
Thr Lys Val Ile Ser Pro Val Val 9540 9545 9550 Thr Ser Ser Ser Ile
Arg Asp Asn Met Val Ser Thr Thr Met Pro Gly 9555 9560 9565 Ser Ser
Gly Ile Thr Arg Ile Glu Ile Glu Ser Met Ser Ser Leu Thr 9570 9575
9580 Pro Gly Leu Arg Glu Thr Arg Thr Ser Gln Asp Ile Thr Ser Ser
Thr9585 9590 9595 9600Glu Thr Ser Thr Val Leu Tyr Lys Met Ser Ser
Gly Ala Thr Pro Glu 9605 9610 9615 Val Ser Arg Thr Glu Val Met Pro
Ser Ser Arg Thr Ser Ile Pro Gly 9620 9625 9630 Pro Ala Gln Ser Thr
Met Ser Leu Asp Ile Ser Asp Glu Val Val Thr 9635 9640 9645 Arg Leu
Ser Thr Ser Pro Ile Met Thr Glu Ser Ala Glu Ile Thr Ile 9650 9655
9660 Thr Thr Gln Thr Gly Tyr Ser Leu Ala Thr Ser Gln Val Thr Leu
Pro9665 9670 9675 9680Leu Gly Thr Ser Met Thr Phe Leu Ser Gly Thr
His Ser Thr Met Ser 9685 9690 9695 Gln Gly Leu Ser His Ser Glu Met
Thr Asn Leu Met Ser Arg Gly Pro 9700 9705 9710 Glu Ser Leu Ser Trp
Thr Ser Pro Arg Phe Val Glu Thr Thr Arg Ser 9715 9720 9725 Ser Ser
Ser Leu Thr Ser Leu Pro Leu Thr Thr Ser Leu Ser Pro Val 9730 9735
9740 Ser Ser Thr Leu Leu Asp Ser Ser Pro Ser Ser Pro Leu Pro Val
Thr9745 9750 9755 9760Ser Leu Ile Leu Pro Gly Leu Val Lys Thr Thr
Glu Val Leu Asp Thr 9765 9770 9775 Ser Ser Glu Pro Lys Thr Ser Ser
Ser Pro Asn Leu Ser Ser Thr Ser 9780 9785 9790 Val Glu Ile Pro Ala
Thr Ser Glu Ile Met Thr Asp Thr Glu Lys Ile 9795 9800 9805 His Pro
Ser Ser Asn Thr Ala Val Ala Lys Val Arg Thr Ser Ser Ser 9810 9815
9820 Val His Glu Ser His Ser Ser Val Leu Ala Asp Ser Glu Thr Thr
Ile9825 9830 9835 9840Thr Ile Pro Ser Met Gly Ile Thr Ser Ala Val
Asp Asp Thr Thr Val 9845 9850 9855 Phe Thr Ser Asn Pro Ala Phe Ser
Glu Thr Arg Arg Ile Pro Thr Glu 9860 9865 9870 Pro Thr Phe Ser Leu
Thr Pro Gly Phe Arg Glu Thr Ser Thr Ser Glu 9875 9880 9885 Glu Thr
Thr Ser Ile Thr Glu Thr Ser Ala Val Leu Tyr Gly Val Pro 9890 9895
9900 Thr Ser Ala Thr Thr Glu Val Ser Met Thr Glu Ile Met Ser Ser
Asn9905 9910 9915 9920Arg Thr His Ile Pro Asp Ser Asp Gln Ser Thr
Met Ser Pro Asp Ile 9925 9930 9935 Ile Thr Glu Val Ile Thr Arg Leu
Ser Ser Ser Ser Met Met Ser Glu 9940 9945 9950 Ser Thr Gln Met Thr
Ile Thr Thr Gln Lys Ser Ser Pro Gly Ala Thr 9955 9960 9965 Ala Gln
Ser Thr Leu Thr Leu Ala Thr Thr Thr Ala Pro Leu Ala Arg 9970 9975
9980 Thr His Ser Thr Val Pro Pro Arg Phe Leu His Ser Glu Met Thr
Thr9985 9990 9995 10000Leu Met Ser Arg Ser Pro Glu Asn Pro Ser Trp
Lys Ser Ser Pro Phe 10005 10010 10015 Val Glu Lys Thr Ser Ser Ser
Ser Ser Leu Leu Ser Leu Pro Val Thr 10020 10025 10030 Thr Ser Pro
Ser Val Ser Ser Thr Leu Pro Gln Ser Ile Pro Ser Ser 10035 10040
10045 Ser Phe Ser Val Thr Ser Leu Leu Thr Pro Gly Met Val Lys Thr
Thr 10050 10055 10060 57858PRTHomo sapiens 57Met Leu Gln Thr Lys
Asp Leu Ile Trp Thr Leu Phe Phe Leu Gly Thr1 5 10 15 Ala Val Ser
Leu Gln Val Asp Ile Val Pro Ser Gln Gly Glu Ile Ser 20 25 30 Val
Gly Glu Ser Lys Phe Phe Leu Cys Gln Val Ala Gly Asp Ala Lys 35 40
45 Asp Lys Asp Ile Ser Trp Phe Ser Pro Asn Gly Glu Lys Leu Thr Pro
50 55 60 Asn Gln Gln Arg Ile Ser Val Val Trp Asn Asp Asp Ser Ser
Ser Thr65 70 75 80 Leu Thr Ile Tyr Asn Ala Asn Ile Asp Asp Ala Gly
Ile Tyr Lys Cys 85 90 95
Val Val Thr Gly Glu Asp Gly Ser Glu Ser Glu Ala Thr Val Asn Val 100
105 110 Lys Ile Phe Gln Lys Leu Met Phe Lys Asn Ala Pro Thr Pro Gln
Glu 115 120 125 Phe Arg Glu Gly Glu Asp Ala Val Ile Val Cys Asp Val
Val Ser Ser 130 135 140 Leu Pro Pro Thr Ile Ile Trp Lys His Lys Gly
Arg Asp Val Ile Leu145 150 155 160 Lys Lys Asp Val Arg Phe Ile Val
Leu Ser Asn Asn Tyr Leu Gln Ile 165 170 175 Arg Gly Ile Lys Lys Thr
Asp Glu Gly Thr Tyr Arg Cys Glu Gly Arg 180 185 190 Ile Leu Ala Arg
Gly Glu Ile Asn Phe Lys Asp Ile Gln Val Ile Val 195 200 205 Asn Val
Pro Pro Thr Ile Gln Ala Arg Gln Asn Ile Val Asn Ala Thr 210 215 220
Ala Asn Leu Gly Gln Ser Val Thr Leu Val Cys Asp Ala Glu Gly Phe225
230 235 240 Pro Glu Pro Thr Met Ser Trp Thr Lys Asp Gly Glu Gln Ile
Glu Gln 245 250 255 Glu Glu Asp Asp Glu Lys Tyr Ile Phe Ser Asp Asp
Ser Ser Gln Leu 260 265 270 Thr Ile Lys Lys Val Asp Lys Asn Asp Glu
Ala Glu Tyr Ile Cys Ile 275 280 285 Ala Glu Asn Lys Ala Gly Glu Gln
Asp Ala Thr Ile His Leu Lys Val 290 295 300 Phe Ala Lys Pro Lys Ile
Thr Tyr Val Glu Asn Gln Thr Ala Met Glu305 310 315 320 Leu Glu Glu
Gln Val Thr Leu Thr Cys Glu Ala Ser Gly Asp Pro Ile 325 330 335 Pro
Ser Ile Thr Trp Arg Thr Ser Thr Arg Asn Ile Ser Ser Glu Glu 340 345
350 Lys Ala Ser Trp Thr Arg Pro Glu Lys Gln Glu Thr Leu Asp Gly His
355 360 365 Met Val Val Arg Ser His Ala Arg Val Ser Ser Leu Thr Leu
Lys Ser 370 375 380 Ile Gln Tyr Thr Asp Ala Gly Glu Tyr Ile Cys Thr
Ala Ser Asn Thr385 390 395 400 Ile Gly Gln Asp Ser Gln Ser Met Tyr
Leu Glu Val Gln Tyr Ala Pro 405 410 415 Lys Leu Gln Gly Pro Val Ala
Val Tyr Thr Trp Glu Gly Asn Gln Val 420 425 430 Asn Ile Thr Cys Glu
Val Phe Ala Tyr Pro Ser Ala Thr Ile Ser Trp 435 440 445 Phe Arg Asp
Gly Gln Leu Leu Pro Ser Ser Asn Tyr Ser Asn Ile Lys 450 455 460 Ile
Tyr Asn Thr Pro Ser Ala Ser Tyr Leu Glu Val Thr Pro Asp Ser465 470
475 480 Glu Asn Asp Phe Gly Asn Tyr Asn Cys Thr Ala Val Asn Arg Ile
Gly 485 490 495 Gln Glu Ser Leu Glu Phe Ile Leu Val Gln Ala Asp Thr
Pro Ser Ser 500 505 510 Pro Ser Ile Asp Gln Val Glu Pro Tyr Ser Ser
Thr Ala Gln Val Gln 515 520 525 Phe Asp Glu Pro Glu Ala Thr Gly Gly
Val Pro Ile Leu Lys Tyr Lys 530 535 540 Ala Glu Trp Arg Ala Val Gly
Glu Glu Val Trp His Ser Lys Trp Tyr545 550 555 560 Asp Ala Lys Glu
Ala Ser Met Glu Gly Ile Val Thr Ile Val Gly Leu 565 570 575 Lys Pro
Glu Thr Thr Tyr Ala Val Arg Leu Ala Ala Leu Asn Gly Lys 580 585 590
Gly Leu Gly Glu Ile Ser Ala Ala Ser Glu Phe Lys Thr Gln Pro Val 595
600 605 Gln Gly Glu Pro Ser Ala Pro Lys Leu Glu Gly Gln Met Gly Glu
Asp 610 615 620 Gly Asn Ser Ile Lys Val Asn Leu Ile Lys Gln Asp Asp
Gly Gly Ser625 630 635 640 Pro Ile Arg His Tyr Leu Val Arg Tyr Arg
Ala Leu Ser Ser Glu Trp 645 650 655 Lys Pro Glu Ile Arg Leu Pro Ser
Gly Ser Asp His Val Met Leu Lys 660 665 670 Ser Leu Asp Trp Asn Ala
Glu Tyr Glu Val Tyr Val Val Ala Glu Asn 675 680 685 Gln Gln Gly Lys
Ser Lys Ala Ala His Phe Val Phe Arg Thr Ser Ala 690 695 700 Gln Pro
Thr Ala Ile Pro Ala Asn Gly Ser Pro Thr Ser Gly Leu Ser705 710 715
720 Thr Gly Ala Ile Val Gly Ile Leu Ile Val Ile Phe Val Leu Leu Leu
725 730 735 Val Val Val Asp Ile Thr Cys Tyr Phe Leu Asn Lys Cys Gly
Leu Phe 740 745 750 Met Cys Ile Ala Val Asn Leu Cys Gly Lys Ala Gly
Pro Gly Ala Lys 755 760 765 Gly Lys Asp Met Glu Glu Gly Lys Ala Ala
Phe Ser Lys Asp Glu Ser 770 775 780 Lys Glu Pro Ile Val Glu Val Arg
Thr Glu Glu Glu Arg Thr Pro Asn785 790 795 800 His Asp Gly Gly Lys
His Thr Glu Pro Asn Glu Thr Thr Pro Leu Thr 805 810 815 Glu Pro Glu
Lys Gly Pro Val Glu Ala Lys Pro Glu Cys Gln Glu Thr 820 825 830 Glu
Thr Lys Pro Ala Pro Ala Glu Val Lys Thr Val Pro Asn Asp Ala 835 840
845 Thr Gln Thr Lys Glu Asn Glu Ser Lys Ala 850 855 58224PRTHomo
sapiens 58Met Glu Lys Leu Leu Cys Phe Leu Val Leu Thr Ser Leu Ser
His Ala1 5 10 15 Phe Gly Gln Thr Asp Met Ser Arg Lys Ala Phe Val
Phe Pro Lys Glu 20 25 30 Ser Asp Thr Ser Tyr Val Ser Leu Lys Ala
Pro Leu Thr Lys Pro Leu 35 40 45 Lys Ala Phe Thr Val Cys Leu His
Phe Tyr Thr Glu Leu Ser Ser Thr 50 55 60 Arg Gly Tyr Ser Ile Phe
Ser Tyr Ala Thr Lys Arg Gln Asp Asn Glu65 70 75 80 Ile Leu Ile Phe
Trp Ser Lys Asp Ile Gly Tyr Ser Phe Thr Val Gly 85 90 95 Gly Ser
Glu Ile Leu Phe Glu Val Pro Glu Val Thr Val Ala Pro Val 100 105 110
His Ile Cys Thr Ser Trp Glu Ser Ala Ser Gly Ile Val Glu Phe Trp 115
120 125 Val Asp Gly Lys Pro Arg Val Arg Lys Ser Leu Lys Lys Gly Tyr
Thr 130 135 140 Val Gly Ala Glu Ala Ser Ile Ile Leu Gly Gln Glu Gln
Asp Ser Phe145 150 155 160 Gly Gly Asn Phe Glu Gly Ser Gln Ser Leu
Val Gly Asp Ile Gly Asn 165 170 175 Val Asn Met Trp Asp Phe Val Leu
Ser Pro Asp Glu Ile Asn Thr Ile 180 185 190 Tyr Leu Gly Gly Pro Phe
Ser Pro Asn Val Leu Asn Trp Arg Ala Leu 195 200 205 Lys Tyr Glu Val
Gln Gly Glu Val Phe Thr Lys Pro Gln Leu Trp Pro 210 215 220
59418PRTHomo sapiens 59Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu
Leu Ala Gly Leu Cys1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu
Asp Pro Gln Gly Asp Ala Ala 20 25 30 Gln Lys Thr Asp Thr Ser His
His Asp Gln Asp His Pro Thr Phe Asn 35 40 45 Lys Ile Thr Pro Asn
Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 50 55 60 Leu Ala His
Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser65 70 75 80 Ile
Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 85 90
95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro
100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr
Leu Asn 115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn
Gly Leu Phe Leu 130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe
Leu Glu Asp Val Lys Lys145 150 155 160 Leu Tyr His Ser Glu Ala Phe
Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170 175 Ala Lys Lys Gln Ile
Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 180 185 190 Ile Val Asp
Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200 205 Val
Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210 215
220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr
Val225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn
Ile Gln His Cys 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met
Lys Tyr Leu Gly Asn Ala 260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp
Glu Gly Lys Leu Gln His Leu Glu 275 280 285 Asn Glu Leu Thr His Asp
Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295 300 Arg Arg Ser Ala
Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr305 310 315 320 Tyr
Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 325 330
335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys
340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu
Lys Gly 355 360 365 Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile
Pro Met Ser Ile 370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe
Val Phe Leu Met Ile Glu385 390 395 400 Gln Asn Thr Lys Ser Pro Leu
Phe Met Gly Lys Val Val Asn Pro Thr 405 410 415 Gln Lys60531PRTHomo
sapiens 60Met Ser Lys Pro His Ser Glu Ala Gly Thr Ala Phe Ile Gln
Thr Gln1 5 10 15 Gln Leu His Ala Ala Met Ala Asp Thr Phe Leu Glu
His Met Cys Arg 20 25 30 Leu Asp Ile Asp Ser Pro Pro Ile Thr Ala
Arg Asn Thr Gly Ile Ile 35 40 45 Cys Thr Ile Gly Pro Ala Ser Arg
Ser Val Glu Thr Leu Lys Glu Met 50 55 60 Ile Lys Ser Gly Met Asn
Val Ala Arg Leu Asn Phe Ser His Gly Thr65 70 75 80 His Glu Tyr His
Ala Glu Thr Ile Lys Asn Val Arg Thr Ala Thr Glu 85 90 95 Ser Phe
Ala Ser Asp Pro Ile Leu Tyr Arg Pro Val Ala Val Ala Leu 100 105 110
Asp Thr Lys Gly Pro Glu Ile Arg Thr Gly Leu Ile Lys Gly Ser Gly 115
120 125 Thr Ala Glu Val Glu Leu Lys Lys Gly Ala Thr Leu Lys Ile Thr
Leu 130 135 140 Asp Asn Ala Tyr Met Glu Lys Cys Asp Glu Asn Ile Leu
Trp Leu Asp145 150 155 160 Tyr Lys Asn Ile Cys Lys Val Val Glu Val
Gly Ser Lys Ile Tyr Val 165 170 175 Asp Asp Gly Leu Ile Ser Leu Gln
Val Lys Gln Lys Gly Ala Asp Phe 180 185 190 Leu Val Thr Glu Val Glu
Asn Gly Gly Ser Leu Gly Ser Lys Lys Gly 195 200 205 Val Asn Leu Pro
Gly Ala Ala Val Asp Leu Pro Ala Val Ser Glu Lys 210 215 220 Asp Ile
Gln Asp Leu Lys Phe Gly Val Glu Gln Asp Val Asp Met Val225 230 235
240 Phe Ala Ser Phe Ile Arg Lys Ala Ser Asp Val His Glu Val Arg Lys
245 250 255 Val Leu Gly Glu Lys Gly Lys Asn Ile Lys Ile Ile Ser Lys
Ile Glu 260 265 270 Asn His Glu Gly Val Arg Arg Phe Asp Glu Ile Leu
Glu Ala Ser Asp 275 280 285 Gly Ile Met Val Ala Arg Gly Asp Leu Gly
Ile Glu Ile Pro Ala Glu 290 295 300 Lys Val Phe Leu Ala Gln Lys Met
Met Ile Gly Arg Cys Asn Arg Ala305 310 315 320 Gly Lys Pro Val Ile
Cys Ala Thr Gln Met Leu Glu Ser Met Ile Lys 325 330 335 Lys Pro Arg
Pro Thr Arg Ala Glu Gly Ser Asp Val Ala Asn Ala Val 340 345 350 Leu
Asp Gly Ala Asp Cys Ile Met Leu Ser Gly Glu Thr Ala Lys Gly 355 360
365 Asp Tyr Pro Leu Glu Ala Val Arg Met Gln His Leu Ile Ala Arg Glu
370 375 380 Ala Glu Ala Ala Ile Tyr His Leu Gln Leu Phe Glu Glu Leu
Arg Arg385 390 395 400 Leu Ala Pro Ile Thr Ser Asp Pro Thr Glu Ala
Thr Ala Val Gly Ala 405 410 415 Val Glu Ala Ser Phe Lys Cys Cys Ser
Gly Ala Ile Ile Val Leu Thr 420 425 430 Lys Ser Gly Arg Ser Ala His
Gln Val Ala Arg Tyr Arg Pro Arg Ala 435 440 445 Pro Ile Ile Ala Val
Thr Arg Asn Pro Gln Thr Ala Arg Gln Ala His 450 455 460 Leu Tyr Arg
Gly Ile Phe Pro Val Leu Cys Lys Asp Pro Val Gln Glu465 470 475 480
Ala Trp Ala Glu Asp Val Asp Leu Arg Val Asn Phe Ala Met Asn Val 485
490 495 Gly Lys Ala Arg Gly Phe Phe Lys Lys Gly Asp Val Val Ile Val
Leu 500 505 510 Thr Gly Trp Arg Pro Gly Ser Gly Phe Thr Asn Thr Met
Arg Val Val 515 520 525 Pro Val Pro 530 611257PRTHomo sapiens 61Met
Lys Ala Ala Asp Glu Pro Ala Tyr Leu Thr Val Gly Thr Asp Val1 5 10
15 Ser Ala Lys Tyr Arg Gly Ala Phe Cys Glu Ala Lys Ile Lys Thr Val
20 25 30 Lys Arg Leu Val Lys Val Lys Val Leu Leu Lys Gln Asp Asn
Thr Thr 35 40 45 Gln Leu Val Gln Asp Asp Gln Val Lys Gly Pro Leu
Arg Val Gly Ala 50 55 60 Ile Val Glu Thr Arg Thr Ser Asp Gly Ser
Phe Gln Glu Ala Ile Ile65 70 75 80 Ser Lys Leu Thr Asp Ala Ser Trp
Tyr Thr Val Val Phe Asp Asp Gly 85 90 95 Asp Glu Arg Thr Leu Arg
Arg Thr Ser Leu Cys Leu Lys Gly Glu Arg 100 105 110 His Phe Ala Glu
Ser Glu Thr Leu Asp Gln Leu Pro Leu Thr Asn Pro 115 120 125 Glu His
Phe Gly Thr Pro Val Ile Ala Lys Lys Thr Asn Arg Gly Arg 130 135 140
Arg Ser Ser Leu Pro Val Thr Glu Asp Glu Lys Glu Glu Glu Ser Ser145
150 155 160 Glu Glu Glu Asp Glu Asp Lys Arg Arg Leu Asn Asp Glu Leu
Leu Gly 165 170 175 Lys Val Val Ser Val Val Ser Ala Thr Glu Arg Thr
Glu Trp Tyr Pro 180 185 190 Ala Leu Val Ile Ser Pro Ser Cys Asn Asp
Asp Ile Thr Val Lys Lys 195 200 205 Asp Gln Cys Leu Val Arg Ser Phe
Ile Asp Ser Lys Phe Tyr Ser Ile 210 215 220 Ala Arg Lys Asp Ile Lys
Glu Val Asp Ile Leu Asn Leu Pro Glu Ser225 230 235 240 Glu Leu Ser
Thr Lys Pro Gly Leu Gln Lys Ala Ser Ile Phe Leu Lys 245 250 255 Thr
Arg Val Val Pro Asp Asn Trp Lys Met Asp Ile Ser Glu Ile Leu 260 265
270 Glu Ser Ser Ser Ser Asp Asp Glu Asp Gly Pro Ala Glu Glu Asn Asp
275 280 285 Glu Glu Lys Glu Lys Glu Ala Lys Lys Thr Glu Glu Glu Val
Pro Glu 290 295 300 Glu Glu Leu Asp Pro Glu Glu Arg Asp Asn Phe Leu
Gln Gln Leu Tyr305 310 315 320 Lys Phe Met Glu Asp Arg Gly Thr Pro
Ile Asn Lys Pro Pro Val Leu 325 330 335 Gly Tyr Lys Asp Leu Asn Leu
Phe Lys Leu Phe Arg Leu Val Tyr His 340 345 350 Gln Gly Gly Cys Asp
Asn Ile Asp Ser Gly Ala Val Trp Lys Gln Ile 355 360
365 Tyr Met Asp Leu Gly Ile Pro Ile Leu Asn Ser Ala Ala Ser Tyr Asn
370 375 380 Val Lys Thr Ala Tyr Arg Lys Tyr Leu Tyr Gly Phe Glu Glu
Tyr Cys385 390 395 400 Arg Ser Ala Asn Ile Gln Phe Arg Thr Val His
His His Glu Pro Lys 405 410 415 Val Lys Glu Glu Lys Lys Asp Leu Glu
Glu Ser Met Glu Glu Ala Leu 420 425 430 Lys Leu Asp Gln Glu Met Pro
Leu Thr Glu Val Lys Ser Glu Pro Glu 435 440 445 Glu Asn Ile Asp Ser
Asn Ser Glu Ser Glu Arg Glu Glu Ile Glu Leu 450 455 460 Lys Ser Pro
Arg Gly Arg Arg Arg Ile Ala Arg Asp Val Asn Ser Ile465 470 475 480
Lys Lys Glu Ile Glu Glu Glu Lys Thr Glu Asp Lys Leu Lys Asp Asn 485
490 495 Asp Thr Glu Asn Lys Asp Val Asp Asp Asp Tyr Glu Thr Ala Glu
Lys 500 505 510 Lys Glu Asn Glu Leu Leu Leu Gly Arg Lys Asn Thr Pro
Lys Gln Lys 515 520 525 Glu Lys Lys Ile Lys Lys Gln Glu Asp Ser Asp
Lys Asp Ser Asp Glu 530 535 540 Glu Glu Glu Lys Ser Gln Glu Arg Glu
Glu Thr Glu Ser Lys Cys Asp545 550 555 560 Ser Glu Gly Glu Glu Asp
Glu Glu Asp Met Glu Pro Cys Leu Thr Gly 565 570 575 Thr Lys Val Lys
Val Lys Tyr Gly Arg Gly Lys Thr Gln Lys Ile Tyr 580 585 590 Glu Ala
Ser Ile Lys Ser Thr Glu Ile Asp Asp Gly Glu Val Leu Tyr 595 600 605
Leu Val His Tyr Tyr Gly Trp Asn Val Arg Tyr Asp Glu Trp Val Lys 610
615 620 Ala Asp Arg Ile Ile Trp Pro Leu Asp Lys Gly Gly Pro Lys Lys
Lys625 630 635 640 Gln Lys Lys Lys Ala Lys Asn Lys Glu Asp Ser Glu
Lys Asp Glu Lys 645 650 655 Arg Asp Glu Glu Arg Gln Lys Ser Lys Arg
Gly Arg Pro Pro Leu Lys 660 665 670 Ser Thr Leu Ser Ser Asn Met Pro
Tyr Gly Leu Ser Lys Thr Ala Asn 675 680 685 Ser Glu Gly Lys Ser Asp
Ser Cys Ser Ser Asp Ser Glu Thr Glu Asp 690 695 700 Ala Leu Glu Lys
Asn Leu Ile Asn Glu Glu Leu Ser Leu Lys Asp Glu705 710 715 720 Leu
Glu Lys Asn Glu Asn Leu Asn Asp Asp Lys Leu Asp Glu Glu Asn 725 730
735 Pro Lys Ile Ser Ala His Ile Leu Lys Glu Asn Asp Arg Thr Gln Met
740 745 750 Gln Pro Leu Glu Thr Leu Lys Leu Glu Val Gly Glu Asn Glu
Gln Ile 755 760 765 Val Gln Ile Phe Gly Asn Lys Met Glu Lys Thr Glu
Glu Val Lys Lys 770 775 780 Glu Ala Glu Lys Ser Pro Lys Gly Lys Gly
Arg Arg Ser Lys Thr Lys785 790 795 800 Asp Leu Ser Leu Glu Ile Ile
Lys Ile Ser Ser Phe Gly Gln Asn Glu 805 810 815 Ala Gly Ser Glu Pro
His Ile Glu Ala His Ser Leu Glu Leu Ser Ser 820 825 830 Leu Asp Asn
Lys Asn Phe Ser Ser Ala Thr Glu Asp Glu Ile Asp Gln 835 840 845 Cys
Val Lys Glu Lys Lys Leu Lys Arg Lys Ile Leu Gly Gln Ser Ser 850 855
860 Pro Glu Lys Lys Ile Arg Ile Glu Asn Gly Met Glu Met Thr Asn
Thr865 870 875 880 Val Ser Gln Glu Arg Thr Ser Asp Cys Ile Gly Ser
Glu Gly Met Lys 885 890 895 Asn Leu Asn Phe Glu Gln His Phe Glu Arg
Glu Asn Glu Gly Met Pro 900 905 910 Ser Leu Ile Ala Glu Ser Asn Gln
Cys Ile Gln Gln Leu Thr Ser Glu 915 920 925 Arg Phe Asp Ser Pro Ala
Glu Glu Thr Val Asn Ile Pro Leu Lys Glu 930 935 940 Asp Glu Asp Ala
Met Pro Leu Ile Gly Pro Glu Thr Leu Val Cys His945 950 955 960 Glu
Val Asp Leu Asp Asp Leu Asp Glu Lys Asp Lys Thr Ser Ile Glu 965 970
975 Asp Val Ala Val Glu Ser Ser Glu Ser Asn Ser Leu Val Ser Ile Pro
980 985 990 Pro Ala Leu Pro Pro Val Val Gln His Asn Phe Ser Val Ala
Ser Pro 995 1000 1005 Leu Thr Leu Ser Gln Asp Glu Ser Arg Ser Val
Lys Ser Glu Ser Asp 1010 1015 1020 Ile Thr Ile Glu Val Asp Ser Ile
Ala Glu Glu Ser Gln Glu Gly Leu1025 1030 1035 1040Cys Glu Arg Glu
Ser Ala Asn Gly Phe Glu Thr Asn Val Ala Ser Gly 1045 1050 1055 Thr
Cys Ser Ile Ile Val Gln Glu Arg Glu Ser Arg Glu Lys Gly Gln 1060
1065 1070 Lys Arg Pro Ser Asp Gly Asn Ser Gly Leu Met Ala Lys Lys
Gln Lys 1075 1080 1085 Arg Thr Pro Lys Arg Thr Ser Ala Ala Ala Lys
Asn Glu Lys Asn Gly 1090 1095 1100 Thr Gly Gln Ser Ser Asp Ser Glu
Asp Leu Pro Val Leu Asp Asn Ser1105 1110 1115 1120Ser Lys Cys Thr
Pro Val Lys His Leu Asn Val Ser Lys Pro Gln Lys 1125 1130 1135 Leu
Ala Arg Ser Pro Ala Arg Ile Ser Pro His Ile Lys Asp Gly Glu 1140
1145 1150 Lys Asp Lys His Arg Glu Lys His Pro Asn Ser Ser Pro Arg
Thr Tyr 1155 1160 1165 Lys Trp Ser Phe Gln Leu Asn Glu Leu Asp Asn
Met Asn Ser Thr Glu 1170 1175 1180 Arg Ile Ser Phe Leu Gln Glu Lys
Leu Gln Glu Ile Arg Lys Tyr Tyr1185 1190 1195 1200Met Ser Leu Lys
Ser Glu Val Ala Thr Ile Asp Arg Arg Arg Lys Arg 1205 1210 1215 Leu
Lys Lys Lys Asp Arg Glu Val Ser His Ala Gly Ala Ser Met Ser 1220
1225 1230 Ser Ala Ser Ser Asp Thr Gly Met Ser Pro Ser Ser Ser Ser
Pro Pro 1235 1240 1245 Gln Asn Val Leu Ala Val Glu Cys Arg 1250
1255 62282PRTHomo sapiens 62Met Gln Arg Leu Arg Trp Leu Arg Asp Trp
Lys Ser Ser Gly Arg Gly1 5 10 15 Leu Thr Ala Ala Lys Glu Pro Gly
Ala Arg Ser Ser Pro Leu Gln Ala 20 25 30 Met Arg Ile Leu Gln Leu
Ile Leu Leu Ala Leu Ala Thr Gly Leu Val 35 40 45 Gly Gly Glu Thr
Arg Ile Ile Lys Gly Phe Glu Cys Lys Pro His Ser 50 55 60 Gln Pro
Trp Gln Ala Ala Leu Phe Glu Lys Thr Arg Leu Leu Cys Gly65 70 75 80
Ala Thr Leu Ile Ala Pro Arg Trp Leu Leu Thr Ala Ala His Cys Leu 85
90 95 Lys Pro Arg Tyr Ile Val His Leu Gly Gln His Asn Leu Gln Lys
Glu 100 105 110 Glu Gly Cys Glu Gln Thr Arg Thr Ala Thr Glu Ser Phe
Pro His Pro 115 120 125 Gly Phe Asn Asn Ser Leu Pro Asn Lys Asp His
Arg Asn Asp Ile Met 130 135 140 Leu Val Lys Met Ala Ser Pro Val Ser
Ile Thr Trp Ala Val Arg Pro145 150 155 160 Leu Thr Leu Ser Ser Arg
Cys Val Thr Ala Gly Thr Ser Cys Leu Ile 165 170 175 Ser Gly Trp Gly
Ser Thr Ser Ser Pro Gln Leu Arg Leu Pro His Thr 180 185 190 Leu Arg
Cys Ala Asn Ile Thr Ile Ile Glu His Gln Lys Cys Glu Asn 195 200 205
Ala Tyr Pro Gly Asn Ile Thr Asp Thr Met Val Cys Ala Ser Val Gln 210
215 220 Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu
Val225 230 235 240 Cys Asn Gln Ser Leu Gln Gly Ile Ile Ser Trp Gly
Gln Asp Pro Cys 245 250 255 Ala Ile Thr Arg Lys Pro Gly Val Tyr Thr
Lys Val Cys Lys Tyr Val 260 265 270 Asp Trp Ile Gln Glu Thr Met Lys
Asn Asn 275 280 63277PRTHomo sapiens 63Met Trp Pro Leu Ala Leu Val
Ile Ala Ser Leu Thr Leu Ala Leu Ser1 5 10 15 Gly Gly Val Ser Gln
Glu Ser Ser Lys Val Leu Asn Thr Asn Gly Thr 20 25 30 Ser Gly Phe
Leu Pro Gly Gly Tyr Thr Cys Phe Pro His Ser Gln Pro 35 40 45 Trp
Gln Ala Ala Leu Leu Val Gln Gly Arg Leu Leu Cys Gly Gly Val 50 55
60 Leu Val His Pro Lys Trp Val Leu Thr Ala Ala His Cys Leu Lys
Glu65 70 75 80 Gly Leu Lys Val Tyr Leu Gly Lys His Ala Leu Gly Arg
Val Glu Ala 85 90 95 Gly Glu Gln Val Arg Glu Val Val His Ser Ile
Pro His Pro Glu Tyr 100 105 110 Arg Arg Ser Pro Thr His Leu Asn His
Asp His Asp Ile Met Leu Leu 115 120 125 Glu Leu Gln Ser Pro Val Gln
Leu Thr Gly Tyr Ile Gln Thr Leu Pro 130 135 140 Leu Ser His Asn Asn
Arg Leu Thr Pro Gly Thr Thr Cys Arg Val Ser145 150 155 160 Gly Trp
Gly Thr Thr Thr Ser Pro Gln Val Asn Tyr Pro Lys Thr Leu 165 170 175
Gln Cys Ala Asn Ile Gln Leu Arg Ser Asp Glu Glu Cys Arg Gln Val 180
185 190 Tyr Pro Gly Lys Ile Thr Asp Asn Met Leu Cys Ala Gly Thr Lys
Glu 195 200 205 Gly Gly Lys Asp Ser Cys Glu Gly Asp Ser Gly Gly Pro
Leu Val Cys 210 215 220 Asn Arg Thr Leu Tyr Gly Ile Val Ser Trp Gly
Asp Phe Pro Cys Gly225 230 235 240 Gln Pro Asp Arg Pro Gly Val Tyr
Thr Arg Val Ser Arg Tyr Val Leu 245 250 255 Trp Ile Arg Glu Thr Ile
Arg Lys Tyr Glu Thr Gln Gln Gln Lys Trp 260 265 270 Leu Lys Gly Pro
Gln 275 64122PRTHomo sapiens 64Met Lys Leu Leu Thr Gly Leu Val Phe
Cys Ser Leu Val Leu Gly Val1 5 10 15 Ser Ser Arg Ser Phe Phe Ser
Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30 Arg Asp Met Trp Arg
Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45 Gly Ser Asp
Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60 Arg
Gly Pro Gly Gly Val Trp Ala Ala Glu Ala Ile Ser Asp Ala Arg65 70 75
80 Glu Asn Ile Gln Arg Phe Phe Gly His Gly Ala Glu Asp Ser Leu Ala
85 90 95 Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro
Asn His 100 105 110 Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 120
6599PRTHomo sapiens 65Met Gln Pro Arg Val Leu Leu Val Val Ala Leu
Leu Ala Leu Leu Ala1 5 10 15 Ser Ala Arg Ala Ser Glu Ala Glu Asp
Ala Ser Leu Leu Ser Phe Met 20 25 30 Gln Gly Tyr Met Lys His Ala
Thr Lys Thr Ala Lys Asp Ala Leu Ser 35 40 45 Ser Val Gln Glu Ser
Gln Val Ala Gln Gln Ala Arg Gly Trp Val Thr 50 55 60 Asp Gly Phe
Ser Ser Leu Lys Asp Tyr Trp Ser Thr Val Lys Asp Lys65 70 75 80 Phe
Ser Glu Phe Trp Asp Leu Asp Pro Glu Val Arg Pro Thr Ser Ala 85 90
95 Val Ala Ala
* * * * *
References