U.S. patent application number 13/963503 was filed with the patent office on 2014-01-23 for protein.
This patent application is currently assigned to OXFORD BIOTHERAPEUTICS, LTD.. The applicant listed for this patent is OXFORD BIOTHERAPEUTICS, LTD.. Invention is credited to Christian Rohlff, Jonathan Alexander Terrett.
Application Number | 20140024048 13/963503 |
Document ID | / |
Family ID | 44353891 |
Filed Date | 2014-01-23 |
United States Patent
Application |
20140024048 |
Kind Code |
A1 |
Rohlff; Christian ; et
al. |
January 23, 2014 |
PROTEIN
Abstract
The present invention provides methods and compositions for the
treatment of and for screening, diagnosis and prognosis of
colorectal cancer, for monitoring the effectiveness of colorectal
cancer treatment, and for drug development.
Inventors: |
Rohlff; Christian; (Oxon,
GB) ; Terrett; Jonathan Alexander; (San Jose,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OXFORD BIOTHERAPEUTICS, LTD. |
Abingdon |
|
GB |
|
|
Assignee: |
OXFORD BIOTHERAPEUTICS,
LTD.
Abingdon
GB
|
Family ID: |
44353891 |
Appl. No.: |
13/963503 |
Filed: |
August 9, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12958373 |
Dec 1, 2010 |
8535677 |
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13963503 |
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12395569 |
Feb 27, 2009 |
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12958373 |
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12329500 |
Dec 5, 2008 |
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12395569 |
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PCT/EP2007/055537 |
Jun 5, 2007 |
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12329500 |
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PCT/US2010/031719 |
Apr 20, 2010 |
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12958373 |
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60811681 |
Jun 7, 2006 |
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61170980 |
Apr 20, 2009 |
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Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
G01N 33/6893 20130101;
G01N 2500/04 20130101; G01N 33/57419 20130101; A61P 35/00 20180101;
A61P 37/04 20180101 |
Class at
Publication: |
435/7.23 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 6, 2006 |
GB |
0611116.5 |
Aug 29, 2006 |
GB |
01616971.8 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
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11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. A method for screening for and/or diagnosis of colorectal
cancer in a human subject, which comprises the step of identifying
the presence or absence of Cadherin-17, or a fragment thereof, in a
biological sample obtained from said human subject, wherein the
method comprises an immunoassay step utilising one or more
antibodies or other affinity reagents such as Affibodies,
Nanobodies or Unibodies against Cadherin-17 or a fragment or
derivative thereof.
19. A method for monitoring and/or assessing colorectal cancer
treatment in a human subject, which comprises the step of
identifying the presence or absence of Cadherin-17, or a fragment
thereof, in a biological sample obtained from said human subject,
wherein the method comprises an immunoassay step utilising one or
more antibodies or other affinity reagents such as Affibodies
Nanobodies or Unibodies against Cadherin-17 or a fragment or
derivative thereof.
20. A method for identifying the presence or absence of metastatic
colorectal cancer cells in a biological sample obtained from a
human subject, which comprises the step of identifying the presence
or absence of Cadherin-17, or a fragment thereof, wherein the
method comprises an immunoassay step utilising one or more
antibodies or other affinity reagents such as Affibodies,
Nanobodies or Unibodies against Cadherin-17 or a fragment or
derivative thereof.
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
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30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. (canceled)
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48. (canceled)
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51. (canceled)
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53. (canceled)
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55. (canceled)
56. (canceled)
57. (canceled)
58. (canceled)
59. (canceled)
60. (canceled)
61. A method as claimed in claim 18, wherein the antibody is
selected from the group consisting of polyclonal, monoclonal,
bispecific, humanized, chimeric and single chain antibodies, Fab
fragments and F(ab').sub.2 fragments.
62. A method as claimed in claim 19, wherein the antibody is
selected from the group consisting of polyclonal, monoclonal,
bispecific, humanized, chimeric and single chain antibodies, Fab
fragments and F(ab').sub.2 fragments.
63. A method as claimed in claim 20, wherein the antibody is
selected from the group consisting of polyclonal, monoclonal,
bispecific, humanized, chimeric and single chain antibodies, Fab
fragments and F(ab').sub.2 fragments.
64. A method as claimed in claim 18, wherein the antibody or
affinity reagent is conjugated to a diagnostic moiety.
65. A method as claimed in claim 19, wherein the antibody or
affinity reagent is conjugated to a diagnostic moiety.
66. A method as claimed in claim 20, wherein the antibody or
affinity reagent is conjugated to a diagnostic moiety.
67. A method as claimed in claim 61, wherein the antibody is
conjugated to a diagnostic moiety.
68. A method as claimed in claim 62, wherein the antibody is
conjugated to a diagnostic moiety.
69. A method as claimed in claim 63, wherein the antibody is
conjugated to a diagnostic moiety.
Description
RELATED APPLICATIONS
[0001] The present application is a Continuation-In-Part of
co-pending U.S. Non-Provisional application Ser. No. 12/395,569,
filed Feb. 27, 2009, which in turn, is a continuation of PCT
Application No. PCT/GB2007/050513 filed Aug. 29, 2007, which in
turn, claims priority from G.B. Application No. 0616971.8 filed
Aug. 29, 2006 and U.S. Provisional Application Ser. No. 60/842,431
filed Sep. 6, 2006.
[0002] The present application is also a Continuation-In-Part of
co-pending U.S. Non-Provisional application Ser. No. 12/329,500,
filed Dec. 5, 2008, which in turn, is a continuation of PCT
Application No. PCT/EP2007/055537 filed Jun. 5, 2007, which in
turn, claims priority from G.B. Application No. 0611116.5 filed
Jun. 6, 2006 and U.S. Provisional Application Ser. No. 60/811,681
filed Jun. 7, 2006.
[0003] The present application is also a Continuation-In-Part of
co-pending PCT Application No. PCT/US2010/031719, filed Apr. 20,
2010, which in turn, claims priority from U.S. Provisional
Application Ser. No. 61/170,980, filed Apr. 20, 2009.
[0004] Applicants claim the benefits of 35 U.S.C. .sctn.120 as to
the U.S. Non-Provisional Applications and the PCT applications, and
priority under 35 U.S.C. .sctn.119 as to the said G.B. and U.S.
Provisional applications, and the entire disclosures of all
applications are incorporated herein by reference in their
entireties.
INTRODUCTION
[0005] The present invention relates to the identification of a
membrane protein associated with colorectal cancer which has
utility as a target for the treatment of colorectal cancer, in
particular using antibodies which bind to said membrane protein,
and has utility as amarker for colorectal cancer and colorectal
cancer metastases and which also forms a biological target against
which therapeutic antibodies (or other affinity reagents such as
Affibodies, Nanobodies or Unibodies) or other pharmaceutical agents
can be made.
BACKGROUND OF THE INVENTION
Colorectal Cancer:
[0006] Colorectal cancer (CRC) is one of the leading causes of
cancer-related morbidity and mortality, responsible for an
estimated half a million deaths per year, mostly in Western, well
developed countries. In these territories, CRC is the third most
common malignancy (estimated number of new cases per annum in USA
and EU is approximately 350,000 per year). Estimated healthcare
costs related to treatment for colorectal cancer in the United
States are more than $8 billion.
Colorectal Cancer Diagnosis:
[0007] Today, the fecal occult blood test and colonoscopy, a highly
invasive procedure, are the most frequently used screening and
diagnostic methods for colorectal cancer.
[0008] Other diagnostic tools include Flexible Sigmoidoscopy
(allowing the observation of only about half of the colon) and
Double Contrast Barium Enema (DCBE, to obtain X-ray images).
Colorectal Cancer Staging:
[0009] CRC has four distinct stages: patients with stage I disease,
have a five-year survival rate of >90%, while those with
metastatic stage IV disease have a <5% survival rate according
to the US National Institutes of Health (NIH).
Colorectal Cancer Treatment:
[0010] Once CRC has been diagnosed, the correct treatment needs to
be selected. Surgery is usually the main treatment for rectal
cancer, although radiation and chemotherapy will often be given
before surgery. Possible side effects of surgery include bleeding
from the surgery, deep vein thrombosis, and damage to nearby organs
during the operation.
[0011] Currently, 60 percent of colorectal cancer patients receive
chemotherapy to treat their disease; however, this form of
treatment only benefits a few percent of the population, while
carrying with it high risks of toxicity, thus demonstrating a need
to better define the patient selection criteria.
[0012] Colorectal cancer has a 30 to 40 percent recurrence rate
within an average of 18 months after primary diagnosis. As with all
cancers, the earlier it is detected the more likely it can be
cured, especially as pathologists have recognised that the majority
of CRC tumours develop in a series of well-defined stages from
benign adenomas.
Colon Cancer Survival by Stage
TABLE-US-00001 [0013] Stage Survival Rate I 93% IIA 85% IIB 72%
IIIA 83% IIIB 64% IIIC 44% IV 8%
Therapeutic Challenges
[0014] The major challenges in colorectal cancer treatment are to
improve early detection rates, to find new non-invasive markers
that can be used to follow disease progression and identify
relapse, and to find improved and less toxic therapies, especially
for more advanced disease where 5 year survival is still very poor.
There is a great need to identify targets which are more specific
to the cancer cells, e.g. ones which are expressed on the surface
of the tumour cells so that they can be attacked by promising new
approaches like immunotherapeutics and targeted toxins.
SUMMARY OF THE INVENTION
[0015] The present invention provides methods and compositions for
screening, diagnosis, prognosis and therapy of colorectal cancer,
for colorectal cancer patients' stratification, for monitoring the
effectiveness of colorectal cancer treatment, and for drug
development for treatment of colorectal cancer.
[0016] We have used mass spectrometry to identify peptides
generated by gel electrophoresis or tagging with iTRAQ reagents and
tryptic digest of membrane proteins extracted from colorectal
cancer tissue samples. Peptide sequences were compared to existing
protein and cDNA databases and the corresponding gene sequences
identified. The protein of the invention has not been previously
reported to originate from colorectal cancer cell membranes and
represents a protein of new diagnostic and therapeutic value.
[0017] Thus, a first aspect of the invention provides methods for
diagnosis of colorectal cancer that comprises analysing a sample of
colon tissue e.g. by gel electrophoresis, iTRAQ or other
appropriate protein separation technique to detect the protein of
the invention. Such methods are also set forth in commonly
assigned, co-pending parent application Ser. No. 12/329,500, filed
Dec. 5, 2008, the disclosure of which is incorporated herein in its
entirety. These methods are also suitable for screening, prognosis,
monitoring the results of therapy, drug development and discovery
of new targets for drug treatment.
[0018] A second aspect of the invention provides methods of
treating colorectal cancer, comprising administering to a patient a
therapeutically effective amount of a compound that modulates
(e.g., upregulates or downregulates) or complements the expression
or the biological activity (or both) of the protein of the
invention in patients having colorectal cancer, in order to (a)
prevent the onset or development of colorectal cancer; (b) prevent
the progression of colorectal cancer; or (c) ameliorate the
symptoms of colorectal cancer.
[0019] A third aspect of the invention provides methods of
screening for compounds that modulate (e.g., upregulate or
downregulate) the expression or biological activity of the protein
of the invention.
[0020] A fourth aspect of the invention provides monoclonal and
polyclonal antibodies or other affinity reagents such as
Affibodies, Nanobodies or Unibodies capable of immunospecific
binding to the protein of the invention.
[0021] Thus, in a fifth aspect, the present invention provides a
method for screening for and/or diagnosis of colorectal cancer in a
human subject, which method comprises the step of identifying the
presence or absence of the protein of the invention, in a
biological sample obtained from said human subject.
[0022] In a sixth aspect, the present invention provides a method
for monitoring and/or assessing colorectal cancer treatment in a
human subject, which comprises the step of identifying the presence
or absence of the protein of the invention, in a biological sample
obtained from said human subject.
[0023] In a seventh aspect, the present invention provides a method
for identifying the presence or absence of metastatic colorectal
cancer cells in a biological sample obtained from a human subject,
which comprises the step of identifying the presence or absence of
the protein of the invention.
[0024] In an eighth aspect, the present invention provides a method
for monitoring and/or assessing colorectal cancer treatment in a
human subject, which comprises the step of determining whether the
protein of the invention is increased/decreased in a biological
sample obtained from a patient.
[0025] The biological sample used can be from any source such as a
serum sample or a tissue sample, e.g. colorectal tissue. For
instance, when looking for evidence of metastatic colorectal
cancer, one would look at major sites of colorectal cancer
metastasis, e.g. the liver, the peritoneal cavity, the pelvis, the
retroperitoneum and the lungs.
[0026] Other aspects of the present invention are set out below and
in the claims herein.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 shows the amino acid sequence of the protein of the
invention isolated from colorectal cancer samples using 1D-gel
electrophoresis technology. The tryptic fragments detected
experimentally by mass spectrometry are highlighted, mass match
peptides are shown in bold and tandem peptides are underlined.
[0028] FIG. 2 is similar to FIG. 1, except the protein of the
invention was isolated from colorectal cancer samples using
iTRAQ.
[0029] FIGS. 3a-3i-show the Protein Index for the protein of the
invention in various diseases. The list of diseases investigated is
shown in Table 3a with Table 3b indicating subcellular
locations.
[0030] FIG. 4 shows ROC curve data for the presence of the protein
of the invention in colorectal cancer patient serum samples.
[0031] FIG. 5 shows results of FACS analysis on CDH17_A4 in LoVo
and LS174T cells.
[0032] FIG. 6A shows results of internalisation of CDH17_A4 by
MabZAP assay in LoVo colon cancer cells.
[0033] FIG. 6B shows results of internalisation of CDH17_A4 by
MabZAP assay in LoVo colon cancer cells.
[0034] FIG. 6C shows results of internalisation of CDH17_A4 by
MabZAP assay in LS174T colon cancer cells.
[0035] FIG. 6D shows results of internalisation of CDH17_A4 by
MabZAP assay in LS174T colon cancer cells.
[0036] FIG. 7A shows results of internalisation of CDH17_A4 by
MabZAP assay in LoVo colon cancer cells.
[0037] FIG. 7B shows results of internalisation of CDH17_A4 by
MabZAP assay in LS174T colon cancer cells.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The invention described in detail below provides methods and
compositions for clinical screening, diagnosis and prognosis of
colorectal cancer in a mammalian subject for identifying patients
most likely to respond to a particular therapeutic treatment, for
monitoring the results of colorectal cancer therapy, for drug
screening and drug development. The invention also encompasses the
administration of therapeutic compositions to a mammalian subject
to treat or prevent colorectal cancer. The mammalian subject may be
a non-human mammal, but is preferably human, more preferably a
human adult, i.e. a human subject at least 21 (more preferably at
least 35, at least 50, at least 60, at least 70, or at least 80)
years old. For clarity of disclosure, and not by way of limitation,
the invention will be described with respect to the analysis of
colon tissue. However, as one skilled in the art will appreciate,
the assays and techniques described below can be applied to other
types of patient samples, including body fluids (e.g. blood, urine
or saliva), a tissue sample from a patient at risk of having
colorectal cancer (e.g. a biopsy such as a colorectal biopsy) or
homogenate thereof. The methods and compositions of the present
invention are specially suited for screening, diagnosis and
prognosis of a living subject, but may also be used for postmortem
diagnosis in a subject, for example, to identify family members at
risk of developing the same disease.
[0039] As used herein, colon tissue refers to the colon itself, as
well as the tissue adjacent to and/or within the strata underlying
the colon.
OGTA001
[0040] In one aspect of the invention, one-dimensional
electrophoresis or isobaric tags for relative and absolute
quantification (iTRAQ) are used to analyze colorectal cancer tissue
samples from a subject, preferably a living subject, in order to
measure the expression of the protein of the invention for
screening or diagnosis of colorectal cancer, to determine the
prognosis of a colorectal cancer patient, to monitor the
effectiveness of colorectal cancer therapy, or for drug
development.
[0041] As used herein, the term "Protein of the invention", or
"OGTA001", refers to the protein illustrated in FIG. 1 detected
experimentally by 1D gel electrophoresis and iTRAQ analysis of
colorectal tissue samples. Protein derivatives of this sequence may
also be useful for the same purposes as described herein.
[0042] This protein has been identified in membrane protein
extracts of colorectal tissue samples from colorectal cancer
patients, through the methods and apparatus of the Preferred
Technologies (1D gel electrophoresis or iTRAQ together with tryptic
digest of membrane protein extracts). Peptide sequences were
compared to the SWISS-PROT and trEMBL databases (held by the Swiss
Institute of Bioinformatics (SIB) and the European Bioinformatics
Institute (EBI) which are available at www.expasy.com), and the
following entry: Q12864, Cadherin-17, was identified.
[0043] According to SWISS-PROT, Cadherin-17 is known to be
predominantly expressed in the gastrointestinal tract and
pancreatic duct. It is not detected in kidney, lung, liver, brain,
adrenal gland or skin. Cadherins are calcium dependent cell
adhesion proteins. They preferentially interact with themselves in
a homophilic manner in connecting cells; cadherins may thus
contribute to the sorting of heterogeneous cell types. Cadherin-17
may have a role in the morphological organization of liver and
intestine. It is involved in intestinal peptide transport.
[0044] The protein of the invention is useful as are fragments e.g.
antigenic or immunogenic fragments thereof and derivatives thereof.
Antigenic or immunogenic fragments will typically be of length 12
amino acids or more e.g. 20 amino acids or more e.g. 50 or 100
amino acids or more. Fragments may be 10% or more of the length of
the full protein e.g. 25% or more e.g. 50% or 75% or 90% or 95% or
more of the length of the full protein.
[0045] Antigenic or immunogenic fragments will be capable of
eliciting a relevant immune response in a patient. DNA encoding the
protein of the invention is also useful as are fragments thereof
e.g. DNA encoding fragments of the protein of the invention such as
immunogenic fragments thereof. Fragments of nucleic acid (e.g. DNA)
encoding the protein of the invention may be 10% or more of the
length of the full coding region e.g. 25% or more e.g. 50% or 75%
or 90% or 95% or more of the length of the full coding region.
Fragments of nucleic acid (e.g. DNA) may be 36 nucleotides or more
e.g. 60 nucleotides or more e.g. 150 or 300 nucleotides or more in
length.
[0046] Derivatives of the protein of the invention include variants
on the sequence in which one or more (e.g. 1-20 such as 15 amino
acids, or up to 20% such as up to 10% or 5% or 1% by number of
amino acids based on the total length of the protein) deletions,
insertions or substitutions have been made. Substitutions may
typically be conservative substitutions. For example derivatives
may have sequence identity of 80% or more e.g. 90% or more e.g. 95%
or more as compared with the reference sequence over the full
length of the reference sequence. Derivatives will typically have
essentially the same biological function as the protein from which
they are derived. Derivatives will typically be comparably
antigenic or immunogenic to the protein from which they are
derived.
[0047] Table 1 below illustrates the different occurrences of
OGTA001 as detected by 1D gel electrophoresis and mass spectrometry
of membrane protein extracts of colorectal tissue samples from
colorectal cancer patients. The first column provides the molecular
weight, the second column gives information on the subfractionation
protocol used, if any (see Example 1 below), and the last column
provides a list of the sequences observed by mass spectrometry and
the corresponding SEQ ID Nos.
[0048] Table 2 below illustrates the different occurrences of
OGTA001 as detected by iTRAQ and mass spectrometry of membrane
protein extracts of colorectal tissue samples from colorectal
cancer patients. The first column provides the sample number, the
second column gives information on the iTRAQ experiment number for
that sample and the last column provides a list of the sequences
observed by mass spectrometry and the corresponding SEQ ID Nos.
TABLE-US-00002 TABLE 1 Colorectal cancer 1D gel MW Subfraction-
(Da) ation Tryptics identified [SEQ ID No] 91099 DEENTANSFLNYR [4],
DNVESAQASEVKPLR [7], WNDPGAQYSLVDK [13] 114588 Nucleotide
AENPEPLVFGVK [2], DAYVFYAVAK [3], Binding DEENTANSFLNYR [4],
DINDNRPTFLQSK [6], DNVESAQASEVKPLR [7] 117144 Nucleotide
DEENTANSFLNYR [4], DEYGKPLSYPLEIHVK [5], Binding DNVESAQASEVKPLR
[7], IDHVTGEIFSVAPLDR [10] 119837 Nucleotide AENPEPLVFGVK [2],
DEENTANSFLNYR [4], Binding DNVESAQASEVKPLR [7], TGAISLTR [13]
125678 Nucleotide DEENTANSFLNYR [4], DNVESAQASEVKPLR [7]
Binding
TABLE-US-00003 TABLE 2 Colorectal cancer iTRAQ Sample Experiment
no. no. Tryptics identified [SEQ ID No] Sample 1 Experiment 1
AENPEPLVFGVK [2], DEYGKPLSYPLEIHVK [5], GWLK [9], IDHVTGEIFSVAPLDR
[10], KPLDFETAAVSNIVFK [11], LGVDTDPHTNTGYVIIK [12],
VKDINDNPPTCPSPVTVFEVQENER [14], VSEDVAIGTK [15], WNDPGAQYSLVDKEKLPR
[17] Sample 1 Experiment 2 AENPEPLVFGVK [2], IDHVTGEIFSVAPLDR [10]
Sample 2 Experiment 1 EGSQELNPAK [8]
[0049] For OGTA001, the detected level obtained upon analyzing
tissue from subjects having colorectal cancer relative to the
detected level obtained upon analyzing tissue from subjects free
from colorectal cancer will depend upon the particular analytical
protocol and detection technique that is used. Accordingly, the
present invention contemplates that each laboratory will establish
a reference range in subjects free from colorectal cancer according
to the analytical protocol and detection technique in use, as is
conventional in the diagnostic art. Preferably, at least one
control positive tissue sample from a subject known to have
colorectal cancer or at least one control negative tissue sample
from a subject known to be free from colorectal cancer (and more
preferably both positive and negative control samples) are included
in each batch of test samples analysed.
[0050] OGTA001 can be used for detection, prognosis, diagnosis, or
monitoring of colorectal cancer or for drug development. In one
embodiment of the invention, tissue from a subject (e.g., a subject
suspected of having colorectal cancer) is analysed by 1D
electrophoresis or iTRAQ for detection of OGTA001. An increased
abundance of OGTA001 in the tissue from the subject relative to
tissue from a subject or subjects free from colorectal cancer
(e.g., a control sample) or a previously determined reference range
indicates the presence of colorectal cancer.
[0051] In relation to fragments, immunogenic fragments or antigenic
fragments of OGTA001, for colorectal cancer applications,
preferably these comprise one or more of the sequences identified
as tryptic sequences in the 3.sup.rd column of Table 1 or the
3.sup.rd column of Table 2.
[0052] OGTA001 may, in particular, be characterized as an isoform
having a MW substantially as recited (eg +/-10%, particularly +/-5%
of the value) in column 1 of any of the rows of Table 1.
[0053] The present invention additionally provides: (a) a
preparation comprising isolated OGTA001; (b) a preparation
comprising one or more fragments of OGTA001; and (c) antibodies or
other affinity reagents such as Affibodies, Nanobodies or Unibodies
that bind to OGTA001, to said fragments, or both to OGTA001 and to
said fragments. As used herein, OGTA001 is "isolated" when it is
present in a preparation that is substantially free of
contaminating proteins, i.e., a preparation in which less than 10%
(preferably less than 5%, more preferably less than 1%) of the
total protein present is contaminating protein(s). A contaminating
protein is a protein having a significantly different amino acid
sequence from that of isolated OGTA001, as determined by mass
spectral analysis. As used herein, a "significantly different"
sequence is one that permits the contaminating protein to be
resolved from OGTA001 by mass spectral analysis, performed
according to the Reference Protocols.
[0054] OGTA001 can be assayed by any method known to those skilled
in the art, including but not limited to, the Preferred
Technologies described herein, kinase assays, enzyme assays,
binding assays and other functional assays, immunoassays, and
western blotting. In one embodiment, OGTA001 is separated on a 1-D
gel by virtue of its MW and visualized by staining the gel. In one
embodiment, OGTA001 is stained with a fluorescent dye and imaged
with a fluorescence scanner. Sypro Red (Molecular Probes, Inc.,
Eugene, Oreg.) is a suitable dye for this purpose. A preferred
fluorescent dye is disclosed in U.S. application Ser. No.
09/412,168, filed on Oct. 5, 1999, which is incorporated herein by
reference in its entirety. In another embodiment, OGTA001 is
analysed using isobaric tags for relative and absolute
quantification (iTRAQ).
[0055] Alternatively, OGTA001 can be detected in an immunoassay. In
one embodiment, an immunoassay is performed by contacting a sample
from a subject to be tested with an anti-OGTA001 antibody (or other
affinity reagent such as an Affibody, Nanobody or Unibody) under
conditions such that immunospecific binding can occur if OGTA001 is
present, and detecting or measuring the amount of any
immunospecific binding by the affinity reagent. Anti-OGTA001
affinity reagents can be produced by the methods and techniques
taught herein.
[0056] OGTA001 may be detected by virtue of the detection of a
fragment thereof e.g. an immunogenic or antigenic fragment thereof.
Fragments may have a length of at least 10, more typically at least
20 amino acids eg at least 50 or 100 amino acids eg at least 200 or
500 amino acids.
[0057] In one embodiment, binding of antibody (or other affinity
reagent such as an Affibody, Nanobody or Unibody) in tissue
sections can be used to detect aberrant OGTA001 localization or an
aberrant level of OGTA001. In a specific embodiment, an antibody
(or other affinity reagent such as an Affibody, Nanobody or
Unibody) to OGTA001 can be used to assay a patient tissue (e.g., a
colon tissue) for the level of OGTA001 where an aberrant level of
OGTA001 is indicative of colorectal cancer. As used herein, an
"aberrant level" means a level that is increased compared with the
level in a subject free from colorectal cancer or a reference
level.
[0058] Any suitable immunoassay can be used, including, without
limitation, competitive and non-competitive assay systems using
techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays and protein A immunoassays.
[0059] For example, OGTA001 can be detected in a fluid sample
(e.g., blood, urine, or saliva) by means of a two-step sandwich
assay. In the first step, a capture reagent (e.g., an anti-OGTA001
antibody or other affinity reagent such as an Affibody, Nanobody or
Unibody) is used to capture OGTA001. The capture reagent can
optionally be immobilized on a solid phase. In the second step, a
directly or indirectly labeled detection reagent is used to detect
the captured OGTA001. In one embodiment, the detection reagent is a
lectin. Any lectin can be used for this purpose that preferentially
binds to OGTA001 rather than to other isoforms that have the same
core protein as OGTA001 or to other proteins that share the
antigenic determinant recognized by the affinity reagent. In a
preferred embodiment, the chosen lectin binds OGTA001 with at least
2-fold greater affinity, more preferably at least 5-fold greater
affinity, still more preferably at least 10-fold greater affinity,
than to said other isoforms that have the same core protein as
OGTA001 or to said other proteins that share the antigenic
determinant recognized by the affinity reagent. Based on the
present description, a lectin that is suitable for detecting
OGTA001 can readily be identified by methods well known in the art,
for instance upon testing one or more lectins enumerated in Table I
on pages 158-159 of Sumar et al., Lectins as Indicators of
Disease-Associated Glycoforms, In: Gabius H-J & Gabius S
(eds.), 1993, Lectins and Glycobiology, at pp. 158-174 (which is
incorporated herein by reference in its entirety). In an
alternative embodiment, the detection reagent is an antibody (or
other affinity reagent such as an Affibody, Nanobody or Unibody),
e.g., an antibody that immunospecifically detects other
post-translational modifications, such as an antibody that
immunospecifically binds to phosphorylated amino acids. Examples of
such antibodies include those that bind to phosphotyrosine (BD
Transduction Laboratories, catalog nos.: P11230-050/P11230-150;
P11120; P38820; P39020), those that bind to phosphoserine (Zymed
Laboratories Inc., South San Francisco, Calif., catalog no.
61-8100) and those that bind to phosphothreonine (Zymed
Laboratories Inc., South San Francisco, Calif., catalogue nos.
71-8200, 13-9200).
[0060] If desired, a gene encoding OGTA001, a related gene, or
related nucleic acid sequences or subsequences, including
complementary sequences, can also be used in hybridization assays.
A nucleotide encoding OGTA001, or subsequences thereof comprising
at least 8 nucleotides, preferably at least 12 nucleotides, and
most preferably at least 15 nucleotides can be used as a
hybridization probe. Hybridization assays can be used for
detection, prognosis, diagnosis, or monitoring of conditions,
disorders, or disease states, associated with aberrant expression
of the gene encoding OGTA001, or for differential diagnosis of
subjects with signs or symptoms suggestive of colorectal cancer. In
particular, such a hybridization assay can be carried out by a
method comprising contacting a subject's sample containing nucleic
acid with a nucleic acid probe capable of hybridizing to a DNA or
RNA that encodes OGTA001, under conditions such that hybridization
can occur, and detecting or measuring any resulting
hybridization.
[0061] The invention also provides diagnostic kits, comprising an
anti-OGTA001 antibody (or other affinity reagent such as an
Affibody, Nanobody or Unibody). In addition, such a kit may
optionally comprise one or more of the following: (1) instructions
for using the anti-OGTA001 affinity reagent for diagnosis,
prognosis, therapeutic monitoring or any combination of these
applications; (2) a labeled binding partner to the affinity
reagent; (3) a solid phase (such as a reagent strip) upon which the
anti-OGTA001 affinity reagent is immobilized; and (4) a label or
insert indicating regulatory approval for diagnostic, prognostic or
therapeutic use or any combination thereof. If no labeled binding
partner to the affinity reagent is provided, the anti-OGTA001
affinity reagent itself can be labeled with a detectable marker,
e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive
moiety.
[0062] The invention also provides a kit comprising a nucleic acid
probe capable of hybridizing to RNA encoding OGTA001. In a specific
embodiment, a kit comprises in one or more containers a pair of
primers (e.g., each in the size range of 6-30 nucleotides, more
preferably 10-30 nucleotides and still more preferably 10-20
nucleotides) that under appropriate reaction conditions can prime
amplification of at least a portion of a nucleic acid encoding
OGTA001, such as by polymerase chain reaction (see, e.g., Innis et
al., 1990, PCR Protocols, Academic Press, Inc., San Diego, Calif.),
ligase chain reaction (see EP 320,308) use of Q13 replicase, cyclic
probe reaction, or other methods known in the art.
[0063] A kit can optionally further comprise a predetermined amount
of OGTA001 or a nucleic acid encoding OGTA001, e.g., for use as a
standard or control.
Use in Clinical Studies
[0064] The diagnostic methods and compositions of the present
invention can assist in monitoring a clinical study, e.g. to
evaluate drugs for therapy of colorectal cancer. In one embodiment,
candidate molecules are tested for their ability to restore OGTA001
levels in a subject having colorectal cancer to levels found in
subjects free from colorectal cancer or, in a treated subject, to
preserve OGTA001 levels at or near non-colorectal cancer
values.
[0065] In another embodiment, the methods and compositions of the
present invention are used to screen candidates for a clinical
study to identify individuals having colorectal cancer; such
individuals can then be excluded from the study or can be placed in
a separate cohort for treatment or analysis.
Production of Protein of the Invention and Corresponding Nucleic
Acid
[0066] A DNA of the present invention can be obtained by isolation
as a cDNA fragment from cDNA libraries using as starter materials
commercial mRNAs and determining and identifying the nucleotide
sequences thereof. That is, specifically, clones are randomly
isolated from cDNA libraries, which are prepared according to Ohara
et al's method (DNA Research Vol. 4, 53-59 (1997)). Next, through
hybridization, duplicated clones (which appear repeatedly) are
removed and then in vitro transcription and translation are carried
out. Nucleotide sequences of both termini of clones, for which
products of 50 kDa or more are confirmed, are determined.
Furthermore, databases of known genes are searched for homology
using the thus obtained terminal nucleotide sequences as queries.
The entire nucleotide sequence of a clone revealed to be novel as a
result is determined. In addition to the above screening method,
the 5' and 3' terminal sequences of cDNA are related to a human
genome sequence. Then an unknown long-chain gene is confirmed in a
region between the sequences, and the full-length of the cDNA is
analyzed. In this way, an unknown gene that is unable to be
obtained by a conventional cloning method that depends on known
genes can be systematically cloned.
[0067] Moreover, all of the regions of a human-derived gene
containing a DNA of the present invention can also be prepared
using a PCR method such as RACE while paying sufficient attention
to prevent artificial errors from taking place in short fragments
or obtained sequences. As described above, clones having DNA of the
present invention can be obtained.
[0068] In another means for cloning DNA of the present invention, a
synthetic DNA primer having an appropriate nucleotide sequence of a
portion of a polypeptide of the present invention is produced,
followed by amplification by the PCR method using an appropriate
library. Alternatively, selection can be carried out by
hybridization of the DNA of the present invention with a DNA that
has been incorporated into an appropriate vector and labeled with a
DNA fragment or a synthetic DNA encoding some or all of the regions
of the polypeptide of the present invention. Hybridization can be
carried out by, for example, the method described in Current
Protocols in Molecular Biology (edited by Frederick M. Ausubel et
al., 1987). DNA of the present invention may be any DNA, as long as
they contain nucleotide sequences encoding the polypeptides of the
present invention as described above. Such a DNA may be a cDNA
identified and isolated from cDNA libraries or the like that are
derived from colorectal tissue. Such a DNA may also be a synthetic
DNA or the like. Vectors for use in library construction may be any
of bacteriophages, plasmids, cosmids, phargemids, or the like.
Furthermore, by the use of a total RNA fraction or a mRNA fraction
prepared from the above cells and/or tissues, amplification can be
carried out by a direct reverse transcription coupled polymerase
chain reaction (hereinafter abbreviated as "RT-PCR method").
[0069] DNA encoding the above polypeptide consisting of an amino
acid sequence that is substantially identical to the amino acid
sequence of OGTA001 or DNA encoding the above polypeptide
consisting of an amino acid sequence derived from the amino acid
sequence of OGTA001 by deletion, substitution, or addition of one
or more amino acids composing a portion of the amino acid sequence
can be easily produced by an appropriate combination of, for
example, a site-directed mutagenesis method, a gene homologous
recombination method, a primer elongation method, and the PCR
method known by persons skilled in the art. In addition, at this
time, a possible method for causing a polypeptide to have
substantially equivalent biological activity is substitution of
homologous amino acids (e.g. polar and nonpolar amino acids,
hydrophobic and hydrophilic amino acids, positively-charged and
negatively charged amino acids, and aromatic amino acids) among
amino acids composing the polypeptide. Furthermore, to maintain
substantially equivalent biological activity, amino acids within
functional domains contained in the polypeptide of the present
invention are preferably conserved.
[0070] Furthermore, examples of DNA of the present invention
include DNA comprising a nucleotide sequence that encodes the amino
acid sequence of OGTA001 and DNA hybridizing under stringent
conditions to the DNA and encoding a polypeptide (protein) having
biological activity (function) equivalent to the function of the
polypeptide consisting of the amino acid sequence of OGTA001. Under
such conditions, an example of such DNA capable of hybridizing to
DNA comprising the nucleotide sequence that encodes the amino acid
sequence of OGTA001 is DNA comprising a nucleotide sequence that
has a degree of overall mean homology with the entire nucleotide
sequence of the DNA, such as approximately 80% or more, preferably
approximately 90% or more, and more preferably approximately 95% or
more. Hybridization can be carried out according to a method known
in the art such as a method described in Current Protocols in
Molecular Biology (edited by Frederick M. Ausubel et al., 1987) or
a method according thereto. Here, "stringent conditions" are, for
example, conditions of approximately "1*SSC, 0.1% SDS, and
37.degree. C., more stringent conditions of approximately "0.5*SSC,
0.1% SDS, and 42.degree. C., or even more stringent conditions of
approximately "0.2*SSC, 0.1% SDS, and 65.degree. C. With more
stringent hybridization conditions, the isolation of a DNA having
high homology with a probe sequence can be expected. The above
combinations of SSC, SDS, and temperature conditions are given for
illustrative purposes. Stringency similar to the above can be
achieved by persons skilled in the art using an appropriate
combination of the above factors or other factors (for example,
probe concentration, probe length, and reaction time for
hybridization) for determination of hybridization stringency.
[0071] A cloned DNA of the present invention can be directly used
or used, if desired, after digestion with a restriction enzyme or
addition of a linker, depending on purposes. The DNA may have ATG
as a translation initiation codon at the 5' terminal side and have
TAA, TGA, or TAG as a translation termination codon at the 3'
terminal side. These translation initiation and translation
termination codons can also be added using an appropriate synthetic
DNA adapter.
[0072] Where it is provided for use with the methods of the
invention OGTA001 is preferably provided in isolated form. More
preferably the OGTA001 polypeptide has been purified to at least to
some extent. OGTA001 polypeptide may be provided in substantially
pure form, that is to say free, to a substantial extent, from other
proteins. OGTA001 polypeptide can also be produced using
recombinant methods, synthetically produced or produced by a
combination of these methods. OGTA001 can be easily prepared by any
method known by persons skilled in the art, which involves
producing an expression vector containing a DNA of the present
invention or a gene containing a DNA of the present invention,
culturing a transformant transformed using the expression vector,
generating and accumulating a polypeptide of the present invention
or a recombinant protein containing the polypeptide, and then
collecting the resultant.
[0073] Recombinant OGTA001 polypeptide may be prepared by processes
well known in the art from genetically engineered host cells
comprising expression systems. Accordingly, the present invention
also relates to expression systems which comprise an OGTA001
polypeptide or nucleic acid, to host cells which are genetically
engineered with such expression systems and to the production of
OGTA001 polypeptide by recombinant techniques. For recombinant
OGTA001 polypeptide production, host cells can be genetically
engineered to incorporate expression systems or portions thereof
for nucleic acids. Such incorporation can be performed using
methods well known in the art, such as, calcium phosphate
transfection, DEAD-dextran mediated transfection, transvection,
microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection (see e.g. Davis et al., Basic Methods in
Molecular Biology, 1986 and Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbour laboratory Press,
Cold Spring Harbour, N.Y., 1989).
[0074] As host cells, for example, bacteria of the genus
Escherichia, Streptococci, Staphylococci, Streptomyces, bacteria of
the genus Bacillus, yeast, Aspergillus cells, insect cells,
insects, and animal cells are used. Specific examples of bacteria
of the genus Escherichia, which are used herein, include
Escherichia coli K12 and DH1 (Proc. Natl. Acad. Sci. U.S.A., Vol.
60, 160 (1968)), JM103 (Nucleic Acids Research, Vol. 9, 309
(1981)), JA221 (Journal of Molecular Biology, Vol. 120, 517
(1978)), and HB101 (Journal of Molecular Biology, Vol. 41, 459
(1969)). As bacteria of the genus Bacillus, for example, Bacillus
subtilis MI114 (Gene, Vol. 24, 255 (1983)) and 207-21 (Journal of
Biochemistry, Vol. 95, 87 (1984)) are used. As yeast, for example,
Saccaromyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, and 20B-12,
Schizosaccaromyces pombe NCYC1913 and NCYC2036, and Pichia pastoris
are used. As insect cells, for example, Drosophila S2 and
Spodoptera Sf9 cells are used. As animal cells, for example, COS-7
and Vero monkey cells, CHO Chinese hamster cells (hereinafter
abbreviated as CHO cells), dhfr-gene-deficient CHO cells, mouse L
cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells,
human FL cells, COS, HeLa, C127,3T3, HEK 293, BHK and Bowes
melanoma cells are used.
[0075] Cell-free translation systems can also be employed to
produce recombinant polypeptides (e.g. rabbit reticulocyte lysate,
wheat germ lysate, SP6/T7 in vitro T&T and RTS 100 E. Coli HY
transcription and translation kits from Roche Diagnostics Ltd.,
Lewes, UK and the TNT Quick coupled Transcription/Translation
System from Promega UK, Southampton, UK).
[0076] The expression vector can be produced according to a method
known in the art. For example, the vector can be produced by (1)
excising a DNA fragment containing a DNA of the present invention
or a gene containing a DNA of the present invention and (2)
ligating the DNA fragment downstream of the promoter in an
appropriate expression vector. A wide variety of expression systems
can be used, such as and without limitation, chromosomal, episomal
and virus-derived systems, e.g. plasmids derived from Escherichia
coli (e.g. pBR322, pBR325, pUC18, and pUC118), plasmids derived
from Bacillus subtilis (e.g. pUB110, pTP5, and pC194), from
bacteriophage, from transposons, from yeast episomes (e.g. pSH19
and pSH15), from insertion elements, from yeast chromosomal
elements, from viruses such as baculoviruses, papova viruses such
as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as those derived from plasmid and
bacteriophage (such as [lambda] phage) genetic elements, such as
cosmids and phagemids. The expression systems may contain control
regions that regulate as well as engender expression. Promoters to
be used in the present invention may be any promoters as long as
they are appropriate for hosts to be used for gene expression. For
example, when a host is Escherichia coli, a trp promoter, a lac
promoter, a recA promoter, a pL promoter, an 1 pp promoter, and the
like are preferred. When a host is Bacillus subtilis, an SPO1
promoter, an SPO2 promoter, a penP promoter, and the like are
preferred. When a host is yeast, a PHOS promoter, a PGK promoter, a
GAP promoter, an ADH promoter, and the like are preferred. When an
animal cell is used as a host, examples of promoters for use in
this case include an SRa promoter, an SV40 promoter, an LTR
promoter, a CMV promoter, and an HSV-TK promoter. Generally, any
system or vector that is able to maintain, propagate or express a
nucleic acid to produce a polypeptide in a host may be used.
[0077] The appropriate nucleic acid sequence may be inserted into
an expression system by any variety of well known and routine
techniques, such as those set forth in Sambrook et al., supra.
Appropriate secretion signals may be incorporated into the OGTA001
polypeptide to allow secretion of the translated protein into the
lumen of the endoplasmic reticulum, the periplasmic space or the
extracellular environment. These signals may be endogenous to the
OGTA001 polypeptide or they may be heterologous signals.
Transformation of the host cells can be carried out according to
methods known in the art. For example, the following documents can
be referred to: Proc. Natl. Acad. Sci. U.S.A., Vol. 69, 2110
(1972); Gene, Vol. 17, 107 (1982); Molecular & General
Genetics, Vol. 168, 111 (1979); Methods in Enzymology, Vol. 194,
182-187 (1991); Proc. Natl. Acad. Sci. U.S.A.), Vol. 75, 1929
(1978); Cell Technology, separate volume 8, New Cell Technology,
Experimental Protocol. 263-267 (1995) (issued by Shujunsha); and
Virology, Vol. 52, 456 (1973). The thus obtained transformant
transformed with an expression vector containing a DNA of the
present invention or a gene containing a DNA of the present
invention can be cultured according to a method known in the art.
For example, when hosts are bacteria of the genus Escherichia, the
bacteria are generally cultured at approximately 15.degree. C. to
43.degree. C. for approximately 3 to 24 hours. If necessary,
aeration or agitation can also be added. When hosts are bacteria of
the genus Bacillus, the bacteria are generally cultured at
approximately 30.degree. C. to 40.degree. C. for approximately 6 to
24 hours. If necessary, aeration or agitation can also be added.
When transformants whose hosts are yeast are cultured, culture is
generally carried out at approximately 20.degree. C. to 35.degree.
C. for approximately 24 to 72 hours using media with pH adjusted to
be approximately 5 to 8. If necessary, aeration or agitation can
also be added. When transformants whose hosts are animal cells are
cultured, the cells are generally cultured at approximately
30.degree. C. to 40.degree. C. for approximately 15 to 60 hours
using media with the pH adjusted to be approximately 6 to 8. If
necessary, aeration or agitation can also be added.
[0078] If an OGTA001 polypeptide is to be expressed for use in
cell-based screening assays, it is preferred that the polypeptide
be produced at the cell surface. In this event, the cells may be
harvested prior to use in the screening assay. If the OGTA001
polypeptide is secreted into the medium, the medium can be
recovered in order to isolate said polypeptide. If produced
intracellularly, the cells must first be lysed before the OGTA001
polypeptide is recovered.
[0079] OGTA001 polypeptide can be recovered and purified from
recombinant cell cultures or from other biological sources by well
known methods including, ammonium sulphate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, affinity
chromatography, hydrophobic interaction chromatography,
hydroxylapatite chromatography, molecular sieving chromatography,
centrifugation methods, electrophoresis methods and lectin
chromatography. In one embodiment, a combination of these methods
is used. In another embodiment, high performance liquid
chromatography is used. In a further embodiment, an antibody which
specifically binds to an OGTA001 polypeptide can be used to deplete
a sample comprising an OGTA001 polypeptide of said polypeptide or
to purify said polypeptide.
[0080] To separate and purify a polypeptide or a protein of the
present invention from the culture products, for example, after
culture, microbial bodies or cells are collected by a known method,
they are suspended in an appropriate buffer, the microbial bodies
or the cells are disrupted by, for example, ultrasonic waves,
lysozymes, and/or freeze-thawing, the resultant is then subjected
to centrifugation or filtration, and then a crude extract of the
protein can be obtained. The buffer may also contain a protein
denaturation agent such as urea or guanidine hydrochloride or a
surfactant such as Triton X-100.TM.. When the protein is secreted
in a culture solution, microbial bodies or cells and a supernatant
are separated by a known method after the completion of culture and
then the supernatant is collected. The protein contained in the
thus obtained culture supernatant or the extract can be purified by
an appropriate combination of known separation and purification
methods. The thus obtained polypeptide (protein) of the present
invention can be converted into a salt by a known method or a
method according thereto. Conversely, when the polypeptide
(protein) of the present invention is obtained in the form of a
salt, it can be converted into a free protein or peptide or another
salt by a known method or a method according thereto. Moreover, an
appropriate protein modification enzyme such as trypsin or
chymotrypsin is caused to act on a protein produced by a
recombinant before or after purification, so that modification can
be arbitrarily added or a polypeptide can be partially removed. The
presence of a polypeptide (protein) of the present invention or a
salt thereof can be measured by various binding assays, enzyme
immunoassays using specific antibodies, and the like.
[0081] Techniques well known in the art may be used for refolding
to regenerate native or active conformations of the OGTA001
polypeptide when the polypeptide has been denatured during
isolation and or purification. In the context of the present
invention, OGTA001 polypeptide can be obtained from a biological
sample from any source, such as and without limitation, a blood
sample or tissue sample, e.g. a colorectal tissue sample.
[0082] OGTA001 polypeptide may be in the form of a "mature protein"
or may be part of a larger protein such as a fusion protein. It is
often advantageous to include an additional amino acid sequence
which contains secretory or leader sequences, a pre-, pro- or
prepro-protein sequence, or a sequence which aids in purification
such as an affinity tag, for example, but without limitation,
multiple histidine residues, a FLAG tag, HA tag or myc tag.
[0083] An additional sequence that may provide stability during
recombinant production may also be used. Such sequences may be
optionally removed as required by incorporating a cleavable
sequence as an additional sequence or part thereof. Thus, an
OGTA001 polypeptide may be fused to other moieties including other
polypeptides or proteins (for example, glutathione S-transferase
and protein A). Such a fusion protein can be cleaved using an
appropriate protease, and then separated into each protein. Such
additional sequences and affinity tags are well known in the art.
In addition to the above, features known in the art, such as an
enhancer, a splicing signal, a polyA addition signal, a selection
marker, and an SV40 replication origin can be added to an
expression vector, if desired.
Production of Affinity Reagents to OGTA001
[0084] According to those in the art, there are three main types of
affinity reagent--monoclonal antibodies, phage display antibodies
and small molecules such as Affibodies, Domain Antibodies (dAbs),
Nanobodies or Unibodies. In general in applications according to
the present invention where the use of antibodies is stated, other
affinity reagents (e.g. Affibodies, domain antibodies, Nanobodies
or Unibodies) may be employed.
Production of Antibodies to OGTA001
[0085] According to the invention OGTA001, an OGTA001 analog, an
OGTA001-related protein or a fragment or derivative of any of the
foregoing may be used as an immunogen to generate antibodies which
immunospecifically bind such an immunogen. Such immunogens can be
isolated by any convenient means, including the methods described
above. The term "antibody" as used herein refers to a peptide or
polypeptide derived from, modeled after or substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, capable of specifically binding an antigen or epitope.
See, e.g. Fundamental Immunology, 3.sup.rd Edition, W.E. Paul, ed.,
Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods
175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
The term antibody includes antigen-binding portions, i.e., "antigen
binding sites," (e.g., fragments, subsequences, complementarity
determining regions (CDRs)) that retain capacity to bind antigen,
including (i) a Fab fragment, a monovalent fragment consisting of
the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a
bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a VH domain; and (vi) an isolated complementarity determining
region (CDR). Single chain antibodies are also included by
reference in the term "antibody." Antibodies of the invention
include, but are not limited to polyclonal, monoclonal, bispecific,
humanized or chimeric antibodies, single chain antibodies, Fab
fragments and F(ab').sub.2 fragments, fragments produced by a Fab
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above. The immunoglobulin
molecules of the invention can be of any class (e.g., IgG, IgE,
IgM, IgD and IgA) or subclass of immunoglobulin molecule.
[0086] The term "specifically binds" (or "immunospecifically
binds") is not intended to indicate that an antibody binds
exclusively to its intended target. Rather, an antibody
"specifically binds" if its affinity for its intended target is
about 5-fold greater when compared to its affinity for a non-target
molecule. Preferably the affinity of the antibody will be at least
about 5 fold, preferably 10 fold, more preferably 25-fold, even
more preferably 50-fold, and most preferably 100-fold or more,
greater for a target molecule than its affinity for a non-target
molecule. In preferred embodiments, Specific binding between an
antibody or other binding agent and an antigen means a binding
affinity of at least 10.sup.6 M.sup.-1. Preferred antibodies bind
with affinities of at least about 10.sup.7 M.sup.-1, and preferably
between about 10.sup.8 M.sup.-1 to about 10.sup.9 M.sup.-1, about
10.sup.9 M.sup.-1 to about 10.sup.10 M.sup.-1, or about 10.sup.10
M.sup.-1 about 10.sup.11 M.sup.-1.
[0087] Affinity is calculated as K.sub.d=k.sub.off/k.sub.on
(k.sub.off is the dissociation rate constant, k.sub.on is the
association rate constant and K.sub.d is the equilibrium constant.
Affinity can be determined at equilibrium by measuring the fraction
bound (r) of labeled ligand at various concentrations (c). The data
are graphed using the Scatchard equation: r/c=K(n-r):
[0088] where
[0089] r=moles of bound ligand/mole of receptor at equilibrium;
[0090] c=free ligand concentration at equilibrium;
[0091] K=equilibrium association constant; and
[0092] n=number of ligand binding sites per receptor molecule
By graphical analysis, r/c is plotted on the Y-axis versus r on the
X-axis thus producing a Scatchard plot. The affinity is the
negative slope of the line. k.sub.off can be determined by
competing bound labeled ligand with unlabeled excess ligand (see,
e.g., U.S. Pat. No. 6,316,409). The affinity of a targeting agent
for its target molecule is preferably at least about
1.times.10.sup.-6 moles/liter, is more preferably at least about
1.times.10.sup.-7 moles/liter, is even more preferably at least
about 1.times.10.sup.-8 moles/liter, is yet even more preferably at
least about 1.times.10.sup.-9 moles/liter, and is most preferably
at least about 1.times.10.sup.-10 moles/liter. Antibody affinity
measurement by Scatchard analysis is well known in the art. See,
e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and
Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.
[0093] In one embodiment, antibodies that recognize gene products
of genes encoding OGTA001 are publicly available. In another
embodiment, methods known to those skilled in the art are used to
produce antibodies that recognize OGTA001, an OGTA001 analog, an
OGTA001-related polypeptide, or a fragment or derivative of any of
the foregoing. One skilled in the art will recognize that many
procedures are available for the production of antibodies, for
example, as described in Antibodies, A Laboratory Manual, Ed Harlow
and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring
Harbor, N.Y. One skilled in the art will also appreciate that
binding fragments or Fab fragments which mimic antibodies can also
be prepared from genetic information by various procedures
(Antibody Engineering: A Practical Approach (Borrebaeck, C., ed.),
1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920
(1992)).
[0094] In one embodiment of the invention, antibodies to a specific
domain of OGTA001 are produced. In a specific embodiment,
hydrophilic fragments of OGTA001 are used as immunogens for
antibody production.
[0095] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.
ELISA (enzyme-linked immunosorbent assay). For example, to select
antibodies which recognize a specific domain of OGTA001, one may
assay generated hybridomas for a product which binds to an OGTA001
fragment containing such domain. For selection of an antibody that
specifically binds a first OGTA001 homolog but which does not
specifically bind to (or binds less avidly to) a second OGTA001
homolog, one can select on the basis of positive binding to the
first OGTA001 homolog and a lack of binding to (or reduced binding
to) the second OGTA001 homolog. Similarly, for selection of an
antibody that specifically binds OGTA001 but which does not
specifically bind to (or binds less avidly to) a different isoform
of the same protein (such as a different glycoform having the same
core peptide as OGTA001), one can select on the basis of positive
binding to OGTA001 and a lack of binding to (or reduced binding to)
the different isoform (e.g., a different glycoform). Thus, the
present invention provides an antibody (preferably a monoclonal
antibody) that binds with greater affinity (preferably at least
2-fold, more preferably at least 5-fold, still more preferably at
least 10-fold greater affinity) to OGTA001 than to a different
isoform or isoforms (e.g., glycoforms) of OGTA001.
[0096] Polyclonal antibodies which may be used in the methods of
the invention are heterogeneous populations of antibody molecules
derived from the sera of immunized animals. Unfractionated immune
serum can also be used. Various procedures known in the art may be
used for the production of polyclonal antibodies to OGTA001, a
fragment of OGTA001, an OGTA001-related polypeptide, or a fragment
of an OGTA001-related polypeptide. For example, one way is to
purify polypeptides of interest or to synthesize the polypeptides
of interest using, e.g., solid phase peptide synthesis methods well
known in the art. See, e.g., Guide to Protein Purification, Murray
P. Deutcher, ed., Meth. Enzymol. Vol 182 (1990); Solid Phase
Peptide Synthesis, Greg B. Fields ed., Meth. Enzymol. Vol 289
(1997); Kiso et al., Chem. Pharm. Bull. (Tokyo) 38: 1192-99, 1990;
Mostafavi et al., Biomed. Pept. Proteins Nucleic Acids 1: 255-60,
1995; Fujiwara et al., Chem. Pharm. Bull. (Tokyo) 44: 1326-31,
1996. The selected polypeptides may then be used to immunize by
injection various host animals, including but not limited to
rabbits, mice, rats, etc., to generate polyclonal or monoclonal
antibodies. The Preferred Technology described herein in Example 1
provides isolated OGTA001 suitable for such immunization. If
OGTA001 is purified by gel electrophoresis, OGTA001 can be used for
immunization with or without prior extraction from the
polyacrylamide gel. Various adjuvants (i.e. immunostimulants) may
be used to enhance the immunological response, depending on the
host species, including, but not limited to, complete or incomplete
Freund's adjuvant, a mineral gel such as aluminum hydroxide,
surface active substance such as lysolecithin, pluronic polyol, a
polyanion, a peptide, an oil emulsion, keyhole limpet hemocyanin,
dinitrophenol, and an adjuvant such as BCG (bacille
Calmette-Guerin) or corynebacterium parvum. Additional adjuvants
are also well known in the art.
[0097] For preparation of monoclonal antibodies (mAbs) directed
toward OGTA001, a fragment of OGTA001, an OGTA001-related
polypeptide, or a fragment of an OGTA001-related polypeptide, any
technique which provides for the production of antibody molecules
by continuous cell lines in culture may be used. For example, the
hybridoma technique originally developed by Kohler and Milstein
(1975, Nature 256:495-497), as well as the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983, Immunology
Today 4:72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies
may be of any immunoglobulin class including IgG, IgM, IgE, IgA,
IgD and any subclass thereof. The hybridoma producing the mAbs of
the invention may be cultivated in vitro or in vivo. In an
additional embodiment of the invention, monoclonal antibodies can
be produced in germ-free animals utilizing known technology
(PCT/US90/02545, incorporated herein by reference).
[0098] The monoclonal antibodies include but are not limited to
human monoclonal antibodies and chimeric monoclonal antibodies
(e.g., human-mouse chimeras). A chimeric antibody is a molecule in
which different portions are derived from different animal species,
such as those having a human immunoglobulin constant region and a
variable region derived from a murine mAb. (See, e.g., Cabilly et
al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No.
4,816,397, which are incorporated herein by reference in their
entirety.) Humanized antibodies are antibody molecules from
non-human species having one or more complementarity determining
regions (CDRs) from the non-human species and a framework region
from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat.
No. 5,585,089, which is incorporated herein by reference in its
entirety.)
[0099] Chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al., 1988, Science 240:1041-1043; Liu et al.,
1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J.
Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005;
Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J.
Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science
229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al., 1988, J. Immunol.
141:4053-4060.
[0100] Completely human antibodies are particularly desirable for
therapeutic treatment of human subjects. Such antibodies can be
produced using transgenic mice which are incapable of expressing
endogenous immunoglobulin heavy and light chain genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunized in the normal fashion with a selected antigen, e.g.,
all or a portion of OGTA001. Monoclonal antibodies directed against
the antigen can be obtained using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.) and
Genpharm (San Jose, Calif.) can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that described above.
[0101] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al. (1994) Bio/technology 12:899-903).
[0102] The antibodies of the present invention can also be
generated by the use of phage display technology to produce and
screen libraries of polypeptides for binding to a selected target.
See, e.g., Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-82,
1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith,
Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No.
5,571,698. A basic concept of phage display methods is the
establishment of a physical association between DNA encoding a
polypeptide to be screened and the polypeptide. This physical
association is provided by the phage particle, which displays a
polypeptide as part of a capsid enclosing the phage genome which
encodes the polypeptide. The establishment of a physical
association between polypeptides and their genetic material allows
simultaneous mass screening of very large numbers of phage bearing
different polypeptides. Phage displaying a polypeptide with
affinity to a target bind to the target and these phage are
enriched by affinity screening to the target. The identity of
polypeptides displayed from these phage can be determined from
their respective genomes. Using these methods a polypeptide
identified as having a binding affinity for a desired target can
then be synthesized in bulk by conventional means. See, e.g., U.S.
Pat. No. 6,057,098, which is hereby incorporated in its entirety,
including all tables, figures, and claims. 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 the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
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. Phage display methods that can be used to make the
antibodies of the present invention include those disclosed 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.
[0103] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and
F(ab').sub.2 fragments can also be employed using methods known in
the art such as those disclosed in PCT publication WO 92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et
al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043
(1988) (said references incorporated by reference in their
entireties).
[0104] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988).
[0105] The invention further provides for the use of bispecific
antibodies, which can be made by methods known in the art.
Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Milstein et al., 1983, Nature 305:537-539). Because of the random
assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., 1991,
EMBO J. 10:3655-3659.
[0106] According to a different and more preferred approach,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2, and CH3 regions. It is preferred to have
the first heavy-chain constant region (CH1) containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0107] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690 published Mar. 3, 1994. For
further details for generating bispecific antibodies see, for
example, Suresh et al., Methods in Enzymology, 1986, 121:210.
[0108] The invention provides functionally active fragments,
derivatives or analogs of the anti-OGTA001 immunoglobulin
molecules. Functionally active means that the fragment, derivative
or analog is able to elicit anti-anti-idiotype antibodies (i.e.,
tertiary antibodies) that recognize the same antigen that is
recognized by the antibody from which the fragment, derivative or
analog is derived. Specifically, in a preferred embodiment the
antigenicity of the idiotype of the immunoglobulin molecule may be
enhanced by deletion of framework and CDR sequences that are
C-terminal to the CDR sequence that specifically recognizes the
antigen. To determine which CDR sequences bind the antigen,
synthetic peptides containing the CDR sequences can be used in
binding assays with the antigen by any binding assay method known
in the art.
[0109] The present invention provides antibody fragments such as,
but not limited to, F(ab').sub.2 fragments and Fab fragments.
Antibody fragments which recognize specific epitopes may be
generated by known techniques. F(ab').sub.2 fragments consist of
the variable region, the light chain constant region and the CH1
domain of the heavy chain and are generated by pepsin digestion of
the antibody molecule. Fab fragments are generated by reducing the
disulfide bridges of the F(ab').sub.2 fragments. The invention also
provides heavy chain and light chain dimers of the antibodies of
the invention, or any minimal fragment thereof such as Fvs or
single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.
4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,
Nature 334:544-54), or any other molecule with the same specificity
as the antibody of the invention. Single chain antibodies are
formed by linking the heavy and light chain fragments of the Fv
region via an amino acid bridge, resulting in a single chain
polypeptide. Techniques for the assembly of functional Fv fragments
in E. coli may be used (Skerra et al., 1988, Science
242:1038-1041).
[0110] In other embodiments, the invention provides fusion proteins
of the immunoglobulins of the invention (or functionally active
fragments thereof), for example in which the immunoglobulin is
fused via a covalent bond (e.g., a peptide bond), at either the
N-terminus or the C-terminus to an amino acid sequence of another
protein (or portion thereof, preferably at least 10, 20 or 50 amino
acid portion of the protein) that is not the immunoglobulin.
Preferably the immunoglobulin, or fragment thereof, is covalently
linked to the other protein at the N-terminus of the constant
domain. As stated above, such fusion proteins may facilitate
purification, increase half-life in vivo, and enhance the delivery
of an antigen across an epithelial barrier to the immune
system.
[0111] The immunoglobulins of the invention include analogs and
derivatives that are modified, i.e., by the covalent attachment of
any type of molecule as long as such covalent attachment does not
impair immunospecific binding. For example, but not by way of
limitation, the derivatives and analogs of the immunoglobulins
include those that have been further modified, e.g., by
glycosylation, acetylation, pegylation, phosphylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. Any
of numerous chemical modifications may be carried out by known
techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, etc. Additionally, the analog
or derivative may contain one or more non-classical amino
acids.
[0112] The foregoing antibodies can be used in methods known in the
art relating to the localization and activity of OGTA001, e.g., for
imaging this protein, measuring levels thereof in appropriate
physiological samples, in diagnostic methods, etc.
Production of Affibodies to OGTA001
[0113] Affibody molecules represent a new class of affinity
proteins based on a 58-amino acid residue protein domain, derived
from one of the IgG-binding domains of staphylococcal protein A.
This three helix bundle domain has been used as a scaffold for the
construction of combinatorial phagemid libraries, from which
Affibody variants that target the desired molecules can be selected
using phage display technology (Nord K, Gunneriusson E, Ringdahl J,
Stahl S, Uhlen M, Nygren P A, Binding proteins selected from
combinatorial libraries of an .alpha.-helical bacterial receptor
domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H, Uhlen
M, Nygren P A, Human immunoglobulin A (IgA)-specific ligands from
combinatorial engineering of protein A, Eur J Biochem 2002;
269:2647-55.). The simple, robust structure of Affibody molecules
in combination with their low molecular weight (6 kDa), make them
suitable for a wide variety of applications, for instance, as
detection reagents (Ronmark J, Hansson M, Nguyen T, et al,
Construction and characterization of affibody-Fc chimeras produced
in Escherichia coli, Immunol Methods 2002; 261:199-211) and to
inhibit receptor interactions (Sandstorm K, Xu Z, Forsberg G,
Nygren P A, Inhibition of the CD28-CD80 co-stimulation signal by a
CD28-binding Affibody ligand developed by combinatorial protein
engineering, Protein Eng 2003; 16:691-7). Further details of
Affibodies and methods of production thereof may be obtained by
reference to U.S. Pat. No. 5,831,012 which is herein incorporated
by reference in its entirety.
[0114] Labelled Affibodies may also be useful in imaging
applications for determining abundance of Isoforms.
Production of Domain Antibodies to OGTA001
[0115] References to antibodies herein embrace references to Domain
Antibodies. Domain Antibodies (dAbs) are the smallest functional
binding units of antibodies, corresponding to the variable regions
of either the heavy (VH) or light (VL) chains of human antibodies.
Domain Antibodies have a molecular weight of approximately 13 kDa.
Domantis has developed a series of large and highly functional
libraries of fully human V.sub.H and V.sub.L dAbs (more than ten
billion different sequences in each library), and uses these
libraries to select dAbs that are specific to therapeutic targets.
In contrast to many conventional antibodies, Domain Antibodies are
well expressed in bacterial, yeast, and mammalian cell systems.
Further details of domain antibodies and methods of production
thereof may be obtained by reference to U.S. Pat. No. 6,291,158;
U.S. Pat. No. 6,582,915; U.S. Pat. No. 6,593,081; U.S. Pat. No.
6,172,197; U.S. Pat. No. 6,696,245; US Serial No. 2004/0110941;
European patent application No. 1433846 and European Patents
0368684 & 0616640; WO05/035572, WO04/101790, WO04/081026,
WO04/058821, WO04/003019 and WO03/002609, each of which is herein
incorporated by reference in its entirety.
Production of Nanobodies to OGTA001
[0116] Nanobodies are antibody-derived therapeutic proteins that
contain the unique structural and functional properties of
naturally-occurring heavy-chain antibodies. These heavy-chain
antibodies contain a single variable domain (VHH) and two constant
domains (C.sub.H2 and C.sub.H3). Importantly, the cloned and
isolated VIM domain is a perfectly stable polypeptide harbouring
the full antigen-binding capacity of the original heavy-chain
antibody. Nanobodies have a high homology with the VH domains of
human antibodies and can be further humanised without any loss of
activity. Importantly, Nanobodies have a low immunogenic potential,
which has been confirmed in primate studies with Nanobody lead
compounds.
[0117] Nanobodies combine the advantages of conventional antibodies
with important features of small molecule drugs. Like conventional
antibodies, Nanobodies show high target specificity, high affinity
for their target and low inherent toxicity. However, like small
molecule drugs they can inhibit enzymes and readily access receptor
clefts. Furthermore, Nanobodies are extremely stable, can be
administered by means other than injection (see e.g. WO 04/041867,
which is herein incorporated by reference in its entirety) and are
easy to manufacture. Other advantages of Nanobodies include
recognising uncommon or hidden epitopes as a result of their small
size, binding into cavities or active sites of protein targets with
high affinity and selectivity due to their unique 3-dimensional,
drug format flexibility, tailoring of half-life and ease and speed
of drug discovery.
[0118] Nanobodies are encoded by single genes and are efficiently
produced in almost all prokaryotic and eukaryotic hosts e.g. E.
coli (see e.g. U.S. Pat. No. 6,765,087, which is herein
incorporated by reference in its entirety), moulds (for example
Aspergillus or Trichoderma) and yeast (for example Saccharomyces,
Kluyveromyces, Hansenula or Pichia) (see e.g. U.S. Pat. No.
6,838,254, which is herein incorporated by reference in its
entirety). The production process is scalable and multi-kilogram
quantities of Nanobodies have been produced. Because Nanobodies
exhibit a superior stability compared with conventional antibodies,
they can be formulated as a long shelf-life, ready-to-use
solution.
[0119] The Nanoclone method (see e.g. WO 06/079372, which is herein
incorporated bye reference in its entirety) is a proprietary method
for generating Nanobodies against a desired target, based on
automated high-throughout selection of B-cells.
Production of Unibodies to OGTA001
[0120] UniBody is a new proprietary antibody technology that
creates a stable, smaller antibody format with an anticipated
longer therapeutic window than current small antibody formats. IgG4
antibodies are considered inert and thus do not interact with the
immune system. Genmab modified fully human IgG4 antibodies by
eliminating the hinge region of the antibody. Unlike the full size
IgG4 antibody, the half molecule fragment is very stable and is
termed a UniBody. Halving the IgG4 molecule left only one area on
the UniBody that can bind to disease targets and the UniBody
therefore binds univalently to only one site on target cells. This
univalent binding does not stimulate cancer cells to grow like
bivalent antibodies might and opens the door for treatment of some
types of cancer which ordinary antibodies cannot treat.
[0121] The UniBody is about half the size of a regular IgG4
antibody. This small size can be a great benefit when treating some
forms of cancer, allowing for better distribution of the molecule
over larger solid tumors and potentially increasing efficacy.
[0122] Fabs typically do not have a very long half-life. UniBodies,
however, were cleared at a similar rate to whole IgG4 antibodies
and were able to bind as well as whole antibodies and antibody
fragments in pre-clinical studies. Other antibodies primarily work
by killing the targeted cells whereas UniBodies only inhibit or
silence the cells.
[0123] Further details of Unibodies may be obtained by reference to
patent WO2007/059782, which is herein incorporated by reference in
its entirety.
Expression of Affinity Reagents
Expression of Antibodies
[0124] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or by recombinant expression, and
are preferably produced by recombinant expression techniques.
[0125] Recombinant expression of antibodies, or fragments,
derivatives or analogs thereof, requires construction of a nucleic
acid that encodes the antibody. If the nucleotide sequence of the
antibody is known, a nucleic acid encoding the antibody may be
assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., 1994, BioTechniques 17:242), which,
briefly, involves the synthesis of overlapping oligonucleotides
containing portions of the sequence encoding antibody, annealing
and ligation of those oligonucleotides, and then amplification of
the ligated oligonucleotides by PCR.
[0126] Alternatively, the nucleic acid encoding the antibody may be
obtained by cloning the antibody. If a clone containing the nucleic
acid encoding the particular antibody is not available, but the
sequence of the antibody molecule is known, a nucleic acid encoding
the antibody may be obtained from a suitable source (e.g., an
antibody cDNA library, or cDNA library generated from any tissue or
cells expressing the antibody) by PCR amplification using synthetic
primers hybridizable to the 3' and 5' ends of the sequence or by
cloning using an oligonucleotide probe specific for the particular
gene sequence.
[0127] If an antibody molecule that specifically recognizes a
particular antigen is not available (or a source for a cDNA library
for cloning a nucleic acid encoding such an antibody), antibodies
specific for a particular antigen may be generated by any method
known in the art, for example, by immunizing an animal, such as a
rabbit, to generate polyclonal antibodies or, more preferably, by
generating monoclonal antibodies. Alternatively, a clone encoding
at least the Fab portion of the antibody may be obtained by
screening Fab expression libraries (e.g., as described in Huse et
al., 1989, Science 246:1275-1281) for clones of Fab fragments that
bind the specific antigen or by screening antibody libraries (See,
e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997
Proc. Natl. Acad. Sci. USA 94:4937).
[0128] Once a nucleic acid encoding at least the variable domain of
the antibody molecule is obtained, it may be introduced into a
vector containing the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.
5,122,464). Vectors containing the complete light or heavy chain
for co-expression with the nucleic acid to allow the expression of
a complete antibody molecule are also available. Then, the nucleic
acid encoding the antibody can be used to introduce the nucleotide
substitution(s) or deletion(s) necessary to substitute (or delete)
the one or more variable region cysteine residues participating in
an intrachain disulfide bond with an amino acid residue that does
not contain a sulfhydyl group. Such modifications can be carried
out by any method known in the art for the introduction of specific
mutations or deletions in a nucleotide sequence, for example, but
not limited to, chemical mutagenesis, in vitro site directed
mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551), PCT
based methods, etc.
[0129] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda
et al., 1985, Nature 314:452-454) by splicing genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human antibody constant region, e.g., humanized
antibodies.
[0130] Once a nucleic acid encoding an antibody molecule of the
invention has been obtained, the vector for the production of the
antibody molecule may be produced by recombinant DNA technology
using techniques well known in the art. Thus, methods for preparing
the protein of the invention by expressing nucleic acid containing
the antibody molecule sequences are described herein. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing an antibody molecule coding sequences
and appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. See, for example, the techniques described in
Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual,
2.sup.nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in
Molecular Biology, John Wiley & Sons, NY).
[0131] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the
invention.
[0132] The host cells used to express a recombinant antibody of the
invention may be either bacterial cells such as Escherichia coli,
or, preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule. In particular, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus are an effective expression system for
antibodies (Foecking et al., 1986, Gene 45:101; Cockett et al.,
1990, Bio/Technology 8:2).
[0133] A variety of host-expression vector systems may be utilized
to express an antibody molecule of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express the
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing the antibody
coding sequences; plant cell systems infected with recombinant
virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
[0134] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions comprising an antibody molecule,
vectors which direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such
vectors include, but are not limited, to the E. coli expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the
antibody coding sequence may be ligated individually into the
vector in frame with the lac Z coding region so that a fusion
protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be
used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by
adsorption and binding to a matrix glutathione-agarose beads
followed by elution in the presence of free glutathione. The pGEX
vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0135] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter). In mammalian host cells, a number of viral-based
expression systems (e.g., an adenovirus expression system) may be
utilized.
[0136] As discussed above, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein.
[0137] For long-term, high-yield production of recombinant
antibodies, stable expression is preferred. For example, cell lines
that stably express an antibody of interest can be produced by
transfecting the cells with an expression vector comprising the
nucleotide sequence of the antibody and the nucleotide sequence of
a selectable (e.g., neomycin or hygromycin), and selecting for
expression of the selectable marker. Such engineered cell lines may
be particularly useful in screening and evaluation of compounds
that interact directly or indirectly with the antibody
molecule.
[0138] The expression levels of the antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
[0139] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0140] Once the antibody molecule of the invention has been
recombinantly expressed, it may be purified by any method known in
the art for purification of an antibody molecule, for example, by
chromatography (e.g., ion exchange chromatography, affinity
chromatography such as with protein A or specific antigen, and
sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins.
[0141] Alternatively, any fusion protein may be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Janknecht et al., 1991, Proc. Natl.
Acad. Sci. USA 88:8972-897). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
open reading frame of the gene is translationally fused to an
amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix binding domain for the fusion protein. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0142] The antibodies that are generated by these methods may then
be selected by first screening for affinity and specificity with
the purified polypeptide of interest and, if required, comparing
the results to the affinity and specificity of the antibodies with
polypeptides that are desired to be excluded from binding. The
screening procedure can involve immobilization of the purified
polypeptides in separate wells of microtiter plates. The solution
containing a potential antibody or groups of antibodies is then
placed into the respective microtiter wells and incubated for about
30 min to 2 h. The microtiter wells are then washed and a labeled
secondary antibody (for example, an anti-mouse antibody conjugated
to alkaline phosphatase if the raised antibodies are mouse
antibodies) is added to the wells and incubated for about 30 min
and then washed. Substrate is added to the wells and a color
reaction will appear where antibody to the immobilized
polypeptide(s) is present.
[0143] The antibodies so identified may then be further analyzed
for affinity and specificity in the assay design selected. In the
development of immunoassays for a target protein, the purified
target protein acts as a standard with which to judge the
sensitivity and specificity of the immunoassay using the antibodies
that have been selected. Because the binding affinity of various
antibodies may differ; certain antibody pairs (e.g., in sandwich
assays) may interfere with one another sterically, etc., assay
performance of an antibody may be a more important measure than
absolute affinity and specificity of an antibody.
[0144] Those skilled in the art will recognize that many approaches
can be taken in producing antibodies or binding fragments and
screening and selecting for affinity and specificity for the
various polypeptides, but these approaches do not change the scope
of the invention.
[0145] For therapeutic applications, antibodies (particularly
monoclonal antibodies) may suitably be human or humanized animal
(e.g. mouse) antibodies. Animal antibodies may be raised in animals
using the human protein (e.g. OGTA001) as immunogen. Humanisation
typically involves grafting CDRs identified thereby into human
framework regions. Normally some subsequent retromutation to
optimize the conformation of chains is required. Such processes are
known to persons skilled in the art.
Expression of Affibodies
[0146] The construction of affibodies has been described elsewhere
(Ronnmark J, Gronlund H, Uhle'n, M., Nygren P. A.degree., Human
immunoglobulin A (IgA)-specific ligands from combinatorial
engineering of protein A, 2002, Eur. J. Biochem. 269, 2647-2655.),
including the construction of affibody phage display libraries
(Nord, K., Nilsson, J., Nilsson, B., Uhle'n, M. & Nygren, P.
A.degree., A combinatorial library of an a-helical bacterial
receptor domain, 1995, Protein Eng. 8, 601-608. Nord, K.,
Gunneriusson, E., Ringdahl, J., Sta.degree.hl, S., Uhle'n, M. &
Nygren, P. A.degree., Binding proteins selected from combinatorial
libraries of an a-helical bacterial receptor domain, 1997, Nat.
Biotechnol. 15, 772-777.)
[0147] The biosensor analyses to investigate the optimal affibody
variants using biosensor binding studies has also been described
elsewhere (Ronnmark J, Gronlund H, Uhle'n, M., Nygren P. A.degree.,
Human immunoglobulin A (IgA)-specific ligands from combinatorial
engineering of protein A, 2002, Eur. J. Biochem. 269,
2647-2655.).
Conjugated Affinity Reagents
[0148] In a preferred embodiment, anti-OGTA001 affinity reagents
such as antibodies or fragments thereof are conjugated to a
diagnostic or therapeutic moiety. The antibodies can be used for
diagnosis or to determine the efficacy of a given treatment
regimen. Detection can be facilitated by coupling the antibody to a
detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
nuclides, positron emitting metals (for use in positron emission
tomography), and nonradioactive paramagnetic metal ions. See
generally U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Suitable enzymes include horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
suitable prosthetic groups include streptavidin, avidin and biotin;
suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride and phycoerythrin; suitable
luminescent materials include luminol; suitable bioluminescent
materials include luciferase, luciferin, and aequorin; and suitable
radioactive nuclides include .sup.125I, .sup.131I, .sup.111In and
.sup.99Tc. .sup.68Ga may also be employed.
[0149] Anti-OGTA001 antibodies or fragments thereof can be
conjugated to a therapeutic agent or drug moiety to modify a given
biological response. The therapeutic agent or drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, a toxin such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis
factor, -interferon, -interferon, nerve growth factor, platelet
derived growth factor, tissue plasminogen activator, a thrombotic
agent or an anti-angiogenic agent, e.g., angiostatin or endostatin;
or, a biological response modifier such as a lymphokine,
interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),
granulocyte macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), nerve growth factor
(NGF) or other growth factor.
[0150] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2.sup.nd Ed.),
Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);
Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
[0151] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0152] An antibody with or without a therapeutic moiety conjugated
to it can be used as a therapeutic that is administered alone or in
combination with cytotoxic factor(s) and/or cytokine(s).
Diagnosis of Colorectal Cancer
[0153] In accordance with the present invention, test samples of
colorectal tissue, serum, plasma or urine obtained from a subject
suspected of having or known to have colorectal cancer can be used
for diagnosis or monitoring. In one embodiment, a change in the
abundance of OGTA001 in a test sample relative to a control sample
(from a subject or subjects free from colorectal cancer) or a
previously determined reference range indicates the presence of
colorectal cancer. In another embodiment, the relative abundance of
OGTA001 in a test sample compared to a control sample or a
previously determined reference range indicates a subtype of
colorectal cancer (e.g., familial or sporadic colorectal cancer).
In yet another embodiment, the relative abundance of OGTA001 in a
test sample relative to a control sample or a previously determined
reference range indicates the degree or severity of colorectal
cancer (e.g., the likelihood for metastasis). In any of the
aforesaid methods, detection of OGTA001 may optionally be combined
with detection of one or more of additional biomarkers for
colorectal cancer. Any suitable method in the art can be employed
to measure the level of OGTA001, including but not limited to the
Preferred Technologies described herein, kinase assays,
immunoassays to detect and/or visualize the OGTA001 (e.g., Western
blot, immunoprecipitation followed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis, immunocytochemistry, etc.). In
a further embodiment, a change in the abundance of mRNA encoding
OGTA001 in a test sample relative to a control sample or a
previously determined reference range indicates the presence of
colorectal cancer. Any suitable hybridization assay can be used to
detect OGTA001 expression by detecting and/or visualizing mRNA
encoding the OGTA001 (e.g., Northern assays, dot blots, in situ
hybridization, etc.).
[0154] In another embodiment of the invention, labeled antibodies
(or other affinity reagents such as Affibodies, Nanobodies or
Unibodies), derivatives and analogs thereof, which specifically
bind to OGTA001 can be used for diagnostic purposes to detect,
diagnose, or monitor colorectal cancer. Preferably, colorectal
cancer is detected in an animal, more preferably in a mammal and
most preferably in a human.
Screening Assays
[0155] The invention provides methods for identifying agents (e.g.,
candidate compounds or test compounds) that bind to OGTA001 or have
a stimulatory or inhibitory effect on the expression or activity of
OGTA001. The invention also provides methods of identifying agents,
candidate compounds or test compounds that bind to an
OGTA001-related polypeptide or an OGTA001 fusion protein or have a
stimulatory or inhibitory effect on the expression or activity of
an OGTA001-related polypeptide or an OGTA001 fusion protein.
Examples of agents, candidate compounds or test compounds include,
but are not limited to, nucleic acids (e.g., DNA and RNA),
carbohydrates, lipids, proteins, peptides, peptidomimetics, small
molecules and other drugs. Agents can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145;
U.S. Pat. No. 5,738,996; and U.S. Pat. No. 5,807,683, each of which
is incorporated herein in its entirety by reference).
[0156] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al., 1993, Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678;
Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem.
37:1233, each of which is incorporated herein in its entirety by
reference.
[0157] Libraries of compounds may be presented, e.g., presented in
solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421), or on
beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature
364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat.
Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al.,
1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and
Smith, 1990, Science 249:386-390; Devlin, 1990, Science
249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA
87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310), each of
which is incorporated herein in its entirety by reference.
[0158] In one embodiment, agents that interact with (i.e., bind to)
OGTA001, an OGTA001 fragment (e.g. a functionally active fragment),
an OGTA001-related polypeptide, a fragment of an OGTA001-related
polypeptide, or an OGTA001 fusion protein are identified in a
cell-based assay system. In accordance with this embodiment, cells
expressing OGTA001, a fragment of an OGTA001, an OGTA001-related
polypeptide, a fragment of the OGTA001-related polypeptide, or an
OGTA001 fusion protein are contacted with a candidate compound or a
control compound and the ability of the candidate compound to
interact with OGTA001 is determined. If desired, this assay may be
used to screen a plurality (e.g. a library) of candidate compounds.
The cell, for example, can be of prokaryotic origin (e.g., E. coli)
or eukaryotic origin (e.g., yeast or mammalian). Further, the cells
can express OGTA001, a fragment of OGTA001, an OGTA001-related
polypeptide, a fragment of the OGTA001-related polypeptide, or an
OGTA001 fusion protein endogenously or be genetically engineered to
express OGTA001, a fragment of OGTA001, an OGTA001-related
polypeptide, a fragment of the OGTA001-related polypeptide, or an
OGTA001 fusion protein. In certain instances, OGTA001, a fragment
of the OGTA001, an OGTA001-related polypeptide, a fragment of the
OGTA001-related polypeptide, or an OGTA001 fusion protein or the
candidate compound is labeled, for example with a radioactive label
(such as .sup.32P, .sup.35S, and .sup.125I) or a fluorescent label
(such as fluorescein isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to
enable detection of an interaction between OGTA001 and a candidate
compound. The ability of the candidate compound to interact
directly or indirectly with OGTA001, a fragment of OGTA001, an
OGTA001-related polypeptide, a fragment of an OGTA001-related
polypeptide, or an OGTA001 fusion protein can be determined by
methods known to those of skill in the art. For example, the
interaction between a candidate compound and OGTA001, an
OGTA001-related polypeptide, a fragment of an OGTA001-related
polypeptide, or an OGTA001 fusion protein can be determined by flow
cytometry, a scintillation assay, immunoprecipitation or western
blot analysis.
[0159] In another embodiment, agents that interact with (i.e., bind
to) OGTA001, an OGTA001 fragment (e.g., a functionally active
fragment), an OGTA001-related polypeptide, a fragment of an
OGTA001-related polypeptide, or an OGTA001 fusion protein are
identified in a cell-free assay system. In accordance with this
embodiment, a native or recombinant OGTA001 or fragment thereof, or
a native or recombinant OGTA001-related polypeptide or fragment
thereof, or an OGTA001-fusion protein or fragment thereof, is
contacted with a candidate compound or a control compound and the
ability of the candidate compound to interact with OGTA001 or
OGTA001-related polypeptide, or OGTA001 fusion protein is
determined. If desired, this assay may be used to screen a
plurality (e.g. a library) of candidate compounds. Preferably,
OGTA001, an OGTA001 fragment, an OGTA001-related polypeptide, a
fragment of an OGTA001-related polypeptide, or an OGTA001-fusion
protein is first immobilized, by, for example, contacting OGTA001,
an OGTA001 fragment, an OGTA001-related polypeptide, a fragment of
an OGTA001-related polypeptide, or an OGTA001 fusion protein with
an immobilized antibody (or other affinity reagent such as an
Affibody, Nanobody or Unibody) which specifically recognizes and
binds it, or by contacting a purified preparation of OGTA001, an
OGTA001 fragment, an OGTA001-related polypeptide, a fragment of an
OGTA001-related polypeptide, or an OGTA001 fusion protein with a
surface designed to bind proteins. OGTA001, an OGTA001 fragment, an
OGTA001-related polypeptide, a fragment of an OGTA001-related
polypeptide, or an OGTA001 fusion protein may be partially or
completely purified (e.g., partially or completely free of other
polypeptides) or part of a cell lysate. Further, OGTA001, an
OGTA001 fragment, an OGTA001-related polypeptide, a fragment of an
OGTA001-related polypeptide may be a fusion protein comprising
OGTA001 or a biologically active portion thereof, or
OGTA001-related polypeptide and a domain such as
glutathionine-5-transferase. Alternatively, OGTA001, an OGTA001
fragment, an OGTA001-related polypeptide, a fragment of an
OGTA001-related polypeptide or an OGTA001 fusion protein can be
biotinylated using techniques well known to those of skill in the
art (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).
The ability of the candidate compound to interact with OGTA001, an
OGTA001 fragment, an OGTA001-related polypeptide, a fragment of an
OGTA001-related polypeptide, or an OGTA001 fusion protein can be
determined by methods known to those of skill in the art.
[0160] In another embodiment, a cell-based assay system is used to
identify agents that bind to or modulate the activity of a protein,
such as an enzyme, or a biologically active portion thereof, which
is responsible for the production or degradation of OGTA001 or is
responsible for the post-translational modification of OGTA001. In
a primary screen, a plurality (e.g., a library) of compounds are
contacted with cells that naturally or recombinantly express: (i)
OGTA001, an isoform of OGTA001, an OGTA001 homolog, an
OGTA001-related polypeptide, an OGTA001 fusion protein, or a
biologically active fragment of any of the foregoing; and (ii) a
protein that is responsible for processing of OGTA001, the OGTA001
isoform, the OGTA001 homolog, the OGTA001-related polypeptide, the
OGTA001 fusion protein, or fragment in order to identify compounds
that modulate the production, degradation, or post-translational
modification of OGTA001, the OGTA001 isoform, the OGTA001 homolog,
the OGTA001-related polypeptide, the OGTA001 fusion protein or
fragment. If desired, compounds identified in the primary screen
can then be assayed in a secondary screen against cells naturally
or recombinantly expressing OGTA001. The ability of the candidate
compound to modulate the production, degradation or
post-translational modification of OGTA001, isoform, homolog,
OGTA001-related polypeptide, or OGTA001 fusion protein can be
determined by methods known to those of skill in the art, including
without limitation, flow cytometry, a scintillation assay,
immunoprecipitation and western blot analysis.
[0161] In another embodiment, agents that competitively interact
with (i.e., bind to) OGTA001, an OGTA001 fragment, an
OGTA001-related polypeptide, a fragment of an OGTA001-related
polypeptide, or an OGTA001 fusion protein are identified in a
competitive binding assay. In accordance with this embodiment,
cells expressing OGTA001, an OGTA001 fragment, an OGTA001-related
polypeptide, a fragment of an OGTA001-related polypeptide, or an
OGTA001 fusion protein are contacted with a candidate compound and
a compound known to interact with OGTA001, an OGTA001 fragment, an
OGTA001-related polypeptide, a fragment of an OGTA001-related
polypeptide or an OGTA001 fusion protein; the ability of the
candidate compound to preferentially interact with OGTA001, the
OGTA001 fragment, the OGTA001-related polypeptide, the fragment of
the OGTA001-related polypeptide, or the OGTA001 fusion protein is
then determined. Alternatively, agents that preferentially interact
with (i.e., bind to) OGTA001, an OGTA001 fragment, an
OGTA001-related polypeptide or fragment of an OGTA001-related
polypeptide are identified in a cell-free assay system by
contacting OGTA001, an OGTA001 fragment, an OGTA001-related
polypeptide, a fragment of an OGTA001-related polypeptide, or an
OGTA001 fusion protein with a candidate compound and a compound
known to interact with OGTA001, the OGTA001-related polypeptide or
the OGTA001 fusion protein. As stated above, the ability of the
candidate compound to interact with OGTA001, an OGTA001 fragment,
an OGTA001-related polypeptide, a fragment of an OGTA001-related
polypeptide, or an OGTA001 fusion protein can be determined by
methods known to those of skill in the art. These assays, whether
cell-based or cell-free, can be used to screen a plurality (e.g., a
library) of candidate compounds.
[0162] In another embodiment, agents that modulate (i.e.,
upregulate or downregulate) the expression or activity of OGTA001,
or an OGTA001-related polypeptide are identified by contacting
cells (e.g., cells of prokaryotic origin or eukaryotic origin)
expressing OGTA001, or the OGTA001-related polypeptide with a
candidate compound or a control compound (e.g., phosphate buffered
saline (PBS)) and determining the expression of OGTA001, the
OGTA001-related polypeptide, or the OGTA001 fusion protein, mRNA
encoding OGTA001, or mRNA encoding the OGTA001-related polypeptide.
The level of expression of OGTA001, the OGTA001-related
polypeptide, mRNA encoding OGTA001, or mRNA encoding the
OGTA001-related polypeptide in the presence of the candidate
compound is compared to the level of expression of OGTA001, the
OGTA001-related polypeptide, mRNA encoding OGTA001, or mRNA
encoding the OGTA001-related polypeptide in the absence of the
candidate compound (e.g., in the presence of a control compound).
The candidate compound can then be identified as a modulator of the
expression of OGTA001, or the OGTA001-related polypeptide based on
this comparison. For example, when expression of OGTA001 or mRNA is
significantly greater in the presence of the candidate compound
than in its absence, the candidate compound is identified as a
stimulator of expression of OGTA001 or mRNA. Alternatively, when
expression of OGTA001 or mRNA is significantly less in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of the expression of OGTA001
or mRNA. The level of expression of OGTA001 or the mRNA that
encodes it can be determined by methods known to those of skill in
the art. For example, mRNA expression can be assessed by Northern
blot analysis or RT-PCR, and protein levels can be assessed by
western blot analysis.
[0163] In another embodiment, agents that modulate the activity of
OGTA001 or an OGTA001-related polypeptide are identified by
contacting a preparation containing OGTA001 or the OGTA001-related
polypeptide or cells (e.g., prokaryotic or eukaryotic cells)
expressing OGTA001 or the OGTA001-related polypeptide with a test
compound or a control compound and determining the ability of the
test compound to modulate (e.g., stimulate or inhibit) the activity
of OGTA001 or the OGTA001-related polypeptide. The activity of
OGTA001 or an OGTA001-related polypeptide can be assessed by
detecting induction of a cellular signal transduction pathway of
OGTA001 or the OGTA001-related polypeptide (e.g., intracellular
Ca.sup.2+, diacylglycerol, IP3, etc.), detecting catalytic or
enzymatic activity of the target on a suitable substrate, detecting
the induction of a reporter gene (e.g., a regulatory element that
is responsive to OGTA001 or an OGTA001-related polypeptide and is
operably linked to a nucleic acid encoding a detectable marker,
e.g., luciferase), or detecting a cellular response, for example,
cellular differentiation, or cell proliferation. Based on the
present description, techniques known to those of skill in the art
can be used for measuring these activities (see, e.g., U.S. Pat.
No. 5,401,639, which is incorporated herein by reference). The
candidate compound can then be identified as a modulator of the
activity of OGTA001 or an OGTA001-related polypeptide by comparing
the effects of the candidate compound to the control compound.
Suitable control compounds include phosphate buffered saline (PBS)
and normal saline (NS).
[0164] In another embodiment, agents that modulate (i.e.,
upregulate or downregulate) the expression, activity or both the
expression and activity of OGTA001 or an OGTA001-related
polypeptide are identified in an animal model. Examples of suitable
animals include, but are not limited to, mice, rats, rabbits,
monkeys, guinea pigs, dogs and cats. Preferably, the animal used
represent a model of colorectal cancer (e.g., xenografts of human
colorectal cancer cell lines such as MDA-MB-345 in
oestrogen-deprived Severe Combined Immunodeficient (SCID) mice,
Eccles et al. 1994 Cell Biophysics 24/25, 279). These can be
utilized to test compounds that modulate OGTA001 levels, since the
pathology exhibited in these models is similar to that of
colorectal cancer. In accordance with this embodiment, the test
compound or a control compound is administered (e.g., orally,
rectally or parenterally such as intraperitoneally or
intravenously) to a suitable animal and the effect on the
expression, activity or both expression and activity of OGTA001 or
an OGTA001-related polypeptide is determined. Changes in the
expression of OGTA001 or an OGTA001-related polypeptide can be
assessed by the methods outlined above.
[0165] In yet another embodiment, OGTA001 or an OGTA001-related
polypeptide is used as a "bait protein" in a two-hybrid assay or
three hybrid assay to identify other proteins that bind to or
interact with OGTA001 or an OGTA001-related polypeptide (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) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene
8:1693-1696; and PCT Publication No. WO 94/10300). As those skilled
in the art will appreciate, such binding proteins are also likely
to be involved in the propagation of signals by OGTA001 as, for
example, upstream or downstream elements of a signaling pathway
involving OGTA001.
[0166] This invention further provides novel agents identified by
the above-described screening assays and uses thereof for
treatments as described herein. In addition, the invention also
provides the use of an agent which interacts with, or modulates the
activity of, OGTA001 in the manufacture of a medicament for the
treatment of colorectal cancer.
Therapeutic Use of OGTA001
[0167] The invention provides for treatment or prevention of
various diseases and disorders by administration of a therapeutic
compound. Such compounds include but are not limited to: OGTA001,
OGTA001 analogs, OGTA001-related polypeptides and derivatives
(including fragments) thereof; antibodies (or other affinity
reagents such as Affibodies, Nanobodies or Unibodies) to the
foregoing; nucleic acids encoding OGTA001, OGTA001 analogs,
OGTA001-related polypeptides and fragments thereof; antisense
nucleic acids to a gene encoding OGTA001 or an OGTA001-related
polypeptide; and modulator (e.g., agonists and antagonists) of a
gene encoding OGTA001 or an OGTA001-related polypeptide. An
important feature of the present invention is the identification of
genes encoding OGTA001 involved in colorectal cancer. Colorectal
cancer can be treated (e.g. to ameliorate symptoms or to retard
onset or progression) or prevented by administration of a
therapeutic compound that reduces function or expression of OGTA001
in the serum or tissue of subjects having colorectal cancer.
[0168] In one embodiment, one or more antibodies (or other affinity
reagents such as Affibodies, Nanobodies or Unibodies) each
specifically binding to OGTA001 are administered alone or in
combination with one or more additional therapeutic compounds or
treatments.
[0169] Preferably, a biological product such as an antibody (or
other affinity reagent such as an Affibody, Nanobody or Unibody) is
allogeneic to the subject to which it is administered. In a
preferred embodiment, a human OGTA001 or a human OGTA001-related
polypeptide, a nucleotide sequence encoding a human OGTA001 or a
human OGTA001-related polypeptide, or an antibody (or other
affinity reagent such as an Affibody, Nanobody or Unibody) to a
human OGTA001 or a human OGTA001-related polypeptide, is
administered to a human subject for therapy (e.g. to ameliorate
symptoms or to retard onset or progression) or prophylaxis.
[0170] Without being limited by theory, it is conceived that the
therapeutic activity of antibodies (or other affinity reagents such
as Affibodies, Nanobodies or Unibodies) which specifically bind to
OGTA001 may be achieved through the phenomenon of
Antibody--Dependent Cell-mediated Cytotoxicity (ADCC) (see e.g.
Janeway Jr. C. A. et al., Immunobiology, 5th ed., 2001, Garland
Publishing, ISBN 0-8153-3642-X; Pier G. B. et al., Immunology,
Infection, and Immunity, 2004, p246-5; Albanell J. et al., Advances
in Experimental Medicine and Biology, 2003, 532:p2153-68 and Weng,
W.-K. et al., Journal of Clinical Oncology, 2003, 21:p
3940-3947).
Treatment and Prevention of Colorectal Cancer
[0171] Colorectal cancer is treated or prevented by administration
to a subject suspected of having or known to have colorectal cancer
or to be at risk of developing colorectal cancer of a compound that
modulates (i.e., increases or decreases) the level or activity
(i.e., function) of OGTA001 that is differentially present in the
serum or tissue of subjects having colorectal cancer compared with
serum or tissue of subjects free from colorectal cancer. In one
embodiment, colorectal cancer is treated or prevented by
administering to a subject suspected of having or known to have
colorectal cancer or to be at risk of developing colorectal cancer
a compound that upregulates (i.e., increases) the level or activity
(i.e., function) of OGTA001 that are decreased in the serum or
tissue of subjects having colorectal cancer. Examples of such a
compound include, but are not limited to, OGTA001 antisense
oligonucleotides, ribozymes, antibodies (or other affinity reagents
such as Affibodies, Nanobodies or Unibodies) directed against
OGTA001, and compounds that inhibit the enzymatic activity of
OGTA001. Other useful compounds e.g., OGTA001 antagonists and small
molecule OGTA001 antagonists, can be identified using in vitro
assays.
[0172] Colorectal cancer is also treated or prevented by
administration to a subject suspected of having or known to have
colorectal cancer or to be at risk of developing colorectal cancer
of a compound that downregulates the level or activity (i.e.
function) of OGTA001 that are increased in the serum or tissue of
subjects having colorectal cancer. Examples of such a compound
include but are not limited to: OGTA001, OGTA001 fragments and
OGTA001-related polypeptides; nucleic acids encoding OGTA001, an
OGTA001 fragment and an OGTA001-related polypeptide (e.g., for use
in gene therapy); and, for those OGTA001 or OGTA001-related
polypeptides with enzymatic activity, compounds or molecules known
to modulate that enzymatic activity. Other compounds that can be
used, e.g., OGTA001 agonists, can be identified using in in vitro
assays.
[0173] In a preferred embodiment, therapy or prophylaxis is
tailored to the needs of an individual subject. Thus, in specific
embodiments, compounds that promote the level or function of
OGTA001 are therapeutically or prophylactically administered to a
subject suspected of having or known to have colorectal cancer, in
whom the levels or functions of OGTA001 are absent or are decreased
relative to a control or normal reference range. In further
embodiments, compounds that promote the level or function of
OGTA001 are therapeutically or prophylactically administered to a
subject suspected of having or known to have colorectal cancer in
whom the levels or functions of OGTA001 are increased relative to a
control or to a reference range. In further embodiments, compounds
that decrease the level or function of OGTA001 are therapeutically
or prophylactically administered to a subject suspected of having
or known to have colorectal cancer in whom the levels or functions
of OGTA001 are increased relative to a control or to a reference
range. In further embodiments, compounds that decrease the level or
function of OGTA001 are therapeutically or prophylactically
administered to a subject suspected of having or known to have
colorectal cancer in whom the levels or functions of OGTA001 are
decreased relative to a control or to a reference range. The change
in OGTA001 function or level due to the administration of such
compounds can be readily detected, e.g., by obtaining a sample
(e.g., blood or urine) and assaying in vitro the levels or
activities of OGTA001, or the levels of mRNAs encoding OGTA001, or
any combination of the foregoing. Such assays can be performed
before and after the administration of the compound as described
herein.
[0174] The compounds of the invention include but are not limited
to any compound, e.g., a small organic molecule, protein, peptide,
antibody (or other affinity reagent such as an Affibody, Nanobody
or Unibody), nucleic acid, etc. that restores the OGTA001 profile
towards normal. The compounds of the invention may be given in
combination with any other chemotherapy drugs.
Vaccine Therapy
[0175] OGTA001 may be useful as antigenic material, and may be used
in the production of vaccines for treatment or prophylaxis of
colorectal cancer. Such material can be "antigenic" and/or
"immunogenic". Generally, "antigenic" is taken to mean that the
protein is capable of being used to raise antibodies (or other
affinity reagents such as Affibodies, Nanobodies or Unibodies) or
indeed is capable of inducing an antibody response in a subject or
experimental animal. "Immunogenic" is taken to mean that the
protein is capable of eliciting a protective immune response in a
subject or experimental animal. Thus, in the latter case, the
protein may be capable of not only generating an antibody response
but, in addition, non-antibody based immune responses.
"Immunogenic" also embraces whether the protein may elicit an
immune-like response in an in-vitro setting eg a T-cell
proliferation assay.
[0176] The skilled person will appreciate that homologues or
derivatives of OGTA001 will also find use as antigenic/immunogenic
material. Thus, for instance proteins which include one or more
additions, deletions, substitutions or the like are encompassed by
the present invention. In addition, it may be possible to replace
one amino acid with another of similar "type". For instance,
replacing one hydrophobic amino acid with another. One can use a
program such as the CLUSTAL program to compare amino acid
sequences. This program compares amino acid sequences and finds the
optimal alignment by inserting spaces in either sequence as
appropriate. It is possible to calculate amino acid identity or
similarity (identity plus conservation of amino acid type) for an
optimal alignment. A program like BLASTx will align the longest
stretch of similar sequences and assign a value to the fit. It is
thus possible to obtain a comparison where several regions of
similarity are found, each having a different score. Both types of
analysis are contemplated in the present invention.
[0177] In the case of homologues and derivatives, the degree of
identity with a protein as described herein is less important than
that the homologue or derivative should retain its antigenicity
and/or immunogenicity. However, suitably, homologues or derivatives
having at least 60% similarity (as discussed above) with the
proteins or polypeptides described herein are provided. Preferably,
homologues or derivatives having at least 70% similarity, more
preferably at least 80% similarity, are provided. Most preferably,
homologues or derivatives having at least 90% or even 95%
similarity are provided.
[0178] In an alternative approach, the homologues or derivatives
could be fusion proteins, incorporating moieties which render
purification easier, for example by effectively tagging the desired
protein or polypeptide. It may be necessary to remove the "tag" or
it may be the case that the fusion protein itself retains
sufficient antigenicity to be useful.
[0179] It is well known that it is possible to screen an antigenic
protein or polypeptide to identify epitopic regions, i.e. those
regions which are responsible for the protein or polypeptide's
antigenicity or immunogenicity. Methods well known to the skilled
person can be used to test fragments and/or homologues and/or
derivatives for antigenicity. Thus, the fragments of the present
invention should include one or more such epitopic regions or be
sufficiently similar to such regions to retain their
antigenic/immunogenic properties. Thus, for fragments according to
the present invention the degree of identity is perhaps irrelevant,
since they may be 100% identical to a particular part of a protein
or polypeptide, homologue or derivative as described herein. The
key issue, once again, is that the fragment retains the
antigenic/immunogenic properties of the protein from which it is
derived.
[0180] What is important for homologues, derivatives and fragments
is that they possess at least a degree of the
antigenicity/immunogenicity of the protein or polypeptide from
which they are derived. Thus, in an additional aspect of the
invention, there is provided antigenic/or immunogenic fragments of
OGTA001, or of homologues or derivatives thereof.
[0181] OGTA001, or antigenic fragments thereof, can be provided
alone, as a purified or isolated preparation. They may be provided
as part of a mixture with one or more other proteins or antigenic
fragments thereof. In a further aspect, therefore, the invention
provides an antigen composition comprising OGTA001 and/or one or
more antigenic fragments thereof. Such a composition can be used
for the detection and/or diagnosis of colorectal cancer.
[0182] In a sixth aspect, the present invention provides a method
of detecting and/or diagnosing colorectal cancer which
comprises:
[0183] bringing into contact with a sample to be tested an
antigenic OGTA001, or an antigenic fragment thereof, or an antigen
composition of the invention; and
[0184] detecting the presence of antibodies (or other affinity
reagents such as Affibodies, Nanobodies or Unibodies) to colorectal
cancer.
[0185] In particular, the protein, antigenic fragment thereof or
antigen composition of the present invention can be used to detect
IgA, IgM or IgG antibodies. Suitably, the sample to be tested will
be a biological sample, e.g. a sample of blood or saliva.
[0186] In a further aspect, the invention provides the use of an
antigenic OGTA001, antigenic fragment thereof or an antigenic
composition of the present invention in detecting and/or diagnosing
colorectal cancer. Preferably, the detecting and/or diagnosing are
carried out in vitro.
[0187] The antigenic OGTA001, antigenic fragments thereof or
antigenic composition of the present invention can be provided as a
kit for use in the in vitro detection and/or diagnosis of
colorectal cancer. Thus, in a still further aspect, the present
invention provides a kit for use in the detection and/or diagnosis
of colorectal cancer, which kit comprises an antigenic OGTA001, an
antigenic fragment thereof or an antigenic composition of the
present invention.
[0188] In addition, the antigenic OGTA001, antigenic fragment
thereof or antigen composition of the invention can be used to
induce an immune response against colorectal cancer. Thus, in a yet
further aspect, the invention provides the use of an antigenic
OGTA001, an antigenic fragment thereof or an antigen composition of
the invention in medicine.
[0189] In a further aspect, the present invention provides a
composition capable of eliciting an immune response in a subject,
which composition comprises OGTA001, an antigenic fragment thereof,
or an antigen composition of the invention. Suitably, the
composition will be a vaccine composition, optionally comprising
one or more suitable adjuvants. Such a vaccine composition may be
either a prophylactic or therapeutic vaccine composition.
[0190] The vaccine compositions of the invention can include one or
more adjuvants. Examples well-known in the art include inorganic
gels, such as aluminium hydroxide, and water-in-oil emulsions, such
as incomplete Freund's adjuvant. Other useful adjuvants will be
well known to the skilled person.
[0191] In yet further aspects, the present invention provides:
[0192] (a) the use of OGTA001, an antigenic fragment thereof, or an
antigen composition of the invention in the preparation of an
immunogenic composition, preferably a vaccine;
[0193] (b) the use of such an immunogenic composition in inducing
an immune response in a subject; and
[0194] (c) a method for the treatment or prophylaxis of colorectal
cancer in a subject, or of vaccinating a subject against colorectal
cancer which comprises the step of administering to the subject an
effective amount of OGTA001, at least one antigenic fragment
thereof or an antigen composition of the invention, preferably as a
vaccine.
[0195] In a specific embodiment, a preparation of OGTA001 or
OGTA001 peptide fragments is used as a vaccine for the treatment of
colorectal cancer. Such preparations may include adjuvants or other
vehicles.
[0196] In another embodiment, a preparation of oligonucleotides
comprising 10 or more consecutive nucleotides complementary to a
nucleotide sequence encoding OGTA001 or OGTA001 peptide fragments
is used as vaccines for the treatment of colorectal cancer. Such
preparations may include adjuvants or other vehicles.
Inhibition of OGTA001 to Treat Colorectal Cancer
[0197] In one embodiment of the invention, colorectal cancer is
treated or prevented by administration of a compound that
antagonizes (inhibits) the level(s) and/or function(s) of OGTA001
which are elevated in the serum or tissue of subjects having
colorectal cancer as compared with serum or tissue of subjects free
from colorectal cancer.
[0198] Compounds useful for this purpose include but are not
limited to anti-OGTA001 antibodies (or other affinity reagents such
as Affibodies, Nanobodies or Unibodies, and fragments and
derivatives containing the binding region thereof), OGTA001
antisense or ribozyme nucleic acids, and nucleic acids encoding
dysfunctional OGTA001 that are used to "knockout" endogenous
OGTA001 function by homologous recombination (see, e.g., Capecchi,
1989, Science 244:1288-1292). Other compounds that inhibit OGTA001
function can be identified by use of known in vitro assays, e.g.,
assays for the ability of a test compound to inhibit binding of
OGTA001 to another protein or a binding partner, or to inhibit a
known OGTA001 function. Preferably such inhibition is assayed in
vitro or in cell culture, but genetic assays may also be employed.
The Preferred Technologies can also be used to detect levels of
OGTA001 before and after the administration of the compound.
Preferably, suitable in vitro or in vivo assays are utilized to
determine the effect of a specific compound and whether its
administration is indicated for treatment of the affected tissue,
as described in more detail below.
[0199] In a specific embodiment, a compound that inhibits OGTA001
function is administered therapeutically or prophylactically to a
subject in whom an increased serum or tissue level or functional
activity of OGTA001 (e.g., greater than the normal level or desired
level) is detected as compared with serum or tissue of subjects
free from colorectal cancer or a predetermined reference range.
Methods standard in the art can be employed to measure the increase
in OGTA001 level or function, as outlined above. Preferred OGTA001
inhibitor compositions include small molecules, i.e., molecules of
1000 daltons or less. Such small molecules can be identified by the
screening methods described herein.
Assays for Therapeutic or Prophylactic Compounds
[0200] The present invention also provides assays for use in drug
discovery in order to identify or verify the efficacy of compounds
for treatment or prevention of colorectal cancer. Test compounds
can be assayed for their ability to restore OGTA001 levels in a
subject having colorectal cancer towards levels found in subjects
free from colorectal cancer or to produce similar changes in
experimental animal models of colorectal cancer. Compounds able to
restore OGTA001 levels in a subject having colorectal cancer
towards levels found in subjects free from colorectal cancer or to
produce similar changes in experimental animal models of colorectal
cancer can be used as lead compounds for further drug discovery, or
used therapeutically. OGTA001 expression can be assayed by the
Preferred Technologies, immunoassays, gel electrophoresis followed
by visualization, detection of OGTA001 activity, or any other
method taught herein or known to those skilled in the art. Such
assays can be used to screen candidate drugs, in clinical
monitoring or in drug development, where abundance of OGTA001 can
serve as a surrogate marker for clinical disease.
[0201] In various specific embodiments, in vitro assays can be
carried out with cells representative of cell types involved in a
subject's disorder, to determine if a compound has a desired effect
upon such cell types.
[0202] Compounds for use in therapy can be tested in suitable
animal model systems prior to testing in humans, including but not
limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For in
vivo testing, prior to administration to humans, any animal model
system known in the art may be used. Examples of animal models of
colorectal cancer include, but are not limited to xenografts of
human colorectal cancer cell lines such as MDA-MB-435 in
oestrogen-deprived Severe Combined Immunodeficient (SCID) mice
(Eccles et al., 1994 Cell Biophysics 24/25, 279). These can be
utilized to test compounds that modulate OGTA001 levels, since the
pathology exhibited in these models is similar to that of
colorectal cancer. It is also apparent to the skilled artisan that
based upon the present disclosure, transgenic animals can be
produced with "knock-out" mutations of the gene or genes encoding
OGTA001. A "knock-out" mutation of a gene is a mutation that causes
the mutated gene to not be expressed, or expressed in an aberrant
form or at a low level, such that the activity associated with the
gene product is nearly or entirely absent. Preferably, the
transgenic animal is a mammal; more preferably, the transgenic
animal is a mouse.
[0203] In one embodiment, test compounds that modulate the
expression of OGTA001 are identified in non-human animals (e.g.,
mice, rats, monkeys, rabbits, and guinea pigs), preferably
non-human animal models for colorectal cancer, expressing OGTA001.
In accordance with this embodiment, a test compound or a control
compound is administered to the animals, and the effect of the test
compound on expression of OGTA001 is determined. A test compound
that alters the expression of OGTA001 can be identified by
comparing the level of OGTA001 (or mRNA encoding the same) in an
animal or group of animals treated with a test compound with the
level of OGTA001 or mRNA in an animal or group of animals treated
with a control compound. Techniques known to those of skill in the
art can be used to determine the mRNA and protein levels, for
example, in situ hybridization. The animals may or may not be
sacrificed to assay the effects of a test compound.
[0204] In another embodiment, test compounds that modulate the
activity of OGTA001 or a biologically active portion thereof are
identified in non-human animals (e.g., mice, rats, monkeys,
rabbits, and guinea pigs), preferably non-human animal models for
colorectal cancer, expressing OGTA001. In accordance with this
embodiment, a test compound or a control compound is administered
to the animals, and the effect of a test compound on the activity
of OGTA001 is determined. A test compound that alters the activity
of OGTA001 can be identified by assaying animals treated with a
control compound and animals treated with the test compound. The
activity of OGTA001 can be assessed by detecting induction of a
cellular second messenger of OGTA001 (e.g., intracellular
Ca.sup.2+, diacylglycerol, 1P3, etc.), detecting catalytic or
enzymatic activity of OGTA001 or binding partner thereof, detecting
the induction of a reporter gene (e.g., a regulatory element that
is responsive to OGTA001 operably linked to a nucleic acid encoding
a detectable marker, such as luciferase or green fluorescent
protein), or detecting a cellular response (e.g., cellular
differentiation or cell proliferation). Techniques known to those
of skill in the art can be utilized to detect changes in the
activity of OGTA001 (see, e.g., U.S. Pat. No. 5,401,639, which is
incorporated herein by reference).
[0205] In yet another embodiment, test compounds that modulate the
level or expression of OGTA001 are identified in human subjects
having colorectal cancer, preferably those having severe colorectal
cancer. In accordance with this embodiment, a test compound or a
control compound is administered to the human subject, and the
effect of a test compound on OGTA001 expression is determined by
analyzing the expression of OGTA001 or the mRNA encoding the same
in a biological sample (e.g., serum, plasma, or urine). A test
compound that alters the expression of OGTA001 can be identified by
comparing the level of OGTA001 or mRNA encoding the same in a
subject or group of subjects treated with a control compound to
that in a subject or group of subjects treated with a test
compound. Alternatively, alterations in the expression of OGTA001
can be identified by comparing the level of OGTA001 or mRNA
encoding the same in a subject or group of subjects before and
after the administration of a test compound. Techniques known to
those of skill in the art can be used to obtain the biological
sample and analyze the mRNA or protein expression. For example, the
Preferred Technologies described herein can be used to assess
changes in the level of OGTA001.
[0206] In another embodiment, test compounds that modulate the
activity of OGTA001 are identified in human subjects having
colorectal cancer, (preferably those with severe colorectal
cancer). In this embodiment, a test compound or a control compound
is administered to the human subject, and the effect of a test
compound on the activity of OGTA001 is determined. A test compound
that alters the activity of OGTA001 can be identified by comparing
biological samples from subjects treated with a control compound to
samples from subjects treated with the test compound.
Alternatively, alterations in the activity of OGTA001 can be
identified by comparing the activity of OGTA001 in a subject or
group of subjects before and after the administration of a test
compound. The activity of OGTA001 can be assessed by detecting in a
biological sample (e.g., serum, plasma, or urine) induction of a
cellular signal transduction pathway of OGTA001 (e.g.,
intracellular Ca.sup.2+, diacylglycerol, IP3, etc.), catalytic or
enzymatic activity of OGTA001 or a binding partner thereof, or a
cellular response, for example, cellular differentiation, or cell
proliferation. Techniques known to those of skill in the art can be
used to detect changes in the induction of a second messenger of
OGTA001 or changes in a cellular response. For example, RT-PCR can
be used to detect changes in the induction of a cellular second
messenger.
[0207] In a preferred embodiment, a test compound that changes the
level or expression of OGTA001 towards levels detected in control
subjects (e.g., humans free from colorectal cancer) is selected for
further testing or therapeutic use. In another preferred
embodiment, a test compound that changes the activity of OGTA001
towards the activity found in control subjects (e.g., humans free
from colorectal cancer) is selected for further testing or
therapeutic use.
[0208] In another embodiment, test compounds that reduce the
severity of one or more symptoms associated with colorectal cancer
are identified in human subjects having colorectal cancer,
preferably subjects with severe colorectal cancer. In accordance
with this embodiment, a test compound or a control compound is
administered to the subjects, and the effect of a test compound on
one or more symptoms of colorectal cancer is determined. A test
compound that reduces one or more symptoms can be identified by
comparing the subjects treated with a control compound to the
subjects treated with the test compound. Techniques known to
physicians familiar with colorectal cancer can be used to determine
whether a test compound reduces one or more symptoms associated
with colorectal cancer. For example, a test compound that reduces
tumour burden in a subject having colorectal cancer will be
beneficial for subjects having colorectal cancer.
[0209] In a preferred embodiment, a test compound that reduces the
severity of one or more symptoms associated with colorectal cancer
in a human having colorectal cancer is selected for further testing
or therapeutic use.
Therapeutic and Prophylactic Compositions and their Use
[0210] The invention provides methods of treatment (and
prophylaxis) comprising administering to a subject an effective
amount of a compound of the invention. In a preferred aspect, the
compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably human.
In a specific embodiment, a non-human mammal is the subject.
[0211] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid are described
above; additional appropriate formulations and routes of
administration are described below.
[0212] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction can be enteral or parenteral and include
but are not limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The compounds may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection; intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Ommaya reservoir. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent.
[0213] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved, for example, and
not by way of limitation, by local infusion during surgery, topical
application, e.g., by injection, by means of a catheter, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers. In one embodiment, administration can be by
direct injection into colorectal tissue or at the site (or former
site) of a malignant tumor or neoplastic or pre-neoplastic
tissue.
[0214] In another embodiment, the compound can be delivered in a
vesicle, in particular a liposome (see Langer, 1990, Science
249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0215] In yet another embodiment, the compound can be delivered in
a controlled release system. In one embodiment, a pump may be used
(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989,
N. Engl. J. Med. 321:574). In another embodiment, polymeric
materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
(1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger
and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61;
see also Levy et al., 1985, Science 228:190; During et al., 1989,
Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In
yet another embodiment, a controlled release system can be placed
in proximity of the therapeutic target, i.e., the colon, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)).
[0216] Other controlled release systems are discussed in the review
by Langer (1990, Science 249:1527-1533).
[0217] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., 1991, Proc.
Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0218] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the subject. The formulation should suit the mode
of administration.
[0219] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lidocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0220] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0221] The amount of the compound of the invention which will be
effective in the treatment of colorectal cancer can be determined
by standard clinical techniques. In addition, in vitro assays may
optionally be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation will also depend on
the route of administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of the
practitioner and each subject's circumstances. However, suitable
dosage ranges for intravenous administration are generally about
20-500 micrograms of active compound per kilogram body weight.
Suitable dosage ranges for intranasal administration are generally
about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective
doses may be extrapolated from dose-response curves derived from in
vitro or animal model test systems.
[0222] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0223] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects (a) approval by the agency of manufacture,
use or sale for human administration, (b) directions for use, or
both.
Determining Abundance of OGTA001 by Imaging Technology
[0224] An advantage of determining abundance of OGTA001 by imaging
technology may be that such a method is non-invasive (save that
reagents may need to be administered) and there is no need to
extract a sample from the subject.
[0225] Suitable imaging technologies include positron emission
tomography (PET) and single photon emission computed tomography
(SPECT). Visualisation of OGTA001 using such techniques requires
incorporation or binding of a suitable label e.g. a radiotracer
such as .sup.18F, .sup.11C or .sup.123I (see e.g. NeuroRx--The
Journal of the American Society for Experimental NeuroTherapeutics
(2005) 2(2), 348-360 and idem pages 361-371 for further details of
the techniques). Radiotracers or other labels may be incorporated
into OGTA001 by administration to the subject (e.g. by injection)
of a suitably labelled specific ligand. Alternatively they may be
incorporated into a binding affinity reagent (antibody, Affibody,
Nanobody, Unibody etc.) specific for OGTA001 which may be
administered to the subject (e.g. by injection). For discussion of
use of Affibodies for imaging see e.g. Orlova A, Magnusson M,
Eriksson T L, Nilsson M, Larsson B, Hoiden-Guthenberg I, Widstrom
C, Carlsson J, Tolmachev V, Stahl S, Nilsson F Y, Tumor imaging
using a picomolar affinity HER2 binding affibody molecule, Cancer
Res. 2006 Apr. 15; 66(8):4339-48).
Diagnosis and Treatment of Colorectal Cancer Using
Immunohistochemistry
[0226] Immunohistochemistry is an excellent detection technique and
may therefore be very useful in the diagnosis and treatment of
colorectal cancer. Immunohistochemistry may be used to detect,
diagnose, or monitor colorectal cancer through the localization of
OGTA001 antigens in tissue sections by the use of labeled
antibodies (or other affinity reagents such as Affibodies,
Nanobodies or Unibodies), derivatives and analogs thereof, which
specifically bind to OGTA001, as specific reagents through
antigen-antibody interactions that are visualized by a marker such
as fluorescent dye, enzyme, radioactive element or colloidal
gold.
[0227] The advancement of monoclonal antibody technology has been
of great significance in assuring the place of immunohistochemistry
in the modern accurate microscopic diagnosis of human neoplasms.
The identification of disseminated neoplastically transformed cells
by immunohistochemistry allows for a clearer picture of cancer
invasion and metastasis, as well as the evolution of the tumour
cell associated immunophenotype towards increased malignancy.
Future antineoplastic therapeutical approaches may include a
variety of individualized immunotherapies, specific for the
particular immunophenotypical pattern associated with each
individual patient's neoplastic disease. For further discussion see
e.g. Bodey B, The significance of immunohistochemistry in the
diagnosis and therapy of neoplasms, Expert Opin Biol Ther. 2002
April; 2(4):371-93.
[0228] Preferred features of each aspect of the invention are as
for each of the other aspects mutatis mutandis. The prior art
documents mentioned herein are incorporated to the fullest extent
permitted by law.
Example 1
Identification of Membrane Proteins Expressed in Colorectal Cancer
Blood and Tissue Samples Using 1D Gel Electrophoresis
[0229] Using the following Reference Protocol, membrane proteins
extracted from colorectal cancer tissue samples were separated by
1D gel and analysed.
1.1 Materials and Methods
1.1.1--Plasma Membrane Fractionation
[0230] The cells recovered from the epithelium of a colorectal
adenocarcinoma were lysed and submitted to centrifugation at 1000G.
The supernatant was taken, and it was subsequently centrifuged at
3000G. Once again, the supernatant was taken, and it was then
centrifuged at 100 000G.
[0231] The resulting pellet was recovered and put on 15-60% sucrose
gradient.
[0232] A Western blot was used to identify sub cellular markers,
and the Plasma Membrane fractions were pooled.
[0233] The pooled solution was either run directly on 1D gels (see
section 1.1.4 below), or further fractionated into heparin binding
and nucleotide binding fractions as described below.
1.1.2--Plasma Membrane Heparin-Binding Fraction
[0234] The pooled solution from 1.1.1 above was applied to a
Heparin column, eluted from column and run on 1D gels (see section
1.1.4 below).
1.1.3--Plasma Nucleotide-Binding Fraction
[0235] The pooled solution from 1.1.1 above was applied to a
Cibacrom Blue 3GA column, eluted from column and run on 1D gels
(see section 1.1.4 below).
1.1.4-1D Gel Technology Protein or membrane pellets were
solubilised in 1D sample buffer (1-2 .mu.g/.mu.l). The sample
buffer and protein mixture was then heated to 95.degree. C. for 3
min.
[0236] A 9-16% acrylamide gradient gel was cast with a stacking gel
and a stacking comb according to the procedure described in Ausubel
F. M. et al., eds., 1989, Current Protocols in Molecular Biology,
Vol. II, Green Publishing Associates, Inc., and John Wiley &
Sons, Inc., New York, section 10.2, incorporated herein by
reference in its entirety.
[0237] 30-50 micrograms of the protein mixtures obtained from
detergent and the molecular weight standards (66, 45, 31, 21, 14
kDa) were added to the stacking gel wells using a 10 microlitre
pipette tip and the samples run at 40 mA for 5 hours.
[0238] The plates were then prised open, the gel placed in a tray
of fixer (10% acetic acid, 40% ethanol, 50% water) and shaken
overnight. Following this, the gel was primed by 30 minutes shaking
in a primer solution (7.5% acetic acid (75 ml), 0.05% SDS (5 ml of
10%)). The gel was then incubated with a fluorescent dye (7.5%
acetic acid, 0.06% OGS in-house dye (600 .mu.l)) with shaking for 3
hrs. Sypro Red (Molecular Probes, Inc., Eugene, Oreg.) is a
suitable dye for this purpose. A preferred fluorescent dye is
disclosed in U.S. application Ser. No. 09/412,168, filed on Oct. 5,
1999, which is incorporated herein by reference in its
entirety.
[0239] A computer-readable output was produced by imaging the
fluorescently stained gels with an Apollo 3 scanner (Oxford
Glycosciences, Oxford, UK). This scanner is developed from the
scanner described in WO 96/36882 and in the Ph.D. thesis of David
A. Basiji, entitled "Development of a High-throughput Fluorescence
Scanner Employing Internal Reflection Optics and Phase-sensitive
Detection (Total Internal Reflection, Electrophoresis)", University
of Washington (1997), Volume 58/12-B of Dissertation Abstracts
International, page 6686, the contents of each of which are
incorporated herein by reference. The latest embodiment of this
instrument includes the following improvements: The gel is
transported through the scanner on a precision lead-screw drive
system. This is preferable to laying the glass plate on the
belt-driven system that is defined in the Basiji thesis as it
provides a reproducible means of accurately transporting the gel
past the imaging optics.
[0240] The gel is secured into the scanner against three alignment
stops that rigidly hold the glass plate in a known position. By
doing this in conjunction with the above precision transport system
and the fact that the gel is bound to the glass plate, the absolute
position of the gel can be predicted and recorded. This ensures
that accurate co-ordinates of each feature on the gel can be
communicated to the cutting robot for excision. This cutting robot
has an identical mounting arrangement for the glass plate to
preserve the positional accuracy.
[0241] The carrier that holds the gel in place has integral
fluorescent markers (Designated M1, M2, M3) that are used to
correct the image geometry and are a quality control feature to
confirm that the scanning has been performed correctly.
[0242] The optical components of the system have been inverted. The
laser, mirror, waveguide and other optical components are now above
the glass plate being scanned. The embodiment of the Basiji thesis
has these underneath. The glass plate is therefore mounted onto the
scanner gel side down, so that the optical path remains through the
glass plate. By doing this, any particles of gel that may break
away from the glass plate will fall onto the base of the instrument
rather than into the optics.
[0243] In scanning the gels, they were removed from the stain,
rinsed with water and allowed to air dry briefly and imaged on the
Apollo 3. After imaging, the gels were sealed in polyethylene bags
containing a small volume of staining solution, and then stored at
4.degree. C.
[0244] Apparent molecular weights were calculated by interpolation
from a set of known molecular weight markers run alongside the
samples.
1.1.5--Recovery and Analysis of Selected Proteins
[0245] Proteins were robotically excised from the gels by the
process described in U.S. Pat. No. 6,064,754, Sections 5.4 and 5.6,
5.7, 5.8 (incorporated herein by reference), as is applicable to
1D-electrophoresis, with modification to the robotic cutter as
follows: the cutter begins at the top of the lane, and cuts a gel
disc 1.7 mm in diameter from the left edge of the lane. The cutter
then moves 2 mm to the right, and 0.7 mm down and cuts a further
disc. This is then repeated. The cutter then moves back to a
position directly underneath the first gel cut, but offset by 2.2
mm downwards, and the pattern of three diagonal cuts are repeated.
This is continued for the whole length of the gel.
[0246] NOTE: If the lane is observed to broaden significantly then
a correction can be made also sideways i.e. instead of returning to
a position directly underneath a previous gel cut, the cut can be
offset slightly to the left (on the left of the lane) and/or the
right (on the right of the lane). The proteins contained within the
gel fragments were processed to generate tryptic peptides; partial
amino acid sequences of these peptides were determined by mass
spectroscopy as described in WO98/53323 and application Ser. No.
09/094,996, filed Jun. 15, 1998.
[0247] Proteins were processed to generate tryptic digest peptides.
Tryptic peptides were analyzed by mass spectrometry using a
PerSeptive Biosystems Voyager--DETM STR Matrix-Assisted Laser
Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometer,
and selected tryptic peptides were analyzed by tandem mass
spectrometry (MS/MS) using a Micromass Quadrupole Time-of-Flight
(Q-TOF) mass spectrometer (Micromass, Altrincham, U.K.) equipped
with a Nanoflow.TM. electrospray Z-spray source. For partial amino
acid sequencing and identification of OGTA001, uninterpreted tandem
mass spectra of tryptic peptides were searched using the SEQUEST
search program (Eng et al., 1994, J. Am. Soc. Mass Spectrom.
5:976-989), version v.C.1. Criteria for database identification
included: the cleavage specificity of trypsin; the detection of a
suite of a, b and y ions in peptides returned from the database,
and a mass increment for all Cys residues to account for
carbamidomethylation. The database searched was a database
constructed of protein entries in the non-redundant database held
by the National Centre for Biotechnology Information (NCBI) which
is accessible at www.ncbi.nlm.nih.gov. Following identification of
proteins through spectral-spectral correlation using the SEQUEST
program, masses detected in MALDI-TOF mass spectra were assigned to
tryptic digest peptides within the proteins identified. In cases
where no amino acid sequences could be identified through searching
with uninterpreted MS/MS spectra of tryptic digest peptides using
the SEQUEST program, tandem mass spectra of the peptides were
interpreted manually, using methods known in the art. (In the case
of interpretation of low-energy fragmentation mass spectra of
peptide ions see Gaskell et al., 1992, Rapid Commun. Mass Spectrom.
6:658-662).
1.1.6--Discrimination of Colorectal Cancer Associated Proteins
[0248] The process to identify OGTA001 uses the peptide sequences
obtained experimentally by mass spectrometry described above of
naturally occurring human proteins to identify and organize coding
exons in the published human genome sequence.
[0249] Recent dramatic advances in defining the chemical sequence
of the human genome have led to the near completion of this immense
task (Venter, J. C. et al. (2001). The sequence of the human
genome. Science 16: 1304-51; International Human Genome Sequencing
Consortium. (2001). Initial sequencing and analysis of the human
genome Nature 409: 860-921). There is little doubt that this
sequence information will have a substantial impact on our
understanding of many biological processes, including molecular
evolution, comparative genomics, pathogenic mechanisms and
molecular medicine. For the full medical value inherent in the
sequence of the human genome to be realised, the genome needs to be
`organised` and annotated. By this, is meant at least the following
three things: (i) The assembly of the sequences of the individual
portions of the genome into a coherent, continuous sequence for
each chromosome. (ii) The unambiguous identification of those
regions of each chromosome that contain genes. (iii) Determination
of the fine structure of the genes and the properties of its mRNA
and protein products. While the definition of a `gene` is an
increasingly complex issue (H Pearson: What is a gene? Nature
(2006) 24: 399-401), what is of immediate interest for drug
discovery and development is a catalogue of those genes that encode
functional, expressed proteins. A subset of these genes will be
involved in the molecular basis of most if not all pathologies.
Therefore an important and immediate goal for the pharmaceutical
industry is to identify all such genes in the human genome and
describe their fine structure.
Processing and Integration of Peptide Masses, Peptide Signatures,
ESTs and Public Domain Genomic Sequence Data to Form OGAP.RTM.
Database
[0250] Discrete genetic units (exons, transcripts and genes) were
identified using the following sequential steps: [0251] 1. A
`virtual transcriptome` is generated, containing the tryptic
peptides which map to the human genome by combining the gene
identifications available from Ensembl and various gene prediction
programs. This also incorporates SNP data (from dbSNP) and all
alternate splicing of gene identifications. Known contaminants were
also added to the virtual transcriptome. [0252] 2. All tandem
spectra in the OGeS Mass Spectrometry Database are interpreted in
order to produce a peptide that can be mapped to one in the virtual
transcriptome. A set of automated spectral interpretation
algorithms were used to produce the peptide identifications. [0253]
3. The set of all mass-matched peptides in the OGeS Mass
Spectrometry Database is generated by searching all peptides from
transcripts hit by the tandem peptides using a tolerance based on
the mass accuracy of the mass spectrometer, typically 20 ppm.
[0254] 4. All tandem and mass-matched peptides are combined in the
form of "protein clusters". This is done using a recursive process
which groups sequences into clusters based on common peptide hits.
Biological sequences are considered to belong to the same cluster
if they share one or more tandem or mass-matched peptide. [0255] 5.
After initial filtering to screen out incorrectly identified
peptides, the resulting clusters are then mapped on the human
genome. [0256] 6. The protein clusters are then aggregated into
regions that define preliminary gene boundaries using their
proximity and the co-observation of peptides within protein
clusters. Proximity is defined as the peptide being within 80,000
nucleotides on the same strand of the same chromosome. Various
elimination rules, based on cluster observation scoring and
multiple mapping to the genome are used to refine the output. The
resulting `confirmed genes` are those which best account for the
peptides and masses observed by mass spectrometry in each cluster.
Nominal co-ordinates for the gene are also an output of this stage.
[0257] 7. The best set of transcripts for each confirmed gene are
created from the protein clusters, peptides, ESTs, candidate exons
and molecular weight of the original protein spot. [0258] 8. Each
identified transcript was linked to the sample providing the
observed peptides. [0259] 9. Use of an application for viewing and
mining the data. The result of steps 1-8 was a database containing
genes, each of which consisted of a number of exons and one or more
transcripts. An application was written to display and search this
integrated genome/proteome data. Any features (OMIM disease locus,
InterPro etc.) that had been mapped to the same Golden Path
co-ordinate system by Ensembl could be cross-referenced to these
genes by coincidence of location and fine structure.
Results
[0260] The process was used to generate approximately 1 million
peptide sequences to identify protein-coding genes and their exons
resulted in the identification of protein sequences for 18083 genes
across 67 different tissues and 57 diseases including 506 genes in
Bladder cancer, 4,713 genes in Breast cancer, 766 genes in
Burkitt's lymphoma, 1,371 genes in Cervical cancer, 949 genes in
Colorectal cancer, 1,782 genes in Hepatocellular carcinoma, 2,424
genes in CLL, 978 genes in Lung cancer, 1,764 genes in Melanoma,
1,033 genes in Ovarian Cancer, 2,961 genes in Pancreatic cancer and
3,307 genes in Prostate cancer, illustrated here by OGTA001
isolated and identified from colorectal cancer samples. Following
comparison of the experimentally determined sequences with
sequences in the OGAP.RTM. database, OGTA001 showed a high degree
of specificity to colorectal cancer indicative of the prognostic
and diagnostic nature.
1.2 Results
[0261] These experiments identified OGTA001, as further described
herein. The full-length OGTA001 was detected in the plasma membrane
of colorectal cancer samples and was not detected in the
cytosol.
[0262] FIG. 3 shows the Protein Index for OGTA001. For each gene,
the protein index uses the mass spectrometry data to assign a score
to each disease, relative to the global database. The Protein Index
can then be used to identify cancer specific genes with a high
score in cancer indications and low/negligible scores in normal and
other diseases. The index contains .about.1 million peptides
sequenced via mass spectrometry from 56 diseases. For each gene,
this yields a score for each disease and subcellular location. The
results are summarized below:
TABLE-US-00004 Protein Index Report for OGTA001 Indications
positive: Colorectal cancer Disease controls Acute monocytic
leukaemia Acute T-cell leukaemia Alzheimer's Disease Arthritis
Asthma Atherosclerosis B-cell non-Hodgkin's lymphoma Bladder
carcinoma Breast cancer Breast diseases, benign Burkitt's lymphoma
Bursitis Cancer, unspecified Cervical cancer Chronic lymphocytic
leukaemia Chronic obstructive pulmonary disease Colorectal cancer
Dementia, vascular Depression Diabetes and Obesity Diverticulitis
Dyslipidaemia Emphysema Focal apocrine metaplasia Gastric cancer
Gaucher disease Glioblastoma Hepatoblastoma Hepatocellular
carcinoma Hypertension Lactational foci Leukaemia, unspecified
Liver cirrhosis Lung cancer Lymphoma, histiocytic Melanoma
Metabolic syndrome X Migraine, acute Multiple sclerosis
Neuroblastoma Normal Obesity Osteoarthritis Osteosarcoma Ovarian
cancer Pancreatic cancer Prostate cancer Prostatic diseases, benign
Prostatitis Renal cell cancer Retinoblastoma Schizophrenia Skin
ulcer Smoker Teratocarcinoma Subcellular location Birbeck Granules
Cell surface digest Chromatin Fraction Crude Cell Membrane Cytosol
Golgi/Mitochondrial Membrane Membrane Glycoprotein Binding Fraction
Mitochondria Nucleus Peroxisomes Plasma Membrane Secreted Soluble
Fraction Supernatant Whole Cell
[0263] FIG. 3 shows the Protein Index for OGTA001 is very high in
colorectal cancer plasma membrane and very low in normal plasma
membrane. OGTA001 was not detected in any other diseases. This
indicates that OGTA001 is potentially a good marker for colorectal
cancer.
Example 2
Identification of Membrane Proteins Expressed in Colorectal Cancer
Blood and Tissue Samples Using Isotope Tagging For Absolute and
Relative Quantitation (iTRAQ)
[0264] Using the following Reference Protocol, membrane proteins
extracted from colorectal cancer tissue and normal adjacent
colorectal tissue samples were digested, labelled with Isotope
Tagging for Absolute & Relative Quantitation reagents (iTRAQ;
Applied Biosystems, Foster City, Calif., USA) and resulting
peptides sequenced by tandem mass spectrometry.
2.1 Materials and Methods
2.1.1--Plasma Membrane Fractionation
[0265] The cells recovered from a colorectal cancer or normal
adjacent tissue were lysed and submitted to centrifugation at
1000G. The supernatant was taken, and it was subsequently
centrifuged at 3000G. Once again, the supernatant was taken, and it
was then centrifuged at 100 000G.
[0266] The resulting pellet was recovered and put on 15-60% sucrose
gradient.
[0267] A Western blot was used to identify sub cellular markers,
and the Plasma Membrane fractions were pooled.
[0268] The pooled solution was then analysed directly by iTRAQ (see
section 2.1.2 below).
2.1.2--iTRAQ Methodology
[0269] Membrane protein pellets from colorectal cancer and normal
adjacent tissue were solubilised in sample buffer (2-4 .mu.g/.mu.l
in 0.5% SDS) by the addition of buffer and then heating to
95.degree. C. for 3 min.
[0270] To a volume of each protein solution equating to 50 .mu.g,
150 .mu.l of 0.5M triethylammonium bicarbonate (TEAB) solution was
added. To each sample, 3 .mu.l of 50 mM
tris-(2-carboxyethyl)phosphine was added and the mixture was
incubated at 60.degree. C. for 1 hour. 1 .mu.l of cysteine blocking
reagent, 200 mM methyl methanethiosulphonate (MMTS) in isopropanol,
was then added. After incubation at room temperature for 10
minutes, 15 .mu.l of 1 .mu.g/.mu.l trypsin was added to each sample
followed by incubation at 37.degree. C. overnight.
[0271] The digested samples were dried under a vacuum and
re-constituted with 30 .mu.l of 0.5M TEAB solution. 70 .mu.l
ethanol was added to each of the four iTRAQ reagents
(114/115/116/117) and one reagent added to each of the four samples
analysed (two colorectal cancer samples and two corresponding
normal adjacent tissue samples) and left at room temperature for 1
hour. The specific reagent added to each sample was recorded. The
four labeled samples were combined & vortexed.
[0272] The combined sample was reduced to dryness under a vacuum
and de-salted by loading onto a C 18 spin column, washing with
aqueous solvent and then eluting with 70% acetonitrile. The sample
fraction was again reduced to dryness and then re-dissolved in 40
.mu.l of solvent A (97.9 water, 2% acetonitrile, 0.1% formic acid)
prior to ion exchange fractionation.
2.1.3--Fractionation and Analysis of Labeled Peptides
[0273] The sample was fractionated by strong cation exchange
chromatography using an Agilent 1200 chromatograph (Agilent, Santa
Clara, Calif., USA). Samples were eluted off an Agilent Zorbax
Bio-SCXII column (3.5 .mu.m; 50.times.0.8 mm) using a 20 .mu.l/min
gradient of 0-100 mM sodium acetate over 20 minutes and then to 1M
over 10 minutes. 1 minute fractions were collected over the 30
minute run.
[0274] Each fraction was analysed by liquid chromatography/mass
spectrometry using an Agilent 1200 chromatograph fitted with a
Zorbax 300SB-C18 (150 mm.times.75 .mu.m) and an Agilent 6510
quadrupole--time-of-flight instrument (Agilent, Santa Clara,
Calif., USA). Peptides were eluted with a 300n1/min gradient
increasing from 15% to 45% acetonitrile in 60 minutes. Data was
acquired in auto MS/MS mode such that up to 3 precursor ions above
the intensity threshold were selected and product ion spectra
accumulated to facilitate the sequencing of the labeled peptides.
Raw was processed to create peak lists using Spectrum Mill software
(Agilent, Santa Clara, Calif., USA).
2.1.4--Amino Acid Sequence Analysis of Labeled Peptides
[0275] For partial amino acid sequencing and identification of
OGTA001, uninterpreted tandem mass spectra of tryptic peptides were
searched using the SEQUEST search program (Eng et al., 1994, J. Am.
Soc. Mass Spectrom. 5:976-989). Criteria for database
identification included: the cleavage specificity of trypsin; the
detection of a suite of a, b and y ions in peptides returned from
the database, and a mass increment for all cysteine residues to
account for modification with methyl methanethiosulphonate and the
addition of iTRAQ labels to free amines (N-terminus & lysine).
The data was searched through IPI Human v3.23
(www.ebi.ac.uk/IPI/IPIhuman.html).
2.1.5--Discrimination of Colorectal Cancer Associated Proteins
[0276] The process described in Example 1 section 1.1.6 was
employed to discriminate the colorectal cancer associated proteins
in the experimental samples.
2.2 Results
[0277] These experiments identified OGTA001, as further described
herein. The full-length OGTA001 was detected in the plasma membrane
of colorectal cancer samples. The iTRAQ analysis showed that levels
of OGTA001 in the colorectal cancer samples were higher than in the
matched normal adjacent tissue samples.
[0278] FIG. 2 shows the Protein Index for OGTA001. See Example 1
section 1.2 for a description of the Protein Index for OGTA001.
Example 3
Multiplex Assay to Detect Soluble OGTA001 in Patient Serum Using
Luminex Technology
[0279] Using the following Reference Protocol, multiplex assays
using the Luminex technology were performed using antibodies to
soluble OGTA001.
3.1 Materials and Methods
[0280] Antibodies to OGTA001 (as defined by SEQ ID No: 1) were
developed at Biosite. Each primary antibody to soluble OGTA001 (as
defined by SEQ ID No: 1) was conjugated to a unique Luminex
magnetic microsphere (Mug beads, Luminex Corporation, Austin,
Tex.). Mag bead cocktail (50 ul) was added to a 96 black well round
bottom Costar plate (Corning Incorporated, Corning N.Y.). Using a
96 well magnetic ring stand, the Mag beads were pulled down for 1
minute and washed with wash/assay buffer (PBS with 1% BSA and 0.02%
Tween 20). 50 ul of sample or standard was added along with an
additional 50 ul of wash/assay buffer and allowed to incubate on a
shaker for 1 hour at room temperature. Plate was placed on magnetic
ring stand and allowed to sit for 1 minute. Mag beads were then
washed again. Biotin labeled antibody was then added at 50 ul per
well with an additional 50 ul of wash/assay buffer and allowed to
incubate on a shaker for 1 hour at room temperature. The plate
again was placed on a magnetic stand and the Mag beads were washed.
Streptavidin-RPE (Prozyme, San Leandro, Calif., Phycolin,
Code#PJ31S) was diluted to 1 ug/ml in wash/assay buffer and 50 ul
was added to each well along with an additional 50 ul of wash/assay
buffer and allowed to incubate on a shaker for 1 hour at room
temperature. Final wash was performed and the beads were
re-suspended with 100 ul of wash/assay buffer and each well was
then read in a Luminex 200 reader using Xponent software 3.0. All
reagent dilutions were made in wash/assay buffer. Biotin-antibody
varied for each assay to optimal concentration. Initial Mag bead
amounts added were approximately 50,000 for each assay. Magnetic
beads were allowed 1 minute pull down time prior to each wash. Each
wash step was 3 times washed with 100 ul of wash/assay buffer.
Assay standard curves were made in a normal donor patient serum
pool. Luminex reader and Mag beads were used and prepared according
to manufacturer guidelines. Standard curves were calculated using a
5 parameter log-logistic fit and each sample concentration was
determined from this curve fit.
[0281] Final Box and ROC results were analyzed using Analyse-it
General +Clinical Laboratory 1.73 (Analyse-it Software Ltd., Leeds
England).
3.2 Results
[0282] Experiments using 61 normal samples and 65 colorectal cancer
resulted in further evidence that soluble OGTA001 can be detected
in colorectal cancer patient serum samples and also that the
concentration of soluble OGTA001 is higher in colorectal cancer
patient serum than in normal serum samples. These results
demonstrate that soluble OGTA001 may be used as a serum diagnostic
for colorectal cancer.
[0283] FIG. 3 shows ROC curve data for soluble OGTA001 in
colorectal cancer patient serum samples. The ROC curves plot
sensitivity (true positives) against 1-specificity (false
positives). An area under the ROC curve of greater than 0.5
indicates good discrimination between disease and normal. This is
the case in the data shown in FIG. 4, which, along with the low p
values, indicate that the concentration of soluble OGTA001 is
significantly higher in colorectal cancer patient serum samples
than in normal serum samples.
[0284] The following examples and the antibodies prepared and used
therein are based in part, upon disclosure found in co-pending
parent International Application No. PCT/US2010/031739, having an
international filing date of Apr. 20, 2010, and the disclosure of
this application is accordingly incorporated herein by reference in
its entirety.
Example 4
Construction of a Phage-Display Library
[0285] A recombinant protein composed of the extracellular domain
of the CDH17 (SEQ ID NO:1) was eurkaryotically synthesized by
standard recombinant methods and used as antigen for
immunization.
[0286] Immunization and mRNA Isolation
[0287] A phage display library for identification of the
CDH17-binding molecules was constructed as follows. A/J mice
(Jackson Laboratories, Bar Harbor, Me.) were immunized
intraperitoneally with the recombinant CDH17 antigen (the
extracellular domain), using 100 .mu.g protein in Freund's complete
adjuvant, on day 0, and with 100 .mu.g antigen on day 28. Test
bleeds of mice were obtained through puncture of the retro-orbital
sinus. If, by testing the titers, they were deemed high by ELISA
using the biotinylated CDH17 antigen immobilized via neutravidin
(Reacti-Bind.TM.) NeutrAvidin.TM.-Coated Polystyrene Plates,
Pierce, Rockford, Ill.), the mice were boosted with 100 .mu.g of
protein on day 70, 71 and 72, with subsequent sacrifice and
splenectomy on day 77. If titers of antibody were not deemed
satisfactory, mice were boosted with 100 .mu.g antigen on day 56
and a test bleed taken on day 63. If satisfactory titers were
obtained, the animals were boosted with 100 .mu.g of antigen on day
98, 99, and 100 and the spleens harvested on day 105.
[0288] The spleens were harvested in a laminar flow hood and
transferred to a petri dish, trimming off and discarding fat and
connective tissue. The spleens were macerated quickly with the
plunger from a sterile 5 cc syringe in the presence of 1.0 ml of
solution D (25.0 g guanidine thiocyanate (Boehringer Mannheim,
Indianapolis, Ind.), 29.3 ml sterile water, 1.76 ml 0.75 M sodium
citrate pH 7.0, 2.64 ml 10% sarkosyl (Fisher Scientific,
Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol (Fisher Scientific,
Pittsburgh, Pa.). This spleen suspension was pulled through an 18
gauge needle until all cells were lysed and the viscous solution
was transferred to a microcentrifuge tube. The petri dish was
washed with 100 .mu.l of solution D to recover any remaining
spleen. This suspension was then pulled through a 22 gauge needle
an additional 5-10 times.
[0289] The sample was divided evenly between two microcentrifuge
tubes and the following added, in order, with mixing by inversion
after each addition: 50 .mu.l 2 M sodium acetate pH 4.0, 0.5 ml
water-saturated phenol (Fisher Scientific, Pittsburgh, Pa.), 100
.mu.l chloroform/isoamyl alcohol 49:1 (Fisher Scientific,
Pittsburgh, Pa.). The solution was vortexed for 10 sec and
incubated on ice for 15 min. Following centrifugation at 14 krpm
for 20 min at 2-8.degree. C., the aqueous phase was transferred to
a fresh tube. An equal volume of water saturated
phenol:chloroform:isoamyl alcohol (50:49:1) was added, and the tube
vortexed for ten seconds. After 15 min incubation on ice, the
sample was centrifuged for 20 min at 2-8.degree. C., and the
aqueous phase transferred to a fresh tube and precipitated with an
equal volume of isopropanol at -20.degree. C. for a minimum of 30
min. Following centrifugation at 14 krpm for 20 min at 4.degree.
C., the supernatant was aspirated away, the tubes briefly spun and
all traces of liquid removed from the RNA pellet.
[0290] The RNA pellets were each dissolved in 300 .mu.l of solution
D, combined, and precipitated with an equal volume of isopropanol
at -20.degree. C. for a minimum of 30 min. The sample was
centrifuged 14 krpm for 20 min at 4.degree. C., the supernatant
aspirated as before, and the sample rinsed with 100 .mu.l of
ice-cold 70% ethanol. The sample was again centrifuged 14 krpm for
20 min at 4.degree. C., the 70% ethanol solution aspirated, and the
RNA pellet dried in vacuo. The pellet was resuspended in 100 .mu.l
of sterile diethyl pyrocarbonate-treated water. The concentration
was determined by A260 using an absorbance of 1.0 for a
concentration of 40 .mu.g/ml. The RNAs were stored at -80.degree.
C.
[0291] Preparation of Complementary DNA (cDNA)
[0292] The total RNA purified from mouse spleens as described above
was used directly as template for cDNA preparation. RNA (50 .mu.g)
was diluted to 100 .mu.L with sterile water, and 10 .mu.L of 130
ng/.mu.L oligo dT12 (synthesized on Applied Biosystems Model 392
DNA synthesizer) was added. The sample was heated for 10 min at
70.degree. C., then cooled on ice. Forty .mu.L 5* first strand
buffer was added (Gibco/BRL, Gaithersburg, Md.), along with 20
.mu.L 0.1 M dithiothreitol (Gibco/BRL, Gaithersburg, Md.), 10 .mu.L
20 mM deoxynucleoside triphosphates (dNTP's, Boehringer Mannheim,
Indianapolis, Ind.), and 10 .mu.L water on ice. The sample was then
incubated at 37.degree. C. for 2 min. Ten .mu.L reverse
transcriptase (Superscript.TM.) II, Gibco/BRL, Gaithersburg, Md.)
was added and incubation was continued at 37.degree. C. for 1 hr.
The cDNA products were used directly for polymerase chain reaction
(PCR).
[0293] Amplification of Antibody Genes by PCR
[0294] To amplify substantially all of the H and L chain genes
using PCR, primers were chosen that corresponded to substantially
all published sequences. Because the nucleotide sequences of the
amino termini of H and L contain considerable diversity, 33
oligonucleotides were synthesized to serve as 5' primers for the H
chains, and 29 oligonucleotides were synthesized to serve as 5'
primers for the kappa L chains as described in U.S. Pat. No.
6,555,310. The constant region nucleotide sequences for each chain
required only one 3' primer for the H chains and one 3' primer for
the kappa L chains.
[0295] A 50 .mu.L reaction was performed for each primer pair with
50 .mu.mol of 5' primer, 50 .mu.mol of 3' primer, 0.25 .mu.L Taq
DNA Polymerase (5 units/4, Boehringer Mannheim, Indianapolis,
Ind.), 3 .mu.L cDNA (prepared as described), 5 .mu.L 2 mM dNTP's, 5
.mu.L 10*Taq DNA polymerase buffer with MgC12 (Boehringer Mannheim,
Indianapolis, Ind.), and H.sub.2O to 50 .mu.L. Amplification was
done using a GeneAmp(R) 9600 thermal cycler (Perkin Elmer, Foster
City, Calif.) with the following thermocycle program: 94.degree. C.
for 1 min; 30 cycles of 94.degree. C. for 20 sec, 55.degree. C. for
30 sec, and 72.degree. C. for 30 sec, 72.degree. C. for 6 min;
4.degree. C.
[0296] The dsDNA products of the PCR process were then subjected to
asymmetric PCR using only a 3' primer to generate substantially
only the anti-sense strand of the target genes. A 100 .mu.L
reaction was done for each dsDNA product with 200 .mu.mol of 3'
primer, 2 .mu.L of ds-DNA product, 0.5 .mu.L Taq DNA Polymerase, 10
.mu.L 2 mM dNTP's, 10 .mu.L 10*Taq DNA polymerase buffer with
MgCl.sub.2 (Boehringer Mannheim, Indianapolis, Ind.), and H.sub.2O
to 100 .mu.L. The same PCR program as that described above was used
to amplify the single-stranded (ss)-DNA.
[0297] 1. Purification of Single-Stranded DNA by High Performance
Liquid Chromatography and Kinasing Single-Stranded DNA
[0298] The H chain ss-PCR products and the L chain single-stranded
PCR products were ethanol precipitated by adding 2.5 volumes
ethanol and 0.2 volumes 7.5 M ammonium acetate and incubating at
-20.degree. C. for at least 30 min. The DNA was pelleted by
centrifuging in an Eppendorf centrifuge at 14 krpm for 10 min at
2-8.degree. C. The supernatant was carefully aspirated, and the
tubes were briefly spun a 2nd time. The last drop of supernatant
was removed with a pipette. The DNA was dried in vacuo for 10 min
on medium heat. The H chain products were pooled in 210 .mu.L water
and the L chain products were pooled separately in 210 .mu.L water.
The single-stranded DNA was purified by high performance liquid
chromatography (HPLC) using a Hewlett Packard 1090 HPLC and a
Gen-Pak.TM.) FAX anion exchange column (Millipore Corp., Milford,
Mass.). The gradient used to purify the single-stranded DNA is
shown in Table 3, and the oven temperature was 60.degree. C.
Absorbance was monitored at 260 nm. The single-stranded DNA eluted
from the HPLC was collected in 0.5 min fractions. Fractions
containing single-stranded DNA were ethanol precipitated, pelleted
and dried as described above. The dried DNA pellets were pooled in
200 .mu.L sterile water.
TABLE-US-00005 TABLE 3 HPLC gradient for purification of ss-DNA
Time (min) % A % B % C Flow (ml/min) 0 70 30 0 0.75 2 40 60 0 0.75
17 15 85 0 0.75 18 0 100 0 0.75 23 0 100 0 0.75 24 0 0 100 0.75 28
0 0 100 0.75 29 0 100 0 0.75 34 0 100 0 0.75 35 70 30 0 0.75 Buffer
A is 25 mM Tris, 1 mM EDTA, pH 8.0 Buffer B is 25 mM Tris, 1 mM
EDTA, 1M NaCl, pH 8.0 Buffer C is 40 mm phosphoric acid
[0299] The single-stranded DNA was 5'-phosphorylated in preparation
for mutagenesis. Twenty-four .mu.L 10* kinase buffer (United States
Biochemical, Cleveland, Ohio), 10.4 .mu.L 10 mM
adenosine-5'-triphosphate (Boehringer Mannheim, Indianapolis,
Ind.), and 2 .mu.L polynucleotide kinase (30 units/4, United States
Biochemical, Cleveland, Ohio) was added to each sample, and the
tubes were incubated at 37.degree. C. for 1 hr. The reactions were
stopped by incubating the tubes at 70.degree. C. for 10 min. The
DNA was purified with one extraction of Tris equilibrated phenol
(pH>8.0, United States Biochemical, Cleveland,
Ohio):chloroform:isoamyl alcohol (50:49:1) and one extraction with
chloroform:isoamyl alcohol (49:1). After the extractions, the DNA
was ethanol precipitated and pelleted as described above. The DNA
pellets were dried, then dissolved in 50 .mu.L sterile water. The
concentration was determined by measuring the absorbance of an
aliquot of the DNA at 260 nm using 33 .mu.g/ml for an absorbance of
1.0. Samples were stored at -20.degree. C.
[0300] 2. Preparation of Uracil Templates Used in Generation of
Spleen Antibody Phage Libraries
[0301] One ml of E. coli CJ236 (BioRAD, Hercules, Calif.) overnight
culture was added to 50 ml 2*YT in a 250 ml baffled shake flask.
The culture was grown at 37.degree. C. to OD600=0.6, inoculated
with 10 .mu.l of a 1/100 dilution of BS45 vector phage stock
(described in U.S. Pat. No. 6,555,310) and growth continued for 6
hr. Approximately 40 ml of the culture was centrifuged at 12 krpm
for 15 min at 4.degree. C. The supernatant (30 ml) was transferred
to a fresh centrifuge tube and incubated at room temperature for 15
min after the addition of 15 .mu.l of 10 mg/ml RNaseA (Boehringer
Mannheim, Indianapolis, Ind.). The phages were precipitated by the
addition of 7.5 ml of 20% polyethylene glycol 8000 (Fisher
Scientific, Pittsburgh, Pa.)/3.5M ammonium acetate (Sigma Chemical
Co., St. Louis, Mo.) and incubation on ice for 30 min. The sample
was centrifuged at 12 krpm for 15 min at 2-8.degree. C. The
supernatant was carefully discarded, and the tube briefly spun to
remove all traces of supernatant. The pellet was resuspended in 400
.mu.l of high salt buffer (300 mM NaCl, 100 mM Tris pH 8.0, 1 mM
EDTA), and transferred to a 1.5 ml tube.
[0302] The phage stock was extracted repeatedly with an equal
volume of equilibrated phenol:chloroform:isoamyl alcohol (50:49:1)
until no trace of a white interface was visible, and then extracted
with an equal volume of chloroform:isoamyl alcohol (49:1). The DNA
was precipitated with 2.5 volumes of ethanol and 1/5 volume 7.5 M
ammonium acetate and incubated 30 min at -20.degree. C. The DNA was
centrifuged at 14 krpm for 10 min at 4.degree. C., the pellet
washed once with cold 70% ethanol, and dried in vacuo. The uracil
template DNA was dissolved in 30 .mu.l sterile water and the
concentration determined by A260 using an absorbance of 1.0 for a
concentration of 40 .mu.g/ml. The template was diluted to 250
ng/.mu.L with sterile water, aliquoted and stored at -20.degree.
C.
[0303] Mutagenesis of Uracil Template with Ss-DNA and
Electroporation into E. Coli to Generate Antibody Phage
Libraries
[0304] Antibody phage display libraries were generated by
simultaneously introducing single-stranded heavy and light chain
genes onto a phage display vector uracil template. A typical
mutagenesis was performed on a 2 .mu.g scale by mixing the
following in a 0.2 ml PCR reaction tube: 8 .mu.l of (250 ng/.mu.L)
uracil template, 8 .mu.L of 10* annealing buffer (200 mM Tris pH
7.0, 20 mM MgCl.sub.2, 500 mM NaCl), 3.33 .mu.l of kinased
single-stranded heavy chain insert (100 ng/4), 3.1 .mu.l of kinased
single-stranded light chain insert (100 ng/4), and sterile water to
80 .mu.l. DNA was annealed in a GeneAmp(R) 9600 thermal cycler
using the following thermal profile: 20 sec at 94.degree. C.,
85.degree. C. for 60 sec, 85.degree. C. to 55.degree. C. ramp over
30 min, hold at 55.degree. C. for 15 min. The DNA was transferred
to ice after the program finished. The extension/ligation was
carried out by adding 8 .mu.l of 10* synthesis buffer (5 mM each
dNTP, 10 mM ATP, 100 mM Tris pH 7.4, 50 mM MgCl2, 20 mM DTT), 8
.mu.L T4 DNA ligase (1 U/.mu.L, Boehringer Mannheim, Indianapolis,
Ind.), 8 .mu.L diluted T7 DNA polymerase (1 U/.mu.L, New England
BioLabs, Beverly, Mass.) and incubating at 37.degree. C. for 30
min. The reaction was stopped with 300 .mu.L of mutagenesis stop
buffer (10 mM Tris pH 8.0, 10 mM EDTA). The mutagenesis DNA was
extracted once with equilibrated phenol
(pH>8):chloroform:isoamyl alcohol (50:49:1), once with
chloroform:isoamyl alcohol (49:1), and the DNA was ethanol
precipitated at -20.degree. C. for at least 30 min. The DNA was
pelleted and the supernatant carefully removed as described above.
The sample was briefly spun again and all traces of ethanol removed
with a pipetman. The pellet was dried in vacuo. The DNA was
resuspended in 4 .mu.L of sterile water.
[0305] One .mu.L of mutagenesis DNA (500 ng) was transferred into
40 .mu.l electrocompetent E. coli DH12S (Gibco/BRL, Gaithersburg,
Md.) using electroporation. The transformed cells were mixed with
approximately 1.0 ml of overnight XL-1 cells which were diluted
with 2*YT broth to 60% the original volume. This mixture was then
transferred to a 15-ml sterile culture tube and 9 ml of top agar
added for plating on a 150-mm LB agar plate. Plates were incubated
for 4 hr at 37.degree. C. and then transferred to 20.degree. C.
overnight. First round antibody phage were made by eluting phage
off these plates in 10 ml of 2*YT, spinning out debris, and taking
the supernatant. These samples are the antibody phage display
libraries used for selecting antibodies against the CDH17.
Efficiency of the electroporations was measured by plating 10 .mu.l
of a 10.sup.-4 dilution of suspended cells on LB agar plates,
follow by overnight incubation of plates at 37.degree. C. The
efficiency was calculated by multiplying the number of plaques on
the 10.sup.-4 dilution plate by 106. Library electroporation
efficiencies are typically greater than 1*10.sup.7 phages under
these conditions.
[0306] Transformation of E. Coli by Electroporation
[0307] Electrocompetent E. coli cells were thawed on ice. DNA was
mixed with 40 L of these cells by gently pipetting the cells up and
down 2-3 times, being careful not to introduce an air bubble. The
cells were transferred to a Gene Pulser cuvette (0.2 cm gap,
BioRAD, Hercules, Calif.) that had been cooled on ice, again being
careful not to introduce an air bubble in the transfer. The cuvette
was placed in the E. coli Pulser (BioRAD, Hercules, Calif.) and
electroporated with the voltage set at 1.88 kV according to the
manufacturer's recommendation. The transformed sample was
immediately resuspended in 1 ml of 2*YT broth or 1 ml of a mixture
of 400 .mu.l 2*YT/600 .mu.l overnight XL-1 cells and processed as
procedures dictated.
[0308] Plating M13 Phage or Cells Transformed with Antibody
Phage-Display Vector Mutagenesis Reaction
[0309] Phage samples were added to 200 .mu.L of an overnight
culture of E. coli XL1-Blue when plating on 100 mm LB agar plates
or to 600 .mu.L of overnight cells when plating on 150 mm plates in
sterile 15 ml culture tubes. After adding LB top agar (3 ml for 100
mm plates or 9 ml for 150 mm plates, top agar stored at 55.degree.
C. (see, Appendix A1, Sambrook et al., supra.), the mixture was
evenly distributed on an LB agar plate that had been pre-warmed
(37.degree. C.-55.degree. C.) to remove any excess moisture on the
agar surface. The plates were cooled at room temperature until the
top agar solidified. The plates were inverted and incubated at
37.degree. C. as indicated.
[0310] 3. Preparation of Biotinylated Cadherin-17 and Biotinylated
Antibodies
[0311] The concentrated recombinant CDH17 antigen (full length
extracellular domain SEQ ID No: 67) (Cadherin domains 1-2 SEQ ID
No: 68) was extensively dialyzed into BBS (20 mM borate, 150 mM
NaCl, 0.1% NaN.sub.3, pH 8.0). After dialysis, 1 mg of the CDH17 (1
mg/ml in BBS) was reacted with a 15 fold molar excess of
biotin-XX-NHS ester (Molecular Probes, Eugene, Oreg., stock
solution at 40 mM in DMSO). The reaction was incubated at room
temperature for 90 min and then quenched with taurine (Sigma
Chemical Co., St. Louis, Mo.) at a final concentration of 20 mM.
The biotinylation reaction mixture was then dialyzed against BBS at
2-8.degree. C. After dialysis, the biotinylated CDH17 was diluted
in panning buffer (40 mM Tris, 150 mM NaCl, 20 mg/ml BSA, 0.1%
Tween 20, pH 7.5), aliquoted, and stored at -80.degree. C. until
needed.
[0312] Antibodies were reacted with
3-(N-maleimidylpropionyl)biocytin (Molecular Probes, Eugene, Oreg.)
using a free cysteine located at the carboxy terminus of the heavy
chain. Antibodies were reduced by adding DTT to a final
concentration of 1 mM for 30 min at room temperature. Reduced
antibody was passed through a Sephadex G50 desalting column
equilibrated in 50 mM potassium phosphate, 10 mM boric acid, 150 mM
NaCl, pH 7.0. 3-(N-maleimidylpropionyl)-biocytin was added to a
final concentration of 1 mM and the reaction allowed to proceed at
room temperature for 60 min. Samples were then dialyzed extensively
against BBS and stored at 2-8.degree. C.
[0313] Preparation of Avidin Magnetic Latex
[0314] The magnetic latex (Estapor, 10% solids, Bangs Laboratories,
Fishers, Ind.) was thoroughly resuspended and 2 ml aliquoted into a
15 ml conical tube. The magnetic latex was suspended in 12 ml
distilled water and separated from the solution for 10 min using a
magnet (PerSeptive Biosystems, Framingham, Mass.). While
maintaining the separation of the magnetic latex with the magnet,
the liquid was carefully removed using a 10 ml sterile pipette.
This washing process was repeated an additional three times. After
the final wash, the latex was resuspended in 2 ml of distilled
water. In a separate 50 ml conical tube, 10 mg of avidin-HS
(NeutrAvidin, Pierce, Rockford, Ill.) was dissolved in 18 ml of 40
mM Tris, 0.15 M sodium chloride, pH 7.5 (TBS). While vortexing, the
2 ml of washed magnetic latex was added to the diluted avidin-HS
and the mixture mixed an additional 30 sec. This mixture was
incubated at 45.degree. C. for 2 hr, shaking every 30 min. The
avidin magnetic latex was separated from the solution using a
magnet and washed three times with 20 ml BBS as described above.
After the final wash, the latex was resuspended in 10 ml BBS and
stored at 4.degree. C.
[0315] Immediately prior to use, the avidin magnetic latex was
equilibrated in panning buffer (40 mM Tris, 150 mM NaCl, 20 mg/ml
BSA, 0.1% Tween 20, pH 7.5). The avidin magnetic latex needed for a
panning experiment (200 .mu.l/sample) was added to a sterile 15 ml
centrifuge tube and brought to 10 ml with panning buffer. The tube
was placed on the magnet for 10 min to separate the latex. The
solution was carefully removed with a 10 ml sterile pipette as
described above. The magnetic latex was resuspended in 10 ml of
panning buffer to begin the second wash. The magnetic latex was
washed a total of 3 times with panning buffer. After the final
wash, the latex was resuspended in panning buffer to the starting
volume.
Example 5
Selection of Recombinant Polyclonal Antibodies to CDH17 Antigen
[0316] Binding reagents that specifically bind to the CDH17 were
selected from the phage display libraries created from
hyperimmunized mice as described in Example 4.
Panning
[0317] First round antibody phage were prepared as described in
Example 4 using BS45 uracil template. Electroporations of
mutagenesis DNA were performed yielding phage samples derived from
different immunized mice. To create more diversity in the
recombinant polyclonal library, each phage sample was panned
separately.
[0318] Before the first round of functional panning with the
biotinylated CDH17 antigen, antibody phage libraries were selected
for phage displaying both heavy and light chains on their surface
by panning with 7F11-magnetic latex (as described in Examples 21
and 22 of U.S. Pat. No. 6,555,310). Functional panning of these
enriched libraries was performed in principle as described in
Example 16 of U.S. Pat. No. 6,555,310. Specifically, 10 .mu.L of
1*10.sup.-6 M biotinylated CDH17 antigen was added to the phage
samples (approximately 1*10.sup.-8 M final concentration of the
CDH17), and the mixture allowed to come to equilibrium overnight at
2-8.degree. C.
[0319] After reaching equilibrium, samples were panned with avidin
magnetic latex to capture antibody phage bound to the CDH17.
Equilibrated avidin magnetic latex (Example 4), 200 .mu.L latex per
sample, was incubated with the phage for 10 min at room
temperature. After 10 min, approximately 9 ml of panning buffer was
added to each phage sample, and the magnetic latex separated from
the solution using a magnet. After a ten minute separation, unbound
phage was carefully removed using a 10 ml sterile pipette. The
magnetic latex was then resuspended in 10 ml of panning buffer to
begin the second wash. The latex was washed a total of three times
as described above. For each wash, the tubes were in contact with
the magnet for 10 min to separate unbound phage from the magnetic
latex. After the third wash, the magnetic latex was resuspended in
1 ml of panning buffer and transferred to a 1.5 mL tube. The entire
volume of magnetic latex for each sample was then collected and
resuspended in 200 .mu.l 2*YT and plated on 150 mm LB plates as
described in Example 1 to amplify bound phage. Plates were
incubated at 37.degree. C. for 4 hr, then overnight at 20.degree.
C.
[0320] The 150 mm plates used to amplify bound phage were used to
generate the next round of antibody phage. After the overnight
incubation, second round antibody phage were eluted from the 150 mm
plates by pipetting 10 mL of 2*YT media onto the lawn and gently
shaking the plate at room temperature for 20 min. The phage samples
were then transferred to 15 ml disposable sterile centrifuge tubes
with a plug seal cap, and the debris from the LB plate pelleted by
centrifuging the tubes for 15 min at 3500 rpm. The supernatant
containing the second round antibody phage was then transferred to
a new tube.
[0321] A second round of functional panning was set up by diluting
100 .mu.L of each phage stock into 900 .mu.L of panning buffer in
15 ml disposable sterile centrifuge tubes. The biotinylated CDH17
antigen was then added to each sample as described for the first
round of panning, and the phage samples incubated for 1 hr at room
temperature. The phage samples were then panned with avidin
magnetic latex as described above. The progress of panning was
monitored at this point by plating aliquots of each latex sample on
100 mm LB agar plates to determine the percentage of kappa
positives. The majority of latex from each panning (99%) was plated
on 150 mm LB agar plates to amplify the phage bound to the latex.
The 100 mm LB agar plates were incubated at 37.degree. C. for 6-7
hr, after which the plates were transferred to room temperature and
nitrocellulose filters (pore size 0.45 mm, BA85 Protran, Schleicher
and Schuell, Keene, N.H.) were overlaid onto the plaques.
[0322] Plates with nitrocellulose filters were incubated overnight
at room temperature and then developed with a goat anti-mouse kappa
alkaline phosphatase conjugate to determine the percentage of kappa
positives as described below. Phage samples with lower percentages
(<70%) of kappa positives in the population were subjected to a
round of panning with 7F11-magnetic latex before performing a third
functional round of panning overnight at 2-8.degree. C. using the
biotinylated CDH17 antigen at approximately 2*10.sup.-9 M. This
round of panning was also monitored for kappa positives. Individual
phage samples that had kappa positive percentages greater than 80%
were pooled and subjected to a final round of panning overnight at
2-8.degree. C. at 5*10.sup.-9 M. The CDH17 antibody genes contained
within the eluted phage from this fourth round of functional
panning were subcloned into the expression vector, pBRncoH3.
[0323] The subcloning process was done generally as described in
Example 18 of U.S. Pat. No. 6,555,310. After subcloning, the
expression vector was electroporated into DH10B cells and the
mixture grown overnight in 2*YT containing 1% glycerol and 10
.mu.g/ml tetracycline. After a second round of growth and selection
in tetracycline, aliquots of cells were frozen at -80.degree. C. as
the source for the CDH17 polyclonal antibody production. Monoclonal
antibodies were selected from these polyclonal mixtures by plating
a sample of the mixture on LB agar plates containing 10 .mu.g/ml
tetracycline and screening for antibodies that recognized the
CDH17.
[0324] Expression and Purification of Recombinant Antibodies
Against Cadherin-17
[0325] A shake flask inoculum was generated overnight from a
-70.degree. C. cell bank in an Innova 4330 incubator shaker (New
Brunswick Scientific, Edison, N.J.) set at 37.degree. C., 300 rpm.
The inoculum was used to seed a 20 L fermentor (Applikon, Foster
City, Calif.) containing defined culture medium [Pack et al. (1993)
BioTechnology 11: 1271-1277] supplemented with 3 g/L L-leucine, 3
g/L L-isoleucine, 12 g/L casein digest (Difco, Detroit, Mich.),
12.5 g/L glycerol and 10 .mu.g/ml tetracycline. The temperature, pH
and dissolved oxygen in the fermentor were controlled at 26.degree.
C., 6.0-6.8 and 25% saturation, respectively. Foam was controlled
by addition of polypropylene glycol (Dow, Midland, Mich.). Glycerol
was added to the fermentor in a fed-batch mode. Fab expression was
induced by addition of L(+)-arabinose (Sigma, St. Louis, Mo.) to 2
g/L during the late logarithmic growth phase. Cell density was
measured by optical density at 600 nm in an UV-1201
spectrophotometer (Shimadzu, Columbia, Md.). Following run
termination and adjustment of pH to 6.0, the culture was passed
twice through an M-210B-EH Microfluidizer (Microfluidics, Newton,
Mass.) at 17,000 psi. The high pressure homogenization of the cells
released the Fab into the culture supernatant.
[0326] The first step in purification was expanded bed immobilized
metal affinity chromatography (EB-IMAC). Streamline.TM. chelating
resin (Pharmacia, Piscataway, N.J.) was charged with 0.1 M
NiCl.sub.2 and was then expanded and equilibrated in 50 mM acetate,
200 mM NaCl, 10 mM imidazole, 0.01% NaN.sub.3, pH 6.0 buffer
flowing in the upward direction. A stock solution was used to bring
the culture homogenate to 10 mM imidazole, following which it was
diluted two-fold or higher in equilibration buffer to reduce the
wet solids content to less than 5% by weight. It was then loaded
onto the Streamline column flowing in the upward direction at a
superficial velocity of 300 cm/hr. The cell debris passed through
unhindered, but the Fab was captured by means of the high affinity
interaction between nickel and the hexahistidine tag on the Fab
heavy chain. After washing, the expanded bed was converted to a
packed bed and the Fab was eluted with 20 mM borate, 150 mM NaCl,
200 mM imidazole, 0.01% NaN.sub.3, pH 8.0 buffer flowing in the
downward direction.
[0327] The second step in the purification used ion-exchange
chromatography (IEC). Q Sepharose FastFlow resin (Pharmacia,
Piscataway, N.J.) was equilibrated in 20 mM borate, 37.5 mM NaCl,
0.01% NaN.sub.3, pH 8.0. The Fab elution pool from the EB-IMAC step
was diluted four-fold in 20 mM borate, 0.01% NaN.sub.3, pH 8.0 and
loaded onto the IEC column. After washing, the Fab was eluted with
a 37.5-200 mM NaCl salt gradient. The elution fractions were
evaluated for purity using an Xcell II.TM. SDS-PAGE system (Novex,
San Diego, Calif.) prior to pooling. Finally, the Fab pool was
concentrated and diafiltered into 20 mM borate, 150 mM NaCl, 0.01%
NaN.sub.3, pH 8.0 buffer for storage. This was achieved in a
Sartocon Slice.TM. system fitted with a 10,000 MWCO cassette
(Sartorius, Bohemia, N.Y.). The final purification yields were
typically 50%. The concentration of the purified Fab was measured
by UV absorbance at 280 nm, assuming an absorbance of 1.6 for a 1
mg/ml solution.
Example 6
Specificity of Monoclonal Antibodies to CDH17 Determined By Flow
Cytometry Analysis
[0328] The specificity of antibodies against Cadherin-17 selected
in Example 5 was tested by flow cytometry. To test the ability of
the antibodies to bind to cell surface Cadherin-17 protein, the
antibodies were incubated with Cadherin-17-expressing cells: LoVo
and LS174T, human colorectal cancer lines. Cells were washed and
resuspended in PBS. Four microliters of the suspensions were
applied to wells of an eight well microscope slide and allowed to
air dry. The slides were lightly heated to fix the smears to the
slide and covered with 0.1 mg/ml of antibody diluted in PBS
containing 1% BSA. The smears were incubated with antibody for 1 h
at 37.degree. C. in a moist chamber. After washing the slides three
times by soaking in PBS for 5 min each, the smears were covered
with fluorescein isothiocyanate conjugated rabbit anti-mouse IgG
(H&L) F(ab')2 (Zymed Laboratories, Inc., South San Francisco,
Calif.) diluted 1:80 in PBS, 1% BSA, 0.05% Evans Blue (Sigma). The
slides were incubated for 1 h at 37.degree. C. in a moist chamber
then washed as described above. After a final wash in deionized
water, the slides were allowed to air dry in the dark. Coverslips
were mounted using a 90% glycerol mounting medium containing 10
mg/ml p-phenylenediamine, pH 8.0.
[0329] 4. The results of the flow cytometry analysis demonstrated
that 14 monoclonal antibodies designated CDH17_A1, CDH17_A2,
CDH17_A3, CDH17_A4, CDH17_A5, CDH17_A6, CDH17_A7, CDH17_A8,
CDH17_A9, CDH17_A10, CDH17_A11, CDH17_A12, CDH17_A13 and CDH17_A14
bind effectively to cell-surface human Cadherin-17.
Example 7
Structural Characterization of Monoclonal Antibodies To
Cadherin-17
[0330] The cDNA sequences encoding the heavy and light chain
variable regions of the CDH17_A1, CDH17_A2, CDH17_A3, CDH17_A4,
CDH17_A5, CDH17_A6, CDH17_A7, CDH17_A8, CDH17_A9, CDH17_A10,
CDH17_A11, CDH17_A12, CDH17_A13 and CDH17_A14 monoclonal antibodies
were obtained using standard PCR techniques and were sequenced
using standard DNA sequencing techniques.
[0331] The antibody sequences may be mutagenized to revert back to
germline residues at one or more residues.
[0332] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A1 are SEQ ID NO: 54 and 30,
respectively.
[0333] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A1 are SEQ ID NO: 42 and 18,
respectively.
[0334] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A2 are SEQ ID NO: 55 and 31,
respectively.
[0335] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A2 are SEQ ID NO: 43 and 19,
respectively.
[0336] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A3 are SEQ ID NO: 55 and 31,
respectively.
[0337] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A3 are SEQ ID NO: 44 and 20,
respectively.
[0338] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A4 are SEQ ID NO: 56 and 32,
respectively.
[0339] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A4 are SEQ ID NO: 45 and 21,
respectively.
[0340] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A5 are SEQ ID NO: 57 and 33,
respectively.
[0341] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A5 are SEQ ID NO: 46 and 22,
respectively.
The nucleotide and amino acid sequences of the light chain variable
region of CDH17_A6 are SEQ ID NO: 58 and 34, respectively.
[0342] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A6 are SEQ ID NO: 47 and 23,
respectively.
[0343] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A7 are SEQ ID NO: 59 and 35,
respectively.
[0344] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A7 are SEQ ID NO: 48 and 24,
respectively.
[0345] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A8 are SEQ ID NO: 60 and 36,
respectively.
[0346] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A8 are SEQ ID NO: 49 and 25,
respectively.
[0347] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A9 are SEQ ID NO: 61 and 37,
respectively.
[0348] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A9 are SEQ ID NO: 47 and 23,
respectively.
[0349] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A10 are SEQ ID NO: 62 and 38,
respectively.
[0350] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A10 are SEQ ID NO: 47 and 23,
respectively.
[0351] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A11 are SEQ ID NO: 63 and 39,
respectively.
[0352] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A11 are SEQ ID NO: 50 and 26,
respectively.
[0353] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A12 are SEQ ID NO: 64 and 38,
respectively.
[0354] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A12 are SEQ ID NO: 51 and 27,
respectively.
[0355] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A13 are SEQ ID NO: 65 and 40,
respectively.
[0356] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A13 are SEQ ID NO: 52 and 28,
respectively.
[0357] The nucleotide and amino acid sequences of the light chain
variable region of CDH17_A14 are SEQ ID NO: 66 and 41,
respectively.
[0358] The nucleotide and amino acid sequences of the heavy chain
variable region of CDH17_A14 are SEQ ID NO: 53 and 29,
respectively.
Example 8
Immunohistochemistry on FFPE Sections Using Anti-Cadherin-17
Antibodies
[0359] Immunohistochemistry was performed on FFPE sections of
colorectal tumor and normal adjacent tissue using the
anti-Cadherin-17 antibodies CDH17_A3, CDH17_A4, CDH17_A6, CDH17_A8
and CDH17_A9.
[0360] EX-De-Wax was from BioGenex, CA, USA. Tissue sections and
arrays were from Biomax, MD, USA. Slides were heated for 2 h at
60.degree. C. in 50 ml Falcons in a water bath with no buffer. Each
Falcon had one slide or two slides back-to back with long gel
loading tip between them to prevent slides from sticking to each
other. Slides were deparaffinised in EZ-DeWax for 5 min in black
slide rack, then rinsed well with the same DeWax solution using 1
ml pipette, then washed with water from the wash bottle. Slides
were placed in a coplin jar filled with water until the pressure
cooker was ready; the water was changed a couple of times. Water
was exchanged for antigen retrieval solution=1.times.citrate
buffer, pH 6 (DAKO). Antigen was retrieved by the pressure cooker
method. The slides in the plastic coplin jar in antigen retrieval
solution were placed into a pressure cooker which was then heated
up to position 6 (the highest setting). 15-20 min into the
incubation, the temperature was reduced to position 3 and left at
that (when the temperature inside the pressure cooker was
117.degree. C.) for another 20-25 minutes. Then the hob was
switched off and the cooker was placed onto the cold hob and the
pressure was released by carefully moving the handle into the
position between "open" and "closed". The whole system was left to
release the pressure and to cool down for another 20 minutes. The
lid was opened and samples taken out to rest on the bench. The
slides were washed 1.times.5 min with PBS-3T (0.5 L PBS+3 drops of
Tween-20) and placed in PBS.
[0361] After antigen retrieval, slides were mounted in the Shandon
Coverplate system. Trapping of air bubbles between the slide and
plastic coverplate was prevented by placing the coverplate into the
coplin jar filled with PBS and gently sliding the slide with tissue
sections into the coverplate. The slide was pulled out of the
coplin jar while holding it tightly together with the coverplate.
The assembled slide was placed into the rack, letting PBS trapped
in the funnel and between the slide and coverplate to run through.
Slides were washed with 2.times.2 ml (or 4.times.1 ml) PBS-3T, 1x2
ml PBS, waiting until all PBS had gone through the slide and
virtually no PBS was left in the funnel.
[0362] Endogenous peroxide blockade was performed using 1-4 drops
of peroxide solution per slide; the incubation time was 5 minutes.
The slides were rinsed with water and then once with 2 ml PBS-3T
and once with 2 ml PBS; it was important to wait until virtually no
liquid was left in the funnel before adding a new portion of wash
buffer.
[0363] The primary antibody was diluted with an Antibody diluent
reagent (DAKO). Optimal dilution was determined to be 1:400. Up to
200 .mu.l of diluted primary antibody was applied to each slide and
incubated for 45 minutes at room temperature. Slides were washed
with 2.times.2 ml (or 4.times.1 ml) PBS-3T and then 1x2 ml PBS.
[0364] The goat anti-mouse kappa HRP secondary (1 mg/ml,
cat.1050-05, Southern Biotech) was applied 2.times.2 drops per
slide and incubated for 35 min at room temperature. The slides were
washed as above.
[0365] The DAB substrate was made up in dilution buffer; 2 ml
containing 2 drops of substrate was enough for 10 slides. The DAB
reagent was applied to the slides by applying a few drops at a time
and left for 10 min. The slides were washed 1.times.2 ml (or
2.times.1 ml) with PBS-3T and 1x2 ml (or 2.times.1 ml) with
PBS.
[0366] Hematoxylin (DAKO) was applied; 1 ml was enough for 10
slides and slides were incubated for 1 min at room temperature. The
funnels of the Shandon Coverplate system were filled with 2 ml of
water and let to run through. When slides were clear of the excess
of hematoxylin, the system was disassembled, tissue sections and/or
arrays were washed with water from the wash bottle and placed into
black slide rack. Tissues were dehydrated by incubating in EZ-DeWax
for 5 min and then in 95% ethanol for 2-5 min.
[0367] Slides were left to dry on the bench at room temperature and
then mounted in mounting media and covered with coverslip.
[0368] Immunohistochemical analysis on antibodies CDH17_A3,
CDH17_A4, CDH17_A6, CDH17_A8 and CDH17_A9 revealed specific
membrane staining of tumor cells in colorectal cancer and no
appreciable staining of normal adjacent tissue in all cases.
Antibody CDH17_A4, in particular, showed clear specific membrane
staining of tumor cells.
Example 9
Immunohistochemistry on Frozen Sections Using Anti-Cadherin-17
Antibodies
[0369] Immunohistochemistry was performed on frozen paired tumor
and normal adjacent tissues using the anti-Cadherin-17 antibodies
CDH17_A4, CDH17_A6, CDH17_A8 and CDH17_A9.
[0370] Tissue sections were from BioChain Institute Inc., CA,
USA.
[0371] Frozen sections were washed with PBS twice for 3 minutes
each and were then placed in PBS.
[0372] Endogenous peroxide blockade was performed using Peroxidase
Blocker (S2001, DAKO). 1-4 drops of peroxidase blocker was added to
each slide and incubated for 5 minutes. The slides were rinsed
three times with 3 ml PBS.
[0373] The primary antibody was diluted with an Antibody diluent
reagent (DAKO). 150 .mu.l of diluted primary antibody was applied
to each slide and incubated for 45 minutes at room temperature.
Slides were washed with twice for 3 minutes with PBS-3T (500 ml
PBS+3 drops of Tween-20) and then once for 3 minutes with PBS.
[0374] The goat anti-mouse kappa HRP secondary was applied at
1:1000 (1 mg/ml, cat.1050-05, Southern Biotech) and incubated for
35 min at room temperature. The slides were washed as above.
[0375] The DAB substrate was made up in dilution buffer; 2 ml
containing 2 drops of substrate was enough for 10 slides. The DAB
reagent was applied to the slides by applying a few drops at a time
and incubated for 10 min. The slides were washed once for 3 minutes
with PBS-3T and twice for 3 minutes with water.
[0376] Hematoxylin (DAKO) was applied; 1 ml was enough for 10
slides and slides were incubated for 1 min at room temperature.
[0377] Slides were left to dry on the bench at room temperature and
then mounted in water-based mounting media from Vector and covered
with coverslip.
[0378] Immunohistochemical analysis on antibodies CDH17_A4,
CDH17_A6, CDH17_A8 and CDH17_A9 on three colorectal cancer samples
along with the paired normal adjacent tissue samples revealed
strong specific membrane staining of tumor cells in colorectal
cancer and some weak staining of normal adjacent tissue. Antibody
CDH17_A4, in particular, showed clear specific membrane staining of
tumor cells.
Example 10
Internalization and MabZAP of CDH17_A4 in LS174T and LoVo Cells
[0379] CDH17_A4 was shown to be internalized by LoVo cells upon
binding to the cells using a Immunofluorescence microscopy assay.
The Immunofluorescence microscopy assay showed internalization of
the anti-Cadherin-17 monoclonal antibodies through binding of an
anti-human IgG secondary antibody conjugated to Fluorescein
isothiocyanate (GamK-FITC). First, CDH17_A4 were bound to the
surface of the LoVo cells. Then, the secondary antibody conjugated
to Fluorescein isothiocyanate were bound to the primary antibodies.
Next, the CDH17_A4/secondary antibody FITC conjugate complex was
internalized by the cells.
[0380] The Immunofluorescence microscopy assay was conducted as
follows. LoVo cell were incubated at 37.degree. C. for 12 hours for
cells to adhere to each other. CDH17_A4 and secondary antibody
conjugated to Fluorescein isothiocyanate were serially diluted,
washed with FACS buffer (PBS, 2% FBS) and then added to the culture
media. The media was then washed again with FACS buffer (PBS, 2%
FBS) and incubated at 37%, after which 200 ul 2% PFA was added.
Coverslips were mounted using a 9 ul aqueous mountaing media and
the cells were then visualized at regular time intervals using
Leica fluorescent microscope.
[0381] The monoclonal antibody, CDH17_A4, was shown to be
internalized by LS147T and LoVo cells upon binding to the cells
using a MabZap assay. The MabZAP assay showed internalization of
the anti-CDH17 monoclonal antibodies through binding of an
anti-human IgG secondary antibody conjugated to the toxin saporin.
(Advanced Targeting System, San Diego, Calif., IT-22-100). First,
CDH17_A4 was bound to the surface of the LS147T and LoVo cells.
Then, the MabZAP antibodies were bound to the primary antibodies.
Next, the MabZAP complex was internalized by the cells. The
entrance of Saporin into the cells resulted in protein synthesis
inhibition and eventual cell death.
[0382] The MabZAP assay was conducted as follows. Each of the cells
was seeded at a density of 5.times.103 cells per well. The
anti-CDH17 monoclonal antibodies or an isotype control human IgG
were serially diluted then added to the cells. The MabZAP was then
added at a concentration of 50 .mu.g/ml and the plates allowed too
incubate for 48 and 72 hours. Cell viability in the plates was
detected by CellTiter-Glo.RTM. Luminescent Cell Viability Assay kit
(Promega, G7571) and the plates were read at 490 nM by a
Luminomitor (Tuner BioSystems, Sunnyvale, Calif.). The data was
analyzed by Prism (Graphpad). Cell death was proportional to the
concentration of CDH17_A4 and monoclonal antibody. FIGS. 6A, 6B,
6C, 6D, 7A and 7B show that the anti-CDH17 monoclonal antibodies
were efficiently internalized by LS174T and LoVo cells respectively
as compared to the anti-human IgG isotype control antibody.
[0383] All references referred to in this application, including
patent and patent applications, are incorporated herein by
reference to the fullest extent possible.
[0384] Throughout the specification and the claims which follow,
unless the context requires otherwise, the word `comprise`, and
variations such as `comprises` and `comprising`, will be understood
to imply the inclusion of a stated integer, step, group of integers
or group of steps but not to the exclusion of any other integer,
step, group of integers or group of steps.
TABLE-US-00006 SEQ ID SEQUENCE NO DESCRIPTION SEQUENCE 1 Cadherin-
MILQAHLHSLCLLMLYLATGYGQEGKFSGPLKPMTFSIYEGQEPSQIIFQFKANPPAVTFELTGETDNIFVIE-
REGLL 17
YYNRALDRETRSTHNLQVAALDANGIIVEGPVPITIEVKDINDNRPTFLQSKYEGSVRQNSRPGKPFLYV-
NATDLD
DPATPNGQLYYQIVIQLPMINNVMYFQINNKTGAISLTREGSQELNPAKNPSYNLVISVKDMGGQSENSFSD-
TTSV
DIIVTENIWKAPKPVEMVENSTDPHPIKITQVRWNDPGAQYSLVDKEKLPRFPFSIDQEGDIYVTQPLDREE-
KDAY
VFYAVAKDEYGKPLSYPLEIHVKVKDINDNPPTCPSPVTVFEVQENERLGNSIGTLTAHDRDEENTANSFLN-
YRIV
EQTPKLPMDGLFLIQTYAGMLQLAKQSLKKQDTPQYNLTIEVSDKDFKTLCFVQINVIDINDQIPIFEKSDY-
GNLTL
AEDTNIGSTILTIQATDADEPFTGSSKILYHIIKGDSEGRLGVDTDPHTNTGYVIIKKPLDFETAAVSNIVF-
KAENPEP
LVFGVKYNASSFAKFTLIVTDVNEAPQFSQHVFQAKVSEDVAIGTKVGNVTAKDPEGLDISYSLRGDTRGWL-
KID
HVTGEIFSVAPLDREAGSPYRVQVVATEVGGSSLSSVSEFHLILMDVNDNPPRLAKDYTGLFFCHPLSAPGS-
LIFEA
TDDDQHLFRGPHFTFSLGSGSLQNDWEVSKINGTHARLSTRHTDFEERAYVVLIRINDGGRPPLEGIVSLPV-
TFCSC
VEGSCFRPAGHQTGIPTVGMAVGILLTTLLVIGIILAVVFIRIKKDKGKDNVESAQASEVKPLRS 2
CDH17 AENPEPLVFGVK peptide 3 CDH17 DAYVFYAVAK peptide 4 CDH17
DEENTANSFLNYR peptide 5 CDH17 DEYGKPLSYPLEIHVK peptide 6 CDH17
DINDNRPTFLQSK peptide 7 CDH17 DNVESAQASEVKPLR peptide 8 CDH17
EGSQELNPAK peptide 9 CDH17 GWLK peptide 10 CDH17 IDHVTGEIFSVAPLDR
peptide 11 CDH17 KPLDFETAAVSNIVFK peptide 12 CDH17
LGVDTDPHTNTGYVIIK peptide 13 CDH17 TGAISLTR peptide 14 CDH17
VKDINDNPPTCPSPVTVFEVQENER peptide 15 CDH17 VSEDVAIGTK peptide 16
CDH17 WNDPGAQYSLVDK peptide 17 CDH17 WNDPGAQYSLVDKEKLPR peptide 18
VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAEVQLLETGGGVVKPGGSLKLSCAASGFTFSNYGMSW-
V acid A1
RQTPEKRLEWVAAINRDGGTTYYTDNVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARQFLL-
WDGWYFDV
WGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDL-
YTL SSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 19 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKVSGYTFSDHAIHW-
M acid A2
SQRPGQGLKWIGYIYPRHGTTNYNENFKGKATLTADTSSSTAYMQLNSLTSEDSAVYFCARMRNY-
FYVMDYWG
QGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT-
LSSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 20 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVLLQQSDAELVKPGASVKISCKASGYTFTDHAIHW-
VK acid A3
QRPEQGLEWIGYIYPEHGTIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTNYF-
YVMEYWGQG
TSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLS-
SSVT VPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 21 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAEVQLQQSVAELVKPGASVKMSCKVSGYTLTDHTIHW-
M acid A4
KQRPEQGLEWIGYIYPRDGITGYNEKFKGKATLTADTSSSTAYMQLNSLTSEDSAVYFCARWGYS-
YRNYAYYYD
YWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD-
LYT LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 22 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDADLVKPGASVKISCKASGYTFTDHAIHW-
V acid A5
KQRPEQGLEWIGYIYPEHGTIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARLRNY-
LYIMDYWGQ
GTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTL-
SSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 23 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHW-
V acid A6,
KQRPEQGLEWIGYIYPEHGTIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTN-
YFYVMEYWGQ A9, A10
GTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVL-
QSDLYTLSSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 24 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKVSGYTFTDHAIHW-
M acid A7
KQRPEQGLEWIGYIYPRDGFTKYNEKFKGKATLTADTSSSTAYMQLNSLTSEDSTVYFCARMTNY-
FYTMDYWG
QGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT-
LSSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 25 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDADLVKPGASVKISCKASGYTFTDHAIHW-
V acid A8
KQRPEQGLEWIGYIYPEHGTIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTNY-
FYVMEYWGQ
GTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTL-
SSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 26 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHW-
V acid A11
KQRPEQGLEWIGYIYPEHGSITYNEKFKGKATLTADKSSSTVYMHLNSLTSEDSAVYFCARLRN-
YLYVMDYWGQ
GTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTL-
SSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 27 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSEAELVKPGASVKLSCKASGYTFTDHAIHW-
M acid A12
KQRPEQGLEWIGYIYPRDDFAKVNEKFKGKATLTADTSSSTAYMQLNSLTSEDSAVYFCARMTN-
YLYIMDYWG
QGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT-
LSSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 28 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHW-
V acid A13
KQRPEQGLEWIGYIYPEHGTITYNEKFKGKATLTADKSSSTVYMHLNSLTSEDSAVYFCARLRN-
YLYIMDYWGQ
GTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTL-
SSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 29 VH amino
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAALVKPGASVKISCKVSGYTFSDHAIHW-
M acid A14
KQRPEQGLEWIGYIFPRDAFSLNNEKFKGKATLSADTSSSTAYMELTSLTFEDSAVYFCARMRN-
YFYVMDYWGQ
GTSVTVSSAKTTPPSVYTLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTL-
SSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 30 VK amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADVVLTQTPLSLPVTLGDQASISCRSS-
QSL acid A1
LHSNGNTYLHWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYFCS-
QSTHVLTFGAG
TKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY-
SMS STLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 31 VK amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCTSS-
KSL acid A2,
LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC-
MQHLEYPFTFG A3
SGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK-
DSTYS MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 32 VK
amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMSQSPSSLAVSVGEKVTMSCKSS-
QS acid A4
LLHSSNQKNYLAWYQQKPGQSPKVLIYWASTRESGVPDRFTGSGSGTDFTLTITSVKSEDLAVYY-
CQQYYSYPW
TFGGGTRLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS-
KDST YSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 33 VK
amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSS-
KSL acid A5
LRSNGNTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCM-
QHLEYPFTFGS
GTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST-
YSM SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 34 VK amino
RILPDAFYRNSLLFLHTRFFGWSETIKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCTSS-
KSLL acid A6
RSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQ-
HLEYPFTFGS
GTKLEIKRADAAPTVSILPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST-
YSM SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 35 VK amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSS-
KSL acid A7
LRTNGNTYLHWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTVFTLRISRVEAEDVGVYYCM-
QHLEYPFTFG
SGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS-
TYS MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 36 VK amino
RILPYAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSS-
KSL acid A8
LRSNGNTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCM-
QHLEYPFTFGS
GTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST-
YSM SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 37 VK amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCTSS-
KSL
acid A9
LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCM-
QHLEYPFTFG
SGTKLEIKRADAAPTVSISPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS-
TYS MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 38 VK amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSS-
KSL acid A10,
LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYY-
CMQHLEYPFTFG A12
GGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS-
KDSTYS MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 39 VK
amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVSVTPGESVSISCRST-
KSL acid A11
LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC-
MQHLEYPFTFG
GGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS-
TYS MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 40 VK amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSS-
KSL acid A13
LRSNGNTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC-
MQHLEYPFTFGS
GTKLEIKRADAAPTVSIFPQYSEQLTTGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST-
YSM SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 41 VK amino
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCTSS-
KSL acid A14
LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC-
MQHLEYPFTFG SGTNLEIKRADAAPTVSIFTTSREQLTSGGASVVCFLNNFYPKDINVK 42 VH
n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A1
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCGAAGTGCAGCTGTTGGAGACTGGGGGA-
GG
CGTAGTGAAGCCCGGAGGGTCCCTTAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGGCAT-
G
TCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAGCCATTAATCGTGATGGTGGTACCACC
TACTATACAGACAATGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACAGCCTGTACCTGCA
AATGAGCAGTCTGAGGTCTGAGGACACAGCCTTGTATTACTGTGCAAGACAGTTCCTTCTCTGGGACGGCTG
GTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTA
TCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTT-
C
CCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTG
CAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACC
TGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 43
VH n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A2
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCT-
GA
GTTGGTGAAACCTGGAGCTTCAGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCAGTGACCATGCTAT-
T
CACTGGATGAGTCAGAGACCTGGACAGGGCCTGAAATGGATTGGATATATTTATCCTAGACATGGGACTACT
AACTACAATGAGAACTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTACATGCA
GCTCAACAGCCTGACATCTGAAGATTCTGCCGTCTATTTCTGTGCAAGAATGAGAAACTACTTCTATGTTAT-
G
GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG
GCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAG
CCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCT
GACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAAC
GTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 44 VH
n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A3
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCTGCTGCAACAGTCTGACGCT-
GAG
TTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATT-
C
ACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGAACTATTA
AGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCTATATGCAGC
TCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTTCAAGACTCACTAACTACTTCTATGTTATGG-
A
GTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGC
CCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCC
AGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGA
CCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGT
TGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 45 VH n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A4
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCGAGGTTCAGCTGCAGCAGTCTGTCGCT-
GAG
TTGGTGAAACCTGGAGCTTCAGTGAAGATGTCATGCAAGGTTTCTGGCTACACCCTCACTGACCATACTATT-
C
ACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTACCCTAGAGATGGAATAACT
GGGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACACTTCTTCCAGCACAGCCTACATGCA
GCTCAACAGCCTGACATCTGAGGATTCTGCAGTCTATTTCTGTGCCAGATGGGGCTATAGTTACAGGAATTA
CGCGTACTACTATGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATC
TGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGG
CTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGC-
T
GTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACC
GTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT
46 VH n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A5
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCT-
GAC
TTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATT-
C
ACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGAACTATTA
AGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCTATATGCAGC
TCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTGCAAGACTCAGGAACTATTTGTATATTATGG
ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGC
CAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTG
ACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACG
TTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 47 VH n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A6, A9,
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCT-
GACGCTGA A10
GTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGC-
TATT
CACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGAACTATT
AAGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCTATATGCA
GCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTTCAAGACTCACTAACTACTTCTATGTTAT-
G
GAGTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG
GCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAG
CCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCT
GACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAAC
GTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 48 VH
n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A7
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCT-
GA
GTTGGTGAAACCTGGAGCCTCAGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCACTGACCATGCTAT-
T
CACTGGATGAAACAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTAGAGATGGTTTTACT
AAGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTACATGCA
GCTCAACAGCCTGACATCTGAGGATTCTACAGTCTATTTCTGTGCAAGAATGACTAACTACTTCTATACTAT-
G
GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG
GCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAG
CCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCT
GACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAAC
GTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 49 VH
n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A8
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCT-
GAC
TTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATT-
C
ACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGAACTATTA
AGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCTATATGCAGC
TCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTTCAAGACTCACTAACTACTTCTATGTTATGG-
A
GTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGC
CCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCC
AGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGA
CCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGT
TGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 50 VH n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A11
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGC-
TGA
GTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTAT-
T
CACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGTAGTATT
ACGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGTACTGTCTATATGCAC
CTCAATAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTGCAAGACTCAGGAACTACTTGTATGTTATG-
G
ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGC
CAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTG
ACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACG
TTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 51 VH n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A12
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGAGGC-
TGA
GCTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTAT-
T
CACTGGATGAAACAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATCTACCCCAGAGATGATTTTGCT
AAGGTGAATGAGAAGTTCAAGGGCAAGGCCACACTGACAGCAGACACATCCTCCAGCACAGCCTACATGCA
GCTCAACAGCCTGACATCTGAGGATTCTGCAGTCTATTTCTGTGCAAGAATGACTAACTACCTCTATATTAT-
G
GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG
GCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAG
CCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCT
GACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAAC
GTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 52 VH
n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A13
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGC-
TGA
GTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTAT-
T
CACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGTACTATT
ACGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGTACTGTCTATATGCAC
CTCAATAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTGCAAGACTCAGGAACTATTTGTATATTATG-
G
ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCCGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGC
CAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTG
ACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACG
TTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 53 VH n.t.
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGC
A14
ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGC-
CGC
GTTGGTGAAACCTGGAGCTTCAGTGAAGATATCGTGCAAGGTTTCTGGCTACACCTTCAGTGACCATGCTAT
TCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTTTCCTAGAGATGCTTTTAG
TTTGAACAATGAGAAGTTCAAGGGCAAGGCCACACTGAGTGCAGACACATCCTCCAGCACAGCCTACATGG
AGCTCACCAGCCTGACATTTGAGGATTCTGCAGTCTATTTCTGTGCAAGAATGAGAAACTACTTCTATGTTA-
T
GGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATACACT
GGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGA
GCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTC
TGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAA
CGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 54 VK
n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A1
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCC-
AT
GGCCGATGTTGTGCTGACCCAGACTCCACTCTCCCTGCCTGTCACTCTTGGAGATCAAGCCTCCATCTCTTG-
C
AGATCTAGTCAGAGCCTTTTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCTGAAGCCAGGCCAG
TCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGA
TCAGGGACAGATTTCACACTCAAGATCACCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAA
AGTACACATGTGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTA
TCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTC-
T
ACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGG
ACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGA
ACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAG
GAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 55 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A2, A3
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACC-
AGCCAT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTG-
C
ACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGG
TCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAA
CATCTAGAATATCCTTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACT
GTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC-
T
TCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT
TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA
TGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAA
CAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 56 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A4
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCC-
AT
GGCCGACATCGTTATGTCTCAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTG-
C
AAGTCCAGCCAGAGCCTTTTACATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGG
GCAGTCTCCTAAAGTGCTGATTTACTGGGCATCCACTAGAGAATCTGGGGTCCCTGATCGCTTCACAGGCAG
TGGATCTGGGACAGATTTCACTCTCACCATCACCAGTGTGAAGTCTGAAGACCTGGCAGTTTATTACTGTCA
GCAATATTATAGCTATCCGTGGACGTTCGGTGGCGGCACCAGGCTGGAAATCAAACGGGCTGATGCTGCACC
AACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAA-
C
AACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAA
CAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACG
AGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCT
TCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 57 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A5
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCC-
AT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTG-
C
AGGTCTAGTAAGAGTCTCCTGCGCAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGCTGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGG-
T
CTGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAAC
ATCTAGAATATCCTTTCACATTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTG
TATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACT-
T
CTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTT
GGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTAT
GAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAAC
AGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 58 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A6
GAGTGAAACGATAAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCC-
AT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTG-
C
ACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGG
TCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAA
CATCTAGAATATCCTTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACT
GTATCCATCCTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC-
T
TCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT
TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA
TGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAA
CAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 59 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A7
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCC-
AT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTTTCCATCTCCTG-
C
AGGTCTTCTAAGAGTCTCCTGCGTACTAATGGCAACACTTACTTGCATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGG
TCAGGAACTGTTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAA
CATCTAGAATATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAAGGGCTGATGCTGCACCAACT
GTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC-
T
TCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT
TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA
TGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAA
CAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 60 VK n.t.
TAAGATTAGCGGATCCTACCTTACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A8
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCC-
AT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAATCAGTATCCATCTCCTG-
C
AGGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGCTGTCTAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGG-
T
CAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAAC
ATCTAGAATATCCTTTCACATTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTG
TATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACT-
T
CTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTT
GGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTAT
GAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAAC
AGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 61 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A9
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCC-
AT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTG-
C
ACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGG
TCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAA
CATCTAGAATATCCTTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACT
GTATCCATCTCCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC-
T
TCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT
TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA
TGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAA
CAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 62 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A10
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGC-
CAT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTG-
C
AGGTCCAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTCATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGG-
T
CAGGAACTGCCTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAAC
ATCTAGAATATCCTTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACT
GTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC-
T
TCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT
TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA
TGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAA
CAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 63 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTTCTGTTTCTCCATACCCGTTTTTTTGGAT-
G A11
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGC-
CAT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTATCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTG-
C
AGGTCTACTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTCCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGG
TCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAA
CATCTAGAATATCCTTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACT
GTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC-
T
TCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT
TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA
TGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAA
CAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 64 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A12
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGC-
CAT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTG-
C
AGGTCTAGTAAGAGTCTCCTACGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGG
TCAGGAACTGCCTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAA
CATCTAGAATATCCTTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACT
GTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC-
T
TCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT
TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA
TGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAA
CAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 65 VK n.t.
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGAT-
G A13
GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGC-
CAT
GGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTG-
C
AGGTCTAGTAAGAGTCTCCTGCGCAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAG
TCTCCTCAGCTCCTGATATATCGGCTGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGG-
T
CAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAAC
ATCTAGAATATCCTTTCACATTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTG
TATCCATCTTCCCACAATACAGTGAGCAGTTAACAACTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACT-
T
CTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTT
GGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTAT
GAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAAC
AGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 66 VK n.t.
CGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGGAGTGAAA-
C A14
GATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA-
TAT
TGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCACGTCTAG-
T
AAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAG
CTCCTGATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACT
GCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAA
TATCCTTTCACGTTCGGCTCGGGGACAAATTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATC
TTCACAACATCCAGAGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCC
AAAGACATCAATGTCAAG 67 Cadherin-17
QEGKFSGPLKPMTFSIYEGQEPSQIIFQFKANPPAVTFELTGETDNIFVIEREGLLYYNRALDRETRSTHNLQ-
VAAL ECD
DANGIIVEGPVPITIKVKDINDNRPTFLQSKYEGSVRQNSRPGKPFLYVNATDLDDPATPNGQLYYQIV-
IQLPMINN
VMYFQINNKTGAISLTREGSQELNPAKNPSYNLVISVKDMGGQSENSFSDTTSVDIIVTENIWKAPKPVEMV-
ENST
DPHPIKITQVRWNDPGAQYSLVDKEKLPRFPFSIDQEGDIYVTQPLDREEKDAYVFYAVAKDEYGKPLSYPL-
EIHV
KVKDINDNPPTCPSPVTVFEVQENERLGNSIGTLTAHDRDEENTANSFLNYRIVEQTPKLPMDGLFLIQTYA-
GMLQ
LAKQSLKKQDTPQYNLTIEVSDKDFKTLCFVQINVIDINDQIPIFEKSDYGNLTLAEDTNIGSTILTIQATD-
ADEPFTG
SSKILYHIIKGDSEGRLGVDTDPHTNTGYVIIKKPLDFETAAVSNIVFKAENPEPLVFGVKYNASSFAKFTL-
IVTDVN
EAPQFSQHVFQAKVSEDVAIGTKVGNVTAKDPEGLDISYSLRGDTRGWLKIDHVTGEIFSVAPLDREAGSPY-
RVQ
VVATEVGGSSLSSVSEFHLILMDVNDNPPRLAKDYTGLFFCHPLSAPGSLIFEATDDDQHLFRGPHFTFSLG-
SGSLQ
NDWEVSKINGTHARLSTRHTDFEEREYVVLIRINDGGRPPLEGIVSLPVTFCSCVEGSCFRPAGHQTGIPTV-
GM 68 Cadherin-
QEGKFSGPLKPMTFSIYEGQEPSQIIFQFKANPPAVTFELTGETDNIFVIEREGLLYYNRALDRETRSTHNLQ-
VAAL 17 ECD
DANGIIVEGPVPITIKVKDINDNRPTFLQSKYEGSVRQNSRPGKPFLYVNATDLDDPATPNGQLYY-
QIVIQLPMINN domains 1-2
VMYFQINNKTGAISLTREGSQELNPAKNPSYNLVISVKDMGGQSENSFSDTTSVDIIVTENIWKAPKP
Sequence CWU 1
1
171832PRTHomo SapiensMISC_FEATURE(1)..(832)Swiss Prot Q12864,
Cadherin 17 1Met Ile Leu Gln Ala His Leu His Ser Leu Cys Leu Leu
Met Leu Tyr 1 5 10 15 Leu Ala Thr Gly Tyr Gly Gln Glu Gly Lys Phe
Ser Gly Pro Leu Lys 20 25 30 Pro Met Thr Phe Ser Ile Tyr Glu Gly
Gln Glu Pro Ser Gln Ile Ile 35 40 45 Phe Gln Phe Lys Ala Asn Pro
Pro Ala Val Thr Phe Glu Leu Thr Gly 50 55 60 Glu Thr Asp Asn Ile
Phe Val Ile Glu Arg Glu Gly Leu Leu Tyr Tyr 65 70 75 80 Asn Arg Ala
Leu Asp Arg Glu Thr Arg Ser Thr His Asn Leu Gln Val 85 90 95 Ala
Ala Leu Asp Ala Asn Gly Ile Ile Val Glu Gly Pro Val Pro Ile 100 105
110 Thr Ile Glu Val Lys Asp Ile Asn Asp Asn Arg Pro Thr Phe Leu Gln
115 120 125 Ser Lys Tyr Glu Gly Ser Val Arg Gln Asn Ser Arg Pro Gly
Lys Pro 130 135 140 Phe Leu Tyr Val Asn Ala Thr Asp Leu Asp Asp Pro
Ala Thr Pro Asn 145 150 155 160 Gly Gln Leu Tyr Tyr Gln Ile Val Ile
Gln Leu Pro Met Ile Asn Asn 165 170 175 Val Met Tyr Phe Gln Ile Asn
Asn Lys Thr Gly Ala Ile Ser Leu Thr 180 185 190 Arg Glu Gly Ser Gln
Glu Leu Asn Pro Ala Lys Asn Pro Ser Tyr Asn 195 200 205 Leu Val Ile
Ser Val Lys Asp Met Gly Gly Gln Ser Glu Asn Ser Phe 210 215 220 Ser
Asp Thr Thr Ser Val Asp Ile Ile Val Thr Glu Asn Ile Trp Lys 225 230
235 240 Ala Pro Lys Pro Val Glu Met Val Glu Asn Ser Thr Asp Pro His
Pro 245 250 255 Ile Lys Ile Thr Gln Val Arg Trp Asn Asp Pro Gly Ala
Gln Tyr Ser 260 265 270 Leu Val Asp Lys Glu Lys Leu Pro Arg Phe Pro
Phe Ser Ile Asp Gln 275 280 285 Glu Gly Asp Ile Tyr Val Thr Gln Pro
Leu Asp Arg Glu Glu Lys Asp 290 295 300 Ala Tyr Val Phe Tyr Ala Val
Ala Lys Asp Glu Tyr Gly Lys Pro Leu 305 310 315 320 Ser Tyr Pro Leu
Glu Ile His Val Lys Val Lys Asp Ile Asn Asp Asn 325 330 335 Pro Pro
Thr Cys Pro Ser Pro Val Thr Val Phe Glu Val Gln Glu Asn 340 345 350
Glu Arg Leu Gly Asn Ser Ile Gly Thr Leu Thr Ala His Asp Arg Asp 355
360 365 Glu Glu Asn Thr Ala Asn Ser Phe Leu Asn Tyr Arg Ile Val Glu
Gln 370 375 380 Thr Pro Lys Leu Pro Met Asp Gly Leu Phe Leu Ile Gln
Thr Tyr Ala 385 390 395 400 Gly Met Leu Gln Leu Ala Lys Gln Ser Leu
Lys Lys Gln Asp Thr Pro 405 410 415 Gln Tyr Asn Leu Thr Ile Glu Val
Ser Asp Lys Asp Phe Lys Thr Leu 420 425 430 Cys Phe Val Gln Ile Asn
Val Ile Asp Ile Asn Asp Gln Ile Pro Ile 435 440 445 Phe Glu Lys Ser
Asp Tyr Gly Asn Leu Thr Leu Ala Glu Asp Thr Asn 450 455 460 Ile Gly
Ser Thr Ile Leu Thr Ile Gln Ala Thr Asp Ala Asp Glu Pro 465 470 475
480 Phe Thr Gly Ser Ser Lys Ile Leu Tyr His Ile Ile Lys Gly Asp Ser
485 490 495 Glu Gly Arg Leu Gly Val Asp Thr Asp Pro His Thr Asn Thr
Gly Tyr 500 505 510 Val Ile Ile Lys Lys Pro Leu Asp Phe Glu Thr Ala
Ala Val Ser Asn 515 520 525 Ile Val Phe Lys Ala Glu Asn Pro Glu Pro
Leu Val Phe Gly Val Lys 530 535 540 Tyr Asn Ala Ser Ser Phe Ala Lys
Phe Thr Leu Ile Val Thr Asp Val 545 550 555 560 Asn Glu Ala Pro Gln
Phe Ser Gln His Val Phe Gln Ala Lys Val Ser 565 570 575 Glu Asp Val
Ala Ile Gly Thr Lys Val Gly Asn Val Thr Ala Lys Asp 580 585 590 Pro
Glu Gly Leu Asp Ile Ser Tyr Ser Leu Arg Gly Asp Thr Arg Gly 595 600
605 Trp Leu Lys Ile Asp His Val Thr Gly Glu Ile Phe Ser Val Ala Pro
610 615 620 Leu Asp Arg Glu Ala Gly Ser Pro Tyr Arg Val Gln Val Val
Ala Thr 625 630 635 640 Glu Val Gly Gly Ser Ser Leu Ser Ser Val Ser
Glu Phe His Leu Ile 645 650 655 Leu Met Asp Val Asn Asp Asn Pro Pro
Arg Leu Ala Lys Asp Tyr Thr 660 665 670 Gly Leu Phe Phe Cys His Pro
Leu Ser Ala Pro Gly Ser Leu Ile Phe 675 680 685 Glu Ala Thr Asp Asp
Asp Gln His Leu Phe Arg Gly Pro His Phe Thr 690 695 700 Phe Ser Leu
Gly Ser Gly Ser Leu Gln Asn Asp Trp Glu Val Ser Lys 705 710 715 720
Ile Asn Gly Thr His Ala Arg Leu Ser Thr Arg His Thr Asp Phe Glu 725
730 735 Glu Arg Ala Tyr Val Val Leu Ile Arg Ile Asn Asp Gly Gly Arg
Pro 740 745 750 Pro Leu Glu Gly Ile Val Ser Leu Pro Val Thr Phe Cys
Ser Cys Val 755 760 765 Glu Gly Ser Cys Phe Arg Pro Ala Gly His Gln
Thr Gly Ile Pro Thr 770 775 780 Val Gly Met Ala Val Gly Ile Leu Leu
Thr Thr Leu Leu Val Ile Gly 785 790 795 800 Ile Ile Leu Ala Val Val
Phe Ile Arg Ile Lys Lys Asp Lys Gly Lys 805 810 815 Asp Asn Val Glu
Ser Ala Gln Ala Ser Glu Val Lys Pro Leu Arg Ser 820 825 830
212PRTHomo Sapiens 2Ala Glu Asn Pro Glu Pro Leu Val Phe Gly Val Lys
1 5 10 310PRTHomo Sapiens 3Asp Ala Tyr Val Phe Tyr Ala Val Ala Lys
1 5 10 413PRTHomo Sapiens 4Asp Glu Glu Asn Thr Ala Asn Ser Phe Leu
Asn Tyr Arg 1 5 10 516PRTHomo Sapiens 5Asp Glu Tyr Gly Lys Pro Leu
Ser Tyr Pro Leu Glu Ile His Val Lys 1 5 10 15 613PRTHomo Sapiens
6Asp Ile Asn Asp Asn Arg Pro Thr Phe Leu Gln Ser Lys 1 5 10
715PRTHomo Sapiens 7Asp Asn Val Glu Ser Ala Gln Ala Ser Glu Val Lys
Pro Leu Arg 1 5 10 15 810PRTHomo Sapiens 8Glu Gly Ser Gln Glu Leu
Asn Pro Ala Lys 1 5 10 94PRTHomo Sapiens 9Gly Trp Leu Lys 1
1016PRTHomo Sapiens 10Ile Asp His Val Thr Gly Glu Ile Phe Ser Val
Ala Pro Leu Asp Arg 1 5 10 15 1116PRTHomo Sapiens 11Lys Pro Leu Asp
Phe Glu Thr Ala Ala Val Ser Asn Ile Val Phe Lys 1 5 10 15
1217PRTHomo Sapiens 12Leu Gly Val Asp Thr Asp Pro His Thr Asn Thr
Gly Tyr Val Ile Ile 1 5 10 15 Lys 138PRTHomo Sapiens 13Thr Gly Ala
Ile Ser Leu Thr Arg 1 5 1425PRTHomo Sapiens 14Val Lys Asp Ile Asn
Asp Asn Pro Pro Thr Cys Pro Ser Pro Val Thr 1 5 10 15 Val Phe Glu
Val Gln Glu Asn Glu Arg 20 25 1510PRTHomo Sapiens 15Val Ser Glu Asp
Val Ala Ile Gly Thr Lys 1 5 10 1613PRTHomo Sapiens 16Trp Asn Asp
Pro Gly Ala Gln Tyr Ser Leu Val Asp Lys 1 5 10 1718PRTHomo Sapiens
17Trp Asn Asp Pro Gly Ala Gln Tyr Ser Leu Val Asp Lys Glu Lys Leu 1
5 10 15 Pro Arg
* * * * *
References