U.S. patent application number 12/353913 was filed with the patent office on 2009-11-19 for cripto blocking antibodies and uses thereof.
This patent application is currently assigned to BIOGEN IDEC MA INC.. Invention is credited to Paul RAYHORN, Michele SANICOLA-NADEL, Susan Gail SCHIFFER, Kevin P. WILLIAMS.
Application Number | 20090285818 12/353913 |
Document ID | / |
Family ID | 27501437 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090285818 |
Kind Code |
A1 |
SANICOLA-NADEL; Michele ; et
al. |
November 19, 2009 |
Cripto Blocking Antibodies and Uses Thereof
Abstract
The invention provides Cripto blocking antibodies, or
biologically functional fragments thereof, and uses thereof.
Antibodies which bind Cripto and modulate Cripto signaling are
provided. Antibodies which bind Cripto and block the interaction
between Cripto and ALK4 are provided. Antibodies which bind Cripto
and modulate tumor growth are also provided. Antibodies which bind
Cripto, modulate signaling, and modulate tumor growth are also
provided. Antibodies which bind Cripto, block the interaction
between Cripto and ALK4 and modulate tumor growth are provided. The
invention also provides methods of using these antibodies in
therapeutic, diagnostic, and research applications.
Inventors: |
SANICOLA-NADEL; Michele;
(Winchester, MA) ; WILLIAMS; Kevin P.; (Chapel
Hill, NC) ; SCHIFFER; Susan Gail; (Lexington, MA)
; RAYHORN; Paul; (Foxborough, MA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
27501437 |
Appl. No.: |
12/353913 |
Filed: |
January 14, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10693538 |
Oct 23, 2003 |
7531174 |
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12353913 |
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PCT/US02/11950 |
Apr 17, 2002 |
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10693538 |
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60367002 |
Mar 22, 2002 |
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60301091 |
Jun 26, 2001 |
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60293020 |
May 17, 2001 |
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60286782 |
Apr 26, 2001 |
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Current U.S.
Class: |
424/138.1 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 2319/30 20130101; C07K 16/30 20130101; C07K 16/28 20130101;
C07K 2317/73 20130101; C07K 2319/00 20130101; A61K 2039/505
20130101; C07K 16/18 20130101; C07K 2317/34 20130101; A61P 35/00
20180101 |
Class at
Publication: |
424/138.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1-61. (canceled)
62. A method of treating cancer in a subject having cancer cells
that over-express Cripto-1 as compared to non-cancerous cells,
comprising administering to said subject a therapeutically
effective amount of a monoclonal antibody or fragment thereof that
binds a Cripto-1, whereby growth or spread of cancer cells in the
subject is inhibited and wherein the mechanism of inhibition of
growth or spread of the cancer cells is through: (a) apoptosis
induced by binding of the antibody or fragment thereof to the
Cripto-1; or (b) cytotoxicity induced by delivery to cancer cells
of a cytotoxic compound conjugated to said monoclonal antibody or
fragment thereof.
63. The method of claim 62, wherein the monoclonal antibody or
fragment thereof is a human monoclonal antibody or fragment
thereof.
64. The method of claim 62, wherein the monoclonal antibody or
fragment thereof is a chimeric monoclonal antibody or fragment
thereof.
65. The method of claim 62, wherein the cancer is a colon
cancer.
66. The method of claim 62, wherein the cancer is a breast
cancer.
67. The method of claim 62, wherein the cancer is a prostate
cancer.
68. The method of claim 62, wherein the cancer is a leukemia.
69. The method of claim 62, wherein the cancer is a lung
cancer.
70. The method of claim 62, wherein the antibody or fragment
thereof induces apoptosis of cancer cells.
71. The method of claim 70, wherein the antibody or fragment
thereof induces apoptosis through activation of a JNK or p38 kinase
cascade.
72. The method of claim 62, wherein the antibody or fragment
thereof binds to an antigenic determinant of a Cripto-1 protein
comprising the amino acid sequence of SEQ ID NO: 1 or an EGF-like
domain region of SEQ ID NO: 1, or amino acids 75-112, 75-85, 80-90,
85-95, 90-100, 95-105, 100-110, or 105-112 of SEQ ID NO: 1.
73. The method of claim 62, wherein the mechanism of inhibition of
grow or spread of cancer cells is apoptosis.
74. The method of claim 62, wherein the mechanism of inhibition of
growth or spread of cancer cells is cytotoxicity.
75. The method of claim 74, wherein the antibody or fragment
thereof is conjugated to a cytotoxic compound.
76. The method of claim 75, wherein the cytotoxic compound is
selected from the group consisting of anthracyclines,
5-fluorouracil, topoisomerase inhibitors, cisplatin, carboplatin
and taxol.
77. The method of claim 76, wherein the cytotoxic compound is an
anthracycline selected from the group consisting of idarubicin,
doxorubicin, daunorubicin and epirubicin.
78. The method of claim 77, wherein the cytotoxic compound is the
topoisomerase inhibitor irinotecan.
79. The method of claim 62, wherein the mechanism of inhibition of
growth or spread of the cancer cells is through apoptosis induced
by binding of the antibody or fragment thereof to the Cripto-1.
80. The method of claim 62, wherein the mechanism of inhibition of
growth or spread of the cancer cells is through cytotoxicity
induced by delivery to cancer cells of a cytotoxic compound
conjugated to said monoclonal antibody or fragment thereof.
Description
RELATED APPLICATIONS
[0001] This is a continuation of PCT/US02/11950, filed Apr. 17,
2002, which claims the benefit of U.S. Ser. No. 60/367,002, filed
Mar. 22, 2002, and U.S. Ser. No. 60/301,091, filed Jun. 26, 2001,
and U.S. Ser. No. 60/293,020, filed on May 17, 2001, and U.S. Ser.
No. 60/286,782, filed on Apr. 26, 2001. The entire disclosure of
each of the aforesaid patent applications are incorporated herein
by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates generally to the fields of
genetics and cellular and molecular biology. More particularly, the
invention relates to antibodies which bind to and modulate the
signaling of Cripto, kits comprising such antibodies, and methods
which use the antibodies.
BACKGROUND OF THE INVENTION
[0003] Cripto is a cell surface protein of 188 amino acid residues
serendipitously isolated in a cDNA screen of a human embryonic
carcinoma library (Ciccodicola et al., 1989, EMBO J., vol. 8, no.
7, pp. 1987-1991). The Cripto protein has at least two notable
domains: a cysteine-rich domain, and a domain first characterized
as similar to the domain found in the epidermal growth factor (EGF)
family. Cripto was originally classified as a member of the EGF
family (Ciccodicola et al., supra); however, subsequent analysis
showed that Cripto did not bind any of the known EGF receptors and
its EGF-like domain was actually divergent from the EGF family
(Bianco et al., 1999, J. Biol. Chem., 274:8624-8629).
[0004] The Cripto signaling pathway has remained elusive despite
continued investigation, with the literature supporting activation
of several different pathways, including a MAP kinase pathway
(DeSantis et al., 1997, Cell Growth Differ., 8:1257-1266; Kannan et
al., 1997, J. Biol. Chem., 272:3330-3335), the TGF-.beta. pathway
(Gritsman et al., 1999, Development, 127:921-932; Schier et al.,
2000, Nature, 403:385-389), possible interactions with the Wnt
pathway (Salomon et al., Endocr Relat Cancer. 2000 December;
7(4):199-226; and cross talk with the EGF pathway (Bianco et al.,
1999, J. Biol. Chem., 274:8624-8629).
[0005] U.S. Pat. No. 5,256,643 and two divisional applications
related thereto (U.S. Pat. Nos. 5,654,140 and 5,792,616), disclose
a human Cripto gene, Cripto protein, and antibodies to Cripto.
[0006] U.S. Pat. No. 5,264,557 and three divisional applications
related thereto (U.S. Pat. Nos. 5,620,866, 5,650,285, and
5,854,399), disclose a human Cripto-related gene and protein. Also
disclosed are antibodies which bind to the Cripto-related protein
but do not cross react by binding to the Cripto protein itself.
[0007] Cripto protein overexpression is associated with many tumor
types (including but not limited to breast, testicular, colon,
lung, ovary, bladder, uterine, cervical, pancreatic, and stomach),
as demonstrated by immunostaining of human tissue with rabbit
polyclonal antibodies raised against small cripto peptides. Panico
et al., 1996, Int. J. Cancer, 65: 51-56; Byrne et al., 1998, J
Pathology, 185:108-111; De Angelis et al., 1999, Int J Oncology,
14:437-440. The art is therefore in need of means of controlling,
restricting, and/or preventing such overexpression, modulating
Cripto signaling, and modulating the consequences of Cripto
expression (i.e., promotion and/or maintenance of cell
transformation).
SUMMARY OF THE INVENTION
[0008] The present invention provides novel antibodies which
specifically bind to Cripto, and methods of making and using such
antibodies. The invention also provides antibodies which bind to
Cripto, and modulate Cripto signaling or protein interaction, e.g.,
an antibody which binds to Cripto such that the signal resulting
from a protein interaction with Cripto is modulated downward. The
invention also provides antibodies which bind to Cripto and block
the interaction between Cripto and ALK4. The invention also
provides antibodies which bind to Cripto and modulate tumor growth.
The invention also provides antibodies which bind to Cripto,
modulate Cripto signaling and modulate tumor growth. The invention
also provides antibodies which bind to Cripto, block the
interaction between Cripto and ALK4 and modulate tumor growth
[0009] In one aspect of the invention, the antibody of the present
invention specifically binds to an epitope selected from the group
of epitopes to which antibodies A6C12.11, A6F8.6 (ATCC ACCESSION
NO. PTA-3318), A7H1.19, A8F1.30, A8G3.5 (ATCC ACCESSION NO.
PTA-3317), A8H3.1 (ATCC ACCESSION NO. PTA-3315), A8H3.2, A19A10.30,
A10B2.18 (ATCC ACCESSION NO. PTA-3311), A27F6.1 (ATCC ACCESSION NO.
PTA-3310), A40G12.8 (ATCC ACCESSION NO. PTA-3316), A2D3.23,
A7A10.29, A9G9.9, A15C12.10, A15E4.14, A17A2.16, A17C12.28,
A17G12.1 (ATCC ACCESSION NO. PTA-3314), A17H6.1, A18B3.11 (ATCC
ACCESSION NO. PTA-3312), A19E2.7, B3F6.17 (ATCC ACCESSION NO.
PTA-3319), B6G7.10 (ATCC ACCESSION NO. PTA-3313), B11H8.4 bind.
[0010] In another aspect of the invention, the antibody of the
present invention specifically binds to an epitope in the
ligand/receptor binding domain of Cripto. Cripto can be selected
from CR-1 (SEQ ID NO:1) or CR-3 (SEQ ID NO:2). In a more particular
embodiment, antibodies that specifically binds to the epitope in
the ligand/receptor binding domain include for example A6C12.11,
A6F8.6 (ATCC ACCESSION NO. PTA-3318), A8G3.5 (ATCC ACCESSION NO.
PTA-3317), A19A10.30, A8H3.1 (ATCC ACCESSION NO. PTA-3315), A27F6.1
(ATCC ACCESSION NO. PTA-3310), A40G12.8 (ATCC ACCESSION NO.
PTA-3316), A17G12.1 (ATCC ACCESSION NO. PTA-3314), A18B3.11 (ATCC
ACCESSION NO. PTA-3312) and B6G7.10 (ATCC ACCESSION NO.
PTA-3313).
[0011] In one embodiment the epitope to which the antibodies of the
present invention bind is in an EGF-like domain. Antibodies that
specifically bind to the epitope in the EGF-like domain include but
are not limited to A40G12.8 (ATCC ACCESSION NO. PTA-3316), A8H3.1
(ATCC ACCESSION NO. PTA-3315), A27F6.1 (ATCC ACCESSION NO.
PTA-3310), B6G7.10 (ATCC ACCESSION NO. PTA-3313), A17G12.1 (ATCC
ACCESSION NO. PTA-3314) and A18B3.11 (ATCC ACCESSION NO.
PTA-3312).
[0012] In another embodiment the epitope to which the antibodies of
the present invention bind is in a cys-rich domain. Antibodies that
specifically bind to the epitope in the cys-rich domain include but
are not limited to A19A10.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317),
A6F8.6 (ATCC ACCESSION NO. PTA-3318) and A6C12.11.
[0013] In another embodiment the epitope to which the antibodies of
the present invention bind is in the domain spanning amino acid
residues 46-62 of Cripto. Antibodies that specifically bind to the
epitope in the domain spanning amino acid residues 46-62 of Cripto
include but are not limited to A10B2.18 (ATCC ACCESSION NO.
PTA-3311), B3F6.17 (ATCC ACCESSION NO. PTA-3319) and A17A2.16.
[0014] The present inventions also contemplate antibodies which
binds specifically to Cripto and are capable of modulating Cripto
signaling. Antibodies that bind specifically to Cripto and are
capable of modulating Cripto signaling, include but are not limited
to, A40G12.8 (ATCC ACCESSION NO. PTA-3316), A8H3.1 (ATCC ACCESSION
NO. PTA-3315), A27F6.1 (ATCC ACCESSION NO. PTA-3310), and A6C12.11.
In one embodiment the antibodies of the present invention which
binds specifically to Cripto and are capable of modulating Cripto
signaling bind to an epitope in an EGF-like domain or a cys-rich
domain of Cripto.
[0015] The present inventions also contemplate antibodies which
binds specifically to Cripto and blocks the interaction between
Cripto and ALK4. Antibodies that bind specifically to Cripto and
are capable of blocking the interaction between Cripto and ALK4,
include but are not limited to, A8G3.5 (ATCC ACCESSION NO.
PTA-3317), A6F8.6 (ATCC ACCESSION NO. PTA-3318) and A6C12.11. In
one embodiment the antibodies of the present invention which binds
specifically to Cripto and are capable of blocking the interaction
between Cripto and ALK4 bind to an epitope in an EGF-like domain or
a cys-rich domain of Cripto.
[0016] In another aspect, the present invention contemplates
antibodies which bind specifically to Cripto and are capable of
modulating tumor growth. Antibodies that specifically bind to
Cripto and are capable of modulating tumor growth include but are
not limited to, A27F6.1 (ATCC ACCESSION NO. PTA-3310), B6G7.10
(ATCC ACCESSION NO. PTA-3313) and A8G3.5 (ATCC ACCESSION NO.
PTA-3317).
[0017] In one embodiment the antibodies of the present invention
which bind specifically to Cripto and are capable of modulating
tumor growth bind to an epitope in an EGF-like domain or a cys-rich
domain of Cripto.
[0018] In yet another aspect, the present invention contemplates
antibodies which bind specifically to Cripto, which are capable of
modulating Cripto signaling, and which are capable of modulating
tumor growth. Antibodies that specifically bind to Cripto, which
are capable of modulating Cripto signaling, and which are capable
of modulating tumor growth, include but are not limited to A27F6.1
(ATCC ACCESSION NO. PTA-3310).
[0019] In one embodiment the antibodies of the present invention
which bind specifically to Cripto, which are capable of modulating
Cripto signaling, and which are capable of modulating tumor growth
bind to an epitope in an EGF-like domain or a cys-rich domain of
Cripto.
[0020] In yet another aspect, the present invention contemplates
antibodies which bind specifically to Cripto, which are capable of
blocking the interaction between Cripto and ALK4, and which are
capable of modulating tumor growth. Antibodies that specifically
bind to Cripto, which are capable of blocking the interaction
between Cripto and ALK4, and which are capable of modulating tumor
growth, include but are not limited to A8G3.5 (ATCC ACCESSION NO.
PTA-3317).
[0021] In another embodiment, the present invention provides an
antibody produced by a hybridoma selected from the group consisting
of A6F8.6 (ATCC Accession No. PTA-3318), A8G3.5 (ATCC Accession No.
PTA-3317), A8H3.1 (ATCC Accession No. PTA-3315), A10B2.18 (ATCC
Accession No. PTA-3311), A27F6.1 (ATCC Accession No. PTA-3310),
A40G12.8 (ATCC Accession No. PTA-3316), A17G12.1 (ATCC Accession
No. PTA-3314), A18B3.11 (ATCC Accession No. PTA-3312), B3F6.17
(ATCC Accession No. PTA-3319), and B6G7.10 (ATCC Accession No.
PTA-3313).
[0022] The antibodies of the present invention include but are not
limited to monoclonal, polyclonal, humanized, chimeric and human
antibodies.
[0023] The present invention also provides for a composition for
administration to a subject having a tumor that expresses Cripto
comprising at least one of the antibodies described above. In a
more particular embodiment the subject is human. The composition
may include a pharmaceutically acceptable excipient. The antibodies
described above can be conjugated to a chemotherapeutic agent or be
provided in combination with a nonconjugated chemotherapeutic.
[0024] Contemplated in another aspect of the invention are methods
of modulating growth of tumor cells in vitro in a sample comprising
the step of adding to the sample the compositions described
above.
[0025] Also comtemplated are methods of modulating growth of tumor
cells in vivo in a subject comprising the step of administering to
the subject an effective amount of the compositions described
above. In a particular embodiment the subject is human.
[0026] Another aspect of the invention are methods of treating
subjects having a tumor that over-expresses Cripto comprising
administering to the subject the compositions described above in an
effective amount. Compositions for administration may include
pharmaceutically acceptable excipients, antibodies conjugated to
chemotherapeutic agents and antibodies administered in combination
with nonconjugated chemotherapeutic agents
[0027] The methods of the present invention are particularly useful
in modulating growth of tumor cells and/or treating a subject (i.e.
a human) having a tumor where the tumor cell is selected from
breast, testicular, colon, lung, ovary, bladder, uterine, cervical,
pancreatic, and stomach tumor cells.
[0028] In yet another embodiment, the present invention
contemplates methods of determining whether a tissue expresses
Cripto, comprising the step of analyzing tissue from the subject in
an immunoassay using any of the antibodies described above. Also
contemplated are methods of determining whether a cell line
overexpresses Cripto, comprising the step of analyzing the cell
line in an immunoassay using any of the antibodies described
above.
[0029] These and other aspects of the invention are set forth in
greater detail below in the Detailed Description of the
Invention.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Antibodies that specifically bind to Cripto and their uses
for modulating Cripto signaling or protein interaction, and/or
block the interaction between Cripto and ALK4, and/or modulate the
growth of tumor cells have been discovered. Various classes of
antibodies that specifically bind to Cripto have been discovered,
including, for example, antibodies that specifically bind to an
epitope in the ligand/receptor binding domain of either a native
Cripto protein or a denatured form of Cripto; antibodies that bind
an EGF-like domain, a cys-rich domain, or a peptide (e.g., from
about 3 to about 20 amino acids) from the region comprising amino
acid residues 46 to 150; antibodies that bind Cripto and modulate
Cripto signalling; antibodies that bind Cripto and modulate tumor
cell growth; and antibodies that bind to Cripto, modulate Cripto
signaling, and modulate tumor cell growth. These antibodies are
selected using conventional in vitro assays for selecting
antibodies which bind the ligand/receptor binding domain, modulate
Cripto signaling, or modulate tumor cell growth.
[0031] The methods of this invention are useful in the therapy of
malignant or benign tumors of mammals where the growth rate of the
tumor (which is an abnormal rate for the normal tissue) is at least
partially dependent upon Cripto. Abnormal growth rate is a rate of
growth which is in excess of that required for normal homeostasis
and is in excess of that for normal tissues of the same origin.
DEFINITIONS
[0032] Various definitions are made throughout this document. Most
words have the meaning that would be attributed to those words by
one skilled in the art. Words specifically defined either below or
elsewhere in this document have the meaning provided in the context
of the present invention as a whole and as are typically understood
by those skilled in the art.
[0033] As used herein, the term "region" means a physically
contiguous portion of the primary structure of a biomolecule. In
the case of proteins, a region is defined by a contiguous portion
of the amino acid sequence of that protein.
[0034] As used herein, the term "domain" refers to a structural
part of a biomolecule that contributes to a known or suspected
function of the biomolecule. Domains may be co-extensive with
regions or portions thereof, domains may also incorporate a portion
of a biomolecule that is distinct from a particular region, in
addition to all or part of that region. Examples of protein domains
include, but are not limited to the extracellular domain (spans
from about residue 31 to about residue 188 of Cripto, including
Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO:2)) and
transmembrane domain (spans from about residue 169 to about residue
188 of Cripto, including Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ
ID NO:2)). A ligand/receptor binding domain of the Cripto protein
spans from about residue 75 to about residue 150 of Cripto,
including Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO:2) and
includes the EGF-like domain of Cripto, which spans, for example,
from about residue 75 to about residue 112 of Cripto, including
Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO:2) and the
cysteine-rich domain of Cripto, which spans, for example, from
about residue 114 to about residue 150 of Cripto, including Cripto,
CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO:2). For example, many
monoclonal antibodies of the present invention have been identified
as binding to the EGF-like or cys-rich domains. Additionally
monoclonal antibody A10B2.18 (ATCC ACCESSION NO. PTA-3311), B3F6.17
(ATCC ACCESSION NO. PTA-3319) and A17A2.16 have been identified as
binding to an epitope formed in a domain in the region spanning
amino acid residues 46-62, upstream of the EGF-like domain. See
Example 3 below. An epitope in the ligand/receptor binding domain
is an epitope, whether formed in the conformational native Cripto
protein, or the denatured Cripto protein, to which antibodies may
bind.
[0035] As used herein, the term "antibody" is meant to refer to
complete, intact antibodies, and Fab, Fab', F(ab)2, and other
fragments thereof. Complete, intact antibodies include, but are not
limited to, monoclonal antibodies such as murine monoclonal
antibodies, polyclonal antibodies, chimeric antibodies, human
antibodies, and humanized antibodies. Various forms of antibodies
may be produced using standard recombinant DNA techniques (Winter
and Milstein, Nature 349: 293-99, 1991). For example, "chimeric"
antibodies may be constructed, in which the antigen binding domain
from an animal antibody is linked to a human constant domain (an
antibody derived initially from a nonhuman mammal in which
recombinant DNA technology has been used to replace all or part of
the hinge and constant regions of the heavy chain and/or the
constant region of the light chain, with corresponding regions from
a human immunoglobulin light chain or heavy chain) (see, e.g.,
Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc.
Natl. Acad. Sci. 81: 6851-55, 1984). Chimeric antibodies reduce the
immunogenic responses elicited by animal antibodies when used in
human clinical treatments.
[0036] In addition, recombinant "humanized" antibodies may be
synthesized. Humanized antibodies are antibodies initially derived
from a nonhuman mammal in which recombinant DNA technology has been
used to substitute some or all of the amino acids not required for
antigen binding with amino acids from corresponding regions of a
human immunoglobulin light or heavy chain. That is, they are
chimeras comprising mostly human immunoglobulin sequences into
which the regions responsible for specific antigen-binding have
been inserted (see, e.g., PCT patent application WO 94/04679).
Animals are immunized with the desired antigen, the corresponding
antibodies are isolated and the portion of the variable region
sequences responsible for specific antigen binding are removed. The
animal-derived antigen binding regions are then cloned into the
appropriate position of the human antibody genes in which the
antigen binding regions have been deleted. Humanized antibodies
minimize the use of heterologous (inter-species) sequences in
antibodies for use in human therapies, and are less likely to
elicit unwanted immune responses. Primatized antibodies can be
produced similarly.
[0037] Another embodiment of the invention includes the use of
human antibodies, which can be produced in nonhuman animals, such
as transgenic animals harboring one or more human immunoglobulin
transgenes. Such animals may be used as a source for splenocytes
for producing hybridomas, as is described in U.S. Pat. No.
5,569,825.
[0038] Antibody fragments and univalent antibodies may also be used
in the methods and compositions of this invention. Univalent
antibodies comprise a heavy chain/light chain dimer bound to the Fc
(or stem) region of a second heavy chain. "Fab region" refers to
those portions of the chains which are roughly equivalent, or
analogous, to the sequences which comprise the Y branch portions of
the heavy chain and to the light chain in its entirety, and which
collectively (in aggregates) have been shown to exhibit antibody
activity. A Fab protein includes aggregates of one heavy and one
light chain (commonly known as Fab'), as well as tetramers which
correspond to the two branch segments of the antibody Y, (commonly
known as F(ab)2), whether any of the above are covalently or
non-covalently aggregated, so long as the aggregation is capable of
specifically reacting with a particular antigen or antigen
family.
[0039] Any of the antibodies of the invention may optionally be
conjugated to a chemotherapeutic, as defined below.
[0040] As used herein, the term "binding" means the physical or
chemical interaction between two proteins or compounds or
associated proteins or compounds or combinations thereof, including
the interaction between an antibody and a protein. Binding includes
ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic
interactions, etc. The physical interaction, the binding, can be
either direct or indirect, indirect being through or due to the
effects of another protein or compound. Direct binding refers to
interactions that do not take place through or due to the effect of
another protein or compound but instead are without other
substantial chemical intermediates. Binding may be detected in many
different manners. Methods of detecting binding are well-known to
those of skill in the art.
[0041] As used herein, "an antibody capable of internalizing
Cripto" means an antibody which enters the cell while removing
Cripto from the cell surface. One can screen for Cripto antibodies
which are capable of internalizing Cripto by using fluorescent
labeled Cripto monoclonal antibodies. In order to determine which
antibodies internalize into the Cripto positive cells one can assay
for the uptake of the fluorescent signal of the antibodies into the
cells by viewing the cells under a fluorescent and/or confocal
microscope. Those antibodies that get internalized will be seen as
fluorescent signals in the cytoplasmic and or cellular vesicles.
Non-limiting examples of Cripto antibodies capable of internalizing
Cripto include A27F6.1 and B3F6.17.
[0042] As used herein, the term "compound" means any identifiable
chemical or molecule, including, but not limited to, ion, atom,
small molecule, peptide, protein, sugar, nucleotide, or nucleic
acid, and such compound can be natural or synthetic.
[0043] As used herein, the terms "modulates" or "modifies" means an
increase or decrease in the amount, quality, or effect of a
particular activity or protein.
[0044] As used herein, the term "modulate Cripto signaling" means
an increase or decrease in the amount, quality, or effect of Cripto
activity, by about 5%, preferably 10%, more preferably 20%, more
preferably 30%, more preferably 40%, more preferably 50%, more
preferably 60%, more preferably 70%, more preferably 80%, more
preferably 90%, and most preferably 100%. Activity may be measured
by assays known in the art, such as the null cell assay shown in
Example 3. In another embodiment, protein interaction between
Cripto and another protein is similarly modulated downward via
binding of the antibodies of the invention.
[0045] As used herein, the term "blocking the interaction between
Cripto and ALK 4" means an increase or decrease in the interaction,
i.e. binding, between Cripto and ALK4, by about 5%, preferably 10%,
more preferably 20%, more preferably 30%, more preferably 40%, more
preferably 50%, more preferably 60%, more preferably 70%, more
preferably 80%, more preferably 90%, and most preferably 100%.
Activity may be measured by assays known in the art, such as the
binding assay shown in Example 8.
[0046] As used herein, the term "modulate growth of tumor cells in
vitro" means an increase or decrease in the number of tumor cells,
in vitro, by about 5%, preferably 10%, more preferably 20%, more
preferably 30%, more preferably 40%, more preferably 50%, more
preferably 60%, more preferably 70%, more preferably 80%, more
preferably 90%, and most preferably 100%. In vitro modulation of
tumor cell growth may be measured by assays known in the art, such
as the GEO cell soft agar assay shown in Example 4.
[0047] As used herein, the term "modulate growth of tumor cells in
vivo" means an increase or decrease in the number of tumor cells,
in vivo, by about 5%, preferably 10%, more preferably 20%, more
preferably 30%, more preferably 40%, more preferably 50%, more
preferably 60%, more preferably 70%, more preferably 80%, more
preferably 90%, and most preferably 100%. In vivo modulation of
tumor cell growth may be measured by assays known in the art, such
as the one shown in Example 5.
[0048] The term "preventing" refers to decreasing the probability
that an organism contracts or develops an abnormal condition.
[0049] The term "treating" refers to having a therapeutic effect
and at least partially alleviating or abrogating an abnormal
condition in the organism. Treating includes maintenance of
inhibited tumor growth, and induction of remission.
[0050] The term "therapeutic effect" refers to the inhibition of an
abnormal condition. A therapeutic effect relieves to some extent
one or more of the symptoms of the abnormal condition. In reference
to the treatment of abnormal conditions, a therapeutic effect can
refer to one or more of the following: (a) an increase or decrease
in the proliferation, growth, and/or differentiation of cells; (b)
inhibition (i.e., slowing or stopping) or promotion of cell death;
(c) inhibition of degeneration; (d) relieving to some extent one or
more of the symptoms associated with the abnormal condition; and
(e) enhancing the function of a population of cells. Compounds
demonstrating efficacy against abnormal conditions can be
identified as described herein.
[0051] The term "administering" relates to a method of
incorporating a compound into cells or tissues of an organism. The
abnormal condition can be prevented or treated when the cells or
tissues of the organism exist within the organism or outside of the
organism. Cells existing outside the organism can be maintained or
grown in cell culture dishes, or in another organism. For cells
harbored within the organism, many techniques exist in the art to
administer compounds, including (but not limited to) oral,
parenteral, dermal, injection, and aerosol applications. For cells
outside of the organism, multiple techniques exist in the art to
administer the compounds, including (but not limited to) cell
microinjection techniques, transformation techniques and carrier
techniques. Administration may be accomplished by the many modes
known in the art, e.g., oral, intravenous, intraperitoneal,
intramuscular, and the like. When used in in vivo therapy, the
antibodies of the subject invention are administered to a patient
in effective amounts. As used herein an "effective amount" is an
amount sufficient to effect beneficial or desired clinical results
(i.e., amounts that eliminate or reduce the patient's tumor
burden). An effective amount can be administered in one or more
administrations. For purposes of this invention, an effective
amount of the antibodies of the present invention is an amount of
the antibodies that is sufficient to ameliorate, stabilize, or
delay the development of the Cripto-associated disease state,
particularly Cripto-associated tumors. Detection and measurement of
these indicators of efficacy are discussed below. An example of a
typical treatment regime includes administering by intravenous
infusion to the subject antibodies of the invention on a weekly
schedule, at a dose of about 2-5 mg/kg. The antibodies are
administered in an outpatient chemoinfusion unit, unless the
patient requires hospitalization. Other administration regimes
known in the art are also contemplated.
[0052] The abnormal condition can also be prevented or treated by
administering an antibody of the invention to a group of cells
having an aberration in a signal transduction pathway to an
organism. The effect of administering a compound on organism
function can then be monitored. The organism is preferably a
human.
[0053] "Cripto overexpression" is intended to mean the expression
of Cripto by a tissue which expression is greater than the Cripto
expression of adjacent normal tissue in a statistically significant
amount.
[0054] "Chemotherapeutics" refers to any agents identified in the
art as having therapeutic effect on the inhibition of tumor growth,
maintenance of inhibited tumor growth, and/or induction of
remission, such as natural compounds, synthetic compounds,
proteins, modified proteins, and radioactive compounds.
Chemotherapeutic agents contemplated herewith include agents that
can be conjugated to the antibodies of the present invention or
alternatively agents that can be used in combination with the
antibodies of the present invention without being conjugated to the
antibody. Exemplary chemotherapeutics that can be conjugated to the
antibodies of the present invention include, but are not limited to
radioconjugates (90Y, 131I, 99mTc, 111In, 186Rh, et al.),
tumor-activated prodrugs (maytansinoids, CC-1065 analogs,
clicheamicin derivatives, anthracyclines, vinca alkaloids, et al.),
ricin, diphtheria toxin, pseudomonas exotoxin.
[0055] Chemotherapeutic agents may be used in combination with the
antibodies of the invention, rather than being conjugated thereto
(i.e. nonconjugated chemotherapeutics), include, but are not
limited to the following: platinums (i.e. cis platinum),
anthracyclines, nucleoside analogs (purine and pyrimidine),
taxanes, camptothecins, epipodophyllotoxins, DNA alkylating agents,
folate antagonists, vinca alkaloids, ribonucleotide reductase
inhibitors, estrogen inhibitors, progesterone inhibitors, androgen
inhibitors, aromatase inhibitors, interferons, interleukins,
monoclonal antibodies, taxol, camptosar, adriamycin (dox), 5-FU and
gemcitabine. Such chemotherapeutics may be employed in the practice
of the invention in combination with the antibodies of the
invention by coadministration of the antibody and the nonconjugated
chemotherapeutic.
[0056] "Pharmaceutically acceptable carrier or excipient" refers to
biologically inert compounds known in the art and employed in the
administration of the antibodies of the invention. Acceptable
carriers are well known in the art and are described, for example,
in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack
Publishing Co., 1990. Acceptable carriers can include
biocompatible, inert or bioabsorbable salts, buffering agents,
oligo- or polysaccharides, polymers, viscoelastic compound such as
hyaluronic acid, viscosity-improving agents, preservatives, and the
like.
[0057] A "subject" refers to vertebrates, particularly members of a
mammalian species, and includes but is not limited to domestic
animals, sports animals, and primates, including humans.
[0058] Antibodies of the Invention
[0059] The antibodies of the invention specifically bind to Cripto:
As used herein, Cripto includes the CR-1 Cripto protein, the CR-3
Cripto protein, and fragments thereof. Such fragments may be entire
domains, such as the extracellular or intracellular domains, the
EGF-like domain, the cys-rich domain, the receptor binding domain,
and the like. Such fragments may also include contiguous and
noncontiguous epitopes in any domain of the Cripto protein.
TABLE-US-00001 The 188 amino acid sequence for CR-1 is as follows
[SEQ ID NO: 1]: MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS
IWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPS
FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD
GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY The 188 amino acid sequence
for CR-3 is as follows [SEQ ID NO: 2]:
MDCRKMVRFSYSVIWIMAISKAFELGLVAGLGHQEFARPSRGDLAFRDDS
IWPQEEPAIRPRSSQRVLPMGIQHSKELNRTCCLNGGTCMLESFCACPPS
FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD
GLVMDEHLVASRTPELPPSARTTTFMLAGICLSIQSYY
[0060] In a one embodiment, the antibodies of the invention bind to
an epitope in the EGF-like domain of Cripto. The EGF-like domain
spans from about amino acid residue 75 to about amino acid residue
112 of the mature Cripto protein. Epitopes in the EGF-like domain
may comprise linear or nonlinear spans of amino acid residues.
Example of linear epitopes contemplated include but are not limited
to about residues 75-85, 80-90, 85-95, 90-100, 95-105, 100-110, or
105-112. In one embodiment, the epitope in the EGF domain is an
epitope formed in the conformational native Cripto protein versus a
denatured Cripto protein.
[0061] In another embodiment, the antibodies of the invention bind
to an epitope in the cys-rich domain of Cripto. The cys-rich domain
spans from about amino acid residue 114 to about amino acid residue
150 of the mature Cripto protein. Epitopes in the cys-rich domain
may comprise linear or nonlinear spans of amino acid residues.
Example of linear epitopes contemplated include but are not limited
to about residues 114-125, 120-130, 125-135, 130-140, 135-145, or
140-150. In one embodiment, the epitope in the cys-rich domain is
an epitope formed in the conformational native Cripto protein
versus a denatured Cripto protein
[0062] Once antibodies are generated, binding of the antibodies to
Cripto may be assayed using standard techniques known in the art,
such as ELISA, while the presence of Cripto on a cell surface may
be assayed using flow cytometry (FACS), as shown in Example 2. Any
other techniques of measuring such binding may alternatively be
used.
[0063] The present invention provides antibodies (e.g., monoclonal
and polyclonal antibodies, single chain antibodies, chimeric
antibodies, bifunctional/bispecific antibodies, humanized
antibodies, human antibodies, and complementary determining region
(CDR)-grafted antibodies, including compounds which include CDR
sequences which specifically recognize a polypeptide of the
invention) specific for Cripto or fragments thereof. Antibody
fragments, including Fab, Fab', F(ab').sub.2, and F.sub.v, are also
provided by the invention. The terms "specific" and "selective,"
when used to describe binding of the antibodies of the invention,
indicates that the variable regions of the antibodies of the
invention recognize and bind Cripto polypeptides. It will be
understood that specific antibodies of the invention may also
interact with other proteins (for example, S. aureus protein A or
other antibodies in ELISA techniques) through interactions with
sequences outside the variable region of the antibodies, and, in
particular, in the constant region of the molecule. Screening
assays to determine binding specificity of an antibody of the
invention (i.e. antibodies that specifically bind to an epitope the
ligand/receptor binding domain and the domain spanning amino acid
residues 46-62) are well known and routinely practiced in the art.
For a comprehensive discussion of such assays, see Harlow et al.
(Eds.), Antibodies A Laboratory Manual; Cold Spring Harbor
Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies
that recognize and bind fragments of Cripto protein are also
contemplated, provided that the antibodies are specific for Cripto
polypeptides. Antibodies of the invention can be produced using any
method well known and routinely practiced in the art.
[0064] In one embodiment, the invention provides an antibody that
specifically binds to an epitope in the ligand/receptor binding
domain of Cripto. Antibody specificity is described in greater
detail below. However, it should be emphasized that antibodies that
can be generated from other polypeptides that have previously been
described in the literature and that are capable of fortuitously
cross-reacting with Cripto (e.g., due to the fortuitous existence
of a similar epitope in both polypeptides) are considered
"cross-reactive" antibodies. Such cross-reactive antibodies are not
antibodies that are "specific" for Cripto. The determination of
whether an antibody specifically binds to an epitope of Cripto is
made using any of several assays, such as Western blotting assays,
that are well known in the art. For identifying cells that express
Cripto and also for modulating Cripto ligand/receptor binding
activity, antibodies that specifically bind to an extracellular
epitope of the Cripto protein (i.e., portions of the Cripto protein
found outside the cell) are particularly useful.
[0065] In one embodiment, the invention provides a cell-free
composition comprising polyclonal antibodies, wherein at least one
of the antibodies is an antibody of the invention specific for
Cripto. Antisera isolated from an animal is an exemplary
composition, as is a composition comprising an antibody fraction of
an antisera that has been resuspended in water or in another
diluent, excipient, or carrier.
[0066] In another embodiment, the invention provides monoclonal
antibodies. Monoclonal antibodies are highly specific, being
directed against a single antigenic site. Further in contrast to
polyclonal preparations which typically include different
antibodies directed against different epitopes, each monoclonal
antibody is directed against a single determinant on the antigen.
Monoclonal antibodies are useful to improve selectivity and
specificity of diagnostic and analytical assay methods using
antigen-antibody binding. Another advantage of monoclonal
antibodies is that they are synthesized by a hybridoma culture,
uncontaminated by other immunoglobulins. Hybridomas that produce
such antibodies are also intended as aspects of the invention.
[0067] In still another related embodiment, the invention provides
an anti-idiotypic antibody specific for an antibody that is
specific for Cripto. For a more detailed discussion of
anti-idiotypic antibodies, see, e.g., U.S. Pat. Nos. 6,063,379 and
5,780,029.
[0068] It is well known that antibodies contain relatively small
antigen binding domains that can be isolated chemically or by
recombinant techniques. Such domains are useful Cripto binding
molecules themselves, and also may be reintroduced into human
antibodies, or fused to a chemotherapeutic or polypeptide. Thus, in
still another embodiment, the invention provides a polypeptide
comprising a fragment of a Cripto-specific antibody, wherein the
fragment and associated molecule, if any, bind to the Cripto. By
way of non-limiting example, the invention provides polypeptides
that are single chain antibodies and CDR-grafted antibodies. For a
more detailed discussion of CDR-grafted antibodies, see, e.g., U.S.
Pat. No. 5,859,205.
[0069] In another embodiment, non-human antibodies may be humanized
by any of the methods known in the art. Humanized antibodies are
useful for in vivo therapeutic applications. In addition,
recombinant "humanized" antibodies may be synthesized. Humanized
antibodies are antibodies initially derived from a nonhuman mammal
in which recombinant DNA technology has been used to substitute
some or all of the amino acids not required for antigen binding
with amino acids from corresponding regions of a human
immunoglobulin light or heavy chain. That is, they are chimeras
comprising mostly human immunoglobulin sequences into which the
regions responsible for specific antigen-binding have been inserted
(see, e.g., PCT patent application WO 94/04679). Animals are
immunized with the desired antigen, the corresponding antibodies
are isolated and the portion of the variable region sequences
responsible for specific antigen binding are removed. The
animal-derived antigen binding regions are then cloned into the
appropriate position of the human antibody genes in which the
antigen binding regions have been deleted. Humanized antibodies
minimize the use of heterologous (inter-species) sequences in
antibodies for use in human therapies, and are less likely to
elicit unwanted immune responses. Primatized antibodies can be
produced similarly using primate (e.g., rhesus, baboon and
chimpanzee) antibody genes. Further changes can then be introduced
into the antibody framework to modulate affinity or immunogenicity.
See, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and
6,180,370.
[0070] Another embodiment of the invention includes the use of
human antibodies, which can be produced in nonhuman animals, such
as transgenic animals harboring one or more human immunoglobulin
transgenes. Such animals may be used as a source for splenocytes
for producing hybridomas, as is described in U.S. Pat. No.
5,569,825, WO00076310, WO00058499 and WO00037504 and incorporated
by reference herein.
[0071] Signal Modulation
[0072] In another embodiment, the antibodies of the invention bind
to Cripto, and modulate Cripto signaling or Cripto-protein
interactions. Over-expression of Cripto activity can lead to a
de-differentiated state promoting mesenchymal cell characteristics,
increased proliferation, and cell migration (Salomon et al.,
BioEssays 21: 61-70, 1999; Ciardiello et al., Oncogene 9: 291-298,
1994; and Baldassarre et al., Int. J. Cancer 66:538-543, 1996),
phenotypes associated with cell transformation seen in
neoplasia.
[0073] One method of testing the activity of anti-Cripto antibodies
and their ability to modulate Cripto signaling is with an F9-Cripto
knock-out (KO) cell line (Minchiotti at al., Mech. Dev. 90:
133-142, 2000). Cripto stimulates smad2 phosphorylation and the
transcription factor FAST in Xenopus embryos, and the activity of
the transcription factor FAST can be monitored by measuring the
luciferase activity from a FAST regulatory element-luciferase
reporter gene (Saijoh et al., Mol. Cell. 5:35-47, 2000). F9-Cripto
KO cells are deleted for the Cripto gene and are thus null for
Cripto and Cripto-dependent signaling (Minchiotti at al., Mech.
Dev. 90: 133-142, 2000). Cripto signaling can be assessed in the F9
Cripto KO cells by transfecting in Cripto, FAST, and the FAST
regulatory element-luciferase gene construct. No Cripto dependent
FAST luciferase activity will be seen in these cell lines unless
Cripto cDNA, and FAST cDNA is transfected into them. Antibodies
capable of blocking Cripto-dependent Nodal signaling are antibodies
that block Cripto signaling function.
[0074] Other assays capable of measuring the activity of Cripto can
be employed by those of skill in the art, such as a growth in soft
agar assay (see Example 4 below). The ability of cells to grow in
soft agar is associated with cell transformation and the assay is a
classical in vitro assay for measuring inhibition of tumor cell
growth. Other assays useful in determining inhibition of activity
include in vitro assays on plastic, and the like.
[0075] Therapeutic Uses
[0076] Antibodies of the invention are also useful for, therapeutic
purposes, such as modulation of tumor cell growth, diagnostic
purposes to detect or quantitate Cripto, and purification of
Cripto.
[0077] In one embodiment of the invention, antibodies are provided
which are capable of binding specifically to Cripto and which
modulate growth of tumor cells in a patient.
[0078] In one embodiment, the tumor cells are testicular, breast,
testicular, colon, lung, ovary, bladder, uterine, cervical,
pancreatic, and stomach tumor cells.
[0079] In another embodiment, antibodies are provided which are
capable of binding specifically to Cripto and which modulate growth
of tumor cells which overexpress Cripto. In one embodiment, the
tumor cells are cell lines which overexpress Cripto, such as cell
lines derived from breast, testicular, colon, lung, ovary, bladder,
uterine, cervical, pancreatic, and stomach cancer.
[0080] Anti-Cripto antibodies may be screened for in vivo activity
as potential anticancer agents following standard protocols used by
those of skill in the art, as illustrated in Example 4 below.
Example of such protocols are outlined by the National Cancer
Institute (NCI) in their "in vivo cancer models screening"
protocols, NIH publication number 84-2635 (February 1984).
[0081] In another embodiment of the invention, the antibodies of
the invention are used to treat a patient having a cancerous
tumor.
[0082] The antibodies of the present invention can be combined with
a pharmaceutically acceptable excipient and administered in a
therapeutically effective dose to the patient. For a discussion of
methods of inhibiting growth of tumors, see, e.g., U.S. Pat. No.
6,165,464.
[0083] Also contemplated are methods of treating a subject
suffering from a disorder associated with abnormal levels (i.e.
elevated or depleted) of Cripto wherein the method comprises
administering to the subject an effective amount of an antibody
that specifically binds to an epitope in the ligand/receptor
binding domain of Cripto, including but not limited to where the
epitope is in an EGF-like domain or a cys-rich domain of
Cripto.
[0084] Also contemplated are methods of treating a subject
suffering from a disorder associated with abnormal levels (i.e.
elevated or depleted) of Cripto wherein the method comprises
administering to the subject an effective amount of an antibody
which specifically forms a complex with Cripto and is directed to
the epitope to which an antibody selected from the group consisting
of A6C 12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318), A7H1.19,
A8F1.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A8H3.1 (ATCC
ACCESSION NO. PTA-3315), A8H3.2, A19A10.30, A10B2.18 (ATCC
ACCESSION NO. PTA-3311), A27F6.1 (ATCC ACCESSION NO. PTA-3310),
A40G12.8 (ATCC ACCESSION NO. PTA-3316), A2D3.23, A7A10.29, A9G9.9,
A15C12.10, A15E4.14, A17A2.16, A17C12.28, A17G12.1 (ATCC ACCESSION
NO. PTA-3314), A17H6.1, A18B3.11 (ATCC ACCESSION NO. PTA-3312),
A19E2.7, B3F6.17 (ATCC ACCESSION NO. PTA-3319), and B6G7.10 (ATCC
ACCESSION NO. PTA-3313) is directed.
[0085] Diagnosis via detection of Cripto is readily accomplished
through standard binding assays using the novel antibodies of the
invention, allowing those of skill in the art to detect the
presence of Cripto specifically in a wide variety of samples,
cultures, and the like.
[0086] Kits comprising an antibody of the invention for any of the
purposes described herein are also comprehended. In general, a kit
of the invention also includes a control antigen for which the
antibody is immunospecific. Embodiments include kits comprising all
reagents and instructions for the use thereof.
[0087] Additional features of the invention will be apparent from
the following illustrative Examples.
EXAMPLES
Example 1
Expression and Purification of Cripto
[0088] An expression plasmid designated pSGS480 was constructed by
sub-cloning a cDNA encoding human Cripto amino acids residues 1 to
169 of Cripto [amino acids 1-169 of SEQ ID NO: 1], fused to human
IgG.sub.1 Fc domain (i.e., "CR(del C)-Fc") into vector pEAG1100.
For a more detailed description of this vector, see copending U.S.
Patent Application Ser. No. 60/233,148, filed Sep. 18, 2000. The
vector pEAG1100 is a derivative of GIBCO-BRL Life Technologies
plasmid pCMV-Sport-betagal, the use of which in CHO transient
transfections was described by Schifferli et al., 1999, Focus 21:
16. It was made by removing the reporter gene beta-galactosidase
NotI fragment from the plasmid pCMV-Sport-Betagal (catalog number
10586-014) as follows: The plasmid was digested with NotI and
EcoRV, the 4.38 kb NotI vector backbone fragment was gel-purified
and ligated. Ligated DNA was transformed into competent E. coli
DH5alpha. pEAG1100 was isolated as a plasmid containing the desired
recombinant from an isolated single colony. The sequence of
pEAG1100 spanning the promoter, polylinker, and transcription
termination signal was confirmed.
[0089] Plasmid pSGS480 was transiently transfected into CHO cells
and the cells were grown at 28.degree. C. for 7 days. The presence
of CR(del C)-Fc protein in these cells and the conditioned media
was examined by Western blot analysis. For Western blot analysis,
conditioned media and cells from Cripto transfected cells were
subjected to SDS-PAGE on 4-20% gradient gels under reducing
conditions, transferred electrophoretically to nitrocellulose, and
the Cripto fusion protein was detected with a rabbit polyclonal
antiserum raised against a Cripto 17-mer peptide (comprising
residues 97-113 of SEQ ID NO: 1)-keyhole limpet hemocyanin
conjugate. After centrifugation to remove the cells, Western blot
analysis showed that the CR(del C)-Fc protein was efficiently
secreted into the conditioned media (supernatant). The supernatant
was applied to a Protein A-Sepaharose (Pharmacia), and bound
protein was eluted with 25 mM sodium phosphate pH 2.8, 100 mM NaCl.
The eluted protein was neutralized with 0.5 M sodium phosphate at
pH 8.6, and analyzed for total protein content from absorbance
measurements at 240-340 nm, and for purity by SDS-PAGE. The eluted
protein was filtered through a 0.2 micron filter, and stored at
-70.degree. C.
Example 2
Generation and Screening of Antibodies
[0090] The eluted CR(del C)-Fc protein is injected into mice, and
standard hybridoma techniques known to those of skill in the art
are used to generate monoclonal antibodies.
[0091] A. Generation of Antibodies
[0092] Particularly, female Robertsonian mice (Jackson Labs) were
immunized intraperitoneally with 25 .mu.g of purified human CR del
C-Fc emulsified with complete fruend's adjuvant (GibcoBRL
#15721-012). They were boosted two times intraperitoneally with 25
.mu.g of CR del C-Fc emulsified with incomplete freunds's adjuvant
(GibcoBRL #15720-014) and once on Protein A beads. The sera were
screened and 3 weeks after the last boost, the mouse with the best
titer was boosted intraperitoneally with 50 .mu.g soluble CR del
C-Fc three days before fusion. The mouse was boosted intravenously
with 50 .mu.g CR del C-Fc the day before fusion. The mouse spleen
cells were fused with FL653 myeloma cell at a 1 spleen:6 myeloma
ratio and were plated at 100,000, 33,000 and 11,000 cells per well
into 96 well tissue culture plates in selection media. Wells
positive for growth were screened by FACS and ELISA a week later.
Two fusions were performed.
[0093] B. Screening of Antibodies
[0094] Supernatants resulting from the first or second fusion were
screened first on ELISA plates for recognition of Cripto del C
and/or Cripto EGF-like domain proteins. A control fusion protein
(LT-beta receptor-Fc) was coated on ELISA plates to discard
monoclonal antibodies that recognized the human Fc epitope. The
ELISA was performed as described below in section C. In the first
fusion, primary supernatants were also screened for their ability
to recognize cell surface Cripto protein on the testicular tumor
cell line, NCCIT by FACS. In the case of the second fusion, the
ability of supernatants to recognize Cripto on two tumor cell
lines, NCCIT and the breast cancer line, DU4475 by FACs was
analyzed. Secondary screens included testing the monoclonal
antibody supernatant's ability to recognize cell surface Cripto on
a panel of tumor cell lines (see Tables 1 and 2 for results),
ability of monoclonal antibodies to recognize human Cripto
immunohistochemically on human breast and colon tumor tissue
sections, ability of monoclonal antibodies to block in Cripto-Nodal
signalling assay, ability to block growth of tumor cell lines on
plastic or in soft agar assays, and ability to internalize cell
surface Cripto.
[0095] C. ELISA
[0096] The ELISA assays were performed as follows:
[0097] Materials: [0098] Plates: Costar high-binding Easy-wash 96W
plates (07-200-642) [0099] 2' antibody: Pierce Gt anti-Ms IgG (H+
L)-HRP (P131430) [0100] Substrate: Pierce TMB Substrate Kit (34021)
[0101] Stop solution: 1N H2SO4
[0102] Buffers: [0103] Binding buffer: 0.1 M NaHPO4 pH 9.0 [0104]
Blocking buffer: PBS+10% Donor Calf Serum [0105] Wash buffer:
PBS+0.1% tween-20
[0106] Antigens CR-del-C-Fc and CR-EGF-fc, control hu IgG1 fusion
protein were diluted in binding buffer to 500 ng/ml. 100 .mu.l were
added per well and incubated for 1 hr at 37.degree. C. or overnight
at 4.degree. C. The liquid was decanted and the plate inverted and
blotted until dry. 250 .mu.l/well blocking buffer was then added,
followed by incubation for 30 min. at 37.degree. C. Again, the
liquid was decanted and the plate inverted and blotted until dry.
Supernatants were diluted 1:50 in wash buffer, and plated at 50
.mu.l/well, followed by incubation for 1 hour at room temperature.
Plates were washed 3.times. vigorously with 250 .mu.l/well wash
buffer. Then 100 .mu.l/well 2' antibody diluted in wash buffer at
1:10,000 was added, followed by incubation for 30 min. at room
temperature. Plates were then washed 3.times. vigorously with 250
.mu.l/well wash buffer, then substrate added at 100 .mu.l/well.
Color was permitted to develop until sufficiently dark, then 100
.mu.l/well stop solution was added and the plates read for
absorbance at 450 nm.
[0107] D. Flow Cytometry
[0108] Cripto positive cell lines may be used to assay the
monoclonal antibodies for binding to Cripto using cell surface
staining and flow cytometry as follows:
[0109] Release cells from T162 flasks with 2 ml PBS.sup.- with 5 mM
EDTA, 10 min., 37.degree. C. Bring up to 20 ml with media with
serum, pipetting up and down several times to unclump cells. Spin
at 1200 rpm for 5 minutes. Wash cells with 5-10 ml 4.degree. C. PBS
with 0.1% BSA (wash buffer). Spin at 1200 rpm for 5 minutes.
Resuspend at 4.times.10.sup.6-10.sup.7/ml in wash buffer. Keep on
ice.
[0110] Prepare antibodies for staining. Purified antibodies are
diluted to 1-10 .mu.g/ml in wash buffer. Add 50 .mu.l of cells to a
96-well Linbro V bottomed plate (ICN 7632105). Plate one well of
cells for each control for each cell line to be analyzed, including
cells for no antibody, 2.degree. antibody only, hybridoma media,
positive control antibody supernatant, if available, or purified,
and an IgG subclass control (if using purified antibodies).
[0111] Plate one well of cells for each experimental sample for
each cell line to be analyzed. Spin plate, 1200 rpm for 5 minutes,
using a table top centrifuge at 4.degree. C. Flick out buffer by
inverting the plate and shaking until the liquid is substantially
discarded. Add 40-50 t of antibodies (or wash buffer for the
no-antibody and 2.degree. antibody-only control wells) to wells.
Incubate at least 30 min.-1 hour at 4.degree. C. Spin plate, 1200
rpm for 5 minutes. Flick out antibody solutions. Wash wells twice
with 200 .mu.l wash buffer per well, spinning after each wash.
Flick out buffer.
[0112] Resuspend cells in each well in 50 .mu.l of 1:200 dilution
(in wash buffer) of R-PE tagged goat anti-mouse IgG, Fc Specific
(Jackson Immunoresearch Laboratories Cat# 115-116-071). Incubate 20
min, 4.degree. C., in the dark. Add 150 .mu.l wash buffer to cells
in each well. Spin plate at 1200 rpm for 5 minutes. Wash once with
200 .mu.l wash buffer per well. Resuspend cells in 150 .mu.l 1% PFA
in PBS. Transfer contents of each well to separate tubes (5 ml
Falcon polystyrene round bottomed tube-352052). Wrap tubes in tin
foil.
[0113] The contents of the tubes are then read by flow
cytometry.
[0114] The results of a two screenings of monoclonal antibodies
produced by this method yielded the following results, summarized
in Tables 1 and 2 below, wherein the first column provides the
designated names for the hybridoma subclones, the next two columns
show the results of ELISA screens, and the remaining columns show
flow cytometry analysis results on four cripto-positive cell lines.
The results are given in units of mean fluorescent index (MFI).
TABLE-US-00002 TABLE 1 Anti-Cripto Monoclonal Antibody
Characterization ELISA ELISA Cripto Cripto EGFlike Hybridoma ATCC
deposit delC domain DU4475 NCCIT GEO Subclone no. Sups Sups MFI MFI
MFI HT3 MFI Control- 0.06 0.07 ELISA Control- 14 9 37 18 MouseIg
A6C12.11 2.21 0.07 11 35 29 8 A6F8.6 PTA-3318 2.32 0.08 11 50 29 10
A7H1.19 2.14 0.09 14 34 27 12 A8F1.30 2.15 0.1 17 27 32 28 A8G3.5
PTA-3317 2.39 0.09 9 30 25 15 A8H3.1 PTA-3315 2.4 1.7 9 44 23 10
A8H3.2 2.54 0.07 13 13 16 14 A19A10.30 2.02 0.09 9 40 20 10
A10B2.18 PTA-3311 2.36 0.07 40 63 100 43 A27F6.1 PTA-3310 2.28 1.19
9 44 26 17 A40G12.8 PTA-3316 2.27 1.59 10 47 26 16
TABLE-US-00003 TABLE 2 Anti-Cripto Monoclonal Antibody
Characterization ELISA ELISA Cripto Hybridoma ATCC deposit Cripto
EGFlike DU4475 NCCIT GEO Subclone no. delC domain MFI MFI MFI HT3
MFI Control- 0.05 0.05 ELISA Control- 10 6 4 6 MouseIg A2D3.23 0.93
0.90 73 138 37 27 A7A10.29 1.37 0.07 75 83 33 83 A9G9.9 1.39 0.07
52 62 32 82 A15C12.10 1.42 0.06 46 55 25 93 A15E4.14 1.38 0.06 50
63 23 95 A17A2.16 1.40 0.06 76 97 41 81 A17C12.28 0.96 0.97 6 16 3
22 A17G12.1 PTA-3314 1.30 1.37 61 66 28 78 A17H6.1 1.38 0.05 35 30
5 28 A18B3.11 PTA-3312 1.36 1.38 50 42 33 65 A19E2.7 1.40 0.06 53
59 26 99 B3F6.17 PTA-3319 1.37 0.06 77 51 39 89 B6G7.10 PTA-3313
1.38 1.40 28 22 22 56 B11H8.4 1.41 0.06 59 101 39 107 B12C12.5 1.10
1.04 27 14 23 59 B15A2.6 1.40 0.06 36 44 22 59 C4A2.16 1.40 0.06 24
36 22 65
Example 3
Null Cell Assay for Inhibition of Cripto Signaling
[0115] The following describes an F9 Cripto null cell signaling
assay used to assess inhibition of Cripto signaling.
[0116] Day 0 Coat 6 welled plates with 0.1% gelatin 2 ml/well at
37.degree. C. for 15 min.
[0117] Seed cells at 6.times.10.sup.5 F9 CRIPTO NULL cells per
well.
[0118] Day 1 Transfection:
[0119] Each of the following samples is added to 300 .mu.l OptiMem1
to yield Solution A for each sample:
Sample 1: 0.5 .mu.g (N.sub.2).sub.7 luciferase FAST reporter cDNA
plus 1.5 .mu.g empty vector cDNA. [0120] Sample 2: 0.5 .mu.g
(N.sub.2).sub.7 luciferase, 0.5 .mu.g FAST, and 1 .mu.g empty
vector cDNAs. Sample 3: 0.5 .mu.g (N.sub.2).sub.7 luciferase, 0.5
.mu.g Cripto ADD 0.5 FAST, and 0.5 .mu.g empty vector cDNAs. Sample
4: 0.5 .mu.g (N.sub.2).sub.7 luciferase, 0.5 .mu.g Cripto, 0.5
FAST, and 0.5 .mu.g empty vector cDNAs Sample 5: 0.5 .mu.g
(N.sub.2).sub.7 luciferase, 0.5 .mu.g Cripto, 0.5 FAST, and 0.5
.mu.g empty vector cDNAs. Sample 6: 0.5 .mu.g (N.sub.2).sub.7
luciferase, 0.5 .mu.g Cripto, 0.5 FAST, and 0.5 .mu.g empty vector
cDNAs. Sample 7: 0.5 .mu.g (N.sub.2).sub.7 luciferase, 0.5 .mu.g
Cripto, 0.5 FAST, and 0.5 .mu.g empty vector cDNAs. Sample 8: 0.5
.mu.g (N.sub.2).sub.7 luciferase, 0.5 .mu.g Cripto, 0.5 FAST, and
0.5 .mu.g empty vector cDNAs. Sample 9: 0.5 .mu.g (N.sub.2).sub.7
luciferase, 0.5 .mu.g Cripto, 0.5 FAST, and 0.5 .mu.g empty vector
cDNAs. Solution B comprises 30 .mu.l of Lipofectamine plus 270
.mu.l of OptiMem1.
[0121] For each sample, mix solution A and solution B together.
Incubate 45 minutes at room temperature. Rinse wells with 2 ml/well
of OptiMem1. Aspirate just before next step.
Add 2.4 ml of OptiMem1 to each mixture of solutions A+B, mix, add
1.5 ml/well to duplicate wells. Incubate 5 hours at 37.degree. C.
Add 1.5 ml/well of DMEM+20% FCS, 2 mM Gln, P/S to wells which
received samples 1-3. Add anti-Cripto antibodies as follows: Sample
4 wells: A27F6.1, 10 .mu.g/ml; Sample 5 wells: A27F6.1, 2 .mu.g/ml;
Sample 6 wells: A40G12.8; 10 .mu.g/ml, Sample 7 wells: A40G12.8 2
.mu.g/ml; Sample 8 wells: A10B2.18, 10 .mu.g/ml; Sample 9 wells:
A10B2.18, 2 .mu.g/ml.
[0122] Day 2 Remove media, wash cells with PBS, 2 ml/well. Add
DMEM+0.5% FCS, 2 mM Gln, P/S with the same amounts of Cripto
antibodies as the previous day, to the same wells.
[0123] Day 3 Develop luciferase signal. Wash wells with
PBS+Ca.sup.2+ and Mg.sup.2+, 2 ml/well. Use LucLite kit, Packard
cat# 6016911. Bring buffer and substrate to room temperature. Dim
lights. Reconstitute substrate with 10 ml of buffer. Dilute 1:1
with PBS+Ca.sup.2+ and Mg.sup.2+. Aspirate wells. Quickly add 250
.mu.l of diluted substrate per well using a repeat pipettor. Swirl
solution and transfer 200 .mu.l to wells of a 96 welled white
opaque bottom plate, Falcon 35-3296. Read plate on luminometer
using Winglow, exporting data to Excel.
[0124] The results of this assay are summarized below in Table
3.
TABLE-US-00004 TABLE 3 Cripto Signaling Assay: Inhibition with
Anti-Cripto Monoclonal Antibodies Relative Luminescent cDNAs
transfected Anti-Cripto Antibody Units (N.sub.2).sub.7 luc none 123
(N.sub.2).sub.7 luc, FAST none 259 (N.sub.2).sub.7 luc, FAST,
Cripto none 3091 (N.sub.2).sub.7 luc, FAST, Cripto A27F6.1 10
.mu.g/ml 1507 (N.sub.2).sub.7 luc, FAST, Cripto A27F6.1 2 .mu.g/ml
2297 (N.sub.2).sub.7 luc, FAST, Cripto A40G12.8 10 .mu.g/ml 1213
(N.sub.2).sub.7 luc, FAST, Cripto A40G12.8 2 .mu.g/ml 2626
(N.sub.2).sub.7 luc, FAST, Cripto A10B2.18 10 .mu.g/ml 3466
(N.sub.2).sub.7 luc, FAST, Cripto A10B2.18 2 .mu.g/ml 3103
Example 4
Assay for In Vitro Inhibition of Tumor Cell Growth
[0125] Inhibition of Cripto Signaling may also be assayed by
measuring the growth of GEO cells in soft agar. See, e.g.,
Ciardiello et al., Oncogene. 1994 January; 9(1):291-8; Ciardiello
et al., Cancer Res. 1991 Feb. 1; 51(3):1051-4.
[0126] First, melt 3% bactoagar. Keep at 42.degree. C. in a water
bath. Then, mix 3% bactoagar solution with prewarmed complete media
to make a solution of 0.6% bactoagar, keeping at 42.degree. C.
Plate 4 mls of the solution in a 6 cm dish and let cool for at
least 30 minutes to form the bottom agar layer. Trypsinize GEO
cells and resuspend to 10.sup.5 cells/ml in complete media. Add
antibodies to be assayed, or controls, to the cell suspensions,
titrating antibodies from 20 .mu.g to 1 .mu.g. Mix equal volumes of
the GEO cell suspensions and 0.6% bactoagar and overlay 2 mls on
top of the bottom agar layer. Let cool for at least 1 hour.
Incubate for 14 days at 37.degree. C. in CO.sub.2 incubator. Count
colonies visible without the use of a microscope. The absence of
colonies, as compared to negative controls, indicates that the
antibody tested inhibits in vitro tumor cell growth.
[0127] This assay was used to yield the results shown in Table 4,
for the antibodies A27F6.1 and B6G7.10, both of which demonstrate
the ability to decrease growth of GEO cell colonies.
TABLE-US-00005 TABLE 4 Results of growth in soft agar assay Average
number Antibody of colonies none 109.0 none 104.3 A27.F6 20
.mu.g/ml 82.0 A27F6.1 10 .mu.g/ml 78.3 A27F6.1 5 .mu.g/ml 79.0
A27F6.1 1 .mu.g/ml 108.7 B6G7.10 20 .mu.g ml 102.3 B6G7.10 10
.mu.g/ml 71.7
Example 5
Assay for In Vivo Inhibition of Tumor Cell Growth
[0128] To assess the inhibition of tumor cell growth, a human tumor
cell line is implanted subcutaneously in athymic nude mice and the
effects of the antibodies of the invention are observed, with and
without additional chemotherapeutic treatments which may provide
synergistic or additive effects on tumor inhibition.
[0129] This assay may be performed alternatively using different
tumor cell lines, such as, for example, GEO (a well differentiated
human colon cancer in-vitro cell line, is obtained from the
American Tissue Type Collection (ATCC)), DU-4475 (a breast cancer
in-vitro cell line obtained from the ATCC), NCCIT (a testicular
tumor cell line obtained from ATCC), or others known in the art.
One example of such assays is as follows:
[0130] Animals are individually marked by ear punches. The GEO cell
line is passed in-vitro or in-vivo for 1-4 passages. Animals are
implanted with GEO cells subcutaneously in the right flank area.
The following groups of animals may be used:
TABLE-US-00006 Group # Treatment # of Mice 1. Saline Control, 0.2
ml/mouse, i.p. three times weekly (M, W, F) 20 2. mAb, low dose,
i.p. 10 3. mAb, middle dose, i.p. 10 4. mAb, high dose, i.p. 10 5.
5-FU, 30 mg/kg/inj, i.p., 3 Rx/wk (M, W, F) 10 6. Cisplatin, 2
mg/kg/inj, s.c., 3 Rx/wk (M, W, F) 10 7. Adriamycin, 1.6 mg/kg/inj,
i.p., 3 Rx/wk (M, W, F) 10 8. Irinotecan, 10 mg/kg/inj., i.p., 5
Rx/wk (M-F) 10 9. mAb, low dose, i.p. + 5-FU (intermediate dose) 10
10. mAb, middle dose, i.p. + 5-FU (intermediate dose) 10 11. mAb,
high dose, i.p. + 5-FU (intermediate dose) 10 12. mAb, low dose,
i.p. + Cisplatin (intermediate dose) 10 13. mAb, middle dose, i.p.
+ Cisplatin (intermediate dose) 10 14. mAb, high dose, i.p. +
Cisplatin (intermediate dose) 10 15. mAb, low dose, i.p. +
Adriamycin (intermediate dose) 10 16. mAb, middle dose, i.p. +
Adriamycin (intermediate dose) 10 17. mAb, high dose, i.p. +
Adriamycin (intermediate dose) 10 18. mAb, low dose, i.p. +
Irinotecan (intermediate dose) 10 19. mAb, middle dose, i.p. +
Irinotecan (intermediate dose) 10 20. mAb, high dose, i.p. +
Irinotecan (intermediate dose) 10
[0131] Day 0: Implant tumor, record initial body weight of
animals.
[0132] Day 1: Initiate treatments as indicated above.
[0133] Day 5: Begin tumor size and body weight measurements and
continue two times weekly until termination of experiment.
[0134] Initial body weight, tumor size and body weight
measurements, histology at sacrifice, and immunohistochemistry
analysis on tumors are examined, analyzing for Cripto expression,
tumor growth, and inhibition thereof.
Example 6
In Vivo Xenograft Tumor Model--Cys-rich Blocking Anti-Cripto
Antibody
[0135] To assess the response of an NCCIT, a human testicular
carcinoma cell line was implanted subcutaneously with an antibody
which binds to a cys-rich domain of Cripto. The experimental
methods are listed below. The results are shown in FIG. 1.
Methods and Materials
[0136] Animals: Athymic nude male mice were used. Animals were
individually numbered by ear punches. Tumor: NCCIT, mediastinal
mixed germ cell human testicular carcinoma in-vitro cell line
originally obtained from the American Tissue Type Collection. Cell
line was passed in-vitro for six passages in RPMI-1640/10% FBS
without antibiotics. Animals implanted subcutaneously with
5.times.10.sup.6 cells/0.2 ml matrigel on the animals right
flank.
TABLE-US-00007 Group # Treatment # of Mice 1 Vehicle Control, (25
mM sodium phosphate, 20 100 mM sodium chloride, pH 7.2), 0.2
ml/mouse, i.p., Q14D Treatments begin on day -1 2. A8G3.5, 1
mg/kg/inj, i.p., Q14D 10 Treatments begin on day -1 3. A8G3.5, 3
mg/kg/inj, i.p., Q14D 10 Treatments begin on day -1 4. A8G3.5, 10
mg/kg/inj, i.p., Q14D 10 Treatments begin on day -1 5.
Cis-platinum, 2 mg/kg/inj, s.c., 3 .times./wk 10 (M, W, F) for 6
treatments
Treatments began on day 1
Testing Schedule
[0137] Day -1: Randomized mice into control and treatments groups.
Recorded initial body weight of animals. Administered first
treatments to antibody groups. Dosing solutions were made.
Treatments were blinded to the technicians until the assay was
terminated. Day 0: Implanted tumor. Ran bacterial cultures on the
tumor implanted into mice. Day 1: Administered first treatment to
the positive chemotherapeutic group. Day 4: Recorded initial tumor
size measurements for tumor baseline on matrigel. Continued to
record tumor size and body weights on mice 2.times./week. Monitored
the study daily and made notations of any unusual observation on
animals. Endpoints: Initial body weight [0138] Tumor size and body
weight measurements
Example 7
In Vivo Xenograft Tumor Model--EGF-Like Domain Blocking Anti-Cripto
Antibody
[0139] To assess the response of an NCCIT, a human testicular
carcinoma cell line was implanted subcutaneously with an antibody
which binds to a EGF-like domain of Cripto. The experimental
methods are listed below. The results are shown in FIG. 2.
Methods and Materials:
[0140] Animals: Athymic nude male mice were used. Animals were
individually numbered by ear punches. Tumor: NCCIT, mediastinal
mixed germ cell human testicular carcinoma in-vitro cell line
originally obtained from the American Tissue Type Collection. Cell
line was passed in-vitro for eight passages in RPMI-1640/10% FBS
without antibiotics. Animals implanted subcutaneously with
5.times.10.sup.6 cells/0.2 ml matrigel on the animals right
flank.
TABLE-US-00008 Group # Treatment # of Mice 1. Vehicle Control, (25
mM sodium phosphate, 100 mM sodium 18 chloride, pH 7.2), 0.2
ml/mouse, i.p., Q14D Treatments begin on day -1 2. A27F6.1, 1
mg/kg/inj, i.p., Q14D 10 Treatments begin on day -1 with a loading
dose of 2.6 mg/kg/mouse 3. A27F6.1, 10 mg/kg/inj, i.p., Q14D 10
Treatments begin on day -1 with a loading dose of 21.2 mg/kg/mouse
4. Cis-platinum, 2 mg/kg/inj, s.c., 3 .times./wk (M, W, F) for 6
treatments 10
Treatments began on day 1. Testing schedule [0141] Day -1:
Randomized mice into control and treatments groups. Recorded
initial body weight of animals. Administered first treatments to
antibody groups. Dosing solutions were made. Treatments were
blinded to the technicians until the assay was terminated. Day 0:
Implant tumor. Ran bacterial cultures on the tumors implanted into
mice. Bacterial culture were negative for contamination at 24 and
48 hours post sampling. Day 1: Administered first treatment to the
positive chemotherapeutic group. Day 4: Recorded initial tumor size
measurements for tumor baseline on matrigel. Continued to record
tumor size and body weights on mice 2.times./week. Monitored the
study daily and made notations of any unusual observation on
animals.
Endpoints:
[0141] [0142] Initial body weight [0143] Tumor size and body weight
measurements
Example 8
Cripto Mabs that Block ALK4 Binding
[0144] In order to assess whether Cripto-specific monoclonal
antibodies can interfere with Cripto's ability to bind to Alk4, the
activin type I receptor, we used flow cytometry analysis using a
293 cell line which stably expresses Alk4. To generate this cell
line, 293 cells were cotransfected with a plasmid that expresses
Alk4 tagged at the C-terminus with a HA epitope and a plasmid that
expresses the drug, puromycin, at a 10:1 ratio. The transfected
cells were then selected in puromycin until colonies formed.
Colonies were then picked, expanded and then analyzed for Alk4
expression using western blotting analysis for HA. Clone 21
(293-Alk4-21) was found to express high levels of Alk4 compared to
control, untransfected 293 cells.
[0145] To analyze Cripto-Alk4 binding by flow cytometry, a
purified, soluble form of human Cripto (aa 1-169) fused to the Fc
portion of human IgG (CrdelC-Fc) was employed. Approximately 5
.mu.g/ml of Crde1C-Fc or control Fc protein was incubated with
3.times.10.sup.5 293-Alk4-21 cells on ice for 30 minutes in 50
.mu.l total volume of FACS buffer (PBS with 0.1% BSA). For samples
containing anti-Cripto antibodies, 5 .mu.g/ml Crde1C-Fc was
preincubated with 50 .mu.g/ml of each Cripto antibody (A10.B2.18,
A40.G12.8, A27.F6.1, A8.H3.1, A19.A10.30, A6.F8.6, A8.G3.5,
A6.C12.11) on ice prior to addition of the cells. The cells were
then washed in FACS buffer and the bound Fc protein was detected by
incubating the cells with a R-phycoerytherin-conjugated goat
anti-human IgG (Fc fragment specific) from Jackson Immunologics.
Samples were then washed again, fixed in 1% paraformaldehyde in
PBS, and analyzed using standard flow cytometry procedures. The
results of the FACS assay are shown in FIG. 3.
[0146] Some of the embodiments of the invention described above are
outlined below and include, but are not limited to, the following
embodiments. As those skilled in the art will appreciate, numerous
changes and modifications may be made to the various embodiments of
the invention without departing from the spirit of the invention.
It is intended that all such variations fall within the scope of
the invention.
[0147] The entire disclosure of each publication cited herein is
hereby incorporated by reference.
Sequence CWU 1
1
21188PRTHomo Sapiens 1Met Asp Cys Arg Lys Met Ala Arg Phe Ser Tyr
Ser Val Ile Trp Ile 1 5 10 15Met Ala Ile Ser Lys Val Phe Glu Leu
Gly Leu Val Ala Gly Leu Gly 20 25 30His Gln Glu Phe Ala Arg Pro Ser
Arg Gly Tyr Leu Ala Phe Arg Asp 35 40 45Asp Ser Ile Trp Pro Gln Glu
Glu Pro Ala Ile Arg Pro Arg Ser Ser 50 55 60Gln Arg Val Pro Pro Met
Gly Ile Gln His Ser Lys Glu Leu Asn Arg65 70 75 80Thr Cys Cys Leu
Asn Gly Gly Thr Cys Met Leu Gly Ser Phe Cys Ala 85 90 95Cys Pro Pro
Ser Phe Tyr Gly Arg Asn Cys Glu His Asp Val Arg Lys 100 105 110Glu
Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu Pro Lys Lys Cys 115 120
125Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg Cys Phe Pro Gln Ala
130 135 140Phe Leu Pro Gly Cys Asp Gly Leu Val Met Asp Glu His Leu
Val Ala145 150 155 160Ser Arg Thr Pro Glu Leu Pro Pro Ser Ala Arg
Thr Thr Thr Phe Met 165 170 175Leu Val Gly Ile Cys Leu Ser Ile Gln
Ser Tyr Tyr 180 1852188PRTHomo Sapiens 2Met Asp Cys Arg Lys Met Val
Arg Phe Ser Tyr Ser Val Ile Trp Ile 1 5 10 15Met Ala Ile Ser Lys
Ala Phe Glu Leu Gly Leu Val Ala Gly Leu Gly 20 25 30His Gln Glu Phe
Ala Arg Pro Ser Arg Gly Asp Leu Ala Phe Arg Asp 35 40 45Asp Ser Ile
Trp Pro Gln Glu Glu Pro Ala Ile Arg Pro Arg Ser Ser 50 55 60Gln Arg
Val Leu Pro Met Gly Ile Gln His Ser Lys Glu Leu Asn Arg65 70 75
80Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Glu Ser Phe Cys Ala
85 90 95Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu His Asp Val Arg
Lys 100 105 110Glu Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu Pro
Lys Lys Cys 115 120 125Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg
Cys Phe Pro Gln Ala 130 135 140Phe Leu Pro Gly Cys Asp Gly Leu Val
Met Asp Glu His Leu Val Ala145 150 155 160Ser Arg Thr Pro Glu Leu
Pro Pro Ser Ala Arg Thr Thr Thr Phe Met 165 170 175Leu Ala Gly Ile
Cys Leu Ser Ile Gln Ser Tyr Tyr 180 185
* * * * *