U.S. patent application number 16/817397 was filed with the patent office on 2020-11-26 for monoclonal antibodies, compositions and methods for detecting mucin-like protein (mlp) as a biomarker for ovarian and pancreatic cancer.
The applicant listed for this patent is Omeros Corporation, University of Leicester. Invention is credited to Gregory A. Demopulos, Hans-Wilhelm Schwaeble.
Application Number | 20200369782 16/817397 |
Document ID | / |
Family ID | 1000005004326 |
Filed Date | 2020-11-26 |
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United States Patent
Application |
20200369782 |
Kind Code |
A1 |
Schwaeble; Hans-Wilhelm ; et
al. |
November 26, 2020 |
Monoclonal Antibodies, Compositions and Methods for Detecting
Mucin-like Protein (MLP) as a Biomarker for Ovarian and Pancreatic
Cancer
Abstract
In various embodiments the invention provides anti-mucin-like
protein (MLP) monoclonal antibodies, compositions and methods for
detecting MLP as a biomarker for mucin-secreting type of cancer
such as ovarian or pancreatic cancer.
Inventors: |
Schwaeble; Hans-Wilhelm;
(Mountsorrel, GB) ; Demopulos; Gregory A.; (Mercer
Island, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Leicester
Omeros Corporation |
Leicester
Seattle |
WA |
GB
US |
|
|
Family ID: |
1000005004326 |
Appl. No.: |
16/817397 |
Filed: |
March 12, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15615588 |
Jun 6, 2017 |
10626184 |
|
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16817397 |
|
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62347824 |
Jun 9, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/56 20130101;
A61K 47/6859 20170801; G01N 33/57492 20130101; G01N 2333/4725
20130101; C07K 16/3092 20130101; A61K 47/6869 20170801; C07K
14/4727 20130101; G01N 33/57438 20130101; C07K 2317/565 20130101;
G01N 33/57449 20130101; C07K 2317/34 20130101; C07K 16/3015
20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C07K 14/47 20060101 C07K014/47; A61K 47/68 20060101
A61K047/68; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method of detecting or diagnosing epithelial cancer by
determining the presence or amount of MLP in a biological sample
from a test subject, the method comprising: (a) contacting a
biological sample from a test subject with an anti-MLP antibody or
antigen-binding fragment thereof in an in vitro immunoassay; and
(b) detecting the presence or absence of binding of said antibody,
wherein the presence of binding indicates the presence or amount of
MLP in the sample, wherein the antibody or fragment thereof binds
to an epitope in the C-terminal region of MLP, set forth as SEQ ID
NO:4.
2. The method of claim 1, wherein said anti-MLP antibody is labeled
with a detectable moiety and step (b) comprises detecting the
presence or amount of said detectable moiety.
3. The method of claim 1, further comprising comparing the amount
of MLP detected in accordance with step (b) with a reference
standard or control sample from a healthy subject, wherein an
increase of at least two-fold or higher (e.g., at least five-fold,
or at least ten-fold) in the level of MLP in the test sample as
compared to the control sample (or reference standard) indicates
the presence of, or increased risk for developing an epithelial
cancer, such as ovarian cancer or pancreatic cancer, in the test
subject.
4. The method of claim 1, wherein the biological sample is selected
from the group consisting of blood, serum, plasma and tissue.
5. The method of claim 1, wherein the anti-MLP antibody or fragment
thereof is a monoclonal antibody that binds to the same epitope or
competes for binding to MLP with a reference antibody selected from
the group consisting of: the monoclonal anti-MLP antibody Clone 11
produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 under the ATCC Designation Number PTA-121699; the
monoclonal anti-MLP antibody Clone B produced by the hybridoma cell
line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation Number PTA-121700; and the monoclonal anti-MLP antibody
clone C produced by the hybridoma cell line deposited at the ATCC
on Oct. 30, 2014 under the ATCC Designation Number PTA-121701.
6. The method of claim 1, wherein the anti-MLP antibody or fragment
thereof is a monoclonal antibody having a heavy chain variable
region CDR-H3 sequence comprising an amino acid sequence set forth
as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:19, and
conservative sequence modifications thereof, and having a light
chain variable region CDR-L3 sequence comprising an amino acid
sequence set forth as SEQ ID NO:23 or SEQ ID NO:27, and
conservative sequence modifications thereof.
7. The method of claim 1, wherein the anti-MLP antibody or fragment
thereof is a monoclonal antibody that comprises a heavy chain
variable region and/or a light chain variable region set forth in
Table 1, and conservative sequence modifications thereof.
8. The method of claim 1, further comprising performing an
immunoassay with one or more additional antibodies that bind to
ovarian and/or pancreatic cancer biomarkers.
9. The method of claim 1, wherein the test subject is: (i)
apparently healthy (ii) has a family history of ovarian or
pancreatic cancer; (iii) experiencing one or more symptoms
associated with ovarian cancer; or (iv) known to be suffering from
ovarian or pancreatic cancer and has had or is currently undergoing
treatment for ovarian or pancreatic cancer.
10. The method of claim 1, wherein the method further comprises
comparing the results of the assay from biological samples obtained
from the test subject at one or more time points to assess the
efficacy of a treatment regimen.
11. The method of claim 1, wherein the anti-MLP antibody is
immobilized on a substrate.
12. The method of claim 1, wherein the immunoassay is an ELISA
assay.
13. A method of detecting or diagnosing the presence of a
mucin-secreting cancer in a test subject comprising (a)
administering to a living test subject a humanized or fully human
anti-MLP antibody or antigen-binding fragment thereof that binds to
an epitope in the C-terminal region of MLP, set forth as SEQ ID
NO:4; and (b) detecting the presence or absence or the amount of
the antibody or fragment thereof bound to MLP, wherein detection of
the presence or amount of MLP in the subject indicates the presence
of a mucin-secreting cancer.
14. The method of claim 13, wherein the mucin-secreting cancer is
selected from the group consisting of ovarian, pancreatic,
colorectal, breast, appendiceal, lung, renal, cervical, biliary,
esophageal and epithelial skin cancer.
15. The method of claim 13, wherein the anti-MLP antibody is
labeled with a detectable moiety suitable for in vivo use and step
(b) comprises detecting the presence or amount of the detectable
moiety.
16. The method of claim 13, wherein method is used in an imaging,
intraoperative, endoscopic or intravascular procedure.
17. The method of claim 13, wherein the anti-MLP antibody or
fragment thereof is a monoclonal antibody that binds to the same
epitope or competes for binding to MLP with a reference antibody
selected from the group consisting of: the monoclonal anti-MLP
antibody Clone 11 produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121699; the monoclonal anti-MLP antibody Clone B produced by
the hybridoma cell line deposited at the ATCC on Oct. 30, 2014
under the ATCC Designation Number PTA-121700; and the monoclonal
anti-MLP antibody clone C produced by the hybridoma cell line
deposited at the ATCC on Oct. 30, 2014 under the ATCC Designation
Number PTA-121701.
18. The method of claim 13, wherein the anti-MLP antibody or
fragment thereof is a monoclonal antibody having a heavy chain
variable region CDR-H3 sequence comprising an amino acid sequence
set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID
NO:19, and conservative sequence modifications thereof, and having
a light chain variable region CDR-L3 sequence comprising an amino
acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27, and
conservative sequence modifications thereof.
19. The method of claim 14, wherein the test subject is: (i)
apparently healthy (ii) has a family history of ovarian or
pancreatic cancer; (iii) experiencing one or more symptoms
associated with ovarian cancer; or (iv) known to be suffering from
ovarian or pancreatic cancer and has had or is currently undergoing
treatment for ovarian or pancreatic cancer.
20. A method of treating a subject suffering from ovarian or
pancreatic cancer comprising administering to an individual
suffering from ovarian or pancreatic cancer a humanized or fully
human anti-MLP antibody or antigen-binding fragment thereof that
binds to an epitope in the C-terminal region of MLP, set forth as
SEQ ID NO:4, wherein the antibody or fragment thereof is coupled to
a therapeutic agent.
21. The method of claim 20, wherein the therapeutic agent is a
chemotherapeutic agent.
22. The method of claim 20, wherein the anti-MLP antibody or
fragment thereof is a monoclonal antibody that binds to the same
epitope or competes for binding to MLP with a reference antibody
selected from the group consisting of: the monoclonal anti-MLP
antibody Clone 11 produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121699; the monoclonal anti-MLP antibody Clone B produced by
the hybridoma cell line deposited at the ATCC on Oct. 30, 2014
under the ATCC Designation Number PTA-121700; and the monoclonal
anti-MLP antibody clone C produced by the hybridoma cell line
deposited at the ATCC on Oct. 30, 2014 under the ATCC Designation
Number PTA-121701.
23. The method of claim 20, wherein the anti-MLP antibody or
fragment thereof is a monoclonal antibody having a heavy chain
variable region CDR-H3 sequence comprising an amino acid sequence
set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID
NO:19, and conservative sequence modifications thereof, and having
a light chain variable region CDR-L3 sequence comprising an amino
acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27, and
conservative sequence modifications thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/615,588, filed Jun. 6, 2017, which claims the benefit
under 35 U.S.C. .sctn. 119(e) of U.S. Provisional Patent
Application No. 62/347,824, filed Jun. 9, 2016, now expired; each
application is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to monoclonal antibodies and
compositions comprising such antibodies for use in detecting
ovarian and pancreatic cancer.
STATEMENT REGARDING SEQUENCE LISTING
[0003] The sequence listing associated with this application is
provided in text format in lieu of a paper copy and is hereby
incorporated by reference into the specification. The name of the
text file containing the sequence listing is
AB.1.0208.US3.Sequence_Listing.20200312_ST25. The text file is 24
KB, was created on Mar. 12, 2020; and is being submitted via
EFS-Web with the filing of the specification.
BACKGROUND
[0004] Amongst female cancer patients there is a high incidence of
ovarian cancer and this is associated with high mortality (Modugno
et al., Am J of Obstetrics and Gynecology 191:733-740, 2004). It is
the fifth most common cancer in women, following breast, bowel,
lung and uterine cancers. It is called a silent killer because it
is asymptomatic until it reaches the late stages (Chauhan et al., J
of Ovarian Research 2:2215, 2009). Early diagnosis of ovarian
cancer is difficult because the first symptoms are non-localized
mild pain, but the symptoms of ovarian malignancies become clearer
in the late stages, and include loss of appetite and weight, strong
pain in the back and pelvis associated with vaginal bleeding after
menopause, frequent urination, constipation or diarrhea and
bloating in the abdomen (American Cancer Society, 2013).
[0005] The early detection of ovarian malignancies represents a so
far unmet need. The presently used tumor associated biomarkers, for
example the relatively unspecific tumor marker CA-125, all fail to
detect ovarian malignancies at an early stage.
[0006] Therefore, there is a pressing need to develop a
non-invasive clinical test for early diagnosis to discover ovarian
malignancies at an early stage when treatment is effective and the
prognosis much better then in later stages of ovarian cancer.
SUMMARY
[0007] This summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This summary is not intended to identify
key features of the claimed subject matter, nor is it intended to
be used as an aid in determining the scope of the claimed subject
matter.
[0008] In one aspect, the invention provides an isolated antibody,
or antigen binding fragment thereof, that specifically binds to an
epitope in the C-terminal region of human mucin-like protein, set
forth as SEQ ID NO:4. In one embodiment, the antibody is a
monoclonal antibody. In one embodiment, said antibody is a
humanized, chimeric or fully human antibody. In one embodiment,
said antibody or antigen binding fragment thereof is capable of
binding to glycosylated human MLP secreted from an epithelial
cancer cell line. In one embodiment, said antibody or antigen
binding fragment thereof is capable of binding to glycosylated
human MLP in an ELISA assay format. In one embodiment, said
antibody or antigen binding fragment thereof binds to human MLP
with a KD of less than 10 nM, such as less than 1 nM. In one
embodiment, said antibody, or antigen binding fragment thereof
recognizes at least part of an epitope recognized by one or more
reference antibodies selected from the group consisting of: [0009]
the monoclonal antibody MLP Clone 11 produced by the hybridoma cell
line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation Number PTA-121699. [0010] (ii) the monoclonal antibody
MLP Clone B produced by the hybridoma cell line deposited at the
ATCC on Oct. 30, 2014 under the ATCC Designation Number PTA-121700;
and [0011] (iii) the monoclonal antibody MLP Clone C produced by
the hybridoma cell line deposited at the ATCC on Oct. 30, 2014
under the ATCC Designation Number PTA-121701.
[0012] In one embodiment, said antibody comprises a variable region
of the heavy chain comprising or consisting of a sequence which is
at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. In
one embodiment, said antibody comprises a variable region of the
light chain comprising or consisting of a sequence which is at
least 90% identical to an amino acid sequence selected from the
group consisting of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12. In
one embodiment, said antibody is labeled with a detectable moiety.
In one embodiment, said antibody is coupled to a therapeutic agent.
In one embodiment, said antibody is immobilized on a substrate.
[0013] In another aspect, the invention provides an antibody, or
antigen binding fragment thereof, that binds to the C-terminal
region of mucin-like protein (MLP), comprising:(i) a heavy chain
variable region comprising CDR-H1, CDR-H2 and CDR-H3 sequences; and
(ii) a light chain variable region comprising CDR-L1, CDR-L2 and
CDR-L3, wherein the heavy chain variable region CDR-H3 sequence
comprises an amino acid sequence set forth as SEQ ID NO:15, SEQ ID
NO:35, SEQ ID NO:36 or SEQ ID NO:19, and conservative sequence
modifications thereof, wherein the light chain variable region
CDR-L3 sequence comprises an amino acid sequence set forth as SEQ
ID NO:23 or SEQ ID NO:27, and conservative sequence modifications
thereof. In one embodiment, said antibody, or antigen binding
fragment thereof, comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ ID NO:14) and CDR-H3
(SEQ ID NO:15 or SEQ ID NO:35 or SEQ ID NO:36) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:21), CDR-L2 (SEQ
ID NO:22) and CDR-L3 (SEQ ID NO:23), and conservative modifications
thereof. In one embodiment, said antibody or antigen binding
fragment thereof comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ ID NO:16) and CDR-H3
(SEQ ID NO:15, SEQ ID NO:35, or SEQ ID NO:36) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:24), CDR-L2 (SEQ
ID NO:22) and CDR-L3 (SEQ ID NO:23), and conservative modifications
thereof. In one embodiment, said antibody, or antigen binding
fragment thereof comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:17), CDR-H2 (SEQ ID NO:18) and CDR-H3
(SEQ ID NO:19) and (ii) a light chain variable region comprising
CDR-L1 (SEQ ID NO:25), CDR-L2 (SEQ ID NO:26) and CDR-L3 (SEQ ID
NO:27), and conservative modifications thereof.
[0014] In another aspect, the invention provides an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO:1, or a
variant thereof having at least 95% identity to SEQ ID NO:1.
[0015] In another aspect, the invention provides an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO:4, or a
variant thereof having at least 95% identity to SEQ ID NO:4.
[0016] In another aspect, the invention provides an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO:5, or a
variant thereof having at least 95% identity to SEQ ID NO:5.
[0017] In another aspect, the invention provides an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO:6, or a
variant thereof having at least 95% identity to SEQ ID NO:6.
[0018] In another aspect, the invention provides a method of
detecting or diagnosing epithelial cancer by determining the
presence or amount of MLP in a biological sample from a test
subject. The method comprises (a) contacting a biological sample
from a test subject with an anti-MLP antibody or antigen-binding
fragment thereof in an in vitro immunoassay; and (b) detecting the
presence or absence of binding of said antibody, wherein the
detection of binding indicates the presence or amount of MLP in the
sample, wherein the antibody or fragment thereof binds to an
epitope in the C-terminal region of MLP, set forth as SEQ ID NO:4,
such as an epitope in SEQ ID NO:5 or SEQ ID NO:6. In some
embodiments, said anti-MLP antibody is labeled with a detectable
moiety and step (b) comprises detecting the presence or amount of
said detectable moiety. In some embodiments, the method further
comprises the step of comparing the amount of MLP detected in
accordance with step (b) with a reference standard or control
sample from a healthy subject, wherein an increase of at least
two-fold or higher in the level of MLP in the test sample as
compared to the control sample or reference standard indicates the
presence of, or increased risk for developing an epithelial cancer,
such as ovarian cancer or pancreatic cancer, in the test subject.
In some embodiments, the biological sample is selected from the
group consisting of blood, serum, plasma and tissue.
[0019] In another aspect, the invention provides a method of
detecting or diagnosing a mucin-secreting type of cancer, such as a
mucin-secreting cancer selected from the group consisting of
ovarian, pancreatic, colorectal, breast, appendiceal, lung, renal,
cervical, biliary, esophageal, epithelial skin and/or other
mucin-secreting types of cancer in a test subject by determining
the presence or amount of MLP comprising (a) administering to a
living test subject a humanized or fully human anti-MLP antibody or
antigen-binding fragment thereof that binds to an epitope in the
C-terminal region of MLP, set forth as SEQ ID NO:4, such as an
epitope in SEQ ID NO:5 or SEQ ID NO:6; and (b) detecting the
presence or absence or the amount of the antibody or fragment
thereof bound to MLP, wherein detection of the presence or amount
of MLP in the subject indicates the presence of ovarian,
pancreatic, colorectal, breast, appendiceal, lung, renal, cervical,
biliary, esophageal, epithelial skin and/or other mucin-secreting
types of cancer in the test subject. In one embodiment, the
anti-MLP antibody is labeled with a detectable moiety suitable for
in vivo use and step (b) comprises detecting the presence or amount
of the detectable moiety. In one embodiment, the method is used in
an imaging, intraoperative, endoscopic or intravascular
procedure.
[0020] In another aspect, the invention provides a method of
treating a subject suffering from a mucin-secreting type of cancer,
such as a mucin-secreting cancer selected from the group consisting
of ovarian, pancreatic, colorectal, breast, appendiceal, lung,
renal, cervical, biliary, esophageal, epithelial skin and/or other
mucin-secreting types of cancer comprising administering to an
individual suffering from a mucin-secreting type of cancer a
humanized or fully human anti-MLP antibody or antigen-binding
fragment thereof that binds to an epitope in the C-terminal region
of MLP, set forth as SEQ ID NO:4, such as an epitope in SEQ ID NO:5
or SEQ ID NO:6, wherein the antibody or fragment thereof is coupled
to a therapeutic agent.
[0021] In another aspect, the invention provides a kit for
detecting the present or amount of MLP in a biological sample, the
kit comprising (a) at least one container, and (b) at least one
anti-MLP antibody that binds to an epitope in the C-terminal region
of MLP, set forth as SEQ ID NO:4, such as an epitope in SEQ ID NO:5
or SEQ ID NO:6.
[0022] The anti-MLP antibodies, compositions and kits of the
invention can be used to practice the methods of the invention.
DESCRIPTION OF THE DRAWINGS
[0023] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
[0024] FIG. 1 depicts the amino acid sequence of full-length human
mucin-like protein (MLP) (SEQ ID NO:1), with the C-terminal region
(aa 279-431) (SEQ ID NO:4) underlined;
[0025] FIG. 2 depicts the amino acid sequence of a recombinant
fusion protein (SEQ ID NO:3) comprising an N-terminal histidine tag
fused to residues 279-431 of the C-terminal region of human MLP,
with the N-terminal vector-derived sequence underlined;
[0026] FIG. 3A graphically illustrates the results of an ELISA
assay carried out with anti-MLP monoclonal antibody clone B tested
against various concentrations of recombinant MLP (rMLP) C-terminal
protein, as described in Example 1;
[0027] FIG. 3B graphically illustrates the results of an ELISA
assay carried out with anti-MLP monoclonal antibody clone 11 tested
against various concentrations of recombinant MLP (rMLP) C-terminal
protein, as described in Example 1;
[0028] FIG. 4 graphically illustrates the results of an ELISA assay
carried out with anti-MLP monoclonal antibody clone 11 tested
against recombinant MLP (rMLP) C-terminal protein, as described in
Example 1;
[0029] FIG. 5 shows the results of a dot blot assay carried out
with anti-MLP monoclonal antibody Clone C, Clone B and Clone 11
against Panc1 cell line supernatant (row A), rMASP-3 polypeptide
(row B) and mMLP C-terminal protein (row C), as described in
Example 2;
[0030] FIG. 6 graphically illustrates the results of an ELISA assay
carried out with anti-MLP monoclonal antibody clones C, 11 and B
against concentrated supernatant from the ovarian cancer cell line
A2780 containing glycosylated full length MLP (gMLP), as described
in Example 2;
[0031] FIG. 7 shows the results of a dot blot assay carried out
with anti-MLP antibody Clone C, Clone B and Clone 11 against
ovarian cancer cell line A2780 supernatant (row A), rMASP-3 protein
(row B) and rMLP C-terminal protein (row C), as described in
Example 2;
[0032] FIG. 8, top panel: shows the results of a Western blot
demonstrating that anti-MLP clone C specifically recognizes gMLP in
A2780 (row 1), and rMLP C-terminal protein (row 3), but not rMASP-3
protein (row 2); middle panel:shows the results of a Western blot
demonstrating that anti-MLP clone B specifically recognizes gMLP in
A2780 (row 1), and rMLP C-terminal protein (row 3), but not rMASP-3
protein (row 2); and lower panel: shows the results of a Western
blot demonstrating that anti-MLP clone 11 specifically recognizes
gMLP in A2780 (row 1), and rMLP C-terminal protein (row 3), but not
rMASP-3 protein (row 2), as described in Example 2;
[0033] FIG. 9 graphically illustrates the results of an ELISA assay
carried out with anti-MLP monoclonal antibody clones C, 5, 11 and B
against concentrated supernatant from the ovarian cancer cell line
A2780 containing glycosylated full length MLP (gMLP), as described
in Example 2;
[0034] FIG. 10A depicts an amino acid sequence alignment between
the variable heavy chain regions of anti-MLP monoclonal antibody
clones 11 (SEQ ID NO:7), B (SEQ ID NO:8) and 2(C) (SEQ ID NO:9), as
described in Example 3; and
[0035] FIG. 10B depicts an amino acid sequence alignment between
the variable light chain regions of anti-MLP monoclonal antibody
clones 11 (SEQ ID NO:10), B (SEQ ID NO:11) and 2(C) (SEQ ID NO:12),
as described in Example 3.
DESCRIPTION OF THE SEQUENCE LISTING
[0036] ANTIGENS
[0037] SEQ ID NO:1: Amino acid sequence of the full length human
mucin-like protein (MLP), 431 amino acids in length.
[0038] SEQ ID NO:2: DNA encoding a fusion protein comprising an
N-terminal histidine tag and a polypeptide region corresponding to
residues 279-431 of the C-terminal region of human MLP.
[0039] SEQ ID NO:3: amino acid sequence of the fusion protein
(encoded by SEQ ID NO:2) comprising the N-terminal histidine
detection tag fused to residues 279-431 of the C-terminal region of
human MLP.
[0040] SEQ ID NO:4: amino acid sequence of the C-terminal region
(aa 279-431) of human MLP.
[0041] SEQ ID NO:5: SSGHRTEKRKRQQPQRRPAAGTGQRRGSEEMEEEG (amino acid
residues 397-431 of human MLP).
[0042] SEQ ID NO:6: EKRKRQQPQRRPAAGTGQRRGSEEMEEEG (amino acid
residues 403-431 of human MLP).
[0043] MONOCLONAL ANTIBODIES VII chains
[0044] SEQ ID NO:7: mAb clone 11: VH amino acid sequence
[0045] SEQ ID NO:8: mAb clone B: VH amino acid sequence
[0046] SEQ ID NO:9: mAb clone C: VH amino acid sequence
[0047] MONOCLONAL ANTIBODIES VL chains
[0048] SEQ ID NO:10: mAb clone 11: VL amino acid sequence
[0049] SEQ ID NO:11: mAb clone B: VL amino acid sequence
[0050] SEQ ID NO:12: mAb clone C: VL amino acid sequence
[0051] MONOCLONAL ANTIBODIES HEAVY CHAIN CDRS
[0052] SEQ ID NO:13: CDR-H1 from Clone 11 and Clone B
[0053] SEQ ID NO:14: CDR-H2 from Clone 11
[0054] SEQ ID NO:15: CDR-H3 from Clone 11
[0055] SEQ ID NO:35: CDR-H3 from Clone B
[0056] SEQ ID NO:16: CDR-H2 from Clone B
[0057] SEQ ID NO:17: CDR-H1 from Clone C
[0058] SEQ ID NO:18: CDR-H2 from Clone C
[0059] SEQ ID NO:19: CDR-H3 from Clone C
[0060] SEQ ID NO:20: CDR-H2 consensus from Clone 11 and Clone B
[0061] SEQ ID NO:36: CDR-H3 consensus from Clone 11 and Clone B
[0062] MONOCLONAL ANTIBODIES LIGHT CHAIN CDRS
[0063] SEQ ID NO:21: CDR-L1 from Clone 11
[0064] SEQ ID NO:22: CDR-L2 from Clone 11 and Clone B
[0065] SEQ ID NO:23: CDR-L3 from Clone 11 and Clone B
[0066] SEQ ID NO:24: CDR-L1 from Clone B
[0067] SEQ ID NO:25: CDR-L1 from Clone C
[0068] SEQ ID NO:26: CDR-L2 from Clone C
[0069] SEQ ID NO:27: CDR-L3 from Clone C
[0070] SEQ ID NO:28: CDR-L1 consensus from Clone 11 and Clone B
DNA encoding VII
[0071] SEQ ID NO:29: DNA encoding VH from Clone 11
[0072] SEQ ID NO:30: DNA encoding VH from Clone B
[0073] SEQ ID NO:31: DNA encoding VH from Clone C
DNA encoding VL
[0074] SEQ ID NO:32: DNA encoding VL from Clone 11
[0075] SEQ ID NO:33: DNA encoding VL from Clone B
[0076] SEQ ID NO:34: DNA encoding VL from Clone C
DETAILED DESCRIPTION
[0077] As described in Examples 1-3, high-affinity monoclonal
antibodies have been identified that specifically bind to the
C-terminal region of mucin-like protein (MLP), set forth as SEQ ID
NO:4, and are useful for early detection of ovarian cancer,
pancreatic cancer and other mucin-secreting malignant neoplasms in
patient serum samples. Accordingly, the present invention is
directed to monoclonal antibodies that specifically bind to the
C-terminal region of mucin-like protein (MLP) and the use of these
antibodies in methods of detecting a mucin-secreting type of
cancer, such as a mucin-secreting cancer selected from the group
consisting of ovarian, pancreatic, colorectal, breast, appendiceal,
lung, renal, cervical, biliary, esophageal, epithelial skin and/or
other mucin-secreting types of cancer mucin-secreting cancer
types.
[0078] I. Definitions
[0079] Unless specifically defined herein, all terms used herein
have the same meaning as would be understood by those of ordinary
skill in the art of the present invention. The following
definitions are provided in order to provide clarity with respect
to the terms as they are used in the specification and claims to
describe the present invention.
[0080] The terms "antibody" and "immunoglobulin" are used
interchangeably herein. These terms are well understood by those in
the field, and refer to a protein consisting of one or more
polypeptides that specifically binds an antigen. One form of
antibody constitutes the basic structural unit of an antibody. This
form is a tetramer and consists of two identical pairs of antibody
chains, each pair having one light and one heavy chain. In each
pair, the light and heavy chain variable regions are together
responsible for binding to an antigen, and the constant regions are
responsible for the antibody effector functions.
[0081] As used herein, the term "antibody" encompasses antibodies
and antibody fragments thereof, derived from any antibody-producing
mammal (e.g., mouse, rat, rabbit, and primate including human), or
from a hybridoma, phage selection, recombinant expression or
transgenic animals (or other methods of producing antibodies or
antibody fragments), that specifically bind to human mucin-like
protein (MLP), set forth as SEQ ID NO:1 or a portion thereof, such
as the C-terminal region of MLP (e.g., a C-terminal region of MLP
comprising or consisting of amino acid residues 279-431, set forth
as SEQ ID NO:4; or a C-terminal region of MLP comprising or
consisting of amino acid residues 397-431, set forth as SEQ ID
NO:5; or a C-terminal region of MLP comprising or consisting of
amino acid residues 403-431, set forth as SEQ ID NO:6). It is not
intended that the term "antibody" be limited as regards to the
source of the antibody or manner in which it is made (e.g., by
hybridoma, phage selection, recombinant expression, transgenic
animal, peptide synthesis, etc). Exemplary antibodies include
polyclonal, monoclonal and recombinant antibodies;
[0082] multispecific antibodies (e.g., bispecific antibodies);
humanized antibodies; fully human antibodies, murine antibodies;
chimeric, mouse-human, mouse-primate, primate-human monoclonal
antibodies; and anti-idiotype antibodies, and may be any intact
molecule or fragment thereof. As used herein, the term "antibody"
encompasses not only intact polyclonal or monoclonal antibodies,
but also fragments thereof (such as dAb, Fab, Fab', F(ab').sub.2,
Fv), single chain (ScFv), synthetic variants thereof, naturally
occurring variants, fusion proteins comprising an antibody portion
with an antigen-binding fragment of the required specificity,
humanized antibodies, chimeric antibodies, and any other modified
configuration of the immunoglobulin molecule that comprises an
antigen-binding site or fragment (epitope recognition site) of the
required specificity.
[0083] As used herein, the term "antigen-binding fragment" refers
to a polypeptide fragment that contains at least one CDR of an
immunoglobulin heavy and/or light chains that specifically binds to
human MLP (SEQ ID NO:1) or a portion thereof, such as the
C-terminal region of MLP (e.g., a region comprising or consisting
of amino acids amino acid residues 279-431, set forth as SEQ ID
NO:4; or a C-terminal region of MLP comprising or consisting of
amino acid residues 397-431, set forth as SEQ ID NO:5; or a
C-terminal region of MLP comprising or consisting of amino acid
residues 403-431, set forth as SEQ ID NO:6). In this regard, an
antigen-binding fragment of the herein described antibodies may
comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence set
forth herein from antibodies that bind MLP.
[0084] As used herein the term "anti-MLP monoclonal antibodies"
refers to a homogenous antibody population, wherein the monoclonal
antibody is comprised of amino acids that are involved in the
selective binding of an epitope on MLP, such as an epitope in the
C-terminal region of MLP (e.g., a region comprising or consisting
of amino acids amino acid residues 279-431, set forth as SEQ ID
NO:4; or a C-terminal region of MLP comprising or consisting of
amino acid residues 397-431, set forth as SEQ ID NO:5; or a
C-terminal region of MLP comprising or consisting of amino acid
residues 403-431, set forth as SEQ ID NO:6). Anti-MLP monoclonal
antibodies are highly specific for the MLP target antigen. The term
"monoclonal antibody" encompasses not only intact monoclonal
antibodies and full-length monoclonal antibodies, but also
fragments thereof (such as Fab, Fab', F(ab').sub.2, Fv), single
chain (ScFv), variants thereof, fusion proteins comprising an
antigen-binding portion, humanized monoclonal antibodies, chimeric
monoclonal antibodies, and any other modified configuration of the
immunoglobulin molecule that comprises an antigen-binding fragment
(epitope recognition site) of the required specificity and the
ability to bind to an epitope.
[0085] As used herein, the modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogenous population of antibodies, and is not intended to be
limited as regards the source of the antibody or the manner in
which it is made (e.g., by hybridoma, phage selection, recombinant
expression, transgenic animals, etc.). The term includes whole
immunoglobulins as well as the fragments etc. described above under
the definition of "antibody". Monoclonal antibodies can be obtained
using any technique that provides for the production of antibody
molecules by continuous cell lines in culture, such as the
hybridoma method described by Kohler, G., et al., Nature 256:495,
1975, or they may be made by recombinant DNA methods (see, e.g.,
U.S. Pat. No. 4,816,567 to Cabilly). Monoclonal antibodies may also
be isolated from phage antibody libraries using the techniques
described in Clackson, T., et al., Nature 352:624-628, 1991, and
Marks, J. D., et al., J. Mol. Biol. 222:581-597, 1991. Such
antibodies can be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof.
[0086] The recognized immunoglobulin polypeptides include the kappa
and lambda light chains and the alpha, gamma (IgG1, IgG2, IgG3,
IgG4), delta, epsilon and mu heavy chains or equivalents in other
species. Full-length immunoglobulin "light chains" (of about 25 kDa
or about 214 amino acids) comprise a variable region of about 110
amino acids at the NH.sub.2-terminus and a kappa or lambda constant
region at the COOH-terminus. Full-length immunoglobulin "heavy
chains" (of about 50 kDa or about 446 amino acids) similarly
comprise a variable region (of about 116 amino acids) and one of
the aforementioned heavy chain constant regions, e.g., gamma (of
about 330 amino acids).
[0087] The basic four-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains. An IgM antibody consists of 5 of the
basic heterotetramer units along with an additional polypeptide
called the J chain, and therefore contains 10 antigen binding
sites. Secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain. Each L chain is linked to an H chain by one covalent
disulfide bond, while the two H chains are linked to each other by
one or more by one or more disulfide bonds, depending on the H
chain isotype. Each H and L chain also has regularly spaced
intrachain disulfide bridges. The pairing of a VH and VL together
forms a single antigen-binding site.
[0088] Each H chain has at the N-terminus, a variable domain (VH),
followed by three constant domains (CH) for each of the a and y
chains, and four CH domains (CH) for .mu. and .epsilon.
isotypes.
[0089] Each L chain has at the N-terminus, a variable domain (VL)
followed by a constant domain (CL) at its other end. The VL is
aligned with the VH and the CL is aligned with the first constant
domain of the heavy chain (CH1). The L chain from any vertebrate
species can be assigned to one of two clearly distinct types,
called kappa (.kappa.) and lambda (.lamda.), based on the amino
acid sequences of their constant domains (CL).
[0090] Depending on the amino acid sequence of the constant domain
of their heavy chains (CH), immunoglobulins can be assigned to
different classes or isotypes. There are five classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains
designated alpha (.alpha.), delta (.delta.), epsilon (.epsilon.),
gamma (.gamma.) and mu (.mu.), respectively. The .gamma. and
.alpha. classes are further divided into subclasses on the basis of
minor differences in CH sequence and function, for example, humans
express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2.
[0091] For the structure and properties of the different classes of
antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition,
Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds);
Appleton and Lange, Norwalk, Conn., 1994, page 71 and Chapter
6.
[0092] The term "variable" refers to that fact that certain
segments of the V domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and defines
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the 110
amino acid span of the variable domains. Rather, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a beta-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the n-sheet
structure. The hypervariable regions in each chain are held
together in close proximity by the FRs and, with the hypervariable
regions from the other chain, contribute to the formation of the
antigen-binding site of antibodies (see Kabat, et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). The constant
domains are not involved directly in binding an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody dependent cellular cytotoxicity
(ADCC).
[0093] As used herein, the term "hypervariable region" refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region generally comprises amino
acid residues from a "complementary determining region" or "CDR"
(i.e., from around about residues 24-34 (L1), 50-56 (L2) and 89-97
(L3) in the light chain variable domain, and around about 31-35
(H1), 50-66 (H2) and 95-102 (H3) in the heavy chain variable domain
when numbering in accordance with the Kabat numbering system as
described in Kabat, et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)); and/or those residues from a
"hypervariable loop" (i.e., residues 24-34 (L1), 50-56 (L2) and
89-97 (L3) in the light chain variable domain, and 26-32 (H1),
52-56 (H2) and 95-101 (H3) in the heavy chain variable domain when
numbered in accordance with the Chothia numbering system, as
described in Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
and/or those residues from a "hypervariable loop"/CDR (e.g.,
residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and
27-38 (H1), 56-65 (H2), and 105-120 (H3) in the VH when numbered in
accordance with the IMGT numbering system as described in Lefranc,
J. P., et al., Nucleic Acids Res 27:209-212; Ruiz, M., et al.,
Nucleic Acids Res 28:219-221 (2000)).
[0094] As used herein, the term "antibody fragment" refers to a
portion derived from or related to a full-length anti-MLP antibody,
generally including the antigen binding or variable region thereof.
Illustrative examples of antibody fragments include Fab, Fab',
F(ab).sub.2, F(ab').sub.2 and Fv fragments, scFv fragments,
diabodies, linear antibodies, single-chain antibody molecules,
bispecific and multispecific antibodies formed from antibody
fragments.
[0095] Where bispecific antibodies are to be used, these may be
conventional bispecific antibodies, which can be manufactured in a
variety of ways (Holliger, P. and Winter G. Current Opinion
Biotechnol. 4, 446-449 (1993)), e.g., prepared chemically or from
hybrid hybridomas, or may be any of the bispecific antibody
fragments mentioned above.
[0096] As used herein, a "single-chain Fv" or "scFv" antibody
fragment comprises the V.sub.H and V.sub.L domains of an antibody,
wherein these domains are present in a single polypeptide chain.
Generally, the Fv polypeptide further comprises a polypeptide
linker between the V.sub.H and V.sub.L domains, which enables the
scFv to form the desired structure for antigen binding. See
Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994). "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and binding site. This fragment
consists of a dimer of one heavy and one light chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (three loops
each from the H and L chain) that contribute the amino acid
residues for antigen binding and confer antigen binding specificity
to the antibody. However, even a single variable domain (or half of
an Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0097] As used herein, the term "specific binding" refers to the
ability of an antibody to preferentially bind to a particular
analyte that is present in a homogeneous mixture of different
analytes. In certain embodiments, a specific binding interaction
will discriminate between desirable and undesirable analytes in a
sample, in some embodiments more than about 10 to 100-fold or more
(e.g., more than about 1000- or 10,000-fold). In certain
embodiments, the affinity between a capture agent and analyte when
they are specifically bound in a capture agent/analyte complex is
characterized by a K.sub.D (dissociation constant) of less than
about 100 nM, or less than about 50 nM, or less than about 25 nM,
or less than about 10 nM, or less than about 5 nM, or less than
about 1 nM.
[0098] As used herein, the term "variant" anti-MLP antibody refers
to a molecule, which differs in amino acid sequence from a "parent"
or reference antibody amino acid sequence by virtue of addition,
deletion, and/or substitution of one or more amino acid residue(s)
in the parent antibody sequence. In one embodiment, a variant
anti-MLP antibody refers to a molecule which contains variable
regions that are identical to the parent variable domains, except
for a combined total of 1, 2, 3, 4, 5, 6, 7, 8 9 or 10 amino acid
substitutions within the CDR regions of the heavy chain variable
region, and/or up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acid substitutions with said CDR regions of the light
chain variable region. In some embodiments, the amino acid
substitutions are conservative sequence modifications.
[0099] As used herein, the term "parent antibody" refers to an
antibody, which is encoded by an amino acid sequence used for the
preparation of the variant. Preferably, the parent antibody has a
human framework region and, if present, has human antibody constant
region(s). For example, the parent antibody may be a humanized or
fully human antibody.
[0100] As used herein, the term "isolated antibody" refers to an
antibody that has been identified and separated and/or recovered
from a component of its natural environment. Contaminant components
of its natural environment are materials, which would interfere
with diagnostic or therapeutic uses for the antibody, and may
include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. In preferred embodiments, the antibody
will be purified (1) to greater than 95% by weight of antibody as
determined by the Lowry method, and most preferably more than 99%
by weight; (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under
reducing or nonreducing conditions using Coomassie blue or,
preferably, silver stain. Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of
the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0101] As used herein, the term "epitope" refers to the portion of
an antigen to which a monoclonal antibody specifically binds.
Epitopic determinants usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and
usually have specific three-dimensional structural characteristics,
as well as specific charge characteristics. More specifically, the
term "MLP epitope," and "C-terminal MLP epitope" as used herein
refers to a portion of the corresponding polypeptide (SEQ ID NO:1
and/or SEQ ID NO:4 and/or SEQ ID NO:5 and/or SEQ ID NO:6) to which
an antibody immunospecifically binds as determined by any method
well known in the art, for example, by immunoassays. Antigenic
epitopes need not necessarily be immunogenic. Such epitopes can be
linear in nature or can be a discontinuous epitope. Thus, as used
herein, the term "conformational epitope" refers to a discontinuous
epitope formed by a spatial relationship between amino acids of an
antigen other than an unbroken series of amino acids.
[0102] As used herein, "a mammalian subject" includes without
limitation, humans, non-human primates, dogs, cats, horses, sheep,
goats, cows, rabbits, pigs and rodents.
[0103] As used herein, a "therapeutic agent" refers to a compound,
molecule or atom, currently known or later developed to act as
anti-neoplastics, anti-inflammatories, cytokines, anti-infectives,
enzyme activators or inhibitors, allosteric modifiers, antibiotics
or other agents administered to induce a desired therapeutic effect
in a patient which is administered separately, concurrently or
sequentially with an antibody moiety or conjugated to an antibody
moiety, i.e., antibody or antibody fragment, or a subfragment, and
is useful in the treatment of a subject suffering from a
pathological condition. The therapeutic agent may also be a toxin,
a chemotherapeutic or a radioisotope, wherein the therapeutic
moiety is intended for the killing of a cancer cell.
[0104] As used herein, a "detectable label" in the context of an
immunoconjugate refers to a portion of the immunoconjugate which is
a moiety having a property rendering its presence detectable, for
example a moiety detectable by spectroscopic, photochemical,
biochemical, immunochemical, chemical and/or other physical means.
A detectable moiety may be coupled either directly and/or
indirectly to the anti-MLP antibodies and antigen binding fragments
thereof of the present invention. For example, the immunoconjugate
may comprise an anti-MLP antibody that is labeled with a
radioactive isotope or enzymatic activity which permits detection
in an immunoassay.
[0105] As used herein, the amino acid residues are abbreviated as
follows: alanine (Ala;A), asparagine (Asn;N), aspartic acid
(Asp;D), arginine (Arg;R), cysteine (Cys;C), glutamic acid (Glu;E),
glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine
(Ile;I), leucine (Leu;L), lysine (Lys;K), methionine (Met;M),
phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine
(Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine
(Val;V).
[0106] In the broadest sense, the naturally occurring amino acids
can be divided into groups based upon the chemical characteristic
of the side chain of the respective amino acids. By "hydrophobic"
amino acid is meant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala,
Cys or Pro. By "hydrophilic" amino acid is meant either Gly, Asn,
Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino
acids can be further subclassed as follows. By "uncharged
hydrophilic" amino acid is meant either Ser, Thr, Asn or Gln. By
"acidic" amino acid is meant either Glu or Asp. By "basic" amino
acid is meant either Lys, Arg or His.
[0107] As used herein the term "conservative amino acid
substitution" is illustrated by a substitution among amino acids
within each of the following groups: (1) glycine, alanine, valine,
leucine, and isoleucine, (2) phenylalanine, tyrosine, and
tryptophan, (3) serine and threonine, (4) aspartate and glutamate,
(5) glutamine and asparagine, and (6) lysine, arginine and
histidine.
[0108] As used herein, an "isolated nucleic acid molecule" is a
nucleic acid molecule (e.g., a polynucleotide) that is not
integrated in the genomic DNA of an organism. For example, a DNA
molecule that encodes a growth factor that has been separated from
the genomic DNA of a cell is an isolated DNA molecule. Another
example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0109] As used herein, a "nucleic acid molecule construct" is a
nucleic acid molecule, either single- or double-stranded, that has
been modified through human intervention to contain segments of
nucleic acid combined and juxtaposed in an arrangement not existing
in nature.
[0110] As used herein, an "expression vector" is a nucleic acid
molecule encoding a gene that is expressed in a host cell.
Typically, an expression vector comprises a transcription promoter,
a gene, and a transcription terminator. Gene expression is usually
placed under the control of a promoter, and such a gene is said to
be "operably linked to" the promoter. Similarly, a regulatory
element and a core promoter are operably linked if the regulatory
element modulates the activity of the core promoter.
[0111] As used herein, the terms "approximately" or "about" in
reference to a number are generally taken to include numbers that
fall within a range of 5% in either direction (greater than or less
than) of the number unless otherwise stated or otherwise evident
from the context (except where such number would exceed 100% of a
possible value). Where ranges are stated, the endpoints are
included within the range unless otherwise stated or otherwise
evident from the context.
[0112] As used herein the singular forms "a", "an" and "the"
include plural aspects unless the context clearly dictates
otherwise. Thus, for example, reference to "a cell" includes a
single cell, as well as two or more cells; reference to "an agent"
includes one agent, as well as two or more agents; reference to "an
antibody" includes a plurality of such antibodies and reference to
"a framework region" includes reference to one or more framework
regions and equivalents thereof known to those skilled in the art,
and so forth.
[0113] Each embodiment in this specification is to be applied
mutatis mutandis to every other embodiment unless expressly stated
otherwise.
[0114] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. These and related techniques and
procedures may be generally performed according to conventional
methods well known in the art and as described in various general
and more specific references that are cited and discussed
throughout the present specification. See e.g., Sambrook et al.,
2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current
Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John
Wiley & Sons, Inc., NY, N.Y.); Current Protocols in Immunology
(Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies,
Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY,
N.Y.); or other relevant Current Protocol publications and other
like references. Unless specific definitions are provided, the
nomenclature utilized in connection with, and the laboratory
procedures and techniques of, molecular biology, analytical
chemistry, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and
commonly used in the art. Standard techniques may be used for
recombinant technology, molecular biological, microbiological,
chemical syntheses, chemical analyses, pharmaceutical preparation,
formulation, and delivery, and treatment of patients.
[0115] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa.
Furthermore, compositions of the invention can be used to achieve
methods of the invention.
[0116] II. Overview
[0117] As described in Examples 1-3 herein, the present invention
provides monoclonal anti-MLP antibodies that bind to human full
length mucin-like protein "MLP" (set forth as SEQ ID NO:1) with
high affinity and are capable of use as a biomarker for detection
of the presence of ovarian, pancreatic, colorectal, breast,
appendiceal, lung, renal, cervical, biliary, esophageal, epithelial
skin and/or other mucin-secreting types of cancer in a serum sample
from a human subject. In particular embodiments, the invention
provides monoclonal antibodies that specifically bind to an epitope
in the C-terminal region of human MLP (set forth as SEQ ID NO:4).
In some embodiments, the invention provides monoclonal antibodies
that specifically bind to an epitope in the C-terminal region of
MLP comprising or consisting of amino acid residues 397-431 of SEQ
ID NO:1 (set forth as SEQ ID NO:5). In some embodiments, the
invention provides monoclonal antibodies that specifically bind to
an epitope in the C-terminal region of MLP comprising or consisting
of amino acid residues 403-431 of SEQ ID NO:1 (set forth as SEQ ID
NO:6).
[0118] As described herein, the subject antibodies bind to
recombinant MLP and also to the naturally occurring glycosylated
form of MLP, referred to as "gMLP" that is secreted by epithelial
cancer cells, such as ovarian cancer cells, pancreatic cancer cells
and, for example, other adenocarcinoma cells. Therefore, the
subject antibodies can be used in diagnostic methods to detect the
presence of MLP in a biological sample obtained from a subject or
in a subject in vivo (e.g., diagnostic imaging) as a biomarker for
the presence or absence of epithelial cancer cells (e.g., ovarian
or pancreatic cancer cells) in the subject.
[0119] III. MLP Antigens
[0120] As described in Example 1 herein, the present inventors have
identified a single exon gene on chromosome 7 which encodes the
full-length sequence of MLP (also referred to as MUC-B), set forth
as SEQ ID NO:1, as shown in FIG. 1, which has a length of 431 amino
acid residues and contains a novel C-terminal region (set forth as
SEQ ID NO:4). As further described in Examples 1-3 herein, the
inventors have used the C-terminal region of MLP as an antigen to
generate anti-MLP antibodies suitable for use in the diagnostic and
therapeutic methods described herein.
[0121] Accordingly, in one aspect, the present invention provides
an isolated polypeptide comprising or consisting of the amino acid
sequence set forth in SEQ ID NO:1 or a variant thereof having an
amino acid sequence that is at least 90%, such as at least 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:1.
[0122] In one embodiment, the present invention provides an
isolated polypeptide comprising or consisting of the amino acid
sequence set forth in SEQ ID NO:4, or a variant thereof having an
amino acid sequence that is at least 90%, such as at least 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:4.
[0123] In one embodiment, the present invention provides an
isolated polypeptide comprising or consisting of the amino acid
sequence set forth in SEQ ID NO:3, or a variant thereof having an
amino acid sequence that is at least 90%, such as at least 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:3.
[0124] In one embodiment, the present invention provides an
isolated polypeptide comprising or consisting of the amino acid
sequence set forth in SEQ ID NO:5, or a variant thereof having an
amino acid sequence that is at least 90%, such as at least 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:5.
[0125] In one embodiment, the present invention provides an
isolated polypeptide comprising or consisting of the amino acid
sequence set forth in SEQ ID NO:6, or a variant thereof having an
amino acid sequence that is at least 90%, such as at least 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:6.
[0126] In one embodiment, the present invention provides an
isolated nucleic acid molecule encoding the amino acid sequence of
the polypeptide set forth as SEQ ID NO:1, or encoding a variant
thereof having an amino acid sequence that is at least 90%, such as
at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO:1. In one embodiment, the present invention provides an
isolated nucleic acid molecule encoding the amino acid sequence of
the polypeptide set forth as SEQ ID NO:4, or encoding a variant
thereof having an amino acid sequence that is at least 90%, such as
at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO:4. In one embodiment, the isolated nucleic acid sequence
comprises or consists of SEQ ID NO:2. In one embodiment, the
present invention provides an isolated nucleic acid molecule
encoding the amino acid sequence of the polypeptide set forth as
SEQ ID NO:5, or encoding a variant thereof having an amino acid
sequence that is at least 90%, such as at least 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5. In one
embodiment, the present invention provides an isolated nucleic acid
molecule encoding the amino acid sequence of the polypeptide set
forth as SEQ ID NO:6, or encoding a variant thereof having an amino
acid sequence that is at least 90%, such as at least 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6.
[0127] In one embodiment, the invention provides an expression
vector encoding at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5 or SEQ ID NO:6. In one embodiment the invention
provides an expression vector comprising SEQ ID NO:2. In one
embodiment, the invention provides a cell comprising a nucleic acid
encoding at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5 or SEQ ID NO:6.
[0128] IV. Anti-MLP Monoclonal Antibodies
[0129] As described in Examples 1 and 2 herein, the inventors have
used the C-terminal region of MLP (SEQ ID NO:4) as an antigen to
generate anti-MLP antibodies suitable for use in the diagnostic and
therapeutic methods described herein. As described in Example 3,
the variable light and heavy chain fragments of several
representative anti-MLP monoclonal antibodies have been cloned and
sequenced. FIG. 10A is an amino acid sequence alignment of the
variable heavy chain regions of three anti-MLP clones that were
identified as having high binding affinity to the C-terminal region
of MLP. FIG. 10B is an amino acid sequence alignment of the
variable light chain regions of three anti-MLP clones that were
identified as having high binding affinity to the C-terminal region
of MLP.
[0130] Substitutable positions of an MLP-specific antibody, as well
the choice of amino acids that may be substituted into those
positions, are revealed by aligning the heavy and light chain amino
acid sequences of the MLP-specific monoclonal antibodies discussed
above, and determining which amino acids occur at which positions
of those antibodies. In one exemplary embodiment, the heavy and
light chain amino acid sequences are aligned, and the identity of
amino acids at each position of the exemplary antibodies is
determined. As illustrated in TABLES 2 and 3 and FIG. 10A and 10B
(illustrating the amino acids present at each position of the heavy
and light chains of the MLP-specific monoclonal antibodies),
several substitutable positions, as well as the amino acid residues
that can be substituted into those positions, are readily
identified.
TABLE-US-00001 TABLE 1 Summary of MLP-specific monoclonal antibody
clones: K.sub.D clone (binding reference# VH VL Ig format to rMLP)
Clone 11 SEQ ID NO: 7 SEQ ID NO: 10 IgG2b, K 0.2 nM (encoded by
(encoded by SEQ ID NO: 29) SEQ ID NO: 32) Clone B SEQ ID NO: 8 SEQ
ID NO: 11 IgG2a, K <1 nM (encoded by (encoded by SEQ ID NO: 30)
SEQ ID NO: 33) Clone C SEQ ID NO: 9 SEQ ID NO: 12 IgG1, K <1 nM
(encoded by (encoded by SEQ ID NO: 31) SEQ ID NO: 34)
[0131] In certain embodiments, a subject MLP-specific monoclonal
antibody has a heavy chain variable domain that is substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical), to that
of any of the heavy chain variable domain sequences set forth in
TABLE 1.
[0132] In some embodiments, a subject MLP-specific monoclonal
antibody has a heavy chain variable domain that is substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical) to clone
11 (VH), set forth as SEQ ID NO:7. In some embodiments, the subject
MLP-specific monoclonal antibody has a heavy chain variable domain
that comprises, or consists of SEQ ID NO:7.
[0133] In some embodiments, a subject MLP-specific monoclonal
antibody has a heavy chain variable domain that is substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 96% identical, at least about 97% identical, at least
about 98% identical, or at least 99% identical) to clone B (VH),
set forth as SEQ ID NO:8. In some embodiments, the subject
MLP-specific monoclonal antibody has a heavy chain variable domain
that comprises, or consists of SEQ ID NO:8.
[0134] In some embodiments, a subject MLP-specific monoclonal
antibody has a heavy chain variable domain that is substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical) to clone C
(VH), set forth as SEQ ID NO:9. In some embodiments, the subject
MLP-specific monoclonal antibody has a heavy chain variable domain
that comprises, or consists of SEQ ID NO:9.
[0135] In some embodiments, a subject MLP-specific monoclonal
antibody has a light chain variable domain that is substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical), to that
of any of the light chain variable domain sequences set forth in
TABLE 1.
[0136] In some embodiments, a subject MLP-specific monoclonal
antibody has a light chain variable domain that is substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical) to clone
11 (VL), set forth as SEQ ID NO:10. In some embodiments, the
subject MLP-specific monoclonal antibody has a light chain that
comprises, or consists of SEQ ID NO:10.
[0137] In some embodiments, a subject MLP-specific monoclonal
antibody has a light chain variable domain that is substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical) to clone B
(VL), set forth as SEQ ID NO:11. In some embodiments, the subject
MLP-specific monoclonal inhibitory antibody has a light chain that
comprises, or consists of SEQ ID NO:11.
[0138] In some embodiments, a subject MLP-specific monoclonal
antibody has a light chain variable domain that is substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical) to clone C
(VL), set forth as SEQ ID NO:12. In some embodiments, the subject
MLP-specific monoclonal antibody has a light chain that comprises,
or consists of SEQ ID NO:12.
[0139] In some embodiments, the MLP-specific monoclonal antibodies
of the invention contain a heavy or light chain that is encoded by
a nucleotide sequence that hybridizes under high stringency
conditions to a nucleotide sequence encoding a heavy or light
chain, as set forth in TABLE 1 (e.g., SEQ ID NO:29, SEQ ID NO:30,
SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34). High
stringency conditions include incubation at 50.degree. C. or higher
in 0.1.times.SSC (15 mM saline/0.15 mM sodium citrate).
[0140] In some embodiments, the MLP-specific monoclonal antibodies
of the invention have a heavy chain variable region comprising one
or more CDRs (CDR1, CDR2 and/or CDR3) that are substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical), or
comprise or consist of the identical sequence as compared to the
amino acid sequence of the CDRs of any of the heavy chain variable
sequences described below in TABLES 2A-G and TABLE 3.
[0141] In some embodiments, the MLP-specific monoclonal antibodies
of the invention have a light chain variable region comprising one
or more CDRs (CDR1, CDR2 and/or CDR3) that are substantially
identical (e.g., at least about 70%, at least 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 96% identical, or at least about 97% identical, or
at least about 98% identical, or at least 99% identical), or
comprise or consist of the identical sequence as compared to the
amino acid sequence of the CDRs of any of the light chain variable
sequences described below in TABLES 4A-F and TABLE 5.
Heavy Chain Variable Region of MLP-Specific Monoclonal
Antibodies
TABLE-US-00002 [0142] TABLE 2A Heavy chain (aa 1-20) Heavy chain aa
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Clone 11 E V Q L
Q Q S G P E L V K P G A S V K I (SEQ NO: 7) Clone B E V Q L Q Q S G
P E L V K P G A S V K I (SEQ NO: 8) Clone C D V K L Q E S G G G L V
Q P G G S M K L (SEQ NO: 9)
TABLE-US-00003 TABLE 2B Heavy chain (aa 21-40) Heavy chain CDR-H1
aa 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Clone 11 S C K A S G Y A F T G F Y I H W M K Q S (SEQ NO: 7) Clone
B S C K A S G Y S F T G F Y I H W V K Q S (SEQ NO: 8) Clone C S C V
A S G F T F S N Y W M N W V R Q S (SEQ NO: 9)
TABLE-US-00004 TABLE 2C Heavy chain (aa 41-57) Heavy chain CDR-H2
aa 41 42 43 44 45 46 47 48 49 50 51 52 52A 52B 52C 53 54 55 56 57
Clone 11 H V K S L E W I G R I H P Y N G A T (SEQ NO: 7) Clone B H
V K S L E W I G R I H P Y N G A P (SEQ NO: 8) Clone C P E K G L E W
V A E I R L K S N N Y A I (SEQ NO: 9)
TABLE-US-00005 TABLE 2D Heavy chain (aa 58-77) Heavy chain CDR-H2
(cont'd) aa 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
76 77 Clone 11 S Y N Q N F K D R A S L T V D E S S S T (SEQ NO: 7)
Clone B T Y N Q N F K D R A R L T V H E S S S T (SEQ NO: 8) Clone C
N Y A E S V K G R F T I S R D D S K S S (SEQ NO: 9)
TABLE-US-00006 TABLE 2E Heavy chain (aa 78-94) Heavy chain aa 78 79
80 81 82 82A 82B 82C 83 84 85 86 87 88 89 90 91 92 93 94 Clone A Y
M E F Y G L T S E D S A V Y Y C A R 11 (SEQ NO: 7) Clone A Y M E F
F G L T S E D S A V Y Y C A R B (SEQ NO: 8) Clone V Y L D M N N L R
A E D T G I Y Y C T S C (SEQ NO: 9)
TABLE-US-00007 TABLE 2F heavy chain (aa 95-108) Heavy chain CDR-H3
aa 95 96 97 98 99 100 100A 100B 100C 100D 100E 100F 101 102 103 104
105 106 107 108 Clone E R V Y Y Y G S T Y E F D S W G Q G T T 11
(SEQ NO: 7) Clone E R V Y Y Y G S T Y E F D F W G Q G T T B (SEQ
NO: 8) Clone Y Y G S S L Y Y L D Y W G Q G T T C (SEQ NO: 9)
TABLE-US-00008 TABLE 2G heavy chain (aa 109-113) Heavy chain aa 109
110 111 112 113 Clone 11 L T V S S (SEQ NO: 7) Clone B L T V S S
(SEQ NO: 8) CloneC L T V S S (SEQ NO: 9)
[0143] Presented below are the heavy chain variable region (VH)
sequences for the monoclonal antibodies listed above in TABLE 1 and
TABLES 2A-G.
[0144] The Kabat CDRs (31-35 (H1), 50-66 (H2) and 95-102 (H3)) are
bolded; and the Chothia CDRs (26-32 (H1), 52-56 (H2) and 95-102
(H3)) are underlined.
TABLE-US-00009 Clone 11 heavy chain variable region (VH) (SEQ ID
NO: 7) EVQLQQSGPELVKPGASVKISCKASGYAFTGFYIHWMKQSHVKSLEWIGR
IHPYNGATSYNQNFKDRASLTVDESSSTAYMEFYGLTSEDSAVYYCARER
VYYYGSTYEFDSWGQGTTLTVSS Clone B heavy chain variable region (VH)
(SEQ ID NO: 8) EVQLQQSGPELVKPGASVKISCKASGYSFTGFYIHWVKQSHVKSLEWIGR
IHPYNGAPTYNQNFKDRARLTVHESSSTAYMEFFGLTSEDSAVYYCARER
VYYYGSTYEFDFWGQGTTLTVSS Clone C heavy chain variable region (VH)
(SEQ ID NO: 9) DVKLQESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE
IRLKSNNYAINYAESVKGRFTISRDDSKSSVYLDMNNLRAEDTGIYYCTS
YYGSSLYYLDYWGQGTTLTVSS
TABLE-US-00010 TABLE 3 Heavy Chain CDRs of MLP-specific monoclonal
antibodies Clone Amino Acid Reference CDR Sequence SEQ ID NO: Clone
11 CDR-H1 (kabat) GFYIH SEQ ID NO: 13 Clone B CDR-H1 (kabat) GFYIH
SEQ ID NO: 13 Clone C CDR-H1 (kabat) NYWMN SEQ ID NO: 17 Clone 11
CDR-H2 (kabat) RIHPYNGATSY SEQ ID NO: 14 NQNFKD Clone B CDR-H2
(kabat) RIHPYNGAPTY SEQ ID NO: 16 NQNFKD Clone C CDR-H2 (kabat)
EIRLKSNNYAI SEQ ID NO: 18 NYAESVKG Consensus CDR-H2 (kabat)
RIHPYNGAXXY SEQ ID NO: 20 from NQNFKD clones 11 (wherein X and B at
position 9 is T or P; and X at position 10 is S or T) Clone 11
CDR-H3 (kabat) ERVYYYGSTY SEQ ID NO: 15 EFDS Clone B CDR-H3 (kabat)
ERVYYYGSTY SEQ ID NO: 35 EFDF Clone C CDR-H3 (kabat) YYGSSLYYLDY
SEQ ID NO: 19 Consensus CDR-H3 (kabat) ERVYYYGSTYE SEQ ID NO: 36
from FDX clones 11 (wherein X and B at position 14 is S or F)
Light Chain Variable Regions of MLP-Specific Monoclonal
Antibodies
TABLE-US-00011 [0145] TABLE 4A Light chain (aa 1-20) Light chain aa
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Clone D V L M T
Q T P L S L P V S L G D Q A S 11 (SEQ NO: 10) Clone D V L M T Q T P
L S L P V S L G D Q A S B (SEQ NO: 11) Clone Q I V L T Q S P V I M
S A S P G E K V T C (SEQ NO: 12)
TABLE-US-00012 TABLE 4B Light chain (aa 21-35) Light chain CDR-L1
aa (Kabat) 21 22 23 24 25 26 27 27A 27B 27C 27D 27E 28 29 30 31 32
33 34 35 aa (Chothia) 21 22 23 24 25 26 27 28 29 30 30A 30B 30C 30D
30E 31 32 33 34 35 Clone 11 I S C R S G Q S I V H T S G V T Y L S W
(SEQ NO: 10) Clone B I S C R S G Q S I V H S S G V T Y L S W (SEQ
NO: 11) Clone C I S C S A S S S V S N M Y W (SEQ NO: 12) (Kabat)
Clone C I S C S A S S S V S N M Y W (SEQ NO: 12) (Chothia)
TABLE-US-00013 TABLE 4C Light chain (aa 36-55) Light chain CDR-L2
aa 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Clone Y L Q K P G Q S P K L L I Y K V F Y R F 11 (SEQ NO: 10) Clone
Y L Q K P G Q S P K L L I Y K V F Y R F B (SEQ NO: 11) Clone Y Q Q
K P G S S P K A W I Y R T S N L A C (SEQ NO: 12)
TABLE-US-00014 TABLE 4D Light chain (aa 56-75) Light chain CDR- L2
(cont'd) aa 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
74 75 Clone S G V P D R F S G S G S G T D F T L K I 11 (SEQ NO: 10)
Clone S G V P D R F S G S G S G T D F T L K I B (SEQ NO: 11) Clone
S G V P A R F S G S G S G T S Y S L T I C (SEQ NO: 12)
TABLE-US-00015 TABLE 4E Light chain (aa 76-95) Light chain CDR-L3
aa 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
Clone S R V E S E D L G V Y Y C F Q G S H V P 11 (SEQ NO: 10) Clone
B S R V E A E D L G V Y Y C F Q G S H V P (SEQ NO: 11) Clone C S S
M E A E D A A T Y Y C H Q Y Q S Y P (SEQ NO: 12)
TABLE-US-00016 TABLE 4F Light chain (aa 96-107) CDR-L3 (cont'd) aa
96 97 98 99 100 101 102 103 104 105 106 107 Clone P T F G A G T K L
E L K 11 (SEQ NO: 10) Clone P T F G T G T K L E L K B (SEQ NO: 11)
Clone R T F G A G T K L E I K C (SEQ NO: 12)
[0146] Presented below are the light chain variable region (VL)
sequences for the MLP-specific monoclonal antibodies listed above
in TABLE 1 and TABLES 4A-F.
[0147] The Kabat CDRs (24-34 (L1); 50-56 (L2); and 89-97 (L3) are
bolded; and the Chothia CDRs (24-34 (L1); 50-56 (L2) and 89-97 (L3)
are underlined. These regions are the same whether numbered by the
Kabat or Chothia system.
TABLE-US-00017 Clone 11 light chain variable region (VL) (SEQ ID
NO: 10) DVLMTQTPLSLPVSLGDQASISCRSGQSIVHTSGVTYLSWYLQKPGQSPK
LLIYKVFYRFSGVPDRFSGSGSGTDFTLKISRVESEDLGVYYCFQGSHVP PTFGAGTKLELK
Clone B light chain variable region (VL) (SEQ ID NO: 11)
DVLMTQTPLSLPVSLGDQASISCRSGQSIVHSSGVTYLSWYLQKPGQSPK
LLIYKVFYRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP PTFGTGTKLELK
Clone C light chain variable region (VL) (SEQ ID NO: 12)
QIVLTQSPVIMSASPGEKVTISCSASSSVSNMYWYQQKPGSSPKAWIYRT
SNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYQSYPRTFGAG TKLEIK
TABLE-US-00018 TABLE 5 Light Chain CDRs (Kabat) of the MLP-specific
antibodies Amino Acid Reference CDR Sequence SEQ ID NO: Clone 11
CDR-L1 RSGQSIVHTSGVTYLS SEQ ID NO: 21 Clone B CDR-L1
RSGQSIVHSSGVTYLS SEQ ID NO: 24 Clone C CDR-L1 SASSSVSNMY SEQ ID NO:
25 Consensus CDR-L1 RSGQSIVHXSGVTYLS SEQ ID NO: 28 of clone
(wherein X at 11 and B position 9 is T or S) Clone 11 CDR-L2
KVFYRFS SEQ ID NO: 22 Clone B CDR-L2 KVFYRFS SEQ ID NO: 22 Clone C
CDR-L2 RTSNLAS SEQ ID NO: 26 Clone 11 CDR-L3 FQGSHVPPT SEQ ID NO:
23 Clone B CDR-L3 FQGSHVPPT SEQ ID NO: 23 Clone C CDR-L3 HQYQSYPRT
SEQ ID NO: 27
[0148] In accordance with the foregoing, in one aspect, the present
invention provides an isolated antibody, or antigen binding
fragment thereof, that specifically binds to an epitope in the
C-terminal region of human mucin-like protein, set forth as SEQ ID
NO:4, such as an epitope in SEQ ID NO:5 or SEQ ID NO:6. In one
embodiment, the antibody is a monoclonal antibody. In one
embodiment, said antibody or antigen binding fragment thereof is
capable of binding to glycosylated human MLP secreted from an
epithelial cancer cell line. In one embodiment, said antibody or
antigen binding fragment thereof is capable of binding to
glycosylated human MLP in an ELISA assay format. In one embodiment,
said antibody or antigen binding fragment thereof binds to human
MLP with an KD of less than 10 nM, such as less than 1 nM. In one
embodiment, said antibody, or antigen binding fragment thereof
recognizes at least part of an epitope recognized by one or more
reference antibodies selected from the group consisting of: [0149]
(i) the monoclonal antibody MLP Clone 11 produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 having the ATCC
Designation Number PTA-121699; [0150] (ii) the monoclonal antibody
MLP Clone B produced by the hybridoma cell line deposited at the
ATCC on Oct. 30, 2014 having the ATCC Designation Number
PTA-121700; and [0151] (iii) the monoclonal antibody MLP Clone C
produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 having the ATCC Designation Number PTA-121701.
[0152] In one embodiment, said antibody comprises a variable region
of the heavy chain comprising or consisting of a sequence which is
at least 90% identical (e.g., at least 91%, or at least 92%, or at
least 93%,or at least 94%, or at least 95%, or at least 96%, or at
least 97%, or at least 98%, or at least 99% identical) to an amino
acid sequence selected from the group consisting of SEQ ID NO:7,
SEQ ID NO:8 and SEQ ID NO:9. In one embodiment, said antibody
comprises a variable region of the light chain comprising or
consisting of a sequence which is at least 90% identical (e.g., at
least 91%, or at least 92%, or at least 93%,or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at
least 99% identical) to an amino acid sequence selected from the
group consisting of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID
NO:12.
[0153] In another aspect, the invention provides an isolated
MLP-specific monoclonal antibody, or antigen binding fragment
thereof, that binds to human MLP (SEQ ID NO:1 or SEQ ID NO:4),
comprising: (i) a heavy chain variable region comprising CDR-H1,
CDR-H2 and CDR-H3 sequences; and (ii) a light chain variable region
comprising CDR-L1, CDR-L2 and CDR-L3, wherein the heavy chain
variable region CDR-H3 sequence comprises an amino acid sequence
set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID
NO:19, and conservative sequence modifications thereof, wherein the
light chain variable region CDR-L3 sequence comprises an amino acid
sequence set forth as SEQ ID NO:23 or SEQ ID NO:27, and
conservative sequence modifications thereof, and wherein the
isolated antibody binds human MLP (SEQ ID NO:1 or SEQ ID NO:4). In
one embodiment, the MLP-specific monoclonal antibody, or
antigen-binding fragment thereof, specifically binds to an epitope
in the C-terminal region of human MLP, set forth as SEQ ID NO:4
(i.e., amino acids 279-431 of SEQ ID NO:1), such as an epitope in
the C-terminal region of human MLP set forth as SEQ ID NO:5 (i.e.,
amino acids 397-431 of SEQ ID NO:1); or such as an epitope in the
C-terminal region of human MLP set forth as SEQ ID NO:6 (i.e, amino
acids 403-431 of SEQ ID NO:1)
[0154] In one embodiment, the heavy chain variable region CDR-H2
sequence comprises an amino acid sequence set forth as SEQ ID NO:
14, 16 or 18, and conservative sequence modifications thereof. In
one embodiment, the heavy chain variable region CDR-H1 sequence
comprises an amino acid sequence set forth as SEQ ID NO:13 or 17,
and conservative modifications thereof. In one embodiment, the
light chain variable region CDR-L2 sequence comprises an amino acid
sequence set forth as SEQ ID NO:22 or SEQ ID NO:26 and conservative
modifications thereof. In one embodiment, the light chain variable
region CDR-L1 sequence comprises an amino acid sequence set forth
as SEQ ID NO:21, 24, 25 or 28 and conservative modifications
thereof.
[0155] In one embodiment, the CDR-H2 of the heavy chain variable
region comprises SEQ ID NO:20. In one embodiment, the amino acid
sequence set forth in SEQ ID NO:20 contains a T (Thr) or P (Pro) at
position 9. In one embodiment, the amino acid sequence set forth
in
[0156] SEQ ID NO:20 contains an S (Ser) or T (Thr) at position 10.
In one embodiment, the CDR-H3 of the heavy chain variable region
comprises SEQ ID NO:15 (as shown in TABLE 3). In one embodiment,
the CDR-H3 of the heavy chain variable region comprises SEQ ID
NO:35 (as shown in TABLE 3). In one embodiment, the CDR-H3 of the
heavy chain variable region comprises SEQ ID NO:36 (as shown in
TABLE 3).
[0157] In one embodiment, the CDR-L1 of the light chain variable
region comprises SEQ ID NO:28 (as shown in TABLE 5). In one
embodiment, the amino acid set forth in SEQ ID NO:28 contains a T
(Thr) at position 9. In one embodiment, the amino acid sequence set
forth in SEQ ID NO:28 contains an S (Ser) at position 9.
[0158] In one embodiment, the CDR-L2 of the light chain variable
region comprises SEQ ID NO:22 (as shown in TABLE 5).
[0159] In one embodiment, the CDR-L3 of the light chain variable
region comprises SEQ ID NO:23 (as shown in TABLE 5).
[0160] In one embodiment, said antibody or antigen binding fragment
thereof binds MLP (SEQ ID NO:1 or SEQ ID NO :4) with a K.sub.D of
10 nM or less. In one embodiment, the conservative sequence
modifications thereof comprise or consist of a molecule which
contains variable regions that are identical to the recited
variable domain(s), except for a combined total of 1, 2, 3, 4, 5,
6, 7, 8 9 or 10 amino acid substitutions within the CDR regions of
the heavy chain variable region, and/or up to a combined total of
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions with said
CDR regions of the light chain variable region.
[0161] In another aspect, the invention provides an isolated
monoclonal antibody, or antigen binding fragment thereof, that
binds to human MLP (SEQ ID NO:1 or SEQ ID NO:4) wherein the
antibody comprises: I) a) a heavy chain variable region, numbered
according to Kabat, comprising: i) a heavy chain CDR-H1 comprising
the amino acid sequence from 31-35 of SEQ ID NO:7, SEQ ID NO:8 or
SEQ ID NO:9; and ii) a heavy chain CDR-H2 comprising the amino acid
sequence from 50-66 of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; and
iii) a heavy chain CDR-H3 comprising the amino acid sequence from
95-102 of SEQ ID NO:7; SEQ ID NO:8 or SEQ ID NO:9; and b) a light
chain variable region, numbered according to Kabat, comprising: i)
a light chain CDR-L1 comprising the amino acid sequence from 24-34
of SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12; and ii) a light
chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ
ID NO:10, SEQ ID NO:11 or SEQ ID NO:12; and iii) a light chain
CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID
NO:10, SEQ ID NO:11 or SEQ ID NO:12; or II) a variant thereof that
is otherwise identical to said variable domains, except for up to a
combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid
substitutions within said CDR regions of said heavy chain variable
region and up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acid substitutions within said CDR regions of said light
chain variable region, wherein the antibody or variant thereof
binds to human MLP (SEQ ID NO:1 or SEQ ID NO:4). In one embodiment,
said variant comprises an amino acid substitution at one or more
positions selected from the group consisting of position 28, 37,
57, 58, 68, 72, 82A or 102 of said heavy chain variable region,
numbered according to Kabat. In one embodiment, said variant
comprises an amino acid substitution at one or more positions
selected from the group consisting of position 27E, 80 or 100 of
said light chain variable region, numbered according to Kabat. In
one embodiment, the heavy chain variable region of said antibody
comprises SEQ ID NO:7. In one embodiment, the heavy chain variable
region of said antibody comprises or consists of SEQ ID NO:8. In
one embodiment, the heavy chain variable region comprises or
consists of SEQ ID NO:9. In one embodiment, the light chain
variable region of said antibody comprises or consists of SEQ ID
NO:10. In one embodiment, the light chain variable region of said
antibody comprises or consists of SEQ ID NO:11. In one embodiment,
the light chain variable region of said antibody comprises or
consists of SEQ ID NO:12.
[0162] In another aspect, the invention provides an isolated
monoclonal antibody that binds to human MLP (SEQ ID NO:1 or SEQ ID
NO:4), wherein the antibody comprises: I) a) a heavy chain variable
region, numbered according to Kabat, comprising, i) a heavy chain
CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID
NO:7; and ii) a heavy chain CDR-H2 comprising the amino acid
sequence from 50-66 of SEQ ID NO:7; and iii) a heavy chain CDR-H3
comprising the amino acid sequence from 95-102 of SEQ ID NO:7; and
b) a light chain variable region comprising: i) a light chain
CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID
NO:10; and ii) a light chain CDR-L2 comprising the amino acid
sequence from 50-56 of SEQ ID NO:10; and iii) a light chain CDR-L3
comprising the amino acid sequence from 89-97 of SEQ ID NO:10; or
II) a variant thereof that is otherwise identical to said variable
domains, except for up to a combined total of 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions within said CDR regions of
said heavy chain variable region and up to a combined total of 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions within said
CDR regions of said light chain variable region, wherein the
antibody or variant thereof binds to human MLP (SEQ ID NO:1 or SEQ
ID NO:4). In one embodiment, said variant comprises an amino acid
substitution at one or more positions selected from the group
consisting of position 28, 37, 57, 58, 68, 72, 82A or 102 of said
heavy chain variable region, numbered according to Kabat. In one
embodiment, said variant comprises an amino acid substitution at
one or more positions selected from the group consisting of
position 27E, 80 or 100 of said light chain variable region,
numbered according to Kabat. In one embodiment, the heavy chain of
said antibody comprises SEQ ID NO:7, or a variant thereof
comprising at least 80% identity to SEQ ID NO:7 (e.g., at least
85%, at least 90%, at least 95% or at least 98% identity to SEQ ID
NO:7). In one embodiment, the light chain of said antibody
comprises SEQ ID NO:10, or a variant thereof comprising at least
80% identity to SEQ ID NO:10 (e.g., at least 85%, at least 90%, at
least 95% or at least 98% identity to SEQ ID NO:10). In one
embodiment, said antibody binds MLP (SEQ ID NO:1 or SEQ ID NO:4)
with a K.sub.D of 10 nM or less.
[0163] In another aspect, the invention provides an isolated
monoclonal antibody that binds to human MLP (SEQ ID NO:1 or SEQ ID
NO:4), wherein the antibody, numbered according to Kabat,
comprises: I) a) a heavy chain variable region comprising: i) a
heavy chain CDR-H1 comprising the amino acid sequence from 31-35 of
SEQ ID NO:8; and ii) a heavy chain CDR-H2 comprising the amino acid
sequence from 50-66 of SEQ ID NO:8; and iii) a heavy chain CDR-H3
comprising the amino acid sequence from 95-102 of SEQ ID NO:8; and
b) a light chain variable region comprising: i) a light chain
CDR-L1 comprising the amino acid sequence from 24-34 of SEQ ID
NO:11; and ii) a light chain CDR-L2 comprising the amino acid
sequence from 50-56 of SEQ ID NO:11; and iii) a light chain CDR-L3
comprising the amino acid sequence from 89-97 of SEQ ID NO:11; or
II) a variant thereof that is otherwise identical to said variable
domains, except for up to a combined total of 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions within said CDR regions of
said heavy chain variable region and up to a combined total of 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions within said
CDR regions of said light chain variable region, wherein the
antibody or variant thereof binds to human MLP (SEQ ID NO:1 or SEQ
ID NO:4). In one embodiment, said variant comprises an amino acid
substitution at one or more positions selected from the group
consisting of position 28, 37, 57, 58, 68, 72, 82A or 102 of said
heavy chain variable region, numbered according to Kabat. In one
embodiment, said variant comprises an amino acid substitution at
one or more positions selected from the group consisting of
position 27E, 80 or 100 of said light chain variable region,
numbered according to Kabat.
[0164] In one embodiment, the heavy chain of said antibody
comprises SEQ ID NO:8, or a variant thereof comprising at least 80%
identity to SEQ ID NO:8 (e.g., at least 85%, at least 90%, at least
95% or at least 98% identity to SEQ ID NO:8). In one embodiment,
the light chain of said antibody comprises SEQ ID NO:11, or a
variant thereof comprising at least 80% identity to SEQ ID NO:11
(e.g., at least 85%, at least 90%, at least 95% or at least 98%
identity to SEQ ID NO:11). In one embodiment, said antibody binds
MLP (SEQ ID NO:1 or SEQ ID NO:4) with a K.sub.D of 10 nM or
less.
[0165] In one embodiment, said anti-MLP antibody, or antigen
binding fragment thereof, comprises (i) a heavy chain variable
region comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ ID NO:14) and
CDR-H3 (SEQ ID NO:15 or SEQ ID NO:35 or SEQ ID NO:36) and (ii) a
light chain variable region comprising CDR-L1 (SEQ ID NO:21),
CDR-L2 (SEQ ID NO:22) and CDR-L3 (SEQ ID NO:23), and conservative
modifications thereof.
[0166] In one embodiment, said anti-MLP antibody or antigen binding
fragment thereof comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ ID NO:16) and CDR-H3
(SEQ ID NO:15 or SEQ ID NO:35 or SEQ ID NO:36) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:24), CDR-L2 (SEQ
ID NO:22) and CDR-L3 (SEQ ID NO:23), and conservative modifications
thereof
[0167] In one embodiment, said anti-MLP antibody, or antigen
binding fragment thereof comprises (i) a heavy chain variable
region comprising CDR-H1 (SEQ ID NO:17), CDR-H2 (SEQ ID NO:18) and
CDR-H3 (SEQ ID NO:19) and (ii) a light chain variable region
comprising CDR-L1 (SEQ ID NO:25), CDR-L2 (SEQ ID NO:26) and CDR-L3
(SEQ ID NO:27), and conservative modifications thereof.
[0168] In one embodiment of any aspect of the invention disclosed
herein, said antibody is an antibody fragment selected from the
group consisting of Fv, Fab, Fab', F(ab).sub.2 and F(ab').sub.2. In
one embodiment, said antibody is a single chain molecule. In one
embodiment, said antibody is an IgG2 molecule. In one embodiment,
said antibody is an IgG1 molecule. In one embodiment, said antibody
is an IgG4 molecule. In one embodiment, said antibody is a
humanized, chimeric or fully human antibody.
[0169] In some embodiments, said antibody or antigen binding
fragment thereof specifically recognizes at least part of an
epitope recognized by at least one of (i) a reference antibody
comprising a heavy chain variable region as set forth in SEQ ID
NO:7 and a light chain variable region as set forth in SEQ ID
NO:10, such as reference antibody clone 11; or (ii) a reference
antibody comprising a heavy chain variable region as set forth in
SEQ ID NO:8 and a light chain variable region as set forth in SEQ
ID NO:11, such as reference antibody clone B; or (iii) a reference
antibody comprising a heavy chain variable region as set forth in
SEQ ID NO:9 and a light chain variable region as set forth in SEQ
ID NO:12, such as reference antibody clone C (see Table 1).
[0170] In accordance with the foregoing, an antibody or
antigen-binding fragment thereof according to certain preferred
embodiments of the present application may be one that competes for
binding to human MLP (SEQ ID NO:1 or SEQ ID NO:4) with any antibody
described herein. In some embodiments, the subject antibody both
(i) specifically binds to the antigen and (ii) comprises a VH
and/or VL domain disclosed herein, or comprises a CDR-H3 disclosed
herein, or a variant of any of these. Competition between binding
members may be assayed easily in vitro, for example using ELISA
and/or by tagging a specific reporter molecule to one binding
member which can be detected in the presence of other untagged
binding member(s), to enable identification of specific binding
members which bind the same epitope or an overlapping epitope.
Thus, there is presently provided a specific antibody or
antigen-binding fragment thereof, comprising an antibody
antigen-binding site which competes with an antibody described
herein that binds to human MLP (SEQ ID NO:1 or SEQ ID NO:4), such
as any one of clone 11, clone B or clone C as set forth in TABLE 1,
for binding to human MLP (SEQ ID NO:1 or SEQ ID NO:4).
[0171] Binding Affinity of anti-MLP Antibodies
[0172] The anti-MLP antibodies of the invention bind to human MLP
(SEQ ID NO:1 or SEQ ID NO:4) with a K.sub.D (dissociation constant)
of less than about 100 nM, or less than about 50 nM, or less than
about 25 nM, or less than about 10 nM, or less than about 5 nM, or
less than or equal to about 1 nM, or less than or equal to 0.1 nM.
The binding affinity of the anti-MLP antibodies can be determined
using a suitable binding assay known in the art, such as an ELISA
assay, as described in Examples 1-3 herein.
[0173] Variant anti-MLP Antibodies
[0174] The above-described monoclonal antibodies may be modified to
provide variant antibodies that specifically bind to human MLP. The
variant antibodies may be made by substituting, adding, or deleting
at least one amino acid of an above-described monoclonal antibody.
In general, these variant antibodies have the general
characteristics of the above-described antibodies and contain at
least the CDRs of an above-described antibody, or, in certain
embodiments, CDRs that are very similar to the CDRs of an
above-described antibody.
[0175] In the preferred embodiment, the variant comprises one or
more amino acid substitution(s) in one or more hypervariable
region(s) of the parent antibody. For example, the variant may
comprise at least one, e.g., from about one to about ten, such as
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9 or at least 10
substitutions, and preferably from about two to about six,
substitutions in one or more CDR regions of the parent antibody. In
one embodiment, said variant comprises an amino acid substitution
at one or more positions selected from the group consisting of
position 28, 37, 57, 58, 68, 72, 82A or 102 of said heavy chain
variable region, numbered according to Kabat. In one embodiment,
said variant comprises an amino acid substitution at one or more
positions selected from the group consisting of position 27E, 80 or
100 of said light chain variable region, numbered according to
Kabat.
[0176] In some embodiments, the variant antibodies have an amino
acid sequence that is otherwise identical to the variable domain of
a subject antibody set forth in TABLE 1, except for up to a
combined total of 1, 2, 3, 4, 5 or 6 amino acid substitutions
within said CDR regions of said heavy chain variable region and/or
up to a combined total of 1, 2, 3, 4, 5 or 6 amino acid
substitutions within said CDR regions of said light chain variable
region, wherein the antibody or variant thereof specifically binds
to human MLP (SEQ ID NO:1 or SEQ ID NO:4).
[0177] Ordinarily, the variant will have an amino acid sequence
having at least 75% amino acid sequence identity with the parent
antibody heavy or light chain variable domain sequences, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, and most preferably at least 95%, or at
least 96%, or at least 97%, or at least 98%, or at least 99%
identity. Identity or homology with respect to this sequence is
defined herein as the percentage of amino acid residues in the
candidate sequence that are identical with the parent antibody
residues, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity. None
of N-terminal, C-terminal, or internal extensions, deletions, or
insertions into the antibody sequence (such as, for example, signal
peptide sequences, linker sequences, or tags, such as HIS tags)
shall be construed as affecting sequence identity or homology. The
variant retains the ability to bind human MLP (SEQ ID NO:1 or SEQ
ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6) and preferably has
properties which are superior to those of the parent antibody. For
example, the variant may have a stronger binding affinity for
binding to MLP (SEQ ID NO:1 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID
NO:6).
[0178] To analyze such properties, one can compare a Fab form of
the variant to a Fab form of the parent antibody or a full-length
form of the variant to a full-length form of the parent antibody.
The variant antibody of particular interest herein is one which
displays at least about 10-fold, preferably at least about 20-fold,
and most preferably at least about 50-fold, enhancement in binding
affinity when compared to the parent antibody.
[0179] The antibodies of the invention may be modified to enhance
desirable properties, such as it may be desirable to control serum
half-life of the antibody. In general, complete antibody molecules
have a very long serum persistence, whereas fragments (<60-80
kDa) are filtered very rapidly through the kidney. Hence, if
long-term action of the antibody is desirable, the antibody is
preferably a complete full length IgG antibody (such as IgG2 or
IgG4), whereas if shorter action of the antibody is desirable, an
antibody fragment may be preferred.
[0180] Hybridomas Producing anti-MLP Monoclonal Antibodies (Clones
11, B and C)
[0181] The hybridoma cell line designated "Hybridoma MLP Clone 11"
was deposited with the American Type Culture Collection ("ATCC,"
Manassas, Va., USA) on Oct. 30, 2014, under the terms of the
Budapest Treaty. The ATCC accorded the Hybridoma MLP Clone 11 the
ATCC Designation Number: PTA-121699. This cell line is a mouse
hybridoma cell line derived from mouse spleen cells and secretes a
monoclonal antibody, which is herein referred to as ".alpha.MLP
Clone 11."
[0182] The hybridoma cell line designated "Hybridoma MLP clone B"
was deposited with the American Type Culture Collection ("ATCC",
Manassas, Va., USA) on Oct. 30, 2014, under the terms of the
Budapest Treaty. The ATCC accorded the Hybridoma MLP Clone B the
ATCC Designation Number PTA-121700. This cell line is a mouse
hybridoma cell line derived from mouse spleen cells and secretes a
monoclonal antibody, which is herein referred to as ".alpha.MLP
Clone B."
[0183] The hybridoma cell line designated "Hybridoma MLP clone C"
was deposited with the American Type Culture Collection ("ATCC",
Manassas, VA, USA) on Oct. 30, 2014, under the terms of the
Budapest Treaty. The ATCC accorded the Hybridoma MLP Clone C the
ATCC Designation Number PTA-121701. This cell line is a mouse
hybridoma cell line derived from mouse spleen cells and secretes a
monoclonal antibody, which is herein referred to as ".alpha.MLP
Clone C."
[0184] Accordingly, in one embodiment, the invention provides an
isolated anti-MLP monoclonal antibody produced by a hybridoma cell
line selected from the group of hybidoma cell lines deposited under
the ATCC Designation Numbers PTA-121699, PTA-121700 and
PTA-121701.
[0185] In another embodiment, the present invention provides a
hybridoma cell line selected from the group consisting of the
hybridoma cell line secreting .alpha.MLP Clone 11, the hybridoma
cell line secreting .alpha.MLP Clone B and the hybridoma cell line
secreting .alpha.MLP Clone C.
[0186] Single-Chain anti-MLP Antibodies
[0187] In one embodiment of the present invention, the anti-MLP
antibody is a single-chain antibody, defined as a genetically
engineered molecule containing the variable region of the light
chain, the variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single-chain molecule.
Such single-chain antibodies are also referred to as "single-chain
Fv" or "scFv" antibody fragments. Generally, the Fv polypeptide
further comprises a polypeptide linker between the VH and VL
domains that enables the scFv to form the desired structure for
antigen binding. The scFv antibodies that bind MLP can be oriented
with the variable light region either amino terminal to the
variable heavy region or carboxyl terminal to it.
[0188] Humanized anti-MLP Antibodies
[0189] The anti-MLP antibodies can be modified without changing
their ability to be used for the purposes described herein. As an
initial matter, it is noted that the antibodies described herein
originated from mice immunized with recombinant C-terminal MLP (SEQ
ID NO:4). The antibodies thus have framework regions (regions
outside the complementarity determining regions, or "CDRs") which
contain the amino acid residues usually found in the framework
regions in murine antibodies, and which may be immunogenic when
administered to a human patient. To reduce immunogenicity of murine
antibodies when used in humans, it is common in the art to engineer
the framework regions by replacing residues found at particular
positions in the antibodies of mice with the residues more
typically found at the same position in human antibodies.
Antibodies engineered in these ways are referred to as "humanized
antibodies" and are typically preferred for in vivo use, since they
have a lower risk of inducing side effects and typically can remain
in the circulation longer. Methods of humanizing antibodies are
known in the art and are set forth in detail in, for example, U.S.
Pat. Nos. 6,180,377; 6,407,213; 5,693,762; 5,585,089; and
5,530,101.
[0190] Further, since the CDRs of the variable regions determine
antibody specificity, the CDRs set forth in Tables 2A-G, Table 3,
Tables 4A-F, and Table 5 can be grafted or engineered into an
antibody of choice to confer specificity for binding to MLP upon
that antibody. For example, the CDRs from clones 11, B and C can be
grafted onto a human antibody framework of known three dimensional
structure (see e.g., WO98/45322; Jones et al., Nature 321:522
(1986); Verhoeyen et al., Science 239:1534 (1988); Riechmann et
al., Nature 332:323 (1988) and Winter & Milstein, Nature
349:293 (1991) to generate an anti-MLP antibody with reduced or no
immunogenic responses when administered to humans.
[0191] Methods for Producing anti-MLP Antibodies
[0192] In another aspect, the present invention provides a method
of producing an antibody specifically recognizing and binding an
epitope within the full-length human MLP polypeptide (SEQ ID NO:1),
such as within the C-terminal portion of human MLP (SEQ ID NO:4),
such as an epitope in the C-terminal region of human MLP set forth
as SEQ ID NO:5 (i.e., amino acids 397-431 of SEQ ID NO:1); or such
as an epitope in the C-terminal region of human MLP set forth as
SEQ ID NO:6 (i.e, amino acids 403-431 of SEQ ID NO:1) said method
comprising administering to a mammal a polypeptide comprising or
consisting of SEQ ID NO:1, or a portion thereof, such as a
polypeptide comprising or consisting of SEQ ID NO:4, or such as a
polypeptide comprising or consisting of SEQ ID NO:5, or such as a
polypeptide comprising or consisting of SEQ ID NO:6 and selecting
antibodies recognizing human MLP.
[0193] In many embodiments, the nucleic acids encoding a subject
monoclonal antibody are introduced directly into a host cell, and
the cell incubated under conditions sufficient to induce expression
of the encoded antibody.
[0194] In some embodiments, the invention provides a nucleic acid
molecule encoding an anti-MLP antibody, or fragment thereof, of the
invention, such as an antibody or fragment thereof set forth in
TABLE 1. In some embodiments the invention provides a nucleic acid
molecule comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID
NO:32, SEQ ID NO:33 and SEQ ID NO:34.
[0195] In some embodiments, the invention provides a cell
comprising a nucleic acid molecule encoding an MLP-specific
monoclonal antibody of the invention.
[0196] In some embodiments, the invention provides an expression
cassette comprising a nucleic acid molecule encoding an
MLP-specific monoclonal antibody of the invention.
[0197] In some embodiments, the invention provides a method of
producing MLP-specific monoclonal antibodies comprising culturing a
cell comprising a nucleic acid molecule encoding an MLP-specific
antibody of the invention.
[0198] In one embodiment, the method of producing an antibody
specifically recognizing an epitope within the C-terminal portion
of human MLP comprises culturing a hybridoma cell line selected
from the group consisting of a hybridoma cell line secreting
.alpha.MLP Clone 11, a hybridoma cell line secreting .alpha.MLP
Clone B and a hybridoma cell line secreting .alpha.MLP Clone C.
[0199] According to certain related embodiments there is provided a
recombinant host cell which comprises one or more constructs as
described herein; a nucleic acid encoding any anti-MLP antibody,
CDR, VH or VL domain, or antigen-binding fragment thereof; and a
method of production of the encoded product, which method comprises
expression from encoding nucleic acid therefor. Expression may
conveniently be achieved by culturing under appropriate conditions
recombinant host cells containing the nucleic acid. Following
production by expression, an antibody or antigen-binding fragment
thereof, may be isolated and/or purified using any suitable
technique, and then used as desired.
[0200] For example, any cell suitable for expression of expression
cassettes may be used as a host cell, for example, yeast, insect,
plant, etc., cells. In many embodiments, a mammalian host cell line
that does not ordinarily produce antibodies is used, examples of
which are as follows: monkey kidney cells (COS cells), monkey
kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 165 1); human
embryonic kidney cells (HEK-293, Graham et al., J. Gen Virol. 36:59
(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese
hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.
(USA) 77:4216, (1980); mouse sertoli cells (TM4, Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70);
African green monkey kidney cells (VERO-76, ATCC CRL-1587); human
cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep
G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI
cells (Mather et al., Annals N.Y. Acad. Sci 383:44-68 (1982));
NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1).
Additional cell lines will become apparent to those of ordinary
skill in the art. A wide variety of cell lines are available from
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209.
[0201] Methods of introducing nucleic acids into cells are well
known in the art. Suitable methods include electroporation,
particle gun technology, calcium phosphate precipitation, direct
microinjection, and the like. The choice of method is generally
dependent on the type of cell being transformed and the
circumstances under which the transformation is taking place (i.e.,
in vitro, ex vivo, or in vivo). A general discussion of these
methods can be found in Ausubel, et al., Short Protocols in
Molecular Biology, 3d ed., Wiley & Sons, 1995. In some
embodiments, lipofectamine and calcium mediated gene transfer
technologies are used.
[0202] After the subject nucleic acids have been introduced into a
cell, the cell is typically incubated, normally at 37.degree. C.,
sometimes under selection, for a suitable time to allow for the
expression of the antibody. In most embodiments, the antibody is
typically secreted into the supernatant of the media in which the
cell is growing in.
[0203] In mammalian host cells, a number of viral-based expression
systems may be utilized to express a subject antibody. In cases
where an adenovirus is used as an expression vector, the antibody
coding sequence of interest may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing the antibody molecule in
infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad.
Sci. USA 81:355-359 (1984)). The efficiency of expression may be
enhanced by the inclusion of appropriate transcription enhancer
elements, transcription terminators, etc. (see Bittner et al.,
Methods in Enzymol. 153:51-544 (1987)).
[0204] For long-term, high-yield production of recombinant
antibodies, stable expression may be used. For example, cell lines,
which stably express the antibody molecule, may be engineered.
Rather than using expression vectors, which contain viral origins
of replication, host cells can be transformed with immunoglobulin
expression cassettes and a selectable marker. Following the
introduction of the foreign DNA, engineered cells may be allowed to
grow for 1-2 days in an enriched media, and then are switched to a
selective media. The selectable marker in the recombinant plasmid
confers resistance to the selection and allows cells to stably
integrate the plasmid into a chromosome and grow to form foci,
which in turn can be cloned and expanded into cell lines. Such
engineered cell lines may be particularly useful in screening and
evaluation of compounds that interact directly or indirectly with
the antibody molecule.
[0205] Once an antibody molecule of the invention has been
produced, it may be purified by any method known in the art for
purification of an immunoglobulin molecule, for example, by
chromatography (e.g., ion exchange, affinity, particularly by
affinity for the specific antigen after Protein A, and sizing
column chromatography), centrifugation, differential solubility, or
by any other standard technique for the purification of proteins.
In many embodiments, antibodies are secreted from the cell into
culture medium and harvested from the culture medium. For example,
a nucleic acid sequence encoding a signal peptide may be included
adjacent the coding region of the antibody or fragment. Such a
signal peptide may be incorporated adjacent to the 5' end of the
amino acid sequences set forth herein for the subject antibodies in
order to facilitate production of the subject antibodies.
[0206] Anti-MLP Antibodies Labeled with a Detectable Moiety
[0207] In another aspect, the invention provides anti-MLP
antibodies labeled with a detectable moiety (i.e., a moiety that
permits detection and/or quantitation), which can be used for
diagnostic applications. As used herein "a detectable moiety" is a
moiety detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical and/or other physical means. A detectable
moiety may be coupled either directly and/or indirectly (for
example via a covalent linkage by chemical or recombinant means) to
antibodies and antigen binding fragments thereof of the present
invention using methods well known in the art. These labeled
anti-MLP antibodies can be used, for example, in in-vitro assays to
detect the presence of MLP in a biological sample or in in vivo
assays (e.g., imaging) to detect the presence of MLP-expressing
cells in a living body.
[0208] Numerous labels are available which can be generally grouped
into the following categories:
[0209] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. The anti-MLP antibody can be labeled with
the radioisotope using the techniques described in Current
Protocols in Immunology, Volumes 1 and 2, Gutigen et al., Ed.,
Wiley-Interscience. New York, N.Y. Pubs., (1991) for example and
radioactivity can be measured using scintillation counting.
Radioisotopes may be bound to antibody either directly or
indirectly by using an intermediary functional group. Useful
intermediary functional groups include chelators such as
ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic
acid. For example, see Shih et al., Int J Cancer 46: 1101 (1990)
and U.S. Pat. No. 5,057,313. The subject anti-MLP antibodies, and
antibody fragments also can be labeled with paramagnetic ions and a
variety of radiological contrast agents for purposes of in vivo
diagnosis. Contrast agents that are particularly useful for
magnetic resonance imaging comprise gadolinium, manganese,
dysprosium, lanthanum, or iron ions. Additional agents include
chromium, copper, cobalt, nickel, rhenium, europium, terbium,
holmium, or neodymium. The subject anti-MLP antibodies and
fragments thereof can also be conjugated to ultrasound
contrast/enhancing agents. For example, one ultrasound contrast
agent is a liposome. [0210] (b) Fluorescent labels such as rare
earth chelates (europium chelates) or fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, Lissamine,
phycoerythrin and Texas Red are available. The fluorescent labels
can be conjugated to the antibody using the techniques disclosed in
Current Protocols in Immunology, supra for example. Fluorescence
can be quantified using a fluorimeter. [0211] (c) Various
enzyme-substrate labels are available. The enzyme generally
catalyses a chemical alteration of the chromogenic substrate, which
can be measured using various techniques. For example, the enzyme
may catalyze a color change in a substrate, which can be measured
spectrophotometrically. Alternatively, the enzyme may alter the
fluorescence or chemiluminescence of the substrate. Techniques for
quantifying a change in fluorescence are described above. The
chemiluminescent substrate becomes electronically excited by a
chemical reaction and may then emit light that can be measured
(using a chemiluminometer, for example) or donates energy to a
fluorescent acceptor. Examples of enzymatic labels include
luciferases (e.g, firefly luciferase and bacterial luciferase; U.S.
Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,
malate dehydrogenase, urease, peroxidase such as horseradish
peroxidase (HRPO), alkaline phosphatase. 0-galactosidase,
glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase,
galactose oxidase, and glucose-6-phosphate dehydrogenase),
heterocyclic oxidases (such as uricase and xanthine oxidase),
lactoperoxidase, microperoxidase, and the like. Techniques for
conjugating enzymes to antibodies are described in O'Sullivan et
al., Methods for the Preparation of Enzyme-Antibody Conjugates for
use in Enzyme Immunoassay, in Methods in Enzyme (ed Langone &
H. Van Vunakis), Academic Press, New York, 73: 147-166 (1981).
[0212] Examples of enzyme-substrate combinations include, for
example: [0213] (i) Horseradish peroxidase (HRPO) with hydrogen
peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes
a dye precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)); [0214] (ii)
alkaline phosphatase (AP) with para-Nitrophenyl phosphate as
chromogenic substrate; and [0215] (iii) .beta.-D-galactosidase
(.beta.-D-Gal) with a chromogenic substrate (e.g.,
p-nitrophenyl-.beta.-D-galactosidase) or the fluorogenic substrate
4-methylumbelliferyl-.beta.-D-galactosidase.
[0216] Numerous other enzyme-substrate combinations are available
to those skilled in the art.
[0217] In some embodiments, the label is indirectly conjugated with
the anti-MLP antibody. The skilled artisan will be aware of various
techniques for achieving this. For example, the anti-MLP antibody
can be conjugated with biotin and any of the three broad categories
of labels mentioned above can be conjugated with avidin, or vice
versa. Biotin binds selectively to avidin and thus, the label can
be conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the anti-MLP antibody is conjugated with a small
hapten (e.g., digoxin) and one of the different types of labels
mentioned above is conjugated with an anti-hapten antibody (e.g.,
anti-digoxin antibody). Thus, indirect conjugation of the label
with the antibody can be achieved.
[0218] In another embodiment, the anti-MLP antibody is not labeled
(i.e., is naked), and the presence thereof can be detected using a
labeled antibody which binds to the anti-MLP antibody.
[0219] Anti-MLP Antibodies Coupled to a Therapeutic Agent
[0220] In another aspect, the invention provides anti-MLP
antibodies coupled to a therapeutic agent, which can be used in
vivo to target therapeutic molecules to MLP expressing cells. For
example, an immunoconjugate comprising an anti-MLP antibody and a
therapeutic moiety can be used to inhibit the growth and
proliferation of cancer cells bearing the MLP antigen. As used
herein, the term "therapeutic agent" is a compound, molecule or
atom which is administered separately, concurrently or sequentially
with an antibody moiety or conjugated to an antibody moiety, i.e.,
antibody or antibody fragment, or a subfragment, and is useful in
the treatment of a subject suffering from a pathological condition,
such as, for example, an epithelial cancer. Examples of therapeutic
agents include antibodies, antibody fragments, cytotoxic agents,
drugs, toxins, nucleases, hormones, immunomodulators,
anti-angiogenic agents, boron compounds, photoactive agents or
dyes, radioisotopes, chemotherapeutic drugs such as vinca
alkaloids, anthracyclines, gemcitabine, epipodophyllotoxins,
taxanes, antimetabolites, alkylating agents, antibiotics, SN-38,
COX-2 inhibitors, antimitotics, antiangiogenic and apoptotic
agents, particularly doxorubicin, methotrexate, taxol, CPT-11,
camptothecans, proteosome inhibitors, mTOR inhibitors, HDAC
inhibitors, tyrosine kinase inhibitors, and others from these and
other classes of anticancer agents. Other useful cancer
chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates,
nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors,
antimetabolites, pyrimidine analogs, purine analogs, platinum
coordination complexes, mTOR inhibitors, tyrosine kinase
inhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins
and hormones. Suitable chemotherapeutic agents are described in
REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.
1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF
THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as
revised editions of these publications. Other suitable
chemotherapeutic agents, such as experimental drugs, are known to
those of skill in the art.
[0221] Any of the anti-MLP antibodies, antibody fragments and
fusion proteins disclosed herein can be conjugated with one or more
therapeutic agents, using a variety of techniques known in the art.
One or more therapeutic or diagnostic agents (e.g., a detectable
moiety) may be attached to each antibody, antibody fragment or
fusion protein, for example by conjugating an agent to a
carbohydrate moiety in the Fc region of the antibody. If the Fc
region is absent (for example with certain antibody fragments), it
is possible to introduce a carbohydrate moiety into the light chain
variable region of either an antibody or antibody fragment to which
a therapeutic or diagnostic agent may be attached. See, for
example, Leung et al., J Immunol. 154: 5919 (1995); Hansen et al.,
U.S. Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No.
6,254,868, the Examples section of each patent incorporated herein
by reference.
[0222] Methods for conjugating peptides to antibody components via
an antibody carbohydrate moiety are well-known to those of skill in
the art. See, for example, Shih et al., Int J Cancer 41: 832
(1988); Shih et al., Int J Cancer 46: 1101 (1990); and Shih et al.,
U.S. Pat. No. 5,057,313, the Examples section of which is
incorporated herein by reference. The general method involves
reacting an antibody component having an oxidized carbohydrate
portion with a carrier polymer that has at least one free amine
function and that is loaded with a plurality of therapeutic agents,
such as peptides or drugs. This reaction results in an initial
Schiff base (imine) linkage, which can be stabilized by reduction
to a secondary amine to form the final conjugate.
[0223] As another example, a therapeutic or diagnostic agent can be
attached at the hinge region of a reduced antibody component via
disulfide bond formation. As an alternative, such agents can be
attached to the antibody component using a heterobifunctional
cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate
(SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). General
techniques for such conjugation are well-known in the art. See, for
example, Wong, Chemistry of Protein Conjugation and Cross-linking
(CRC Press 1991); Upeslacis et al., "Modification of Antibodies by
Chemical Methods," in Monoclonal Antibodies: Principles and
Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.
1995); Price, "Production and Characterization of Synthetic
Peptide-Derived Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), pages
60-84 (Cambridge University Press 1995).
V. Methods of Detecting Epithelial Cancer (e.g., Ovarian and/or
Pancreatic Cancer) using Anti-MLP Antibodies
[0224] As described herein, the inventors have generated anti-MLP
antibodies that are suitable for use in a diagnostic assay for
detecting early stage epithelial cancer (e.g., ovarian and/or
pancreatic cancer). A brief overview of ovarian cancer tumor types,
risk factors, diagnosis and classification is provided below.
[0225] 1. Overview of Ovarian Cancer Tumor Types, Risk Factors,
Diagnosis and Classification
Ovarian Tumor Types
[0226] Ovarian tumors are divided into benign tumors and malignant
tumors. The benign tumor is an abnormal cell growth, stays local
and does not spread to other organs in the body. There are usually
no symptoms. However, the second type of the tumor is cancerous
(malignant) and is able to metastasize to other parts of the body.
Ovarian tumors are classified histopathologically according to
their cellular origin into epithelial, stroma and germ cell tumors
as shown below in Table 6.
TABLE-US-00019 TABLE 6 Ovarian Tumor Types Type of ovarian % Female
age tumor Incidence groups Epithelial ovarian 90% After tumors
menopause Germ cell tumors 3% Young girl Stromal tumors 6% No
specific ages
Table 6. The incidence of different types of ovarian tumors and the
age groups affected. Metastatic tumors 1% (ovary has tumors) due to
secondary metastases to other parts of the body such as stomach,
colon and breast (Chauhan et al., Jour Ovarian Res 2:212-215,
2009).
[0227] Epithelial tumors constitute 90% of all ovarian tumors
(Chauhan et al., Jour Ovarian Res 2:212-215, 2009; Nguyen et al.,
Women's Health, 9: 171-187, 2013). Most epithelial tumors are
benign tumors, for example, serous adenomas and Brenner tumors.
Malignant epithelial tumors are a real threat to patients in around
90% of cases because they are diagnosed at the late stages. The
incidence of these tumors is great after menopause (Chauhan et al.,
Jour Ovarian Res 2:212-215, 2009; Nguyen et al., Women's Health, 9:
171-187, 2013. Some epithelial ovarian tumors may appear with low
malignant potential (LMP tumors), which are tumors with slow growth
and low spreading rate. These LMP tumors are also known as
borderline epithelial ovarian cancers as this type of tumor is not
clearly visible under the microscope (Altchek et al., Diagnosis and
Management of Ovarian Disorders, 2.sup.nd edition, San Diego
Calif., Academic Press, 2003).
[0228] Germ cells are located in the ovary responsible for the
producing the egg. Germ cell tumors arise from the germinal cells
and they constitute about 3% of all ovarian tumors (Chauhan et al.,
Jour Ovarian Res 2:212-215, 2009). Mostly they are benign rather
than malignant. The incidence of these tumors is greater in young
females. Examples include, mature teratomas and endodermal sinus
(Altchek et al., Diagnosis and Management of Ovarian Disorders,
2.sup.nd Edition, San Diego Calif., Academic Press, 2003).
[0229] Stromal cell tumors originate from connective tissue that
hold the ovaries together and these cells also have the function of
producing hormones such as progesterone and estrogen (Altchek et
al., Diagnosis and Management of Ovarian Disorders, 2.sup.nd
edition, San Diego Calif., Academic Press, 2003). These tumors
constitute 6% of all ovarian tumors (Chauhan et al., Jour Ovarian
Res 2:212-215, 2009). They affect both young and post-menopausal
age groups and include granulose theca tumor and granulose cell
tumors (Altechek et al., 2003).
Risk Factors for Ovarian Cancer
[0230] Risk factors for ovarian cancer include endometriosis
(Modugno et al., American J of Obstetrics and Gynecology
191:733-740, 2004), ovarian cysts (Alteck et al., 2003), age
(post-menopause, with increased risk for women age 65 years or
older (Yancik et al., Cancer 71:517-523, 1993; Guppy et al., 2005),
smoking (Jordan et al., Gynecologic Oncology 103:1122-1129, 2006),
and obesity (McLemore et al., Cancer Nursing 32:281, 2009). Also,
mutations in breast cancer type 1 (BRCA1) located on chromosome 17q
and/or mutations in breast cancer type 2 (BRCA2) located on
chromosome 13q are regarded as high risk factors for breast and
ovarian cancers (Nguyen et al., Women's Health, 9: 171-187,
2013).
Diagnosis and Classification of Ovarian Cancer
[0231] Early diagnosis of ovarian cancer is difficult because the
first symptoms of ovarian cancer are non-localized mild pain. The
symptoms of ovarian malignancies become clearer in the later stages
and include loss of appetite and weight loss, pain in the back and
pelvis, vaginal bleeding after menopause, frequent urination,
constipation or diarrhea and bloating in the abdomen (American
Cancer Society, How is Ovarian Cancer Diagnosed,
http:cancer.org/cancer/ovariancancer/detailedguide/ovarian-cancer-diagnos-
is, accessed on Apr. 22, 2013).
[0232] Ovarian cancer is diagnosed based on the presence of a tumor
as determined by physical examination of the pelvis, medical
imaging (i.e., ultrasound, computerized tomography scan (CT scan),
magnetic resonance imaging (MM), and blood test to measure various
biomarkers that have been associated with late stage ovarian cancer
(i.e., CA-125, IL-6, IL-8, serum amyloid A (SAA) and C-reactive
protein (CRP). For example, OvPlex.TM. is a commercially available
ELISA kit that uses a combination of the above-listed markers for
the detection of ovarian cancer in symptomatic women that have
already undergone surgery to remove primary ovarian tumors.
OvPlex.TM. is not an early detection kit for ovarian cancer since
the inflammatory markers IL-6, IL-8 and the liver acute phase
markers SAA and CRP require a strong stimulus to be raised to
detectable levels (see Healthlinx, O., What is the OvP1ex.TM.
Diagnostic, http:healthlinx.com.au/ovplex-tm, accessed on Apr. 8,
2013).
[0233] Biomarkers Other than MLP that have been studied for Early
Detection of Ovarian Cancer
[0234] Many studies have been carried out to identify biomarkers
for the early detection of ovarian malignances (Costa et al.,
Clinics 64:641-644, 2009). Table 7 summarizes the status of several
biomarkers that have been studied for early detection of ovarian
cancer. In contrast to the mucin biomarker CA-125, MLP expression
is absent in normal tissues and benign tumors.
TABLE-US-00020 TABLE 7 Previous Studies with Biomarkers to Detect
Early Stage Ovarian Tumors Biomarker Sensitivity Specificity
References CA-125 50%-60% 90% Kim et al., PLoS One 7, e44960, 2012;
Su et al., Clinica ChimicaActa, 2012 HE4 72% 95% Rossing et al.,
Cancer Causes and Control: CCC 19:1357-1364, 2008 HE4 PLUS 76%
95.7% Moore et al., International CA-125 Journal of Gynecological
Cancer 21(7):1185-1190, 2011 Mesothelin 60% 98% Nguyen et al.,
Women's Health 9:171-187, 2013 Mesothelin 12% in 95% in Nguyen et
al., Women's Health and serum serum 9:171-187, 2013; Sarojini et
al., CA-125 42% in 95 in Journal of Oncology, 2012 urine urine
Kallikreins 21%-26% 95% Nguyen et al., Women's Health 9:171-187,
2013; Sarojini et al., Journal of Oncology, 2012 Kallikreins 42%
90% Diamandis et al., Journal of plus Clinical Oncology
21:1035-1043, AC-125 2003 Osteopontin 93.8% 33% Jordan et al.,
Gynecologic Oncology 103:1122-1129, 2006 B7-H4 45% 97% Singh I.,
Anatomy and Physiology for Physiotherpists, Anshan, 2006 B7-H4 65%
97% Singh I., Anatomy and Physiology combined for Physiotherpists,
Anshan, 2006 with AC 125
[0235] Classification
[0236] There are four stages of ovarian cancer. Stage I includes
cancer that is limited within the ovary. It can occur in one or
both ovaries. Ovarian cancer is given stage II when cancer growth
involves one or both ovaries with extension or metastasis to the
pelvis. In the third and fourth stages there is extension of the
disease beyond the pelvis, which includes intra-abdominal to lymph
node in the case of stage III or distant metastasis past the
abdominal cavity in the case of stage IV (Chauhan et al., Jour
Ovarian Res 2:212-215, 2009; Guppy et al., Clinial Oncology
17:339-411, 2005).
[0237] If ovarian cancer is diagnosed in the early stage (stage I),
the five-year survival rate ranges from 60% to 90%, whereas if
diagnosed in stage II the five year survival rate decreases to a
range from 37% to 66%. Ovarian cancer patients diagnosed in stage
III have a five year survival rate in the range of 5% to 50% and
those diagnosed in stage IV have a five year survival rate between
0%-17% (Stimpfl et al., Cancer Letters 145:133-141, 1999).
[0238] Treatment
[0239] The standard treatment of ovarian cancer is by surgical
removal of the ovary, followed by chemotherapy, which is typically
a combination of platinum-based drugs and paclitaxel (see Cohen et
al., Jour of Molecular Medicine, 91(3): 357-368, 2013).
Radiotherapy is used in cases where cancer cells and metastases are
missed during surgical removal (Sornsukolrat et al., Chotmaihet
Thangphaet 95:1141-1148, 2012). The tumor volume at the diagnosis
is also a crucial prognostic factor in determining the efficacy of
surgery for both primary and secondary tumors with regard to the
progression of free intervals and of prolonged survival (Weidner et
al., Modern Surgical Pathology 2, Saunders/Elsevier, 2009).
[0240] 2. In Vitro Assays for the Presence of the MLP Antigen as a
Biomarker for the Presence of Epithelial Cancer (e.g., Ovarian
Cancer or Pancreatic Cancer)
[0241] In one aspect, the anti-MLP antibodies of the present
invention are used in an in vitro immunoassay for screening a
biological sample obtained from a test subject for the presence of
the MLP antigen, wherein the presence of MLP, or an increased
amount of MLP as compared to a control from a healthy subject, or
reference standard, indicates the subject has, or is at an
increased risk of developing an epithelial cancer (e.g., ovarian or
pancreatic cancer). The test subject can be an apparently healthy
subject, or a subject at risk for developing an epithelial cancer,
or a subject suspected of having early stage epithelial cancer, or
a subject suffering from an epithelial cancer such as ovarian,
pancreatic, colorectal, breast, appendiceal, lung, renal, cervical,
biliary, esophageal, epithelial skin and/or other mucin-secreting
types of cancer. In such in vitro immunoassays, the anti-MLP
antibody, or antigen-binding fragment thereof, may be naked or may
be labeled with a detectable moiety, as described herein, and may
be utilized in liquid phase or bound to a substrate, as described
below. For purposes of in vitro assays, any type of antibody such
as murine, chimeric, humanized or human may be utilized, since
there is no host immune response to consider.
[0242] The antibodies of the present disclosure may be employed in
any known diagnostic immunoassay method, such as a competitive
binding assay, a direct or indirect sandwich assay, and an
immunoprecipitation assay (see e.g., Zola, Monoclonal Antibodies: A
Manual of Techniques, pp. 147-158 (CRC Press. Inc., 1987).
[0243] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of MLP in the test sample is
inversely proportional to the amount of standard that becomes bound
to the antibodies. To facilitate determining the amount of standard
that becomes bound, the antibodies generally are insolubilized
before or after the competition, so that the standard and analyte
that are bound to the antibodies may conveniently be separated from
the standard and analyte that remain unbound.
[0244] Sandwich assays, involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected (e.g., MLP). In a sandwich assay, the
test sample analyte is bound by a first antibody (e.g., an anti-MLP
antibody), which is immobilized on a solid support, and thereafter
a second antibody binds to the analyte, thus forming an insoluble
three-part complex. The second antibody may itself be labeled with
a detectable moiety (direct sandwich assays) or may be measured
using an anti-immunoglobulin antibody that is labeled with a
detectable moiety (indirect sandwich assay). For example, one
preferable type of sandwich assay is an ELISA assay, in which case
the detectable moiety is an enzyme.
[0245] For immunohistochemistry, the tissue sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin, for example.
[0246] In one embodiment, the anti-MLP antibodies of the invention
are used to detect the presence of the MLP antigen in a biological
sample using an enzyme-linked immunosorbent assay (ELISA) (see
e.g., Gold et al. J Clin Oncol. 24:252-58, 2006).
[0247] In the direct competitive ELISA, a pure or semipure antigen
preparation is bound to a substrate that is insoluble in the fluid
or cellular extract being tested and a quantity of detectably
labeled soluble antibody is added to permit detection and/or
quantitation of the binary complex formed between substrate-bound
antigen and labeled antibody.
[0248] In contrast, a "double-determinant" ELISA, also known as a
"two-site ELISA" or "sandwich assay," requires small amounts of
antigen and the assay does not require extensive purification of
the antigen. Thus, the double-determinant ELISA is preferred to the
direct competitive ELISA for the detection of an antigen in a
clinical sample. See, for example, the use of the
double-determinant ELISA for quantitation of the c-myc oncoprotein
in biopsy specimens. Field et al., Oncogene 4: 1463 (1989);
Spandidos et al., AntiCancer Res. 9: 821 (1989). In a
double-determinant ELISA, a quantity of unlabeled monoclonal
antibody or antibody fragment (the "capture antibody") is bound to
a substrate (e.g., a solid support), the test sample is brought
into contact with the capture antibody, and a quantity of
detectably labeled soluble antibody (or antibody fragment) is added
to permit detection and/or quantitation of the ternary complex
formed between the capture antibody, antigen, and labeled antibody.
In one embodiment, the capture antibody bound to a substrate (e.g.,
solid support) is an anti-MLP antibody or antigen-binding fragment
thereof as disclosed herein that binds to an epitope in the
C-terminal portion of MLP. Methods of performing a
double-determinant ELISA are well-known by those of skill in the
art. See, for example, Field et al., Oncogene 4: 1463 (1989);
Spandidos et al., AntiCancer Res. 9: 821 (1989); and Moore et al.,
Methods in Molecular Biology Vol 10:273-281 (The Humana Press, Inc.
1992).
[0249] In the double-determinant ELISA, the soluble antibody or
antibody fragment must bind to an MLP epitope that is distinct from
the epitope recognized by the capture antibody. The
double-determinant ELISA can be performed to ascertain whether the
MLP antigen is present in a test biological sample, such as a body
fluid (e.g., blood, plasma or serum) or a biopsy sample.
Alternatively, the assay can be performed to quantitate the amount
of MLP antigen that is present in a clinical sample of body fluid.
The quantitative assay can be performed by including dilutions of
purified MLP antigen.
[0250] In another embodiment, the anti-MLP antibodies of the
invention are used to detect the presence of the MLP antigen in a
test substance (e.g., a biological sample obtained from a subject)
using a radioimmunoassay (RIA). For example, in one form of RIA,
the test biological sample is mixed with an anti-MLP antibody in
the presence of radiolabeled MLP antigen. In this method, the
concentration of the test substance will be inversely proportional
to the amount of labeled MLP antigen bound to the anti-MLP antibody
and directly related to the amount of free, labeled MLP antigen.
Other suitable screening methods will be readily apparent to those
of skill in the art.
[0251] In vitro immunoassays can be performed in which at least one
anti-MLP antibody or antigen-binding fragment thereof is bound to a
substrate (e.g., a solid-phase carrier). For example, anti-MLP
monoclonal antibodies or fragments thereof can be attached to a
polymer, such as aminodextran, in order to link the monoclonal
antibody to an insoluble substrate such as a polymer-coated bead, a
plate, a tube, or a ceramic or metal chip. In one embodiment, the
substrate is suitable for use in an ELISA method (e.g., a multiwell
microtitre plate). Accordingly, the determination of the level of
MLP in the sample may be determined by commercially available
methods such as an ELISA based assay, chemical or enzymatic protein
determination.
[0252] In one embodiment, the methods of the present disclosure use
a solid-state device for determining the level of MLP in a sample
isolated from the patient. The solid-state device comprises a
substrate having an activated surface on to which at least one
anti-MLP antibody is immobilised at discreet areas of the activated
surface. Preferably, the solid state device may perform
multi-analyte assays such that the level of a biomarker of interest
in a sample isolated from the patient may be determined
simultaneously with the level of a further biomarker of interest in
the sample. In this embodiment, the solid state device has a
multiplicity of discrete reaction sites each bearing a desired
antibody covalently bound to the substrate, and in which the
surface of the substrate between the reaction sites is inert with
respect to the target biomarker. The solid-state, multi-analyte
device may therefore exhibit little or no non-specific binding. For
example, a solid-state device suitable for use in the methods of
the present disclosure is the Biochip Array Technology system (BAT)
(available from Randox Laboratories Limited).
[0253] Other suitable in vitro assays will be readily apparent to
those of skill in the art. The specific concentrations of
detectably labeled anti-MLP antibody and MLP antigen, the
temperature and time of incubation, as well as other assay
conditions may be varied, depending on various factors including
the concentration of the MLP antigen in the sample, the nature of
the sample, and the like. The binding activity of a sample of
anti-MLP antibody may be determined according to well-known
methods. Those skilled in the art will be able to determine
operative and optimal assay conditions for each determination by
employing routine experimentation.
[0254] In another embodiment, the subject antibodies and
antigen-binding fragments thereof can be used to detect the
presence of the MLP antigen in tissue sections prepared from a
histological specimen (e.g., a biopsy sample). Such in situ
detection can be used to determine the presence of the MLP antigen
and to determine the distribution of the MLP antigen in the
examined tissue. In situ detection can be accomplished by applying
a detectably-labeled anti-MLP antibody to tissue sections. General
techniques of in situ detection are well-known to those of ordinary
skill. See, for example, Ponder, "Cell Marking Techniques and Their
Application," in Mammalian Development: A Practical Approach 113-38
Monk (ed.) (IRL Press 1987).
[0255] The subject anti-MLP antibodies and antigen-binding
fragments thereof can be labeled with any appropriate detectable
moiety as described herein. Examples of suitable detectable
moieties include radioisotopes, enzymes, fluorescent labels, dyes,
chromogens, chemiluminescent labels, bioluminescent labels and
paramagnetic labels. The above-described in vitro and in situ
detection methods may be used to assist in the detection of MLP
antigen in a biological sample (e.g., a serum sample), or in the
diagnosis or staging of a pathological condition such as the
presence of an epithelial cancer, such as ovarian, pancreatic,
colorectal, breast, appendiceal, lung, renal, cervical, biliary,
esophageal, epithelial skin and/or other mucin-secreting types of
cancer. For example, such methods can be used to detect tumors that
express the MLP antigen such as ovarian, pancreatic, colorectal,
breast, appendiceal, lung, renal, cervical, biliary, esophageal,
epithelial skin and/or other mucin-secreting types of cancer.
[0256] In accordance with the foregoing, in one aspect, the present
invention provides a method of detecting the presence or amount of
MLP in a biological sample from a test subject, the method
comprising (a) contacting a biological sample from a test subject
with an anti-MLP antibody or antigen-binding fragment thereof in an
in vitro immunoassay and (b) detecting the presence or absence of
binding of said antibody, wherein the presence of binding indicates
the presence of MLP in the sample, wherein the antibody or fragment
thereof binds to an epitope in the C-terminal region of MLP, set
forth as SEQ ID NO:4. In one embodiment, the anti-MLP antibody is
labeled with a detectable moiety and step (b) comprises detecting
the presence of said detectable moiety.
[0257] In one embodiment, the method further comprises comparing
the amount of MLP detected in accordance with step (b) with a
reference standard or control sample from a healthy subject,
wherein an increase of at least two-fold or higher (e.g., at least
five-fold, or at least ten-fold, or at least 20-fold, or at least
50-fold, or at least 100-fold or higher) in the level of MLP in the
test sample as compared to the control sample (or reference
standard) indicates the presence of, or increased risk for
developing an epithelial cancer, such as ovarian cancer or
pancreatic cancer, in the test subject.
[0258] Although the details of an immunoassay may vary with the
particular format employed, the method of detecting MLP in a
biological sample comprises the steps of contacting the biological
sample with an antibody that specifically binds to MLP. The
antibody is allowed to bind to MLP in the biological sample under
immunologically reactive conditions, and the presence of the bound
antibody is detected directly or indirectly. The MLP-specific
antibodies may be used, for example, as the capture antibody of an
ELISA, or as a second antibody to bind to MLP captured by the
capture antibody. As is known in the art, the presence of the
second antibody is typically then detected.
[0259] A biological sample may be any sample of biological tissue
or fluid obtained from a mammalian subject. Examples of biological
samples include, but are not limited to, tissue from biopsy,
sputum, amniotic fluid, ascites, blood or serum. In one embodiment,
the biological sample is selected from the group consisting of
blood, serum, plasma and tissue.
[0260] In one embodiment of the method, the anti-MLP antibody or
fragment thereof is a monoclonal antibody that binds to the same
epitope or competes for binding to MLP with a reference antibody
selected from the group consisting of: (i) the monoclonal anti-MLP
antibody Clone 11 produced by the cell line deposited at the ATCC
on Oct. 30, 2014 under the ATCC Designation number PTA-121699; (ii)
the monoclonal anti-MLP antibody Clone B produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation number PTA-121700 ; and (iii) the monoclonal anti-MLP
antibody Clone C produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation number
PTA-121701.
[0261] In one embodiment of the method, the anti-MLP antibody or
fragment thereof is a monoclonal antibody having a heavy chain
variable region CDR-H3 sequence comprising an amino acid sequence
set forth as SEQ ID NO:15, or SEQ ID NO:35 or SEQ ID NO:36 or SEQ
ID NO:19, and conservative sequence modifications thereof, and
having a light chain variable region CDR-L3 sequence comprising an
amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27, and
conservative sequence modifications thereof.
[0262] In one embodiment, said antibody, or antigen binding
fragment thereof, comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ ID NO:14) and CDR-H3
(SEQ ID NO:15 or SEQ ID NO:35 or SEQ ID NO:36) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:21), CDR-L2 (SEQ
ID NO:22) and CDR-L3 (SEQ ID NO:23), and conservative modifications
thereof. In one embodiment, said antibody or antigen binding
fragment thereof comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ ID NO:16) and CDR-H3
(SEQ ID NO: 15 or SEQ ID NO:35 or SEQ ID NO:36) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:24), CDR-L2 (SEQ
ID NO:22) and CDR-L3 (SEQ ID NO:23), and conservative modifications
thereof. In one embodiment, said antibody, or antigen binding
fragment thereof comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:17), CDR-H2 (SEQ ID NO:18) and CDR-H3
(SEQ ID NO:19) and (ii) a light chain variable region comprising
CDR-L1 (SEQ ID NO:25), CDR-L2 (SEQ ID NO:26) and CDR-L3 (SEQ ID
NO:27), and conservative modifications thereof.
[0263] In one embodiment, the anti-MLP antibody or fragment thereof
is a monoclonal antibody that comprises a heavy chain variable
region and/or a light chain variable region set forth in Table 1,
and conservative sequence modifications thereof.
[0264] In one embodiment, the anti-MLP antibody or fragment thereof
is a monoclonal anti-MLP antibody produced by the hybridoma cell
line secreting .alpha.MLP Clone 11 deposited at the ATCC on Oct.
30, 2014 under the ATCC Designation number PTA-121699. In one
embodiment, the anti-MLP antibody or fragment thereof is a
monoclonal antibody produced by the hybridoma cell line secreting
.alpha.MLP Clone B deposited at the ATCC on Oct. 30, 2014 under the
ATCC Designation number PTA-121700. In one embodiment, the anti-MLP
antibody or fragment thereof is a monoclonal antibody produced by
the hybridoma cell line secreting .alpha.MLP Clone C deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation number
PTA-121701.
[0265] In one embodiment, the method further comprises performing
an immunoassay with one or more additional antibodies that bind to
ovarian and/or pancreatic cancer biomarkers, such as the biomarkers
shown in Table 7 (e.g., CA-125, HE4, Mesothelin, Kallikreins,
Osteopontin, and B7-H4). Additional biomarkers for all stages of
ovarian cancer include: CRP, EGF-R, CA-19-9, Apo-A1, Apo-CIII,
IL-6, IL-18, MIP-1a, Tenascin C, Myoglobin, vWF, Haptoglobin,
IL-10, IGF-I, IGF-II, Prolactin, ACE, ASP, Resistin and
Carcinoembryonic antigen (CEA).
[0266] In one embodiment, the test subject is apparently
healthy.
[0267] In one embodiment, the test subject has a family history of
ovarian or pancreatic cancer.
[0268] In one embodiment, the test subject is experiencing one or
more symptoms associated with ovarian cancer. For example, symptoms
associated with ovarian cancer in a human female subject include
one or more of the following: pelvic mass, ascites, abdominal
distention, abdominal pressure, swelling or bloating, pelvic pain
or discomfort, nausea, constipation, gas, indigestion, diarrhea,
urinary problems with as frequent urination or urgent need to
urinate, loss of appetite or a quick feeling of fullness; increased
abdominal circumference or tight-fitting clothing, persistent lack
of energy, weight gain or loss with no known reason, abnormal
bleeding from the vagina and low back pain.
[0269] In one embodiment, the test subject is known to be suffering
from ovarian, pancreatic, colorectal, breast, appendiceal, lung,
renal, cervical, biliary, esophageal, epithelial skin and/or other
mucin-secreting types of cancer and has had or is currently
undergoing treatment for ovarian, pancreatic, colorectal, breast,
appendiceal, lung, renal, cervical, biliary, esophageal, epithelial
skin and/or other mucin-secreting types of cancer. In one
embodiment, more than one sample is obtained from a particular test
subject at more than one point in time. In one embodiment, the
method further comprises comparing the results of the assay at one
or more time points to assess the efficacy of a treatment regimen.
As will be appreciated by persons skilled in the art, the relative
expression levels of MLP in specimens taken from a subject prior to
and again after treatment, or, optionally, at progressive stages
during treatment may be determined to assess the efficacy of
treatment, wherein a decrease in the level of MLP over time is
indicative of treatment efficacy.
[0270] 3. In Vivo Assays for Determining the Presence of the MLP
Antigen in a Living Subject
[0271] In another aspect, the anti-MLP antibodies of the present
invention are used to detect the presence of MLP-expressing cells
in a living subject, such as an apparently healthy subject, or a
subject at risk for developing an epithelial cancer, or a subject
suspected of having early stage epithelial cancer, or a subject
suffering from an epithelial cancer such as ovarian, pancreatic,
colorectal, breast, appendiceal, lung, renal, cervical, biliary,
esophageal, epithelial skin and/or other mucin-secreting types of
cancer. Various methods of in vivo diagnostic imaging with
radiolabeled monoclonal antibodies are well known in the art. For
diagnostic imaging, the subject anti-MLP antibodies or
antigen-binding fragments thereof are labeled with a detectable
moiety suitable for use in vivo, as described herein, (e.g., a
gamma-emitting radioisotope) and introduced into a patient. For
example, in the technique of immunoscintigraphy, anti-MLP
antibodies are labeled with a gamma-emitting radioisotope and
introduced into a patient and a gamma camera is used to detect the
location and distribution of gamma-emitting radioisotopes. See, for
example, Srivastava (ed.), Radiolabeled Monoclonal Antibodies for
Imaging and Therapy (Plenum Press 1988); Chase, "Medical
Applications of Radioisotopes," in Remington's Pharmaceutical
Sciences, 18th Edition, Gennaro et al. (eds.), pp. 624-652 (Mack
Publishing Co., 1990); and Brown, "Clinical Use of Monoclonal
Antibodies," in Biotechnology and Pharmacy, Pezzuto et al. (eds.)
(Chapman & Hall 1993). The radiation dose delivered to the
patient is maintained at as low a level as possible through the
choice of isotope for the best combination of minimum half-life,
minimum retention in the body, and minimum quantity of isotope
which will permit detection and accurate measurement.
[0272] In a preferred embodiment, the anti-MLP antibodies for in
vivo use in a human subject are humanized or human antibodies in
order to reduce the human antimouse antibody (HAMA) response.
Humanized or human anti-MLP monoclonal antibodies are suitable for
use in the in vitro and in vivo diagnostic and therapeutic methods
described herein.
[0273] In accordance with the foregoing, in one aspect, the present
invention provides a method of detecting or diagnosing ovarian,
pancreatic, colorectal, breast, appendiceal, lung, renal, cervical,
biliary, esophageal, epithelial skin and/or other mucin-secreting
types of cancer comprising (a) administering to a living subject a
humanized or fully human anti-MLP antibody or antigen-binding
fragment thereof that binds to an epitope in the C-terminal region
of MLP, set forth as SEQ ID NO:4; and (b) detecting the presence or
absence or amount of the antibody or fragment thereof bound to MLP,
wherein detection of the presence of MLP in the subject indicates
the presence of ovarian, pancreatic, colorectal, breast,
appendiceal, lung, renal, cervical, biliary, esophageal, epithelial
skin and/or other mucin-secreting types of cancer. In one
embodiment, the anti-MLP antibody is labeled with a detectable
moiety suitable for in vivo use. In one embodiment, the method is
used in an imaging, intraoperative, endoscopic or intravascular
procedure.
[0274] In one embodiment, the anti-MLP antibody or fragment thereof
is a monoclonal antibody that binds to the same epitope or competes
for binding to MLP with a reference antibody selected from the
group consisting of: (i) the monoclonal anti-MLP antibody Clone 11
produced by the cell line deposited at the ATCC on Oct. 30, 2014
under the ATCC Designation number PTA-121699; (ii) the monoclonal
anti-MLP antibody Clone B produced by the hybridoma cell line
deposited at the ATCC on Oct. 30, 2014 under the ATCC Designation
number PTA-121700 ; and (iii) the monoclonal anti-MLP antibody
Clone C produced by the hybridoma cell line deposited at the ATCC
on Oct. 30, 2014 under the ATCC Designation number PTA-121701.
[0275] In one embodiment, the anti-MLP antibody or fragment thereof
is a monoclonal antibody having a heavy chain variable region
CDR-H3 sequence comprising an amino acid sequence set forth as SEQ
ID NO:15 or SEQ ID NO:35 or SEQ ID NO:36 or SEQ ID NO:19, and
conservative sequence modifications thereof, and having a light
chain variable region CDR-L3 sequence comprising an amino acid
sequence set forth as SEQ ID NO:23 or SEQ ID NO:27, and
conservative sequence modifications thereof. In one embodiment,
said antibody, or antigen binding fragment thereof, comprises (i) a
heavy chain variable region comprising CDR-H1 (SEQ ID NO:13),
CDR-H2 (SEQ ID NO:14) and CDR-H3 (SEQ ID NO:15 or SEQ ID NO:35 or
SEQ ID NO:36) and (ii) a light chain variable region comprising
CDR-L1 (SEQ ID NO:21), CDR-L2 (SEQ ID NO:22) and CDR-L3 (SEQ ID
NO:23), and conservative modifications thereof. In one embodiment,
said antibody or antigen binding fragment thereof comprises (i) a
heavy chain variable region comprising CDR-H1 (SEQ ID NO:13),
CDR-H2 (SEQ ID NO:16) and CDR-H3 (SEQ ID NO:15 or SEQ ID NO:35 or
SEQ ID NO:36) and (ii) a light chain variable region comprising
CDR-L1 (SEQ ID NO:24), CDR-L2 (SEQ ID NO:22) and CDR-L3 (SEQ ID
NO:23), and conservative modifications thereof. In one embodiment,
said antibody, or antigen binding fragment thereof comprises (i) a
heavy chain variable region comprising CDR-H1 (SEQ ID NO:17),
CDR-H2 (SEQ ID NO:18) and CDR-H3 (SEQ ID NO:19) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:25), CDR-L2 (SEQ
ID NO:26) and CDR-L3 (SEQ ID NO:27), and conservative modifications
thereof.
[0276] In one embodiment, the test subject is apparently
healthy.
[0277] In one embodiment, the test subject has a family history of
ovarian or pancreatic cancer or other mucin-secreting malignant
neoplasm.
[0278] In one embodiment, the test subject is experiencing one or
more symptoms associated with ovarian cancer. For example, symptoms
associated with ovarian cancer in a human female subject include
one or more of the following: pelvic mass, ascites, abdominal
distention, abdominal pressure, swelling or bloating, pelvic pain
or discomfort, nausea, constipation, gas, indigestion, diarrhea,
urinary problems with as frequent urination or urgent need to
urinate, loss of appetite or a quick feeling of fullness; increased
abdominal circumference or tight-fitting clothing, persistent lack
of energy, weight gain or loss with no known reason, abnormal
bleeding from the vagina and low back pain.
[0279] In one embodiment, the test subject is known to be suffering
from a mucin-secreting cancer type selected from the group
consisting of ovarian, pancreatic, colorectal, breast, appendiceal,
lung, renal, cervical, biliary, esophageal, epithelial skin and/or
other mucin-secreting types of cancer and has had or is currently
undergoing treatment for ovarian, pancreatic, colorectal, breast,
appendiceal, lung, renal, cervical, biliary, esophageal, epithelial
skin and/or other mucin-secreting types of cancer. In one
embodiment, more than one sample is obtained from a particular test
subject at more than one point in time. In one embodiment, the
method further comprises comparing the results of the assay at one
or more time points to assess the efficacy of a treatment regimen.
As will be appreciated by persons skilled in the art, the relative
expression levels of MLP in specimens taken from a subject prior to
and again after treatment, or, optionally, at progressive stages
during treatment may be determined to assess the efficacy of
treatment, wherein a decrease in the level of MLP over time is
indicative of treatment efficacy.
[0280] VI. Therapeutic Methods of Using an anti-MLP Antibody
Coupled to, and/or in Combination with, a Therapeutic Agent
[0281] In another aspect, therapeutic methods are provided for
treating a malignancy comprising administering an anti-MLP
antibody, or antigen-binding fragment thereof, such as the subject
anti-MLP antibodies disclosed herein, to a subject suffering from
an epithelial cancer such as ovarian or pancreatic cancer or other
mucin-secreting malignant neoplasm. In one embodiment, the anti-MLP
antibody or antigen-binding fragment thereof is administered to the
subject in conjunction with one or more other therapeutic agents.
In one embodiment, the anti-MLP antibody or antigen-binding
fragment thereof is coupled to a therapeutic agent, as described
herein. In a preferred embodiment, the anti-MLP antibodies and
fragments thereof are humanized or fully human.
[0282] In embodiments where more than one therapeutic agent is
used, the therapeutic agents may comprise multiple copies of the
same therapeutic agent or else combinations of different
therapeutic agents.
[0283] In one embodiment, an anti-MLP antibody or fragment thereof
is coupled to a therapeutic agent to generate an epithelial cancer
cell-targeting therapeutic agent. A wide variety of therapeutic
reagents can be administered concurrently or sequentially, or
advantageously conjugated to the antibodies of the invention, for
example, drugs, toxins, oligonucleotides (e.g., siRNA),
immunomodulators, hormones, hormone antagonists, enzymes, enzyme
inhibitors, radionuclides, angiogenesis inhibitors, pro-apoptotic
agents, etc. The therapeutic agents recited here are those agents
that are useful for either conjugated to an antibody, fragment or
fusion protein or for administration separately with a naked
antibody as described above.
[0284] In accordance with the foregoing, in one aspect, the present
invention provides a method of treating a subject suffering from a
mucin-secreting cancer selected from the group consisting of
ovarian, pancreatic, colorectal, breast, appendiceal, lung, renal,
cervical, biliary, esophageal, epithelial skin and/or other
mucin-secreting types of cancer comprising administering to an
individual suffering from ovarian, pancreatic, colorectal, breast,
appendiceal, lung, renal, cervical, biliary, esophageal, epithelial
skin and/or other mucin-secreting types of cancer a humanized or
fully human anti-MLP antibody or antigen-binding fragment thereof
that binds to an epitope in the C-terminal region of MLP, set forth
as SEQ ID NO:4, wherein the antibody or fragment thereof is coupled
to a therapeutic agent. In one embodiment, the therapeutic agent is
a chemotherapeutic agent. In one embodiment, the anti-MLP antibody
or fragment thereof is a monoclonal antibody that binds to the same
epitope or competes for binding to MLP with a reference antibody
selected from the group consisting of: (i) the monoclonal anti-MLP
antibody Clone 11 produced by the cell line deposited at the ATCC
on Oct. 30, 2014 under the ATCC Designation number PTA-121699; (ii)
the monoclonal anti-MLP antibody Clone B produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation number PTA-121700 ; and (iii) the monoclonal anti-MLP
antibody Clone C produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation number
PTA-121701.
[0285] In one embodiment, the anti-MLP antibody or fragment thereof
is a monoclonal antibody having a heavy chain variable region
CDR-H3 sequence comprising an amino acid sequence set forth as SEQ
ID NO:15 or SEQ ID NO:35 or SEQ ID NO:36 or SEQ ID NO:19, and
conservative sequence modifications thereof, and having a light
chain variable region CDR-L3 sequence comprising an amino acid
sequence set forth as SEQ ID NO:24 or SEQ ID NO:27, and
conservative sequence modifications thereof.
[0286] In one embodiment, said antibody, or antigen binding
fragment thereof, comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ ID NO:14) and CDR-H3
(SEQ ID NO:15 or SEQ ID NO:35 or SEQ ID NO:36) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:21), CDR-L2 (SEQ
ID NO:22) and CDR-L3 (SEQ ID NO:23), and conservative modifications
thereof. In one embodiment, said antibody or antigen binding
fragment thereof comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ ID NO:16) and CDR-H3
(SEQ ID NO:15 or SEQ ID NO:35 or SEQ ID NO:36) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:24), CDR-L2 (SEQ
ID NO:22) and CDR-L3 (SEQ ID NO:23), and conservative modifications
thereof. In one embodiment, said antibody, or antigen binding
fragment thereof comprises (i) a heavy chain variable region
comprising CDR-H1 (SEQ ID NO:17), CDR-H2 (SEQ ID NO:18) and CDR-H3
(SEQ ID NO:19) and (ii) a light chain variable region comprising
CDR-L1 (SEQ ID NO:25), CDR-L2 (SEQ ID NO:26) and CDR-L3 (SEQ ID
NO:27), and conservative modifications thereof.
Pharmaceutically Suitable Excipients
[0287] Additional pharmaceutical methods may be employed to control
the duration of action of an anti-MLP antibody in a therapeutic
application. Control release preparations can be prepared through
the use of polymers to complex or adsorb the antibody, antibody
fragment or fusion protein. For example, biocompatible polymers
include matrices of poly(ethylene-co-vinyl acetate) and matrices of
a polyanhydride copolymer of a stearic acid dimer and sebacic acid.
Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of
release of an antibody, antibody fragment or fusion protein from
such a matrix depends upon the molecular weight of the antibody,
antibody fragment or fusion protein, the amount of antibody within
the matrix, and the size of dispersed particles. Saltzman et al.,
Biophys. J. 55: 163 (1989);
[0288] Sherwood et al., supra. Other solid dosage forms are
described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG
DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro
(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack
Publishing Company 1990), and revised editions thereof.
[0289] Compositions comprising the subject anti-MLP antibodies, or
fragments thereof may comprise one or more pharmaceutically
suitable excipients, one or more additional ingredients, or some
combination of these. The antibody can be formulated according to
known methods to prepare pharmaceutically useful compositions,
whereby the immunoconjugate or naked antibody is combined in a
mixture with a pharmaceutically suitable excipient. Sterile
phosphate-buffered saline is one example of a pharmaceutically
suitable excipient. Other suitable excipients are well-known to
those in the art. See, for example, Ansel et al., PHARMACEUTICAL
DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea &
Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL
SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised
editions thereof.
[0290] The immunoconjugate or naked antibody can be formulated for
intravenous administration via, for example, bolus injection or
continuous infusion. Formulations for injection can be presented in
unit dosage form, e.g., in ampules or in multi-dose containers,
with an added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0291] The immunoconjugate, naked antibody, or fragment thereof may
also be administered to a mammal subcutaneously or by other
parenteral routes. In a preferred embodiment, the antibody or
fragment thereof is administered in a dosage of 20 to 2000
milligrams protein per dose. Moreover, the administration may be by
continuous infusion or by single or multiple boluses. In general,
the dosage of an administered immunoconjugate or naked antibody for
humans will vary depending upon such factors as the patient's age,
weight, height, sex, general medical condition and previous medical
history. Typically, it is desirable to provide the recipient with a
dosage of immunoconjugate or naked antibody that is in the range of
from about 1 mg/kg to 20 mg/kg as a single intravenous or infusion,
although a lower or higher dosage also may be administered as
circumstances dictate. This dosage may be repeated as needed, for
example, once per week for four to ten weeks, preferably once per
week for eight weeks, and more preferably, once per week for four
weeks. It may also be given less frequently, such as every other
week for several months, or more frequently, such as two- or
three-time weekly. The dosage may be given through various
parenteral routes, with appropriate adjustment of the dose and
schedule.
VII. Compositions and Kits Comprising Anti-MLP Antibodies
[0292] Compositions
[0293] In another aspect, the invention provides compositions for
treating ovarian or pancreatic cancer comprising a therapeutically
effective amount of an MLP-specific monoclonal antibody or fragment
thereof coupled to a therapeutic agent and a pharmaceutically
acceptable carrier. In general, the MLP-specific antibody
compositions of the present invention, coupled to, and/or combined
with any other selected therapeutic agents, are suitably contained
in a pharmaceutically acceptable carrier. The carrier is non-toxic,
biocompatible and is selected so as not to detrimentally affect the
biological activity of the MLP-specific antibody (and any other
therapeutic agents combined therewith). Exemplary pharmaceutically
acceptable carriers for polypeptides are described in U.S. Pat. No.
5,211,657 to Yamada. The MLP-specific antibodies may be formulated
into preparations in solid, semi-solid, gel, liquid or gaseous
forms such as tablets, capsules, powders, granules, ointments,
solutions, depositories, inhalants and injections allowing for
oral, parenteral or surgical administration. The invention also
contemplates local administration of the compositions by coating
medical devices and the like.
[0294] In another aspect, the invention provides a substrate, such
as a solid support (e.g., an insoluble substrate, such as a plate
or slide made of glass, plastic or metal, a polymer-coated bead, a
tube, or a ceramic or metal chip) that comprises immobilized (or
otherwise deposited) monoclonal anti-MLP antibodies. In some
embodiments, the antibodies are immobilized (or deposited) at
discrete locations (e.g., in the wells of a multiwall plate, or
deposited in an array on a biochip). In some embodiments, the
substrate comprising the anti-MLP antibodies may be part of a kit
for detecting MLP in a biological sample obtained from a mammalian
subject.
[0295] Kits
[0296] In another aspect, the invention provides kits (i.e., a
packaged combination of reagents in predetermined amounts) with
instructions for detecting the presence of MLP in a biological
sample. Exemplary kits may contain at least one or more anti-MLP
monoclonal antibody or antigen binding fragment thereof as
described herein. Where the anti-MLP antibody is labeled with a
detectable moiety, such as an enzyme, the kit will include
substrates and cofactors required by the enzyme (e.g., a substrate
precursor which provides the detectable chromophore or
fluorophore). In addition, other additives may be included such as
stabilizers, buffers (e.g., a blocking buffer or lysis buffer) and
the like. The relative amounts of the various reagents may be
varied widely to provide for concentrations in solution of the
reagents, which substantially optimize the sensitivity of the
assay. Particularly, the reagents may be provided as dry powders,
usually lyophilized, including excipients which on dissolution will
provide a reagent solution having the appropriate
concentration.
[0297] In addition, kits may include instructional materials
disclosing means of use of an antibody of the present invention
(e.g., for detection of MLP as a biomarker for ovarian or
pancreatic cancer). The kits may also include additional components
to facilitate the particular application for which the kit is
designed. For example, the kit may additionally contain means of
detecting a label (e.g., enzyme substrates for enzymatic labels,
filter sets to detect fluorescent labels, appropriate secondary
labels such as a sheep anti-mouse-HRP or the like). The kits may
additionally include buffers and other reagents routinely used for
the practice of a particular immunoassay, as is well known in the
art.
[0298] The following examples merely illustrate the best mode now
contemplated for practicing the invention, but should not be
construed to limit the invention.
EXAMPLE 1
[0299] This Example demonstrates that glycosylated mucin-like
protein (gMLP) is a biomarker of ovarian cancer and describes the
recombinant expression and protein production of a recombinant
fusion protein comprising the novel C-terminal region of human MLP
protein and the generation of monoclonal antibodies that
specifically bind to the C-terminal region of human MLP.
[0300] Background/Rationale:
[0301] Mucins, which are high molecular-weight glycoproteins, are
the major structural component of mucus, a gel that covers
epithelial cell surfaces. The mucin family is composed of
glycoproteins that contain a protein backbone conjugated with a
large number of 0-linked oligosaccharide chains and a few N-glycan
chains (Andrianifahanana et al., Biochimica Et Biophysica Acta
Reviews on Cancer, 1765:189-222, 2006). The mucin family is highly
glycosylated, and the molecular weight of these glycoproteins is
typically around 200 kDa to >1000 kDa. The glycoproteins contain
three domains: N-terminal domain, C-terminal domain and in the
middle have tandem repeats. O-glycosidic bound N-terminus bind to
the hydroxyl side chain of a repetitive tandem repeat structure.
Such tandem repeats contain a frequent and rich quantity of Serine
(Ser), Proline (Pro) and Threonine (Thr) amino acid residues (Kim
et al., Glycoconjugate Journal 13:693-707, 1996).
[0302] The mucin family, which includes a total of twenty family
members, is divided into two classes, (i) the secreted mucins (both
gel forming and non-gel forming) and (ii) the membrane-bound
mucins.
[0303] The secreted mucin gel-forming proteins are expressed by
globlet cells (surface epithelium) and mucosa cells (sub mucosal
gland) and include MUC2, MUC5AC, MUC5B, MUC6 and MUC19 (Chauhan et
al., Journal of Ovarian Research 2:212-215, 2009; Chen et al.,
American Journal of Respiratory Cell and Molecular Biology
30:155-165, 2004). Secreted mucin non-gel forming proteins include
MUC7, MUC8 and MUC9, both MUC8 and MUC9 high tandem repeat than
MUC7. MUC7 was found in saliva secretions (Andrianifahanana et al.,
2006, supra).
[0304] The membrane-bound mucins include MUC1, MUC3A, MUC3B, MUC4,
MUC12, MUCI13, MUC15, MUC16, MUCI17, and MUCI20 (Andrianifahanana
et al., 2006, supra). The membrane-bound mucins are expressed in
normal cells, but are overexpressed in cancer conditions, cystic
fibrosis and asthma (Bafna et al., Oncogene 29:2893-2904, 2010). In
normal (noncancerous cells), the functions of mucins include
cytoprotection (protection of the cells against biological agent),
lubrication of the epithelial luminal surfaces, cell adhesion and
interaction between each cell (Brockhausen et al., EMBO Reports
7:599-604, 2006). However, it has been shown that mucins in cancer
cells have an increased level of, as well as altered,
glycosylation. Cancer cells overexpress mucin on the cell surface,
which may affect cellular adhesion and metastasis (Richards et al.,
Cancer Immunology, Immunotherapy 46:245-252, 1998). It has also
been determined that mucins in cancer cells have altered
glycosylation resulting, for example, from the synthesis of
sialyl-Tn antigen truncated oligosaccharide side chains and Tn
antigen leading to the accumulation of core oligosaccharides (see
Yamashita et al., Journal of the National Cancer Institute
87:441-446, 1995). Numerous alterations of mucin antigens have been
described in neoplastic epithelial tissues and in the sera of
patients with, for example, pancreatic, breast, ovarian and colon
cancer, and these antigens (e.g., DF3, CA19-9, CA125, SPan-1 and
DuPan2) have been used as diagnostic markers (Ho. S. B. et al.,
Cancer Res 53:641-651, 1993). In ovarian epithelium malignancy
leads to overexpression of oligosaccharides, which can be used as a
tumor marker for early detection of ovarian cancer (Giuntoli et
al., Cancer Research 58:5546-5550, 1998).
[0305] As described in WO98/48014, a novel mucin-like protein
(MLP), also referred to as "MUC-B1" was identified as a previously
unrecognized member of the human mucin family. It was determined
that MLP is a glycoprotein composed of a protein backbone, which is
highly glycosylated with O-linked oligosaccharides. The high degree
of glycosylation accounts for most of the molecule weight of the
native glycoprotein. In situ hybridization using a MLP-specific DNA
probe indicated that the MLP gene is located on chromosome 7. As
further described in WO 98/48014, two different partial cDNA
transcripts encoding MLP were isolated and it was determined at the
time of cloning that both clones contained two possible ORF's at
the 5' end (ORF1 and ORF2), encoding very similar peptides with
multiple tandem repeat motifs. Both deduced peptides were expressed
in E. coli and were used as antigens to generate antibodies. It was
determined by immunohistological analysis using the antibodies
raised against the peptides encoded by ORF1 and ORF2 that
glycosylated MLP (gMLP) is exclusively expressed and released by a
substantial proportion of epithelial cancers such as ovarian and
pancreatic tumor cells and is not present in normal ovarian tissue
or ovarian cysts with no signs of malignancy. It was also
determined that MLP is not specifically filtered by the kidney, so
tumors may be easily detected by testing for MLP in blood.
[0306] The present inventors have since determined that the
sequence of the predicted protein published in WO 98/48014 is
incorrect, likely due to difficulties with contig assembly during
genomic sequencings due to the highly repetitive central section of
the sequence. The present inventors have carried out genome-derived
sequencing and have now identified a single exon gene on chromosome
7 which encodes the correct full-length sequence of mucin-like
protein (MLP), also referred to as MUC-B), set forth as SEQ ID
NO:1, which has a length of 431 residues and contains a novel
C-terminal region (set forth as SEQ ID NO:4, including SEQ ID NO:5
and SEQ ID NO:6) that was not previously described in WO98/48014.
Knowledge of the correct, complete cDNA sequence coding for MLP
allows for the expression of a recombinant polypeptide
corresponding to the C-terminal region of the protein (e.g., a
polypeptide comprising or consisting of SEQ ID NO:4, SEQ ID NO:5 or
SEQ ID NO:6) for use as an antigen to generate MLP-specific
monoclonal antibodies, as described herein. The novel C-terminal
region of MLP was chosen for use as an antigen to generate anti-MLP
monoclonal antibodies because of the high immunogenicity of this
sequence, the very high degree of specificity for MLP (as described
herein), and the low degree of similarity between this region and
any other peptide sequence in the database. It is further noted
that the MLP protein is heavily glycosylated, however, the extreme
C-terminal 29 amino acid region of MLP (set forth as SEQ ID NO:6)
contains only two predicted glycosylation sites (as determined by
the NetOGlyc prediction program). Therefore, anti-MLP antibodies
raised against this C-terminal region are capable of binding to the
protein backbone of the highly glycosylated MLP glycoprotein
secreted from MLP positive eukaryotic cells as the peptide epitopes
of these C-terminal MLP specific antigen sites are not obscured by
glycosylation.
[0307] As described in this example, the C-terminal region (SEQ ID
NO:4 corresponding to aa 279-431 of full length MLP, shown as the
underlined region in FIG. 1) was expressed as a fusion protein with
an N-terminal histidine tag for use as an antigen to generate MLP
C-terminal specific monoclonal antibodies.
[0308] Methods
[0309] 1. Expression of rMLP C-Terminal Polypeptide Antigen
[0310] A nucleic acid sequence (SEQ ID NO:2) encoding a fusion
protein comprising the C-terminal region (aa 279-431 of human MLP,
set forth as SEQ ID NO:1) fused to a HIS tag as shown in FIG. 2,
was cloned into the pRSETB expression vector (Invitrogen), for
expression of the recombinant (rMLP) C-terminal fusion protein in
E. coli.
[0311] Purification of rMLP C-terminal protein was carried out
using a HIS GraviTrap nickel column. The bound recombinant rMLP
C-terminal protein was washed with 20 ml PBS buffer and then washed
again with 10 mM Imidazole before eluting with 200 mM Imidazole.
Fractions were collected from the column and analyzed by 12%
SDS-PAGE. Fractions containing purified rMLP C-terminal protein
were pooled and concentrated to yield the purified rMLP protein
(0.53 mg/ml). Western blot analysis using a polyhistidine tag
specific antibody showed that the purified rMLP C-terminal protein
ran at the predicted molecule weight of 28 kDa (data not
shown).
[0312] 2. Generation of Monoclonal Antibodies that Specifically
Bind the C-terminal Region of Human MLP
[0313] Mice were immunized with the purified rMLP C-terminal
protein and then boosted with the same protein. Mice with high
titers of antibodies were sacrificed, spleen cells from each mouse
were fused to myeloma cells following standard procedures and
supernatants from clones of hybridoma cells were screening using an
enzyme-linked immuno-sorbent assay (ELISA). Eight candidate murine
hybridomas were selected for analysis (named clones 1, 4, 5, 6, 7,
11, clone B and clone C).
[0314] The hybridomas were grown in Dulbecco's modified eagle's
medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine
serum (FBS, Gibco), 5 mL L-glutamine 200 mM (Sigma-Aldrich) and 5
mL 100 U/mL penicillin-streptomycin (Sigma-Aldrich). The hybridomas
were cultured and mouse IgG antibodies were purified from the
supernatants via protein G sepharose column chromatography (GE
Healthcare), which have the ability to bind with the Fc region of
IgG immunoglobulins. The purified monoclonal antibodies were then
analyzed by 12% SDS PAGE under reducing conditions and stained with
Coomassie Blue, which showed the expected light and heavy chain Ig
bands at 25 kDa and 50 kDa (data not shown).
[0315] 3. Testing Candidate Anti-MLP Monoclonal Antibodies Against
rMLP C-Terminal Protein in an ELISA Assay
[0316] Micro-titer ELISA plates (Maxisorb, Nunc), were coated with
10.0 .mu.g/ml of recombinant MLP C-terminal protein using coating
buffer (15 mM sodium carbonate anhydrous Na.sub.2CO.sub.3 and
sodium hydrogen carbonate NaHCO.sub.3, pH 9.6). The plate was
incubated overnight at 4.degree. C. The next day, residual protein
binding sites were blocked by adding 250 .mu.L of 1% bovine serum
albumen (BSA) in TBS buffer (10 mM Tris-HCL, 140 mM NaCl and 10 mM
CaCl.sub.2, pH 7.4) to each well in the plate and incubated at room
temperature for 2 hours. The plates were then washed three times
with TBS buffer with 0.05% and Tween20. Serial dilution of
monoclonal antibodies in TBS buffer (clone 1, 4, 5, 6, 7, C, 11 and
B) were added to the plates in duplicate starting from 1:100
dilution and incubated for two hours at room temperature. Wells
were then washed three times. 100 of goat anti-mouse antibody
conjugated with alkaline phosphatase (diluted 1:5000) was then
added to each well and incubated for two hours at room temperature.
The plates were washed three times; substrate solution (fast
p-Nitrophenyl phosphate tablet sets, Sigma) was then added and
incubated at room temperature for 20 to 30 minutes. The absorbance
was measured at 405 nm using Biorad ELISA micro-titre plate reader
model 608.
[0317] Results:
[0318] Three of the eight mAb clones tested (clone 11, B and C)
were positive in an ELISA assay against the recombinant MLP-C
terminal protein, although the signal with clone C was much weaker
than the signals seen with clones 11 and B, while five of the mAb
clones were negative in the ELISA assay (data not shown). mAb
clones B and 11 showed good binding against MLP with a titer of
approximately 1 .mu.g/ml.
[0319] To confirm the specificity of clones 11 and B for the MLP
C-terminal protein, they were also tested in an ELISA assay against
an unrelated control protein (plates were coated with 10.0 .mu.g/ml
of recombinant MASP-3) and were all found to be negative (data not
shown).
[0320] 4. Detection of rMLP by anti-MLP clones 11 and B by ELISA
Assay
[0321] Micro-titer ELISA plates (Maxisorb, Nunc), were coated with
anti-MLP monoclonal antibody clone 11 and clone B at serial
dilutions starting at a concentration of 1 .mu.g/mL down to 0.05
.mu.g/mL using coating buffer (15 mM sodium carbonate anhydrous
Na.sub.2CO.sub.3 and sodium hydrogen carbonate NaHCO.sub.3, pH
9.6). The plate was incubated overnight at 4.degree. C. The next
day, residual protein binding sites were blocked by adding 250
.mu.L of 1% bovine serum albumen (BSA) in TBS buffer (10 mM
Tris-HCL, 140 mM NaCl and 10 mM CaCl.sub.2, pH 7.4) to each well in
the plate and incubated at room temperature for 2 hours. The plates
were then washed three times with TBS buffer with 0.05% and
Tween20. Serial dilutions of recombinant MLP C-terminal protein in
a concentration range of from 0.01 .mu.g/mL to 5 .mu.g/mL were
added to the plates in duplicate and incubated for two hours at
room temperature. Wells were then washed three times. 100 .mu.L of
polyclonal goat anti-MLP antibody conjugated with alkaline
phosphatase (diluted 1:5000) was then added to each well and
incubated for two hours at room temperature. The plates were washed
three times; substrate solution (fast p-Nitrophenyl phosphate
tablet sets, Sigma) was then added and incubated at room
temperature for 20 to 30 minutes. The absorbance was measured at
405 nm using Biorad ELISA micro-titre plate reader model 608. As a
negative control, plates were coated with monoclonal anti-MLP
antibody clone B or clone 11 and developed with the polyclonal goat
anti-MLP antibody, but instead of the MLP antigen, only TBS buffer
was added to the mAb-coated plates.
[0322] Results:
[0323] FIG. 3A graphically illustrates the detection of rMLP by
anti-MLP mAb clone B by ELISA assay. As shown in FIG. 3A, the
sensitivity of this assay is very high, showing detection of the
rMLP antigen down to a concentration of 0.01 .mu.g/mL. As further
shown in FIG. 3A, dilution of the primary antibody mAb clone B down
to 0.01625 .mu.g/mL appears to give close to a maximal signal.
[0324] FIG. 3B graphically illustrates the detection of rMLP by
anti-MLP mAb clone 11 by ELISA assay. As shown in FIG. 3B, the
sensitivity of this assay is very high, showing detection of the
rMLP antigen down to a concentration of 0.01 .mu.g/mL. As further
shown in FIG. 3B, dilution of the primary antibody mAb clone 11
down to 0.01625 .mu.g/mL appears to give close to a maximal
signal.
[0325] 5. Development of a Sandwich ELISA Assay to Detect MLP
[0326] Micro-titer ELISA plates (Maxisorb, Nunc), were coated with
anti-MLP monoclonal antibody clone 11 at 0.1 .mu.g per well using
coating buffer (15 mM sodium carbonate anhydrous Na.sub.2CO.sub.3
and sodium hydrogen carbonate NaHCO.sub.3, pH 9.6). The plate was
incubated overnight at 4.degree. C. The next day, residual protein
binding sites were blocked by adding 250 .mu.L of 1% bovine serum
albumen (BSA) in TBS buffer (10 mM Tris-HCL, 140 mM NaCl and 10 mM
CaCl.sub.2, pH 7.4) to each well in the plate and incubated at room
temperature for 2 hours. The plates were then washed three times
with TBS buffer with 0.05% and Tween20. Serial dilutions of
recombinant MLP C-terminal protein in a concentration range of from
0.0001 .mu.g/mL to 1 .mu.g/mL were added to the plates in duplicate
and incubated for two hours at room temperature. Wells were then
washed three times. Polyclonal goat anti-MLP antibody conjugated
with alkaline phosphatase (0.5 .mu.g/mL) was then added to each
well and incubated for two hours at room temperature. The plates
were washed three times; substrate solution (fast p-Nitrophenyl
phosphate tablet sets, Sigma) was then added and incubated at room
temperature for 20 to 30 minutes. The absorbance was measured at
405 nm using Biorad ELISA micro-titre plate reader model 608. As a
negative control, the coating antibody mAb clone 11 was omitted and
the wells were incubated with coating buffer prior to the addition
of rMLP and the polyclonal MLP detection antibody.
[0327] Results:
[0328] FIG. 4 graphically illustrates the detection of rMLP with
anti-MLP mAb clone 11 by ELISA assay. As shown in FIG. 4, the
sensitivity of this sandwich ELISA assay is very high, showing
linearity down to a concentration of less than 10 ng/mL rMLP.
EXAMPLE 2
[0329] This Example describes Analysis of anti-MLP monoclonal
antibody Clones C, 11 and B for Use as an Epithelial Cancer Cell
Biomarker.
Background/Rationale:
[0330] Panc-1 is a human pancreatic carcinoma, epithelial-like cell
line and is used as an in vitro model of non-endocrine pancreatic
cancer for tumorigenicity studies (ATCC CRL-1469). Cell line A2780
is a human ovarian carcinoma cancer cell line (Louie K.G. et al.,
Cancer Res 45(5):2110-5, 1985; Hamilton T. C. et al., Seminars in
Oncology 11(3):285-298, 1984).
[0331] The following experiment was carried out to determine if the
anti-MLP monoclonal antibody clones C, 11 and B are capable of
detecting glycosylated MLP (gMLP) secreted from Panc-1 and A2780
cell lines.
Methods:
[0332] 1. Analysis of Anti-MLP mAb Clones C, 11 and B for Binding
to gMLP Secreted from Panc-1 (Human Pancreatic Cancer Cell
Line):
[0333] The three anti-MLP mAbs (clones C, 11 and B) were tested for
binding to gMLP present in supernatant obtained from a human
pancreatic cell line (Pancl) obtained from the ATCC in a dot blot
and ELISA assay format as follows.
[0334] A. Dot Blot Analysis
[0335] The three anti-MLP mAbs (clones C, 11 and B) generated as
described in Example 1, were tested against a human pancreatic
cancer cell line (Panc-1) obtained from the ATCC. Dot blot analysis
of the purified anti-MLP specific mAbs (clones C, 11 and B), was
carried out with the rMLP C-terminal protein and glycosylated full
length MLP (gMLP) that is present in the supernatant of pancreatic
cancer cell line Panc-1.
[0336] 4 .mu.l of protein (rMLP, gMLP from Pancl supernatant, or
rMASP-3) was dropped onto a nitrocellulose membrane and allowed to
dry at room temperature. The nitrocellulose membrane was then
blocked with 5% skimmed milk in PBS for 45 to 60 minutes with
gently shaking at room temperature. Anti-MLP mAb clones C, 11 and B
(final concentration of mAbs 1:1000 dilution of a 1 mg/ml protein G
Sepharose enriched antibody stock in a volume of 10 mL 5% skimmed
milk in PBS) were then added to the nitrocellulose membrane and
incubated at room temperature for 1 hour with gently shaking. The
membranes were then washed three times with PBS and 0.05% Tween 20.
Anti-mouse antibody conjugated with horseradish peroxidase (HRP)
diluted 1:6000 was added and incubated at room temperature for 1
hour with gently shaking. The membrane was then washed three times
and antibody binding was detected by ECL kit.
Results of Dot Blot Analysis:
[0337] FIG. 5 shows the results of a dot blot assay carried out
with anti-MLP monoclonal antibody Clone C, Clone B and Clone 11
against Panc1 cell line supernatant (row A), rMASP-3 polypeptide
(row B) and mMLP C-terminal protein (row C). As shown in FIG. 5,
all three mAbs (clone C, clone B and clone 11) were found to bind
to the naturally produced glycosylated form of MLP (gMLP) present
in the supernatant of the Panc-1 cell line (row A), and the rMLP
C-terminal protein control (row C), but did not bind to the
negative control rMASP-3 polypeptide (row B).
[0338] B. ELISA Assay using Plates Coated with gMLP from Ovarian
Cancer Cell Line A2789
[0339] The three anti-MLP mAbs (clone 11, B and C) were tested with
ELISA plates coated with 1.0 .mu.g/m1 of glycosylated MLP from
concentrated supernatant obtained from ovarian cancer cell line
A2789 as follows.
[0340] Micro-titer ELISA plates (Maxisorb, Nunc), were coated with
1.0 .mu.g/ml of glycosylated MLP (gMLP) from concentrated
supernatant obtained from ovarian cancer cell line A2789 using
coating buffer (15 mM sodium carbonate anhydrous Na.sub.2CO.sub.3
and sodium hydrogen carbonate NaHCO.sub.3, pH 9.6). The plate was
incubated overnight at 4.degree. C. The next day, residual protein
binding sites were blocked by adding 250 .mu.L of 1% bovine serum
albumen (BSA) in TBS buffer (10 mM Tris-HCL, 140 mM NaCl and 10 mM
CaCl.sub.2, pH 7.4) to each well in the plate and incubated at room
temperature for 2 hours. The plates were then washed three times
with TBS buffer with 0.05% and Tween20. Serial dilution of mAbs
(clone 11, B and C) in TBS buffer were added to the plates in
duplicate starting from a 1:100 dilution and incubated for two
hours at room temperature. Wells were then washed three times. 100
.mu.L of goat anti-mouse antibody conjugated with alkaline
phosphatase (diluted 1:5000) was then added to each well and
incubated for two hours at room temperature. The plates were washed
three times, substrate solution (fast p-Nitrophenyl phosphate
tablet sets, Sigma) was then added and incubated at room
temperature for 20 to 30 minutes. The absorbance was measured at
405 nm using Biorad ELISA micro-titre plate reader model 608.
Results of ELISA Assay with Plates Coated with gMLP from Ovarian
Cancer Cell Line A2789 [0341] As shown in FIG. 6, clones 11 and B
showed significant binding to gMLP, however in this assay format
clone C was only marginally higher for binding to gMLP than binding
to the negative control rMASP-3 protein.
[0342] 2. Analysis of Anti-MLP mAb Clones C, 11 and B for Binding
to gMLP Secreted from Human Ovarian Cancer Cell Line A2780.
[0343] The three anti-MLP mAbs (clones C, 5, 11 and B) were tested
for binding to gMLP present in supernatant obtained from a human
ovarian cancer cell line (A2780) obtained from the ATCC in a dot
blot, Western blot and ELISA assay format as follows.
[0344] A. Dot Blot Analysis
[0345] A dot blot assay was carried out as described above, using
supernatant of ovarian cancer cell line A2780. 4 .mu.l of protein
(rMLP, gMLP from A2780 supernatant, or rMASP-3) was dropped onto a
nitrocellulose membrane and allowed to dry at room temperature. The
nitrocellulose membrane was then blocked with 5% skimmed milk in
PBS for 45 to 60 minutes with gently shaking at room temperature.
Anti-MLP mAb clones C, 11 and B (final concentration of mAbs 1:1000
dilution of a 1 mg/ml protein G Sepharose enriched antibody stock
in a volume of 10 mL 5% skimmed milk in PBS) were then added to the
nitrocellulose membrane and incubated at room temperature for 1
hour with gently shaking. The membranes were then washed three
times with PBS and 0.05% Tween 20. Anti-mouse antibody conjugated
with horseradish peroxidase (HRP) diluted 1:6000 was added and
incubated at room temperature for 1 hour with gently shaking. The
membrane was then washed three times and antibody binding was
detected by ECL kit.
Results of Dot Blot Analysis:
[0346] FIG. 7 shows the results of a dot blot assay carried out
with anti-MLP antibody Clone C, Clone B and Clone 11 against
ovarian cancer cell line A2780 supernatant (row A), rMASP-3 protein
(row B) and rMLP C-terminal protein (row C).
[0347] As shown in FIG. 7, clone C, clone B and clone 11 were each
found to bind to the naturally produced glycosylated form of MLP
(gMLP) present in the supernatant of the
[0348] A2780 cell line (row A), and the rMLP C-terminal protein
control (row C), but did not bind to the negative control
recombinant MASP-3 polypeptide (row B).
[0349] B. Western Blot Analysis
[0350] As shown in FIG. 8, the dot blot results were confirmed by
Western blotting using the concentrated supernatant from the A2780
cell line, where the distinctive 180 kDa gMLP band (row 1) and the
28kDa rMLP C-terminal protein (row 3) were detected with clone C
(top panel), clone B (middle panel) and clone 11 (lower panel), and
the negative control protein rMASP-3 (row 2) was not detected by
the three MLP-specific mAbs.
[0351] C. ELISA Assay using Plates Coated with gMLP from A2780
Cells
[0352] The three anti-MLP mAbs (clone C, 5, 11, and B) were tested
with ELISA plates coated with 1 .mu.g/ml of concentrated
supernatant obtained from A2780 cells which contained trace amounts
of glycosylated native MLP using the methods described above.
Results of ELISA Assay with Plates Coated with gMLP from A2780
Cells
[0353] As shown in FIG. 9, clones 11 and B showed significant
binding to gMLP from A2780 cells, however in this assay format
clone C was only marginally higher for binding to gMLP than binding
to the negative control rMASP-3 protein. The negative control in
FIG. 9 is clone 11 against rMASP-3 protein.
TABLE-US-00021 TABLE 9 Summary of Results: detection of gMLP
secreted from Panc1 and A2780 cancer cell lines ELISA test positive
Hybridoma with both rMLP Clone Western Blot Dot Blot and gMLP Clone
B +++ +++ +++ Clone 11 +++ +++ +++ Clone C +++ +++ +
SUMMARY
[0354] These results demonstrate that MLP-specific monoclonal
antibody clones 11, B and C specifically bind rMLP C-terminal
protein and are capable of detecting gMLP secreted from a
pancreatic cancer cell line and an ovarian cancer cell line, as
determined by Western blot and Dot blot. Further, this example
demonstrates that MLP-specific monoclonal antibody clones 11 and B
are able to detect both rMLP C-terminal protein and gMLP secreted
from an ovarian cancer cell line in an ELISA assay format.
[0355] These MLP specific mAbs can be used in a sensitive detection
ELISA, for example, in the format of a kit to measure MLP levels in
the sera of ovarian cancer patients as a biomarker of early stage
ovarian cancer, which is an urgently needed non-invasive early
diagnosis to discover ovarian malignancies at an early stage when
treatment is effective and the prognosis much better than in later
stages of ovarian cancer.
EXAMPLE 3
[0356] This Example describes the cloning and sequencing of the DNA
encoding the VH and VL regions of anti-MLP monoclonal antibodies
produced by hybridoma clones 11, B and C.
[0357] Methods: Hybridoma cells of the cell lines secreting
anti-MLP monoclonal antibodies referred to as .alpha.MLP Clone 11,
.alpha.MLP Clone B and .alpha.MLP Clone C deposited at the ATCC on
Oct. 30, 2014 under the ATCC Designation numbers PTA-121699,
PTA-121700 and PTA-121701, respectively, were cultured in
Dulbecco's modified eagle's medium (DMEM, Sigma-Aldrich)
supplemented with 10% fetal bovine serum (FBS, Gibco), 5mL
L-glutamine 200 mM (Sigma-Aldrich) and 5 mL 100 U/mL
penicillin-streptomycin (Sigma-Aldrich). DNA was purified from the
hybridoma cell lines, the variable regions were PCR-amplified and
sequence analysis of clones #C, 11 and B was carried out using
standard methods.
[0358] Results:
[0359] Heavy Chain Variable Region (VH) sequences
[0360] The amino acid sequence of the VH region of anti-MLP
monoclonal antibody .alpha.MLP Clone 11, produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 as ATCC
Designation Number PTA-121699 is set forth as SEQ ID NO:7.
[0361] The amino acid sequence of the VH region of anti-MLP
monoclonal antibody .alpha.MLP Clone B, produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 as ATCC
Designation Number PTA-121700 is set forth as SEQ ID NO:8.
[0362] The amino acid sequence of the VH region of anti-MLP
monoclonal antibody .alpha.MLP Clone C, produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 as ATCC
Designation Number PTA-121701 is set forth as SEQ ID NO:9.
[0363] FIG. 10A shows an amino acid sequence alignment between the
variable heavy chain regions of anti-MLP monoclonal antibody clones
11 (SEQ ID NO:7), B (SEQ ID NO:8) and C (SEQ ID NO:9).
[0364] Light Chain Variable Region (VL) sequences
[0365] The amino acid sequence of the VL region of anti-MLP
monoclonal antibody .alpha.MLP Clone 11, produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 as ATCC
Designation Number PTA-121699 is set forth as SEQ ID NO:10.
[0366] The amino acid sequence of the VL region of anti-MLP
monoclonal antibody .alpha.MLP Clone B, produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 as ATCC
Designation Number PTA-121700 is set forth as SEQ ID NO:11.
[0367] The amino acid sequence of the VL region of anti-MLP
monoclonal antibody .alpha.MLP Clone C, produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 as ATCC
Designation Number PTA-121701 is set forth as SEQ ID NO:12.
[0368] FIG. 10B shows an amino acid sequence alignment between the
variable light chain regions of anti-MLP monoclonal antibody clones
11 (SEQ ID NO:10), B (SEQ ID NO:11) and 2(C) (SEQ ID NO:12).
[0369] The high degree of sequence identity between clone 11 and
clone B suggests that both clones originated from the same parent
hybridoma cell binding to the identical MLP epitope.
[0370] In accordance with various embodiments, the present
disclosure provides:
[0371] 1. An isolated antibody, or antigen binding fragment
thereof, that specifically binds to an epitope in the C-terminal
region of human mucin-like protein, set forth as SEQ ID NO:4.
[0372] 2. The antibody of paragraph 1, wherein the antibody is a
monoclonal antibody.
[0373] 3. The antibody of paragraph 2, wherein said antibody is a
recombinant, humanized or chimeric antibody.
[0374] 4. The antibody of paragraph 2, wherein said antibody is a
fully human antibody.
[0375] 5. The antibody of paragraph 1, wherein said antibody or
antigen binding fragment thereof is capable of binding to
glycosylated human MLP secreted from an epithelial cancer cell
line.
[0376] 6. The antibody of paragraph 5, wherein said antibody or
antigen binding fragment thereof is capable of binding to
glycosylated human MLP in an ELISA assay format.
[0377] 7. The antibody of paragraph 1, wherein said antibody or
antigen binding fragment thereof binds to human MLP with an KD of
less than 10 nM.
[0378] 8. The antibody of paragraph 1, wherein said antibody, or
antigen binding fragment thereof recognizes at least part of an
epitope recognized by one or more reference antibodies selected
from the group consisting of: [0379] (i) the monoclonal anti-MLP
antibody Clone 11 produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121699; [0380] (ii) the monoclonal anti-MLP antibody Clone B
produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 under the ATCC Designation Number PTA-121700; and [0381]
(iii) the monoclonal anti-MLP antibody clone C produced by the
hybridoma cell line deposited at the ATCC on Oct. 30, 2014 under
the ATCC Designation Number PTA-121701.
[0382] 9. The antibody of paragraph 1, wherein said antibody
comprises a variable region of the heavy chain comprising or
consisting of a sequence which is at least 90% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NO:7, SEQ ID NO:8 and SEQ ID NO:9.
[0383] 10. The antibody of paragraph 1, wherein said antibody
comprises a variable region of the light chain comprising or
consisting of a sequence which is at least 90% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NO:10, SEQ ID NO:11 and SEQ ID NO:12.
[0384] 11. The antibody of any of paragraphs 1-8, wherein said
antibody is labeled with a detectable moiety.
[0385] 12. The antibody of paragraph 1, wherein said antibody is
coupled to a therapeutic agent.
[0386] 13. The antibody of paragraph 1, wherein said antibody is
immobilized on a substrate.
[0387] 14. An isolated monoclonal antibody, or antigen binding
fragment thereof, that binds to human mucin-like protein (MLP),
comprising:
[0388] (i) a heavy chain variable region comprising CDR-H1, CDR-H2
and CDR-H3 sequences; and
[0389] (ii) a light chain variable region comprising CDR-L1, CDR-L2
and CDR-L3, wherein the heavy chain variable region CDR-H3 sequence
comprises an amino acid sequence set forth as SEQ ID NO:15, SEQ ID
NO:35, SEQ ID NO:36 or SEQ ID NO:19, and conservative sequence
modifications thereof, wherein the light chain variable region
CDR-L3 sequence comprises an amino acid sequence set forth as SEQ
ID NO:23 or SEQ ID NO:27, and conservative sequence modifications
thereof, and wherein the isolated antibody binds to human
mucin-like protein (MLP).
[0390] 15. The isolated antibody or antigen binding fragment of
paragraph 14, wherein the heavy chain variable region CDR-H2
sequence comprises an amino acid sequence set forth as SEQ ID
NO:20, 14, 16 or 18, and conservative sequence modifications
thereof.
[0391] 16. The isolated antibody or antigen binding fragment of
paragraph 15, wherein the heavy chain variable region CDR-H1
sequence comprises an amino acid sequence set forth as SEQ ID NO:13
or SEQ ID NO:17, and conservative modifications thereof.
[0392] 17. The isolated antibody or antigen binding fragment of
paragraph 16, wherein the light chain variable region CDR-L2
sequence comprises an amino acid sequence set forth as SEQ ID NO:22
or SEQ ID NO:26 and conservative modifications thereof.
[0393] 18. The isolated antibody or antigen binding fragment of
paragraph 17, wherein the light chain variable region CDR-L1
sequence comprises an amino acid sequence set forth as SEQ ID
NO:28, SEQ ID NO:21, SEQ ID NO:24 or SEQ ID NO:25 and conservative
modifications thereof.
[0394] 19. The isolated antibody or antigen binding fragment of
paragraph 16, wherein the CDR-H1 of the heavy chain variable region
comprises SEQ ID NO:13.
[0395] 20. The isolated antibody or antigen binding fragment of
paragraph 15, wherein the CDR-H2 of the heavy chain variable region
comprises SEQ ID NO:20.
[0396] 21. The isolated antibody or antigen binding fragment of
paragraph 14, wherein the CDR-H3 of the heavy chain variable region
comprises SEQ ID NO:15.
[0397] 22. The isolated antibody of paragraph 20, wherein the amino
acid sequence set forth in SEQ ID NO:20 contains a T at position
9.
[0398] 23. The isolated antibody of paragraph 18, wherein the
CDR-L1 of the light chain variable region comprises SEQ ID
NO:28.
[0399] 24. The isolated antibody of paragraph 23, wherein the amino
acid sequence set forth in SEQ ID NO:28 contains a T at position
9.
[0400] 25. The isolated antibody of paragraph 23, wherein the amino
acid sequence set forth in SEQ ID NO:28 contains an S at position
9.
[0401] 26. The isolated antibody of paragraph 18, wherein CDR-L2 of
the light chain variable region comprises SEQ ID NO:22.
[0402] 27. The isolated antibody of paragraph 18, wherein CDR-L2 of
the light chain variable region comprises SEQ ID NO:26.
[0403] 28. The isolated antibody of paragraph 14, wherein the
CDR-L3 of the light chain variable region comprises SEQ ID
NO:23.
[0404] 29. The isolated antibody of paragraph 14, wherein the
antibody or antigen-binding fragment thereof is selected from the
group consisting of a Fab, a Fab' fragment, a F(ab')2 fragment and
a whole antibody.
[0405] 30. The isolated antibody of paragraph 14, wherein the
antibody or antigen-binding fragment thereof is selected from the
group consisting of a single chain antibody, an ScFv, and a
univalent antibody lacking a hinge region.
[0406] 31. The isolated antibody of paragraph 14, wherein said
antibody binds human MLP with a K.sub.D of 10 nM or less.
[0407] 32. The isolated antibody of paragraph 14, wherein said
antibody binds glycosylated MLP secreted from an epithelial cancer
cell line.
[0408] 33. The isolated antibody of paragraph 14, wherein said
antibody binds human MLP in an ELISA assay format.
[0409] 34. The isolated antibody of paragraph 14, wherein said
antibody is labeled with a detectable moiety.
[0410] 35. The isolated antibody of paragraph 14, wherein said
antibody is coupled to a therapeutic agent.
[0411] 36. The isolated antibody of paragraph 14, wherein said
antibody is immobilized on a substrate.
[0412] 37. The isolated antibody of paragraph 14, wherein said
antibody is humanized or fully human.
[0413] 38. The isolated antibody of paragraph 14, or antigen
binding fragment thereof, wherein said antibody comprises (i) a
heavy chain variable region comprising CDR-H1 (SEQ ID NO:13),
CDR-H2 (SEQ ID NO:14) and CDR-H3 (SEQ ID NO:15, SEQ ID NO:35 or SEQ
ID NO:36) and (ii) a light chain variable region comprising CDR-L1
(SEQ ID NO:21), CDR-L2 (SEQ ID NO:22) and CDR-L3 (SEQ ID NO:23),
and conservative modifications thereof.
[0414] 39. The isolated antibody of paragraph 14, or antigen
binding fragment thereof, wherein said antibody comprises a heavy
chain variable region comprising CDR-H1 (SEQ ID NO:13), CDR-H2 (SEQ
ID NO:16) and CDR-H3 (SEQ ID NO:15) and (ii) a light chain variable
region comprising CDR-L1 (SEQ ID NO:24), CDR-L2 (SEQ ID NO:22) and
CDR-L3 (SEQ ID NO:23), and conservative modifications thereof.
[0415] 40. The isolated antibody of paragraph 14, or antigen
binding fragment thereof, wherein said antibody comprises (i) a
heavy chain variable region comprising CDR-H1 (SEQ ID NO:17),
CDR-H2 (SEQ ID NO:18) and CDR-H3 (SEQ ID NO:19) and (ii) a light
chain variable region comprising CDR-L1 (SEQ ID NO:25), CDR-L2 (SEQ
ID NO:26) and CDR-L3 (SEQ ID NO:27), and conservative modifications
thereof.
[0416] 41. The isolated antibody of paragraph 14, wherein said
antibody comprises a heavy chain variable domain comprising SEQ ID
NO:7, or a variant thereof having a sequence at least 90% identical
to SEQ ID NO:7.
[0417] 42. The isolated antibody of paragraph 14, wherein said
antibody comprises a heavy chain variable domain comprising SEQ ID
NO:8, or a variant thereof having a sequence that is at least 90%
identical to SEQ ID NO:8.
[0418] 43. The isolated antibody of paragraph 14, wherein said
antibody comprises a heavy chain variable domain comprising SEQ ID
NO:9, or a variant thereof having a sequence that is at least at
least 90% identical to SEQ ID NO:9.
[0419] 44. A nucleic acid molecule encoding the amino acid sequence
of an anti-MLP antibody, or fragment thereof, as set forth in any
of paragraphs 14-43.
[0420] 45. An expression cassette comprising a nucleic acid
molecule encoding an anti-MLP antibody of the invention according
to paragraph 44.
[0421] 46. A cell comprising at least one of the nucleic acid
molecules encoding an anti-MLP antibody of the invention according
to paragraph 44 or paragraph 45.
[0422] 47. An anti-MLP monoclonal antibody designated as Clone 11
produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 having the ATCC Designation Number PTA-121699.
[0423] 48. An anti-MLP monoclonal antibody designated as Clone B
produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 having the ATCC Designation Number PTA-121700.
[0424] 49. An anti-MLP monoclonal antibody designated as Clone C
produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 having the ATCC Designation Number PTA-121701.
[0425] 50. A hybridoma cell line producing an anti-MLP antibody,
wherein the cell line is selected from the group consisting of
[0426] (i) a hybridoma cell line secreting anti-MLP monoclonal
antibody Clone 11 having the ATCC Designation Number
PTA-121699;
[0427] (ii) a hybridoma cell line secreting anti-MLP monoclonal
antibody Clone B having the ATCC Designation Number PTA-121700
and
[0428] (iii) a hybridoma cell line secreting anti-MLP monoclonal
antibody Clone C having the ATCC Designation Number PTA-121701.
[0429] 51. An isolated polypeptide comprising the amino acid
sequence of SEQ ID NO:1, or a variant thereof having at least 95%
identity to SEQ ID NO:1.
[0430] 52. An isolated polypeptide comprising the amino acid
sequence of SEQ ID NO:4, or a variant thereof having at least 95%
identity to SEQ ID NO:4.
[0431] 53. An isolated polypeptide comprising the amino acid
sequence of SEQ ID NO:5, or a variant thereof having at least 95%
identity to SEQ ID NO:5.
[0432] 54. An isolated polypeptide comprising the amino acid
sequence of SEQ ID NO:6, or a variant thereof having at least 95%
identity to SEQ ID NO:6.
[0433] 55. A method of generating an isolated anti-MLP antibody
comprising culturing the cell of paragraph 46 or paragraph 50 under
conditions allowing for expression of the nucleic acid molecules
encoding the anti-MLP antibody and isolating said anti-MLP
antibody.
[0434] 56. A method of detecting or diagnosing epithelial cancer by
determining the presence or amount of MLP in a biological sample
from a test subject, the method comprising
[0435] (a) contacting a biological sample from a test subject with
an anti-MLP antibody or antigen-binding fragment thereof in an in
vitro immunoassay; and
[0436] (b) detecting the presence or absence of binding of said
antibody, wherein the presence of binding indicates the presence or
amount of MLP in the sample, wherein the antibody or fragment
thereof binds to an epitope in the C-terminal region of MLP, set
forth as SEQ ID NO:4.
[0437] 57. The method of paragraph 56, wherein said anti-MLP
antibody is labeled with a detectable moiety and step (b) comprises
detecting the presence or amount of said detectable moiety.
[0438] 58. The method of paragraph 56 or paragraph 57, further
comprising comparing the amount of MLP detected in accordance with
step (b) with a reference standard or control sample from a healthy
subject, wherein an increase of at least two-fold or higher (e.g.,
at least five-fold, or at least ten-fold) in the level of MLP in
the test sample as compared to the control sample (or reference
standard) indicates the presence of, or increased risk for
developing an epithelial cancer, such as ovarian cancer or
pancreatic cancer, in the test subject.
[0439] 59. The method of paragraph 56, wherein the biological
sample is selected from the group consisting of blood, serum,
plasma and tissue.
[0440] 60. The method of paragraph 56, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody that binds to
the same epitope or competes for binding to MLP with a reference
antibody selected from the group consisting of: [0441] (i) the
monoclonal anti-MLP antibody Clone 11 produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation Number PTA-121699; [0442] (ii) the monoclonal anti-MLP
antibody Clone B produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121700; and [0443] (iii) the monoclonal anti-MLP antibody clone
C produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 under the ATCC Designation Number PTA-121701.
[0444] 61. The method of paragraph 56, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody having a
heavy chain variable region CDR-H3 sequence comprising an amino
acid sequence set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36
or SEQ ID NO:19, and conservative sequence modifications thereof,
and having a light chain variable region CDR-L3 sequence comprising
an amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27,
and conservative sequence modifications thereof.
[0445] 62. The method of paragraph 56, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody that
comprises a heavy chain variable region and/or a light chain
variable region set forth in Table 1, and conservative sequence
modifications thereof.
[0446] 63. The method of paragraph 56, further comprising
performing an immunoassay with one or more additional antibodies
that bind to ovarian and/or pancreatic cancer biomarkers.
[0447] 64. The method of paragraph 56, wherein the test subject is:
(i) apparently healthy (ii) has a family history of ovarian or
pancreatic cancer; (iii) experiencing one or more symptoms
associated with ovarian cancer; or (iv) known to be suffering from
ovarian or pancreatic cancer and has had or is currently undergoing
treatment for ovarian or pancreatic cancer.
[0448] 65. The method of paragraph 56, wherein the method further
comprises comparing the results of the assay from biological
samples obtained from the test subject at one or more time points
to assess the efficacy of a treatment regimen.
[0449] 66. The method of paragraph 56, wherein the anti-MLP
antibody is immobilized on a substrate.
[0450] 67. The method of paragraph 56, wherein the immunoassay is
an ELISA assay.
[0451] 68. A method of detecting or diagnosing ovarian or
pancreatic cancer in a test subject comprising (a) administering to
a living test subject a humanized or fully human anti-MLP antibody
or antigen-binding fragment thereof that binds to an epitope in the
C-terminal region of MLP, set forth as SEQ ID NO:4; and (b)
detecting the presence or absence or the amount of the antibody or
fragment thereof bound to MLP, wherein detection of the presence or
amount of MLP in the subject indicates the presence of ovarian or
pancreatic cancer.
[0452] 69. The method of paragraph 68, wherein the anti-MLP
antibody is labeled with a detectable moiety suitable for in vivo
use and step (b) comprises detecting the presence or amount of the
detectable moiety.
[0453] 70. The method of paragraph 68 or paragraph 69, wherein
method is used in an imaging, intraoperative, endoscopic or
intravascular procedure.
[0454] 71. The method of paragraph 68, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody that binds to
the same epitope or competes for binding to MLP with a reference
antibody selected from the group consisting of: [0455] (i) the
monoclonal anti-MLP antibody Clone 11 produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation Number PTA-121699; [0456] (ii) the monoclonal anti-MLP
antibody Clone B produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121700; and [0457] (iii) the monoclonal anti-MLP antibody clone
C produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 under the ATCC Designation Number PTA-121701.
[0458] 72. The method of paragraph 68, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody having a
heavy chain variable region CDR-H3 sequence comprising an amino
acid sequence set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36
or SEQ ID NO:19, and conservative sequence modifications thereof,
and having a light chain variable region CDR-L3 sequence comprising
an amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27,
and conservative sequence modifications thereof.
[0459] 73. The method of paragraph 68, wherein the test subject is:
(i) apparently healthy (ii) has a family history of ovarian or
pancreatic cancer; (iii) experiencing one or more symptoms
associated with ovarian cancer; or (iv) known to be suffering from
ovarian or pancreatic cancer and has had or is currently undergoing
treatment for ovarian or pancreatic cancer.
[0460] 74. A method of treating a subject suffering from ovarian or
pancreatic cancer comprising administering to an individual
suffering from ovarian or pancreatic cancer a humanized or fully
human anti-MLP antibody or antigen-binding fragment thereof that
binds to an epitope in the C-terminal region of MLP, set forth as
SEQ ID NO:4, wherein the antibody or fragment thereof is coupled to
a therapeutic agent.
[0461] 75. The method of paragraph 74, wherein the therapeutic
agent is a chemotherapeutic agent.
[0462] 76. The method of paragraph 74, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody that binds to
the same epitope or competes for binding to MLP with a reference
antibody selected from the group consisting of: [0463] (i) the
monoclonal anti-MLP antibody Clone 11 produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation Number PTA-121699; [0464] (ii) the monoclonal anti-MLP
antibody Clone B produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121700; and [0465] (iii) the monoclonal anti-MLP antibody clone
C produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 under the ATCC Designation Number PTA-121701.
[0466] 77. The method of paragraph 74, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody having a
heavy chain variable region CDR-H3 sequence comprising an amino
acid sequence set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36
or SEQ ID NO:19, and conservative sequence modifications thereof,
and having a light chain variable region CDR-L3 sequence comprising
an amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27,
and conservative sequence modifications thereof.
[0467] 78. A method of detecting or diagnosing a mucin-secreting
type of cancer by determining the presence or amount of MLP in a
biological sample from a test subject, the method comprising:
[0468] (a) contacting a biological sample from a test subject with
an anti-MLP antibody or antigen-binding fragment thereof in an in
vitro immunoassay; and
[0469] (b) detecting the presence or absence of binding of said
antibody, wherein the presence of binding indicates the presence or
amount of MLP in the sample,
[0470] wherein the antibody or fragment thereof binds to an epitope
in the C-terminal region of MLP, set forth as SEQ ID NO:4.
[0471] 79. The method of paragraph 78, wherein said anti-MLP
antibody is labeled with a detectable moiety and step (b) comprises
detecting the presence or amount of said detectable moiety.
[0472] 80. The method of paragraph 78 or paragraph 79, further
comprising comparing the amount of MLP detected in accordance with
step (b) with a reference standard or control sample from a healthy
subject, wherein an increase of at least two-fold or higher (e.g.,
at least five-fold, or at least ten-fold) in the level of MLP in
the test sample as compared to the control sample (or reference
standard) indicates the presence of, or increased risk for
developing an mucin-secreting type of cancer in the test
subject.
[0473] 81. The method of paragraph 78, wherein the biological
sample is selected from the group consisting of blood, serum,
plasma and tissue.
[0474] 82. The method of paragraph 78, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody that binds to
the same epitope or competes for binding to MLP with a reference
antibody selected from the group consisting of: [0475] (i) the
monoclonal anti-MLP antibody Clone 11 produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation Number PTA-121699; [0476] (ii) the monoclonal anti-MLP
antibody Clone B produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121700; and [0477] (iii) the monoclonal anti-MLP antibody clone
C produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 under the ATCC Designation Number PTA-121701.
[0478] 83. The method of paragraph 78, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody having a
heavy chain variable region CDR-H3 sequence comprising an amino
acid sequence set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36
or SEQ ID NO:19, and conservative sequence modifications thereof,
and having a light chain variable region CDR-L3 sequence comprising
an amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27,
and conservative sequence modifications thereof.
[0479] 84. The method of paragraph 78, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody that
comprises a heavy chain variable region and/or a light chain
variable region set forth in Table 1, and conservative sequence
modifications thereof.
[0480] 85. The method of paragraph 78, wherein the mucin-secreting
cancer type is selected from the group consisting of ovarian,
pancreatic, colorectal, breast, appendiceal, lung, renal, cervical,
biliary, esophageal and epithelial skin cancer.
[0481] 86. The method of paragraph 78, wherein the mucin-secreting
cancer type is ovarian or pancreatic cancer.
[0482] 87. The method of paragraph 86, further comprising
performing an immunoassay with one or more additional antibodies
that bind to ovarian and/or pancreatic cancer biomarkers.
[0483] 88. The method of paragraph 78, wherein the test subject is:
(i) apparently healthy (ii) has a family history of cancer; (iii)
experiencing one or more symptoms associated with cancer; or (iv)
known to be suffering from cancer and has had or is currently
undergoing treatment for cancer.
[0484] 89. The method of paragraph 78, wherein the method further
comprises comparing the results of the assay from biological
samples obtained from the test subject at one or more time points
to assess the efficacy of a treatment regimen.
[0485] 90. The method of paragraph 78, wherein the anti-MLP
antibody is immobilized on a substrate.
[0486] 91. The method of paragraph 78, wherein the immunoassay is
an ELISA assay.
[0487] 92. A method of detecting or diagnosing the presence of a
mucin-secreting cancer in a test subject comprising (a)
administering to a living test subject a humanized or fully human
anti-MLP antibody or antigen-binding fragment thereof that binds to
an epitope in the C-terminal region of MLP, set forth as SEQ ID
NO:4; and (b) detecting the presence or absence or the amount of
the antibody or fragment thereof bound to MLP, wherein detection of
the presence or amount of MLP in the subject indicates the presence
of mucin-secreting cancer.
[0488] 93. The method of paragraph 92, wherein the anti-MLP
antibody is labeled with a detectable moiety suitable for in vivo
use and step (b) comprises detecting the presence or amount of the
detectable moiety.
[0489] 94. The method of paragraph 92 or paragraph 93, wherein
method is used in an imaging, intraoperative, endoscopic or
intravascular procedure.
[0490] 95. The method of paragraph 92, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody that binds to
the same epitope or competes for binding to MLP with a reference
antibody selected from the group consisting of: [0491] (i) the
monoclonal anti-MLP antibody Clone 11 produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation Number PTA-121699; [0492] (ii) the monoclonal anti-MLP
antibody Clone B produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121700; and [0493] (iii) the monoclonal anti-MLP antibody clone
C produced by the hybridoma cell line deposited at the ATCC on Oct.
30, 2014 under the ATCC Designation Number PTA-121701.
[0494] 96. The method of paragraph 92, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody having a
heavy chain variable region CDR-H3 sequence comprising an amino
acid sequence set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36
or SEQ ID NO:19, and conservative sequence modifications thereof,
and having a light chain variable region CDR-L3 sequence comprising
an amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27,
and conservative sequence modifications thereof.
[0495] 97. The method of paragraph 92, wherein the test subject is:
(i) apparently healthy (ii) has a family history of cancer; (iii)
experiencing one or more symptoms associated with cancer; or (iv)
known to be suffering from cancer and has had or is currently
undergoing treatment for cancer.
[0496] 98. A method of treating a subject suffering from a
mucin-secreting type of cancer comprising administering to an
individual suffering from a mucin-secreting type of cancer a
humanized or fully human anti-MLP antibody or antigen-binding
fragment thereof that binds to an epitope in the C-terminal region
of MLP, set forth as SEQ ID NO:4, wherein the antibody or fragment
thereof is coupled to a therapeutic agent.
[0497] 99. The method of paragraph 98, wherein the therapeutic
agent is a chemotherapeutic agent.
[0498] 100. The method of paragraph 98, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody that binds to
the same epitope or competes for binding to MLP with a reference
antibody selected from the group consisting of: [0499] (i) the
monoclonal anti-MLP antibody Clone 11 produced by the hybridoma
cell line deposited at the ATCC on Oct. 30, 2014 under the ATCC
Designation Number PTA-121699; [0500] (ii) the monoclonal anti-MLP
antibody Clone B produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation
[0501] Number PTA-121700; and [0502] (iii) the monoclonal anti-MLP
antibody clone C produced by the hybridoma cell line deposited at
the ATCC on Oct. 30, 2014 under the ATCC Designation Number
PTA-121701.
[0503] 101. The method of paragraph 98, wherein the anti-MLP
antibody or fragment thereof is a monoclonal antibody having a
heavy chain variable region CDR-H3 sequence comprising an amino
acid sequence set forth as SEQ ID NO:15, SEQ ID NO:35, SEQ ID NO:36
or SEQ ID NO:19, and conservative sequence modifications thereof,
and having a light chain variable region CDR-L3 sequence comprising
an amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:27,
and conservative sequence modifications thereof.
[0504] 102. The method of paragraph 98, wherein the mucin-secreting
cancer type is selected from the group consisting of ovarian,
pancreatic, colorectal, breast, appendiceal, lung, renal, cervical,
biliary, esophageal and epithelial skin cancer.
[0505] 103. A composition comprising an anti-MLP antibody as set
forth in any of paragraphs 1-43.
[0506] 104. A substrate for use in an immunoassay comprising at
least one anti-MLP antibody as set forth in any of paragraphs
1-43.
[0507] 105. A kit for detecting the presence of MLP in a biological
sample, said kit comprising (a) at least one container, and (b) at
least one anti-MLP antibody as set forth in any of paragraphs
1-43.
[0508] While the preferred embodiment of the invention has been
illustrated and described, it will be appreciated that various
changes can be made therein without departing from the spirit and
scope of the invention.
Sequence CWU 1
1
361431PRTHomo sapiens 1Met Lys Pro Arg Gln Lys Glu Gln Asp Thr Arg
Leu Arg Lys Leu Arg1 5 10 15Glu Ser Ser Glu Gly Asp Gln Trp Leu Glu
Asn Glu Lys Thr Lys Pro 20 25 30Leu Arg Pro Gln Gln Gln Pro Gln Cys
Gln Pro Ala Gly Gly Thr Gly 35 40 45Gln Arg Arg Gly Ser Gly Ser Ser
Pro Ser Ala Asp Gln Gln Gly Ala 50 55 60Gln Asp Arg Glu Glu Glu Ala
Ala Ala Ala Pro Ala Pro Thr Ser Arg65 70 75 80Gly His Arg Thr Glu
Lys Arg Lys Pro Gln Gln Pro Gln Arg Arg Pro 85 90 95Ala Gly Gly Thr
Gly Gln Arg Arg Gly Ser Arg Ser Ser Ser Ser Ala 100 105 110Asp Gln
Gln Gly Ala Gln Asp Arg Glu Glu Glu Ala Ala Ala Ala Pro 115 120
125Ala Pro Thr Ser Ser Gly His Arg Thr Glu Lys Arg Lys Pro Gln Gln
130 135 140Pro Gln Cys Arg Pro Ala Ala Gly Thr Gly Gln Arg Arg Gly
Ser Gly145 150 155 160Cys Ser Pro Ser Ala Asp Gln Gln Arg Ala Gln
Asp Arg Glu Glu Glu 165 170 175Ala Thr Ala Ala Pro Val Pro Thr Ser
Ser Gly His Arg Thr Glu Lys 180 185 190Arg Lys Arg Leu Gln Leu Gln
Cys Gln Pro Ala Gly Gly Thr Gly Gln 195 200 205Arg Arg Gly Ser Arg
Ser Ser Pro Ser Ala Asp Gln Gln Arg Ala Gln 210 215 220Asp Arg Glu
Glu Glu Ala Ala Ala Ala Pro Ala Pro Thr Ser Arg Gly225 230 235
240His Arg Thr Glu Lys Arg Lys Pro Gln Gln Pro Gln Arg Arg Pro Ala
245 250 255Ala Gly Thr Gly Gln Arg Arg Gly Ser Gly Cys Ser Pro Ser
Ala Asp 260 265 270Gln Gln Gly Ala Gln Asp Arg Glu Glu Glu Ala Ala
Ala Ala Pro Ala 275 280 285Pro Thr Ser Arg Gly His Arg Thr Glu Lys
Arg Lys Arg Leu Gln Pro 290 295 300Gln Arg Arg Pro Ala Gly Gly Thr
Gly Gln Arg Arg Gly Ser Arg Ser305 310 315 320Ser Pro Ser Ala Asp
Gln Gln Arg Ala Gln Asp Arg Glu Glu Glu Ala 325 330 335Ala Ala Ala
Pro Val Pro Thr Ser Arg Gly His Arg Thr Glu Lys Arg 340 345 350Lys
Arg Leu Gln Leu Gln Cys Gln Pro Ala Gly Gly Thr Gly Gln Arg 355 360
365Arg Gly Ser Gly Ser Ser Pro Ser Ala Asp Gln Gln Arg Ala Gln Asp
370 375 380Arg Glu Glu Glu Ala Ala Ala Ala Pro Ala Pro Thr Ser Ser
Gly His385 390 395 400Arg Thr Glu Lys Arg Lys Arg Gln Gln Pro Gln
Arg Arg Pro Ala Ala 405 410 415Gly Thr Gly Gln Arg Arg Gly Ser Glu
Glu Met Glu Glu Glu Gly 420 425 4302971DNAArtificialSynthetic
2attttgttta ctttaagaag gagatataca tatgcggggt tctcatcatc atcatcatca
60tggtatggct agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga
120taaggatcga tggggatccc ccgggctgca ggaattcggc acgagagaag
aggaagccgc 180agcagcccca gcgccgacca gcagggggca caggacagag
aagaggaagc ggctacagcc 240ccagcgccga ccagcagggg gcacaggaca
gagaagagga agccgcagca gccccagcgc 300cgaccagcag cgggcacagg
acagagaaga ggaagcggct gcagctccag tgccaaccag 360cagggggcac
aggacagaga agaggaagcg gctgcagctc cagtgccaac cagcaggggg
420cacaggacag agaagaggaa gcggcagcag ccccagtgcc gaccagcagc
gggcacagga 480cagagaagag gaagccgcag cagccccagc gccgaccagc
agcgggcaca ggacagagaa 540gaggaagcgg cagcagcccc agcgccgacc
agcagcgggc acaggacaga gaagaggaag 600tgaggaaatg gaagaagagg
gttgacctga gctgaaggac atgaacttcc acgtgaaata 660gccccagggc
cgggcacacg aggtcaggag ttcaagacca gcctggccaa gatggtgaaa
720ccctgtctct actaaaaata aaaaattagc tgggtgcggt ggcaggcacc
tgtaatccca 780gctactcagg aggctgaggc aggagaatca cttgaaccca
ggaggtggag gttacagtga 840gccaagatct caccactgca ctgtagcctg
ggcaacagag caagactcca tgaaaaaaaa 900aaaaaaaaac tcgagggggg
gcccggtacc atggaattcg aagcttgatc cggctgctaa 960caaagcccga a
9713197PRTArtificialSynthetic 3Met Arg Gly Ser His His His His His
His Gly Met Ala Ser Met Thr1 5 10 15Gly Gly Gln Gln Met Gly Arg Asp
Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30Arg Trp Gly Ser Pro Gly Leu
Gln Glu Phe Gly Thr Arg Glu Glu Glu 35 40 45Ala Ala Ala Ala Pro Ala
Pro Thr Ser Arg Gly His Arg Thr Glu Lys 50 55 60Arg Lys Arg Leu Gln
Pro Gln Arg Arg Pro Ala Gly Gly Thr Gly Gln65 70 75 80Arg Arg Gly
Ser Arg Ser Ser Pro Ser Ala Asp Gln Gln Arg Ala Gln 85 90 95Asp Arg
Glu Glu Glu Ala Ala Ala Ala Pro Val Pro Thr Ser Arg Gly 100 105
110His Arg Thr Glu Lys Arg Lys Arg Leu Gln Leu Gln Cys Gln Pro Ala
115 120 125Gly Gly Thr Gly Gln Arg Arg Gly Ser Gly Ser Ser Pro Ser
Ala Asp 130 135 140Gln Gln Arg Ala Gln Asp Arg Glu Glu Glu Ala Ala
Ala Ala Pro Ala145 150 155 160Pro Thr Ser Ser Gly His Arg Thr Glu
Lys Arg Lys Arg Gln Gln Pro 165 170 175Gln Arg Arg Pro Ala Ala Gly
Thr Gly Gln Arg Arg Gly Ser Glu Glu 180 185 190Met Glu Glu Glu Gly
1954153PRTArtificialSynthetic 4Arg Glu Glu Glu Ala Ala Ala Ala Pro
Ala Pro Thr Ser Arg Gly His1 5 10 15Arg Thr Glu Lys Arg Lys Arg Leu
Gln Pro Gln Arg Arg Pro Ala Gly 20 25 30Gly Thr Gly Gln Arg Arg Gly
Ser Arg Ser Ser Pro Ser Ala Asp Gln 35 40 45Gln Arg Ala Gln Asp Arg
Glu Glu Glu Ala Ala Ala Ala Pro Val Pro 50 55 60Thr Ser Arg Gly His
Arg Thr Glu Lys Arg Lys Arg Leu Gln Leu Gln65 70 75 80Cys Gln Pro
Ala Gly Gly Thr Gly Gln Arg Arg Gly Ser Gly Ser Ser 85 90 95Pro Ser
Ala Asp Gln Gln Arg Ala Gln Asp Arg Glu Glu Glu Ala Ala 100 105
110Ala Ala Pro Ala Pro Thr Ser Ser Gly His Arg Thr Glu Lys Arg Lys
115 120 125Arg Gln Gln Pro Gln Arg Arg Pro Ala Ala Gly Thr Gly Gln
Arg Arg 130 135 140Gly Ser Glu Glu Met Glu Glu Glu Gly145
150535PRTArtificialSynthetic 5Ser Ser Gly His Arg Thr Glu Lys Arg
Lys Arg Gln Gln Pro Gln Arg1 5 10 15Arg Pro Ala Ala Gly Thr Gly Gln
Arg Arg Gly Ser Glu Glu Met Glu 20 25 30Glu Glu Gly
35629PRTArtificialSynthetic 6Glu Lys Arg Lys Arg Gln Gln Pro Gln
Arg Arg Pro Ala Ala Gly Thr1 5 10 15Gly Gln Arg Arg Gly Ser Glu Glu
Met Glu Glu Glu Gly 20 257123PRTArtificialSynthetic 7Glu Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Gly Phe 20 25 30Tyr
Ile His Trp Met Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile 35 40
45Gly Arg Ile His Pro Tyr Asn Gly Ala Thr Ser Tyr Asn Gln Asn Phe
50 55 60Lys Asp Arg Ala Ser Leu Thr Val Asp Glu Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Glu Phe Tyr Gly Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Glu Arg Val Tyr Tyr Tyr Gly Ser Thr Tyr
Glu Phe Asp Ser 100 105 110Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
Ser 115 1208123PRTArtificialSynthetic 8Glu Val Gln Leu Gln Gln Ser
Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Ser Phe Thr Gly Phe 20 25 30Tyr Ile His Trp Val
Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile 35 40 45Gly Arg Ile His
Pro Tyr Asn Gly Ala Pro Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg
Ala Arg Leu Thr Val His Glu Ser Ser Ser Thr Ala Tyr65 70 75 80Met
Glu Phe Phe Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Arg Val Tyr Tyr Tyr Gly Ser Thr Tyr Glu Phe Asp Phe
100 105 110Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115
1209122PRTArtificialSynthetic 9Asp Val Lys Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Val Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Trp Met Asn Trp Val Arg Gln
Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Arg Leu Lys
Ser Asn Asn Tyr Ala Ile Asn Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr Leu
Asp Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95Tyr Cys
Thr Ser Tyr Tyr Gly Ser Ser Leu Tyr Tyr Leu Asp Tyr Trp 100 105
110Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115
12010112PRTArtificialSynthetic 10Asp Val Leu Met Thr Gln Thr Pro
Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys
Arg Ser Gly Gln Ser Ile Val His Thr 20 25 30Ser Gly Val Thr Tyr Leu
Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile
Tyr Lys Val Phe Tyr Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ser Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser
His Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105
11011112PRTArtificialSynthetic 11Asp Val Leu Met Thr Gln Thr Pro
Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys
Arg Ser Gly Gln Ser Ile Val His Ser 20 25 30Ser Gly Val Thr Tyr Leu
Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile
Tyr Lys Val Phe Tyr Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser
His Val Pro Pro Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys 100 105
11012106PRTArtificialSynthetic 12Gln Ile Val Leu Thr Gln Ser Pro
Val Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Ile Ser Cys
Ser Ala Ser Ser Ser Val Ser Asn Met 20 25 30Tyr Trp Tyr Gln Gln Lys
Pro Gly Ser Ser Pro Lys Ala Trp Ile Tyr 35 40 45Arg Thr Ser Asn Leu
Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75 80Asp Ala
Ala Thr Tyr Tyr Cys His Gln Tyr Gln Ser Tyr Pro Arg Thr 85 90 95Phe
Gly Ala Gly Thr Lys Leu Glu Ile Lys 100
105135PRTArtificialSynthetic 13Gly Phe Tyr Ile His1
51417PRTArtificialSynthetic 14Arg Ile His Pro Tyr Asn Gly Ala Thr
Ser Tyr Asn Gln Asn Phe Lys1 5 10 15Asp1514PRTArtificialSynthetic
15Glu Arg Val Tyr Tyr Tyr Gly Ser Thr Tyr Glu Phe Asp Ser1 5
101617PRTArtificialSynthetic 16Arg Ile His Pro Tyr Asn Gly Ala Pro
Thr Tyr Asn Gln Asn Phe Lys1 5 10 15Asp175PRTArtificialSynthetic
17Asn Tyr Trp Met Asn1 51819PRTArtificialSynthetic 18Glu Ile Arg
Leu Lys Ser Asn Asn Tyr Ala Ile Asn Tyr Ala Glu Ser1 5 10 15Val Lys
Gly1911PRTArtificialSynthetic 19Tyr Tyr Gly Ser Ser Leu Tyr Tyr Leu
Asp Tyr1 5 102017PRTArtificialSyntheticMISC_FEATURE(9)..(9)Wherein
Xaa at position 9 is T or PMISC_FEATURE(10)..(10)Wherein Xaa at
position 10 is S or T 20Arg Ile His Pro Tyr Asn Gly Ala Xaa Xaa Tyr
Asn Gln Asn Phe Lys1 5 10 15Asp2116PRTArtificialSynthetic 21Arg Ser
Gly Gln Ser Ile Val His Thr Ser Gly Val Thr Tyr Leu Ser1 5 10
15227PRTArtificialSynthetic 22Lys Val Phe Tyr Arg Phe Ser1
5239PRTArtificialSynthetic 23Phe Gln Gly Ser His Val Pro Pro Thr1
52416PRTArtificialSynthetic 24Arg Ser Gly Gln Ser Ile Val His Ser
Ser Gly Val Thr Tyr Leu Ser1 5 10 152510PRTArtificialSynthetic
25Ser Ala Ser Ser Ser Val Ser Asn Met Tyr1 5
10267PRTArtificialSynthetic 26Arg Thr Ser Asn Leu Ala Ser1
5279PRTArtificialSynthetic 27His Gln Tyr Gln Ser Tyr Pro Arg Thr1
52816PRTArtificialSyntheticMISC_FEATURE(9)..(9)Wherein Xaa at
position 9 is T or S 28Arg Ser Gly Gln Ser Ile Val His Xaa Ser Gly
Val Thr Tyr Leu Ser1 5 10 1529370DNAArtificialSynthetic
29gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata
60tcctgcaagg cttctggtta cgcattcact ggcttctaca tacactggat gaagcaaagc
120catgtaaaga gccttgagtg gattggacgt attcatcctt acaatggtgc
tactagctat 180aatcagaatt tcaaggacag ggccagcttg actgtagatg
agtcctccag tacagcctac 240atggagttct atggcctgac atctgaggac
tctgcagtct attactgtgc aagagagaga 300gtctattact acggtagtac
ttacgagttt gactcctggg gccaaggcac cactctcaca 360gtctcctcag
37030370DNAArtificialSynthetic 30gaggtccagc tgcaacaatc tggacctgag
ctggtgaagc ctggggcttc agtgaagata 60tcctgcaagg cttctggtta ctcattcact
ggcttctaca tacactgggt gaagcaaagc 120catgtaaaga gccttgagtg
gattggacgt attcatcctt acaatggtgc tcctacctac 180aaccagaatt
tcaaggacag ggcccgcttg actgtacatg agtcctccag cacagcctac
240atggagttct ttggcctgac atctgaggac tctgcagtct attactgtgc
aagagagaga 300gtctattact acggtagtac ttacgagttt gacttctggg
gccaaggcac cactctcaca 360gtctcctcag 37031367DNAArtificialSynthetic
31gatgtgaagc ttcaggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactc
60tcctgtgttg cctctggatt cactttcagt aactactgga tgaactgggt ccgccagtct
120ccagagaagg ggcttgagtg ggttgctgaa attagattga aatctaataa
ttatgcaata 180aattatgcgg agtctgtgaa agggaggttt accatctcaa
gagatgattc caaaagcagt 240gtctacctgg acatgaacaa cttaagagct
gaagacactg gcatttatta ctgtacctcc 300tactacggca gtagcctcta
ctaccttgac tactggggcc aaggcaccac tctcacagtc 360tcctcag
36732337DNAArtificialSynthetic 32gatgttttga tgacccaaac tccactctcc
ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctggtca gagcattgtc
catactagtg gcgtcaccta tttatcatgg 120tacctgcaga aaccaggcca
gtctccaaag ctcctgatct acaaagtttt ctaccgattt 180tctggggtcc
cggacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc
240agcagagtgg agtctgagga tctgggagtt tattactgct ttcaaggttc
acatgttcct 300cccacgttcg gtgctgggac caagctggag ctgaaac
33733337DNAArtificialSynthetic 33gatgttttga tgacccaaac tccactctcc
ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctggtca gagcattgtc
catagtagtg gcgtcaccta tttatcatgg 120tacctgcaga aaccaggcca
gtctccaaag ctcctgatct acaaagtttt ctaccgattt 180tctggggtcc
cggacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc
240agcagagtgg aggctgagga tctgggagtt tattactgct ttcagggttc
acatgttcct 300cccacgttcg gtactgggac caagctggag ctgaaac
33734319DNAArtificialSynthetic 34caaattgttc tcacccagtc tccagtaatc
atgtctgcat ctccagggga gaaggtcacc 60atatcctgca gtgccagctc aagtgtaagt
aacatgtact ggtaccagca gaagccagga 120tcctccccca aagcctggat
ttatcgcaca tccaacctgg cttctggagt ccctgctcgc 180ttcagtggca
gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa
240gatgctgcca cttattactg ccaccagtat caaagttacc cacggacgtt
cggtgcaggc 300accaagctgg aaatcaaac 3193514PRTArtificialSynthetic
35Glu Arg Val Tyr Tyr Tyr Gly Ser Thr Tyr
Glu Phe Asp Phe1 5
103614PRTArtificialSyntheticMISC_FEATURE(14)..(14)Wherein Xaa at
position 14 is S or F 36Glu Arg Val Tyr Tyr Tyr Gly Ser Thr Tyr Glu
Phe Asp Xaa1 5 10
* * * * *