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Patent 2480119 Summary

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(12) Patent: (11) CA 2480119
(54) English Title: CRIPTO-SPECIFIC ANTIBODIES
(54) French Title: ANTICORPS SPECIFIQUES AU CRIPTO
Status: Deemed expired
Bibliographic Data
(51) International Patent Classification (IPC):
  • C07K 16/30 (2006.01)
  • A61K 39/395 (2006.01)
  • G01N 33/53 (2006.01)
  • G01N 33/574 (2006.01)
(72) Inventors :
  • SANICOLA-NADEL, MICHELE (United States of America)
  • ADKINS, HEATHER (United States of America)
  • MIKLASZ, STEVEN DONALD (United States of America)
  • RAYHORN, PAUL (United States of America)
  • SCHIFFER, SUSAN GAIL (United States of America)
  • WILLIAMS, KEVIN P. (United States of America)
(73) Owners :
  • BIOGEN IDEC MA INC. (United States of America)
(71) Applicants :
  • BIOGEN, INC. (United States of America)
(74) Agent: GOWLING WLG (CANADA) LLP
(74) Associate agent:
(45) Issued: 2014-04-08
(86) PCT Filing Date: 2002-10-01
(87) Open to Public Inspection: 2003-10-09
Examination requested: 2007-10-01
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2002/031462
(87) International Publication Number: WO2003/083041
(85) National Entry: 2004-09-22

(30) Application Priority Data:
Application No. Country/Territory Date
60/367,002 United States of America 2002-03-22
PCT/US02/11950 United States of America 2002-04-17

Abstracts

English Abstract




The invention provides Cripto-specific antibodies, or biologically functional
fragments thereof, and uses thereof. Antibodies which bind Cripto and inhibit
Cripto activity are provided. Antibodies which bind Cripto and inhibit the
interaction between Cripto and ALK4 and/or between Cripto and Activin B are
provided. Antibodies which bind Cripto and inhibit tumor growth are also
provided. Antibodies which bind Cripto, inhibit Cripto activity, and inhibit
tumor growth are also provided. The invention also provides methods of using
these antibodies in therapeutic, diagnostic, and research application.


French Abstract

L'invention concerne des anticorps spécifiques au cripto ou des fragments biologiquement fonctionnels de ces derniers, et leurs utilisations. L'invention concerne également des anticorps qui lient le cripto et inhibent l'activité du cripto ; des anticorps qui lient le cripto et inhibent l'interaction entre le cripto et ALK4 et/ou entre le cripto et l'activine B ; des anticorps qui lient le cripto et inhibent la croissance tumorale ; des anticorps qui lient le cripto, inhibent l'activité du cripto et inhibent la croissance tumorale. L'invention concerne des procédés utilisant ces anticorps dans les applications thérapeutiques, diagnostiques et de recherche.

Claims

Note: Claims are shown in the official language in which they were submitted.



THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Use of an antibody that specifically binds to amino acids 114 to 150 of
SEQ ID NO:1 or
SEQ ID NO:2 for inhibiting tumor growth, wherein the antibody blocks the
interaction of
Cripto with Activin B and/or Alk4.
2. Use of an antibody that specifically binds to amino acids 114 to 150 of
SEQ ID NO:1 or
SEQ ID NO:2 in the manufacture of a medicament for inhibiting tumor growth,
wherein
the antibody blocks the interaction of Cripto with Activin B and/or Alk4.
3. An antibody that specifically binds to amino acids 114 to 150 of SEQ ID
NO:1 or SEQ
ID NO:2 for use to inhibit tumor growth, wherein the antibody blocks the
interaction of
Cripto with Activin B and/or Alk4.
4. The use of claim 1 or 2, or the antibody of claim 3, wherein the
antibody is conjugated to
a chemotherapeutic agent.
5. The use of claim 1 or 2, or the antibody of claim 3, wherein the
antibody is for
administration in combination with a nonconjugated chemotherapeutic agent.
6. The use of claim 4 or 5, or the antibody of claim 4 or 5, wherein the
chemotherapeutic
agent is selected from the group consisting of a tumor-activated prodrug, a
radionuclide
and a toxin.
7. The use of claim 6, or the antibody of claim 6, wherein the
chemotherapeutic agent is a
tumor-activated prodrug.
8. The use of claim 7, or the antibody of claim 7, wherein the tumor-
activated prodrug is a
maytansinoid.
9. The use of any one of claims 1, 2 and 4 to 8, or the antibody of any one
of claims 3 to 8,
wherein the antibody is monoclonal.



10. An antibody that specifically binds to amino acids 114 to 150 of SEQ ID
NO:1 or SEQ
ID NO:2, wherein the antibody blocks the interaction of Cripto with Activin B
and/or
Alk4.
11. The antibody of claim 10, wherein the antibody is conjugated to a
chemotherapeutic
agent.
12. The antibody of claim 10, wherein the antibody is administrable in
combination with a
nonconjugated chemotherapeutic agent.
13. The antibody of claim 11 or 12, wherein the chemotherapeutic agent is
selected from the
group consisting of a tumor-activated prodrug, a radionuclide and a toxin.
14. The antibody of claim 13, wherein the chemotherapeutic agent is a tumor-
activated
prodrug.
15. The antibody of claim 14, wherein the tumor-activated prodrug is a
maytansinoid.
16. The antibody of any one of claims 10 to 15, wherein the antibody is
monoclonal.

66

Description

Note: Descriptions are shown in the official language in which they were submitted.


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CRIPTO-SPECIFIC ANTIBODIES
10
Technical Field of the Invention
[0002] This invention relates generally to the
fields of genetics and cellular and molecular biology.
More particularly, the invention relates to anti-Cripto
antibodies.
Background of the Invention
[00031 Cripto is a 188-amino-acid cell surface
protein. It was serendipitously isolated in a cDNA
screen of a human embryonic carcinoma library
(Ciccodicola et al., 1989, EMBO J. 8:1987-91). The

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Cripto protein has at least two notable domains: a
cysteine-rich (cys-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. 274:8624-29).
[0004] The Cripto signaling pathway has remained
elusive despite continued investigation. The
literature supports activation of several different
pathways, including a MAP kinase pathway (DeSantis et
al., 1997, Cell Growth Differ. 8:1257-66; Kannan et
al., 1997, J. Biol. Chem. 272:3330-35); the TGF-p
pathway (Gritsman et al., 1999, Development 127:921-32;
Schier et al., 2000, Nature 403:385-89); possible
interactions with the Wnt pathway (Salomon et al.,
2000, Endocr. Relat. Cancer. 7:199-226); and cross-talk
with the EGF pathway (Bianco et al., 1999, J. Biol.
Chem. 274:8624-29).
[0005] U.S. Patent 5,256,643 and two patents related
thereto (U.S. Patents 5,654,140 and 5,792,616) disclose
a human Cripto gene, the Cripto protein, and antibodies
to Cripto.
[0006] U.S. Patent 5,264,557 and three patents
related thereto (U.S. Patents 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.

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[0007] Overexpression of the Cripto protein is
associated with tumors in many tissues (including, but
not limited to brain, 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
185:108-11; De Angelis et al., 1999, Int. J. Oncology
14:437-40. The art is therefore in need of means of
controlling, restricting, and/or preventing such
overexpression, inhibiting Cripto activity, and
inhibiting the consequences of Cripto expression (i.e.,
promotion and/or maintenance of cell transformation).
Summary of the Invention
[0008] This 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 inhibit
Cripto activity 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 block
the interaction between Cripto and Activin B. The
invention also provides antibodies which bind to Cripto
and inhibit tumor growth. The invention also provides
antibodies which bind to Cripto, inhibit Cripto
activity and inhibit tumor growth. The invention also

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provides antibodies which bind to Cripto, block the
interaction between Cripto and ALK4 and/or between
Cripto and Activin B, and inhibit tumor growth.
[0009] In one aspect of the invention, the antibody
of the invention specifically binds to an epitope
selected from the group of epitopes to which antibodies
A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318, deposited on
April 19, 2001), A7H1.19, A8F1.30, A8G3.5 (ATCC ACCESSION
NO. PTA-3317, deposited on April 19, 2001), A8H3.1 (ATCC
ACCESSION NO. PTA-3315, deposited on April 19, 2001),
A8H3.2, A10A10.30, A19A10.30, A10B2.17, A10B2.18 (ATCC
ACCESSION NO. PTA-3311, deposited on April 19, 2001),
A27F6.1 (ATCC ACCESSION NO. PTA-3310, deposited on April 19,
2001), A40G12.8 (ATCC ACCESSION NO. PTA-3316, deposited on
April 19, 2001), A2D3.23, A7A10.29, A9G9.9, A15C12.10,
A15E4.14, A17A2.16, A17012.28, A17G12.1 (ATCC ACCESSION NO.
PTA-3314, deposited on April 19, 2001), A17H6.1, A18B3.11
(ATCC ACCESSION NO. PTA-3312, deposited on April 19, 2001),
A19E2.7, B3F6.17 (ATCC ACCESSION NO. PTA-3319, deposited on
April 19, 2001), 56G7.10 (ATCC ACCESSION NO. PTA-3313,
deposited on April 19, 2001), 1-1A4C.2, 2-2C9.2, 2-3H9.2, 2-
4E5.6, 2-4D1.3, 3-4E8.3, 3-3G1.1, 4-2F6, 4-3A7 and 4-1E2
bind.
[0010] In another aspect of the invention, the
antibody of the 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 bind to the
epitope in the ligand/receptor binding domain include,
for example, A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-
3318), A7H1.19, A8F1.30, A8G3.5 (ATCC ACCESSION NO.
PTA-3317), A8113.1 (ATCC ACCESSION NO. PTA-3315),
A8H3.2, A10A10.30, A19A10.30, A10B2.17, A10B2.18 (ATCC
ACCESSION NO. PTA-3311), A27F6.1 (vrcc ACCESSION NO.
PTA-3310), A40G12.8 (lacc ACCESSION NO. PTA-3316),
A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14,

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Al7A2.16, A17C12.28, Al7G12.1 (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), 1-1A4C.2, 2-2C9.2, 2-
3H9.2, 2-4E5.6, 2-4D1.3, 3-4E8.3, 3-3G1.1, 4-2F6, 4-3A7
and 4-1E2.
[0011] In some embodiments, the epitope to which the
antibodies of the invention bind is in an EGF-like
domain. Antibodies that specifically bind to an
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),
A18B3.11 (ATCC ACCESSION NO. PTA-3312), 1-1A4C.2,
2-2C9.2 and 2-4D1.3.
[0012] In other embodiments the epitope to which the
antibodies of the invention bind is in a cys-rich
domain. Antibodies that specifically bind to an
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), A6C12.11,
1-1A4C.2 and 2-2C9.2.
[0013] In still other embodiments the epitope to
which the antibodies of the invention bind is in the
amino terminus. Antibodies that specifically bind to
an epitope in the amino terminus include, but are not
limited to, A10B2.17.
[0014] In still other embodiments the epitope to
which the antibodies of the 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

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include, but are not limited to, A10B2.18 (ATCC
ACCESSION NO. PTA-3311), B3F6.17 (ATCC ACCESSION NO.
PTA-3319), A17A2.16, 2-3H9.2, 2-4E5.6, 2-4D1.3,
3-4E8.3, 3-1E7.2 and 3-3G1.1.
[0015] In other embodiments the epitope to which the
antibodies of the invention bind is in the CR40 (SEQ ID
NO: 3), CR41 (SEQ ID NO: 4), CR43 (SEQ ID NO: 5), CR44
(SEQ ID NO: 6), CR49 (SEQ ID NO: 7), CR50 (SEQ ID NO:
8) or CR51 (SEQ ID NO: 9) polypeptides. Antibodies
that specifically bind to an epitope in one of these
polypeptides include, but are not limited to, A6C12.11,
A6F8.6, A7H1.19, A8F1.30, A8G3.5, A8H3.1, A8H3.2,
A10A10.30, A19A10.30, A10B2.17, A10B2.18, A27F6.1,
A40G12.8, A2D3.23, A7A10.29, A9G9.9, A15C12.10,
A15E4.14, A17A2.16, A17C12.28, A17G12.1, A17H6.1,
A18B3.11, A19E2.7, B3F6.17, B6G7.10, 1-1A4C.2, 2-2C9.2,
2-3H9.2, 2-4E5.6, 2-4D1.3, 3-4E8.3, 3-3G1.1, 4-2F6,
4-3A7 and 4-1E2.
[0016] This invention also includes antibodies which
bind specifically to Cripto and are capable of
inhibiting Cripto activity. Antibodies that bind
specifically to Cripto and are capable of inhibiting
Cripto activity 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), A6C12.11, 1-1A4C.2 and 2-2C9.2. In some
embodiments, the antibodies of the invention which bind
specifically to Cripto and are capable of inhibiting
Cripto activity bind to an epitope in an EGF-like
domain or a cys-rich domain of Cripto.
[0017] This invention also includes antibodies which
bind specifically to Cripto and block the interaction
between Cripto and ALK4. Antibodies that bind

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specifi cal ly 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), A6C12.11,
1-1A4C.2 and 2-2C9.2. In some embodiments, the
antibodies of the invention which bind 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.
[0018] This invention also includes antibodies which
bind specifically to Cripto and block the interaction
between Cripto and Activin B. Antibodies that bind
specifically to Cripto and are capable of blocking the
interaction between Cripto and Activin B, include but
are not limited to, A8G3.5 (ATCC ACCESSION NO. PTA-
3317) and 1-1A4C.2. In some embodiments, the
antibodies of the invention which bind specifically to
Cripto and are capable of blocking the interaction
between Cripto and Activin B bind to an epitope in a
cys-rich domain of Cripto.
[0019] In another aspect, this invention includes
antibodies which bind specifically to Cripto and are
capable of inhibiting tumor growth. Antibodies that
specifically bind to Cripto and are capable of
inhibiting tumor growth include, but are not limited
to, A27F6.1 (ATCC ACCESSION NO. PTA-3310), 36G7.10
(ATCC ACCESSION NO. PTA-3313) and A8G3.5 (ATCC
ACCESSION NO. PTA-3317), 1-1A4C.2 and 2-2C9.2.
[0020] In some embodiments, the antibodies of the
invention which bind specifically to Cripto and are
capable of inhibiting tumor growth bind to an epitope
in an EGF-like domain or a cys-rich domain of Cripto.

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[0021]
In yet another aspect, this invention
=
includes antibodies which bind specifically to Cripto,
which are capable of inhibiting Cripto activity, and
which are capable of inhibiting tumor growth.
Antibodies that specifically bind to Cripto, which are
capable of inhibiting Cripto activity, and which are
capable of inhibiting tumor growth include, but are not
limited to, A27F6.1 (ATCC ACCESSION NO. PTA-3310),
A8G3.5, 1-1A4C.2 and 2-2C9.2.
[0022] In some embodiments, the antibodies of this
invention which bind specifically to Cripto, which are
capable of inhibiting Cripto activity, and which are
capable of inhibiting tumor growth bind to an epitope
in an EGF-like domain or a cys-rich domain of Cripto.
[0023] In yet another aspect, this invention
includes antibodies which bind specifically to Cripto,
which are capable of blocking the interaction between
Cripto and ALK4, and which are capable of inhibiting
tumor growth. Antibodies that specifically bind to
Cripto, which are capable of blocking the interaction
between Cripto and ALK4, and which are capable of
inhibiting tumor growth include, but are not limited
to, A8G3.5 (ATCC ACCESSION NO. PTA-3317), 1-1A4C.2 and
2-2C9.2.
[0024] In yet another aspect, this invention
includes antibodies which bind specifically to Cripto,
which are capable of blocking the interaction between
Cripto and Activin B, and which are capable of
inhibiting tumor growth. Antibodies that specifically
bind to Cripto, which are capable of blocking the
interaction between Cripto and Activin B, and which are
capable of inhibiting tumor growth include, but are not

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limited to, A8G3.5 (ATCC ACCESSION NO. PTA-3317) and 1 -
=
1A4C . 2.
[0025] In another aspect, the invention includes a
method for inhibiting binding of Cripto to Activin B in
a sample, comprising adding to the sample an antibody
that binds specifically to Cripto and which is capable
of blocking the interaction between Cripto and Activin
B. In a related aspect, the invention includes a
method for inhibiting binding of Cripto to Activin B in
a mammal, comprising administering to the mammal an
antibody which binds specifically to Cripto and which
is capable of blocking the interaction between Cripto
and Activin B.
[0026] In another embodiment, the 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),
B6G7.10 (ATCC Accession No. PTA-3313), 1-1A4C.2,
2-2C9.2, 2-3H9.2, 2-4E5.6, 2-4D1.3, 3-4E8.3, 3-3G1.1,
4-2F6, 4-3A7 and 4-1E2. Several of the antibodies have
alternative designations as follows: 1-1A4C.2 is the
same as 1P1A4-C2.1 (ATCC Accession No. to be assigned);
2-2C9.2 is the same as 2P2C9.2 (ATCC Accession No. to
be assigned); 2-4E5.6 is the same as 2P4E5.6 (ATCC
Accession No. to be assigned); 3-3G1.1 is the same as
3P3G1.1 (ATCC Accession No. to be assigned); and
3-4E8.3 is the same as 3P4E8.3 (ATCC Accession No. to
be assigned).

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[0027] The antibodies of the invention include, but
are not limited to, monoclonal, polyclonal, humanized,
chimeric and human antibodies.
[0028] This invention also provides a composition
for administration to a mammal having a tumor that
expresses Cripto comprising at least one of the
antibodies described above. In some embodiments, the
mammal is human. The composition may include a
pharmaceutically acceptable excipient. The antibodies
described above can be conjugated to a chemotherapeutic
agent or can be provided in combination with a
nonconjugated chemotherapeutic.
[0029] Included in another aspect of the invention
are methods of inhibiting growth of tumor cells in
vitro in a sample comprising the step of adding to the
sample a composition described above.
[0030] Also included are methods of inhibiting
growth of tumor cells in vivo in a mammal comprising
the step of administering to the mammal an effective
amount of a composition described above. In some
embodiments the mammal is human.
[0031] Another aspect of the invention is a method
of treating a mammal having a tumor that overexpresses
Cripto comprising administering to the mammal a
composition described above in an effective amount. A
composition for administration may include
pharmaceutically acceptable excipients, antibodies
conjugated to chemotherapeutic agents and antibodies
administered in combination with nonconjugated
chemotherapeutic agents
[0032] The methods of the invention are particularly
useful in inhibiting growth of tumor cells and/or
treating a mammal (e.g., a human) having a tumor where

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the tumor cell is se1ect4d from brain, breast,
testicular, colon, lung, ovary, bladder, uterine,
cervical, pancreatic and stomach tumor cells.
(0033] In yet another embodiment, the invention
includes methods of determining whether a tissue
expresses Cripto, comprising the step of analyzing
tissue from the mammal in an immunoassay using any of
the antibodies described above. Also included 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.
[0034] In a further aspect, the invention provides a
method of preserving or maintaining Activin B-induced
inhibition of a tumor cell, comprising exposing the
tumor cell to an antibody of the invention. In certain
embodiments, the tumor cell is a human tumor cell. In
some embodiments the tumor cell is selected from brain,
breast, testicular, colon, lung, ovary, bladder,
uterine, cervical, pancreatic and stomach tumor cells.
00353 In still another aspect, the invention
provides a method for identifying a compound which is
capable of blocking the interaction between Cripto and
Activin B, comprising the steps of contacting Cripto
and Activin B in the presence of a candidate compound
and detecting a change the interaction between Cripto
and Activin B. In some embodiments the compound is an
antibody.
- - -

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Thi:3 invnron also includes the use of an antibody
[hat ,TeciEicany hinds to a Cripto amino acid sequence
shown in SEQ ED NO:1 or SEQ ID NO:2 in the manufacture of a
medicament for the treatment of a brain, head, neck or
prostate tumor.
This invention also includes the use of an antibody
that specifically binds to a Cripto amino acid sequence
shown in SEQ ID NO:1 or SEQ ID NO:2 for the treatment of a
brain, head, neck or prostate tumor.
This invention also includes an antibody that
specifically binds to a Cripto amino acid sequence shown in
SEQ ID NO:1 or SEQ ID NO:2 for use in the treatment of a
brain, head, neck or prostate tumor.
This invention also includes use of an antibody that
specifically binds to an epitope comprised in the
ligand/receptor binding domain of Cripto spanning from
amino acid residue 75 to amino acid residue 150 of SEQ ID
NO: 1 or SEQ ID NO:2 in the manufacture of a medicament for
inhibiting angiogenesis of a tumor cell.
This invention also includes use of an antibody that
specifically binds to an epitope comprised in the cysteine-
rich domain of Cripto spanning from amino acid residue 114
to amino acid residue 150 of SEQ ID NO: 1 or SEQ ID NO:2
for inhibiting angiogenesis of a tumor cell, wherein the
antibody blocks the interaction of Cripto with Activin B
and/or A1k4.
This invention also includes an antibody that
specifically binds to an epitope comprised in the
ligand/receptor binding domain of Cripto spanning from
amino acid residue 75 to amino acid residue 150 of the
Ha

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,
sequence shown in SEQ ID NO: 1 or SEQ ID NO:2 for
inhibiting angiogenesis of a tumor cell.
These and other aspects of the invention are set forth
in greater detail below in the Detailed Description of the
Invention.
lib

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Detailed Description of the Invention
[0037] This invention is based on the discovery that
certain Cripto-specific antibodies can affect Cripto
activity by, e.g., inhibiting the interaction between
Cripto and ALK4 and/or the interaction between Cripto
and Activin B, and can be used to inhibit the growth of
tumor cells. Some of these antibodies specifically
bind to an epitope in the ligand/receptor binding
domain of either a native Cripto protein or a denatured
form of Cripto. For instance, they may bind to an EGF-
like domain, a cys-rich domain, or a peptide (e.g.,
from about 3 to about 20 amino acids) from the region
spanning amino acid residues 46 to 150 of Cripto.
[0038] The antibodies of this invention are useful
in the therapy of malignant or benign tumors of mammals
where the growth of the tumor is at least partially
dependent upon Cripto. This growth usually has an
abnormal growth rate that is in excess of that required
for normal homeostasis and is in excess of that for
normal tissues of the same origin.
I. DEFINITIONS
[0039] 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 invention as a whole and are as typically
understood by those skilled in the art.
[0040] As used herein, "region" means a physically
contiguous portion of the primary structure of a
biomolecule. In the case of proteins, a region is

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defined by a contiguous portion of the amino acid
sequence of that protein.
[0041] As used herein, "domain" means 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 CR-1 (SEQ ID NO:1) and CR-3
(SEQ ID NO:2)) and the transmembrane domain (spans from
about residue 169 to about residue 188 of Cripto,
including CR-1 and CR-3). A ligand/receptor binding
domain of the Cripto protein spans from about residue
75 to about residue 150 of Cripto, including CR-1 and
CR-3; this domain includes the EGF-like domain, which
spans, for example, from about residue 75 to about
residue 112 of Cripto, including CR-1 and CR-3, and the
cys-rich domain, which spans, for example, from about
residue 114 to about residue 150 of Cripto, including
CR-1 and CR-3. Many monoclonal antibodies of the
invention were identified as binding to the EGF-like or
cys-rich domain. 'Additionally, monoclonal antibodies
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 of
Cripto upstream of the EGF-like domain. See Example 3
below.
[0042] An epitope to which an anti-Cripto antibody
of the invention binds may be present in the

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conformationally native Cripto protein or the denatured
Cripto protein. In addition, an epitope can be formed
by noncontiguous sequences in the Cripto polypeptide.
[0043] As used herein, an antibody of this invention
can be, for instance, a murine antibody, a humanized
antibody, a fully human antibody, or a chimeric
antibody. It can be a whole antibody (i.e., with two
full length light chains and two full length heavy
chains) of any isotype and subtypes (e.g., IgM, IgD,
IgGl, IgG2, IgG3, IgG4, IgE, IgAl and IgA2; with either
kappa or lambda light chain). Alternatively, the
antibody of this invention refers to an antigen-binding
fragment (e.g., Fab, F(ab')2, and single chain Fv) of a
whole antibody.
[0044] Any of the antibodies of the invention may
optionally be conjugated to a chemotherapeutic, as
defined below.
[0045] 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, for example, using
fluorescently labeled Cripto monoclonal antibodies.
In order to determine which antibodies can be
internalized into the Cripto positive cells one can
assay for the uptake of the fluorescent signal of the
labeled antibodies into the cells by viewing the cells
under a fluorescent and/or confocal microscope. Those
antibodies that are 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,
B3F6.17 and 1-1A4C.2.

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[0046] As used herein, "compound" means any
identifiable chemical or molecule, including, but not
limited to, ion, atom, small molecule, peptide,
protein, sugar, nucleotide, and nucleic acid. Such a
compound can be natural or synthetic.
[0047] As used herein, "modulate" or "modify" means
an increase or decrease in the amount, quality, or
effect of a particular activity or protein.
[0048] As used herein, "inhibit" means a decrease in
the amount, quality or effect of a particular activity
or protein.
[0049] As used herein, "modulate Cripto activity"
means an increase or decrease in the amount, quality,
or effect of Cripto activity. The increase or decrease
in the amount, quality, or effect of Cripto activity
can be by at least about, for example, 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Increases
greater than about 100% are also envisioned, for
example, at least about 3, 4, 5, 10, 20 or more fold.
Activity may be measured by assays known in the art,
such as the null cell assay shown in Example 3. In
some embodiments, protein interaction between Cripto
and another protein is inhibited via binding of the
antibodies of the invention.
[0050] As used herein, "blocking the interaction
between Cripto and ALK 4" or "modulating the
interaction between Cripto and ALK 4" means an increase
or decrease in the interaction, i.e. binding, between
Cripto and ALK4. The increase or decrease in the
interaction can be by at least about, for example, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
Increases greater than about 100% are also envisioned,
for example, about 3, 4, 5, 10, 20 or more fold.

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Activity may be measured by assays known in the art,
such as the binding assay shown in Example 8.
[0051] As used herein, "blocking the interaction
between Cripto and Activin B" or "modulating the
interaction between Cripto and Activin B" means an
increase or decrease in the interaction, i.e. binding,
between Cripto and Activin B. The increase or decrease
in the interaction can be by at least about, for
example, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, or 100%. Increases greater than about 100% are
also envisioned, for example, about 3, 4, 5, 10, 20 or
more fold. Activity may be measured by assays known in
the art, such as the binding assay shown in Example 11
[0052] As used herein, "modulate growth of tumor
cells in vitro" means an increase or decrease in the
number of tumor cells in vitro. The increase or
decrease in the number of tumor cells can be by at
least about, for example, 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 100%. Increases greater than
about 100% are also envisioned, for example, about 3,
4, 5, 10, 20 or more fold. 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.
[0053] As used herein, "modulate growth of tumor
cells in vivo" means a decrease in the number,
angiogenesis, and/or metastasis of tumor cells in vivo.
The decrease in the number of tumor cells can be by at
least about, for example, 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 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.

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[0054] As used herein, "therapeutic effect" means
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 (i.e., increasing or
starting) 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.
[0055] As used herein, "administering" means 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 (in vivo) or outside
of the organism (ex vivo). 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.,

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oral, intravenous, intraperitoneal, intramuscular, and
the like. When used in in vivo therapy, the antibodies
of the 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 an antibody of the invention is an
amount of the antibody that is sufficient to
ameliorate, stabilize, prevent or delay the development
of the Cripto- or activin B-associated disease state,
particularly Cripto- or activin B-associated tumors.
[0056] An example of a typical treatment regime
includes administering by intravenous infusion to the
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
included.
[0057] 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.
[0058] As used herein, "Cripto overexpression" means
the expression of Cripto by a tissue or cell which
expression is greater (e.g., by at least about 5%, 10%,
20%, 30%, 40%, 50%, or even by at least about 2, 3, 4,

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5, or 10 fold) than the Cripto expression of normal
tissue or cells in a statistically significant amount.
[0059] As used herein, "chemotherapeutic" means any
agent 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 included herewith
include agents that can be conjugated to the antibodies
of the invention or alternatively agents that can be
used in combination with the antibodies of the
invention without being conjugated to the antibody.
Exemplary chemotherapeutics that can be conjugated to
the antibodies of the invention include, but are not
limited to radioconjugates (90y, 131-1, 9 -9mTc, mIn, 186Rh,
etc.), tumor-activated prodrugs (maytansinoids, CC-1065
analogs, clicheamicin derivatives, anthracyclines,
vinca alkaloids, etc.), ricin, diptheria toxin, and
pseudomonas exotoxin.
[0060] 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

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gemcitabine. Such chemotherapeutics may be employed in
the practice of the invention in combination with the
antibodies of the invention by adjunctive
administration of the antibody and the nonconjugated
chemotherapeutic.
[0061] As used
herein, "pharmaceutically acceptable
carrier or excipient" means 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,
oligosaccharides, or polysaccharides, polymers,
viscoelastic compound such as hyaluronic acid,
viscosity-improving agents, preservatives, and the
like.
II. ANTIBODIES OF THE INVENTION
[0062] 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. Examples of
antigens used to raise antibodies specific for Cripto
include, but are not limited to, CR40 (SEQ ID NO: 3),
CR41 (SEQ ID NO: 4), CR43 (SEQ ID NO: 5), CR44 (SEQ ID
NO: 6), CR49 (SEQ ID NO: 7), CR50 (SEQ ID NO: 8) and

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CR5 1 (SEQ ID NO: 9), the amino acid sequences of which
are provided in Example 2.
[0063] The 188 amino acid sequence for CR-1 is as
follows (SEQ ID NO: 1):
MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS
IWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPS
FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD
GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY
[0064] The 188 amino acid sequence for CR-3 is as
follows (SEQ ID NO: 2):
MDCRKMVRFSYSVIWIMAISKAFELGLVAGLGHQEFARPSRGDLAFRDDS
IWPQEEPAIRPRSSQRVLPMGIQHSKELNRTCCLNGGTCMLESFCACPPS
FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD
GLVMDEHLVASRTPELPPSARTTTFMLAGICLSIQSYY
[0065] In some embodiments, 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. Examples of linear epitopes include, but are
not limited to, about residues 75-85, 80-90, 85-95, 90-
100, 95-105, 100-110, or 105-112. In some embodiments,
the epitope in the EGF domain is an epitope formed in
the conformationally native Cripto protein versus a
denatured Cripto protein.
[0066] In other embodiments, 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. Examples of linear epitopes include but are
not limited to about residues 114-125, 120-130, 125-

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135, 130-140, 135-145, or 140-150. In certain
embodiments, the epitope in the cys-rich domain is an
epitope formed in the conformationally native Cripto
protein versus a denatured Cripto protein
[0067] 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.
[0068] This 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. The terms
"specific" and "selective," when used to describe
binding of the antibodies of the invention, indicate
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 regions of the antibodies, and, in
particular, in the constant regions of the molecule.
[0069] Screening assays to determine binding
specificity of an antibody of the invention (e.g.,
antibodies that specifically bind to an epitope the

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ligand/receptor binding domain or 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, NY (1988), Chapter 6. Antibodies that
recognize and bind fragments of Cripto protein are also
included, 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.
[0070] In some embodiments, 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 inhibiting 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.

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[0 07 1] The invention also provides a cell-free
composition comprising polyclonal antibodies, wherein
at least one of the antibodies is an antibody of the
invention. Antiserum isolated from an animal is an
exemplary composition, as is a composition comprising
an antibody fraction of an antiserum that has been
resuspended in water or in another diluent, excipient,
or carrier.
[0072] In other embodiments, 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 can be synthesized by cultured
cells such as hybridomas, uncontaminated by other
immunoglobulins. Recombinant cells and hybridomas that
produce such antibodies are also intended as aspects of
the invention. See also discussions below.
[0073] In still other related embodiments, 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. Patents 6,063,379 and 5,780,029.
[0074] 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,

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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. Patent 5,859,205 and
discussion below.
[0075] In other embodiments, 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 in which the
regions responsible for specific antigen-binding have
been replaced.
[0076] Various forms of antibodies may be produced
using standard recombinant DNA techniques (Winter and
Milstein, 1991, Nature 349:293-99). For example, the
monoclonal antibodies of this invention can be
generated by well known hybridoma technology. For
instance, animals (e.g., mice, rats or rabbits) can be
immunized with purified or crude Cripto preparations,
cells transfected with cDNA constructs encoding Cripto,

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cells that constitutively express Cripto, and the like.
In addition, the antigen can be delivered as purified
protein, protein expressed on cells, protein fragment
or peptide thereof, or as naked DNA or viral vectors
encoding the protein, protein fragment, or peptide.
Sera of the immunized animals are then tested for the
presence of anti-Cripto antibodies. B cells are
isolated from animals that test positive, and
hybridomas are made with these B cells.
[0077] Antibodies secreted by the hybridomas are
screened for their ability to bind specifically to
Cripto (e.g., binding to Cripto-transfected cells and
not to untransfected parent cells) and for any other
desired features, e.g., having the desired CDR
consensus sequences, inhibiting (or not in the case of
nonblockers) the binding between Cripto and ALK4 or
between Cripto and Activin B.
[0078] Hybridoma cells that test positive in the
screening assays are cultured in a nutrient medium
under conditions that allow the cells to secrete the
monoclonal antibodies into the culture medium. The
conditioned hybridoma culture supernatant is then
collected and antibodies contained in the supernatant
are purified. Alternatively, the desired antibody may
be produced by injecting the hybridoma cells into the
peritoneal cavity of an unimmunized animal (e.g., a
mouse). The hybridoma cells proliferate in the
peritoneal cavity, secreting the antibody which
accumulates as ascites fluid. The antibody may then be
harvested by withdrawing the ascites fluid from the
peritoneal cavity with a syringe.
[0079] The monoclonal antibodies can also be
generated by isolating the antibody-coding cDNAs from

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the desired hybridomas, transfecting mammalian host
cells (e.g., CHO or NSO cells) with the cDNAs,
culturing the transfected host cells, and recovering
the antibody from the culture medium.
[0080] The monoclonal antibodies of this invention
can also be generated by engineering a cognate
hybridoma (e.g., murine, rat or rabbit) antibody. For
instance, a cognate antibody can be altered by
recombinant DNA technology such that part or all of the
hinge and/or constant regions of the heavy and/or light
chains are replaced with the corresponding components
of an antibody from another species (e.g., human).
Generally, the variable domains of the engineered
antibody remain identical or substantially so to the
variable domains of the cognate antibody. Such an
engineered antibody is called a chimeric antibody and
is less antigenic than the cognate antibody when
administered to an individual of the species from which
the hinge and/or constant region is derived (e.g., a
human). Methods of making chimeric antibodies are well
known in the art. Human constant regions include those
derived from IgG1 and IgG4.
[0081] The monoclonal antibodies of this invention
also include fully human antibodies. They may be
prepared using in vitro-primed human splenocytes, as
described by Boerner et al., 1991, J. Immunol. 147:86-
95, or using phage-displayed antibody libraries, as
described in, e.g., U.S. Patent 6,300,064.
[0082] Alternatively, fully human antibodies may be
prepared by repertoire cloning as described by Persson
et al., 1991 , Proc. Natl. Acad. Sci. USA 88:2432-36;
and Huang and Stollar, 1991, J. Immunol. Methods
141:227-36. In addition, U.S. Patent 5,798,230

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describes preparation of human monoclonal antibodies
from human B cells, wherein human antibody-producing B
cells are immortalized by infection with an Epstein-
Barr virus, or a derivative thereof, that expresses
Epstein-Barr virus nuclear antigen 2 (EBNA2), a protein
required for immortalization. The EBNA2 function is
subsequently shut off, resulting in an increase in
antibody production.
[0083] Some other methods for producing fully human
antibodies involve the use of non-human animals that
have inactivated endogenous Ig loci and are transgenic
for un-rearranged human antibody heavy chain and light
chain genes. Such transgenic animals can be immunized
with Cripto and hybridomas made from B cells derived
therefrom. These methods are described in, e.g., the
various GenPharm/Medarex (Palo Alto, CA)
publications/patents concerning transgenic mice
containing human Ig miniloci (e.g., U.S. Patent
5,789,650); the various Abgenix (Fremont, CA)
publications/patents with respect to XENOMICE' (e.g.,
U.S. Patents 6,075,181, 6,150,584 and 6,162,963; Green
et al., 1994, Nature Genetics 7:13-21; and Mendez et
al., 1997, Nature Genetics 15:146-56); and the various
Kirin (Japan) publications/patents concerning
"transomic" mice (e.g., EP 843 961, and Tomizuka et
al., 1997, Nature Genetics 16:1433-43). See also,
e.g., United States patent 5,569,825, W000076310,
W000058499 and W000037504.
[0084] The monoclonal antibodies of this invention
also include humanized versions of cognate anti-Cripto
antibodies derived from other species. A humanized
antibody is an antibody produced by recombinant DNA

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technology, in which some or all of the amino acids of
a human immunoglobulin light or heavy chain that are
not required for antigen binding (e.g., the constant
regions and the framework regions of the variable
domains) are used to substitute for the corresponding
amino acids from the light or heavy chain of the
cognate, nonhuman antibody. By way of example, a
humanized version of a murine antibody to a given
antigen has on both of its heavy and light chains (1)
constant regions of a human antibody; (2) framework
regions from the variable domains of a human antibody;
and (3) CDRs from the murine antibody. When necessary,
one or more residues in the human framework regions can
be changed to residues at the corresponding positions
in the murine antibody so as to preserve the binding
affinity of the humanized antibody to the antigen.
This change is sometimes called "back mutation."
Humanized antibodies generally are less likely to
elicit an immune response in humans as compared to
chimeric human antibodies because the former contain
considerably fewer non-human components.
[0085] The methods for making humanized antibodies
are described in, e.g., Winter EP 239 400; Jones et
al., 1986, Nature 321:522-25; Riechmann et al., 1988,
Nature 332:323-27 (1988); Verhoeyen et al., 1988,
Science 239:1534-36; Queen et al., 1989, Proc. Natl.
Acad. Sci. USA 86:10029-33; U.S. Patent 6,180,370; and
Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA
86:3833-37. See also, e.g., PCT patent application No.
94/04679. 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

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affinity or immunogenicity. See, e.g., U.S. Patent
Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370.
[0086]
Generally, the transplantation of murine (or
other non-human) CDRs onto a human antibody is achieved
as follows. The cDNAs encoding heavy and light chain
variable domains are isolated from a hybridoma. The
DNA sequences of the variable domains, including the
CDRs, are determined by sequencing. The DNAs encoding
the CDRs are transferred to the corresponding regions
of a human antibody heavy or light chain variable
domain coding sequence by site directed mutagenesis.
Then human constant region gene segments of a desired
isotype (e.g, yl for CH and K for CL) are added. The
humanized heavy and light chain genes are co-expressed
in mammalian host cells (e.g., CHO or NSO cells) to
produce soluble humanized antibody. To facilitate
large scale production of antibodies, it is often
desirable to produce such humanized antibodies in
bioreactors containing the antibody-expressing cells,
or to produce transgenic mammals (e.g., goats, cows, or
sheep) that express the antibody in milk (see, e.g.,
U.S. Patent 5,827,690).
[0087] At
times, direct transfer of CDRs to a human
framework leads to a loss of antigen-binding affinity
of the resultant antibody. This is because in some
cognate antibodies, certain amino acids within the
framework regions interact with the CDRs and thus
influence the overall antigen binding affinity of the
antibody. In such cases, "back mutations" (supra)
should be introduced into the framework regions of the
acceptor antibody in order to retain the antigen-
binding activity of the cognate antibody.

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[0088] The general approach of making back mutations
is known in the art. For instance Queen, et al., 1989,
Proc. Natl. Acad. Sci. USA 86:10029-33, Co et al.,
1991, Proc. Natl. Acad. Sci. USA 88:2869-73, and WO
90/07861 (Protein Design Labs Inc.) describe an
approach that involves two key steps. First, the human
V framework regions are chosen by computer analysis for
optimal protein sequence homology to the V region
framework of the cognate murine antibody. Then, the
tertiary structure of the murine V region is modeled by
computer in order to visualize framework amino acid
residues that are likely to interact with the murine
CDRs, and these murine amino acid residues are then
superimposed on the homologous human framework.
[0089] Under this two-step approach, there are
several criteria for designing humanized antibodies.
The first criterion is to use as the human acceptor the
framework from a particular human immunoglobulin that
is usually homologous to the non-human donor
immunoglobulin, or to use a consensus framework from
many human antibodies. The second criterion is to use
the donor amino acid rather than the acceptor if the
human acceptor residue is unusual and the donor residue
is typical for human sequences at a specific residue of
the framework. The third criterion is to use the donor
framework amino acid residue rather than the acceptor
at positions immediately adjacent to the CDRs.
[0090] One may also use a different approach as
described in, e.g., Tempest, 1991, Biotechnology 9:
266-71. Under this approach, the V region frameworks
derived from NEWM and REI heavy and light chains,
respectively, are used for CDR-grafting without radical
introduction of mouse residues. An advantage of using

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this approach is that the three-dimensional structures
of NEWM and REI variable regions are known from X-ray
crystallography and thus specific interactions between
CDRs and V region framework residues can be readily
modeled.
[0091] The humanized antibody of this invention may
contain a mutation (e.g., deletion, substitution or
addition) at one or more (e.g., 2, 3, 4, 5, 6, 7 or 8)
of certain positions in the heavy chain such that an
effector function of the antibody (e.g., the ability of
the antibody to bind to a Fc receptor or a complement
factor) is altered without affecting the antibody's
ability to bind to Cripto (U.S. Patent 5,648,260).
These heavy chain positions include, without
limitation, residues 234, 235, 236, 237, 297, 318, 320
and 322 (EU numbering system). The humanized antibody
can, for instance, contain the mutations L234A (i.e.,
replacing leucine at position 234 of an unmodified
antibody with alanine) and L235A (EU numbering system)
in its heavy chain.
[0092] In addition, the humanized antibody of this
invention may contain a mutation (e.g., deletion or
substitution) at an amino acid residue that is a site
for glycosylation, such that the glycosylation site is
eliminated. Such an antibody may be clinically
beneficial for having reduced effector functions or
other undesired functions while retaining its Cripto
binding affinity. Mutations of glycosylation sites can
also be beneficial for process development (e.g.,
protein expression and purification). For instance,
the heavy chain of the antibody may contain the
mutation N297Q (EU numbering system) such that the
heavy chain can no longer be glycosylated at this site.

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[0093] In still other embodiments, the heavy and/or
light chains of the antibody of this invention contain
mutations that increase affinity for binding to Cripto
and thereby increase potency for treating Cripto-
mediated disorders.
[0094] The monoclonal antibodies of this invention
may further include other moieties to effect or enhance
a desired function. For instance, the antibodies may
include a toxin moiety (e.g., tetanus toxoid or ricin)
or a radionuclide (e.g., 111In or "Y) for killing of
cells targeted by the antibodies (see, e.g., U.S.
Patent 6,307,026). The antibodies may include a moiety
(e.g., biotin, fluorescent moieties, radioactive
moieties, histidine tag or other peptide tags) for easy
isolation or detection. The antibodies may also
include a moiety that can prolong their serum half
life, for example, a polyethylene glycol (PEG) moiety,
and a member of the immunoglobulin super family or
fragment thereof (e.g., a portion of human IgG1 heavy
chain constant region such as the hinge, CH2 and CH3
regions).
[0095] 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

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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.
III. SIGNAL MODULATION
[0096] The antibodies of the invention can inhibit
Cripto activity and/or Cripto interactions with its
ligands. Overexpression of Cripto activity can lead to
a de-differentiated state promoting mesenchymal cell
characteristics, increased proliferation, and cell
migration (Salomon et al., 1999, BioEssays 21:61-70;
Ciardiello et al., 1994, Oncogene 9:291-98; and
Baldassarre et al., 1996, Int. J. Cancer 66:538-43),
phenotypes associated with cell transformation seen in
neoplasia.
[0097] One method of testing the activity of anti-
Cripto antibodies and their ability to inhibit Cripto
activity is with an F9-Cripto knock-out (KO) cell line
(Minchiotti at al., 2000, Mech. Dev. 90:133-42).
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., 2000, Mol. Cell 5:35-47). F9-Cripto KO
cells have null mutations in the Cripto gene and cannot
transduce Cripto-dependent signaling (Minchiotti et
al., supra). Cripto activity can be assessed in the F9
Cripto KO cells by transfecting them with Cripto, FAST,
and the FAST regulatory element-luciferase gene

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constructs. No Cripto-dependent FAST luciferase
activity will be seen in these cell lines unless Cripto
cDNA and FAST cDNA are transfected into them.
Antibodies capable of blocking Cripto-dependent Nodal
signaling are antibodies that block Cripto activity.
[0098] 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.
[0099] In certain embodiments, the antibodies of the
invention bind to Cripto and inhibit Cripto-Activin B
interactions. We have discovered that Cripto can bind
to Activin B and inhibit the Activin B signaling
pathway. Activin B can inhibit proliferation of tumor
cells (Risbridger et al., 2001, Endocr. Rev. 22:836-
58). Cripto binding to Activin B also can disrupt
Activin B-induced inhibition of proliferation.
[0100] One method for testing the activity of anti-
Cripto antibodies and their ability to inhibit Cripto-
Activin B interactions is by measuring the ability of
the antibody to prevent Cripto-mediated disruption of
Activin B-induced inhibition of proliferation. One
such method is described in Example 9. A method for
directly testing the ability of anti-Cripto antibodies
to inhibit Cripto-Activin B interactions is described
in Example 11.

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IV. THERAPEUTIC USES
[0101] Antibodies of the invention are also useful
for therapeutic purposes, such as inhibition of tumor
cell growth, diagnostic purposes to detect or
quantitate Cripto, and purification of Cripto.
[0102] In some embodiments of the invention,
antibodies are provided which are capable of binding
specifically to Cripto and which inhibit growth of
tumor cells in a patient, especially where the tumor
growth is mediated by the loss or decrease of Activin B
signaling. In certain embodiments, the tumor cells are
brain, head, neck, prostate, breast, testicular, colon,
lung, ovary, bladder, uterine, cervical, pancreatic and
stomach tumor cells.
[0103] In other embodiments, antibodies are provided
which are capable of binding specifically to Cripto and
which inhibit 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 brain, breast, testicular, colon, lung,
ovary, bladder, uterine, cervical, pancreatic and
stomach cancer.
[0104] 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 (Feb 1984).
[0105] In other embodiments of the invention, the
antibodies of the invention are used to treat a patient
having a cancerous tumor.

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[0 106 ] The antibodies of the invention can be
combined with a pharmaceutically acceptable excipient
and administered in a therapeutically effective dose to
a patient. For a discussion of methods of inhibiting
growth of tumors, see, e.g., U.S. Patent 6,165,464.
[0107] Also included are methods of treating a
mammal suffering from a disorder associated with
elevated levels of Cripto or decreased levels of
Activin B wherein the method comprises administering to
the mammal 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.
[0108] Also included are methods of treating a
mammal suffering from a disorder associated with
elevated levels of Cripto wherein the method comprises
administering to the mammal 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 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, Al0A10.30, Al9A10.30, A10B2.17,
A1032.18 (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, Al5C12.10,
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), 1-1A4C.2,
2-2C9.2, 2-3H9.2, 2-4E5.6, 2-4D1.3, 3-4E8.3, 3-3G1.1,
4-2F6, 4-3A7 and 4-1E2 is directed.

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[0109] 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.
(0110] 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.
V. EXAMPLES
[0111] Additional features of the invention will be
apparent from the following Examples. It should be
understood that the following Examples are for purposes
of illustration only, and are not to be construed as
limiting the scope of the invention in any manner.
Example 1: Expression and Purification of Cripto
[0112] An expression plasmid designated pSGS480 was
constructed by sub-cloning a cDNA encoding amino acid
residues 1 to 169 of a human Cripto protein (amino
acids 1-169 of SEQ ID NO: 1), fused to human IgGi Fc
domain (i.e., "CR(del C)-Fc") into vector pEAG1100.
For a more detailed iescription of this vector, see
Publication No. WO 2002/022808, published March 21, 2002.
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

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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.
co/i DH5a. 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.
[0113] Plasmid pSGS480 was transiently transfected
into CHO cells and the cells were grown at 28 C for 7
days. The presence of CR(delC)-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 (KLH)
conjugate. After centrifugation to remove the cells,
western blot analysis showed that the CR(delC)-Fc
protein was efficiently secreted into the conditioned
media (supernatant). The supernatant was applied to
Protein A-Sepharose (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

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filtered through a 0.2 micron filter, and stored at
-70 C.
Example 2: Generation and Screening of Antibodies
[0114] The eluted CR(delC)-Fc protein, as well as
other Cripto polypeptides, are injected into mice, and
standard techniques known to those of skill in the art
are used to generate hybridomas and monoclonal
antibodies.
A. Generation of Antibodies
[0115] Female Robertsonian mice (Jackson Labs) were
immunized intraperitoneally with 25 pg of purified
human CR(delC)-Fc emulsified with complete Freund's
adjuvant ("FCA"; GibcoBRL #15721-012). They were
boosted two times intraperitoneally (IP) with 25 pg of
CR(delC)-Fc emulsified with incomplete Freund's
adjuvant ("FIA"; 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 pg soluble CR(del C) -
Fc three days before fusion. The mouse was boosted
intravenously (IV) with 50 pg CR(delC)-Fc the day
before fusion.
[0116] 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.
B. Screening of Antibodies
[0117] Supernatants resulting from the first or
second fusion were screened first on ELISA plates for

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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 by FACS for their ability to
recognize cell surface Cripto protein on the testicular
tumor cell line NCCIT. 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, was analyzed by FACS. 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 signalling in a 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.
C. ELISA
[0118] The ELISA assays were performed as follows:
Materials:
Plates: Costar high-binding Easy-wash 96W
plates (07-200-642)
Secondary antibody: Pierce Gt anti- Ms IgG
(H+L)- HRP (P131430)
Substrate: Pierce TME3 Substrate Kit (34021)
Stop solution: 1N H2SO4

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Buffers:
Binding buffer: 0.1 M NaHPO4 pH 9.0
Blocking buffer: PBS + 10% Donor Calf Serum
Wash buffer: PBS + 0.1% Tween 20
[0119] Antigens CR(del C)-Fc and CR-EGF-Fc, control
hu IgG1 fusion protein were diluted in binding buffer
to 500ng/ml. 100 pl were added per well and incubated
for 1 hr at 37 C or overnight at 4 C. The liquid was
decanted and the plate inverted and blotted until dry.
250 p1/well blocking buffer was then added, followed by
incubation for 30 min. at 37 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 p1/well, followed by incubation for 1 hour
at room temperature. Plates were washed 3X vigorously
with 250 p1/well wash buffer. Then 100 p1/well
secondary antibody diluted in wash buffer at 1:10,000
was added, followed by incubation for 30 min. at room
temperature. Plates were then washed 3X vigorously
with 250 p1/well wash buffer, then substrate added at
100 p1/well. Color was permitted to develop until
sufficiently dark, then 100 p1/well stop solution was
added and the plates read for absorbance at 450nm.
[0120] In certain experiments, we coated 96 well
plates with Cripto proteins by adding 100 1 of 0.5
g/ml Cripto proteins in 0.1 M NaHPO4 (pH 9.0) and
incubating at 37 C for 1 hr. We blocked the plates with
PBS/10% DCS. We added antibodies diluted in PBS/0.05%
Tween 20 at 50 l/well and incubated at 37 C for 1 hr.
We washed with PBS/0.05% Tween 20 and probed with anti-
mouse-HRP (Pierce). We detected bound antibodies by

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adding TMB, stopped the reaction with 1N H2SO4 and read
at 450 nm.
[0121] In still other experiments, we coated 96 well
plates by adding to each well 100 1 of 3-5 g/ml
Activin B or another ligand in 50 mM carbonate (pH
9.5). We incubated the plates for 1 hr. at room
temperature and blocked overnight with TBS/1% BSA
overnight at 4 C. We incubated the plate with CR-Fc or
ALK4-Fc in blocking buffer at room temperature, washed
in TBST and probed with anti-human-AP (Jackson). We
washed the plate in TBST, once in 10x substrate buffer
(200 mM Tris-HC1, 10 mM MgCl2, pH 9.8) and added CSPD
and Sapphire (Applied Biosystems).
D. Flow Cytometry
[0122] 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:
[0123] Release cells from T162 flasks with 2 ml PBS
with 5 mM EDTA, 10 min., 37 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 C PBS with 0.1% BSA (wash
buffer). Spin at 1200 rpm for 5 minutes. Resuspend at
4x106-107/m1 in wash buffer. Keep on ice.
[0124] Prepare antibodies for staining. Purified
antibodies are diluted to 1-10 pg/ml in wash buffer.
Add 50 pl 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, secondary antibody only,
hybridoma media, positive control antibody supernatant,

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if available, or purified, and an IgG subclass control
(if using purified antibodies).
[0125] 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 C. Flick out buffer by inverting the plate and
shaking until the liquid is substantially discarded.
Add 40-50 pl of antibodies (or wash buffer for the no-
antibody and secondary antibody-only control wells) to
wells. Incubate at least 30 min.-1 hour at 4 C. Spin
plate, 1200 rpm for 5 minutes. Flick out antibody
solutions. Wash wells twice with 200 pl wash buffer
per well, spinning after each wash. Flick out buffer.
[0126] Resuspend cells in each well in 50 pl 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 C,
in the dark. Add 150 pl wash buffer to cells in each
well. Spin plate at 1200 rpm for 5 minutes. Wash once
with 200 pl wash buffer per well. Resuspend cells in
150 pl 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.
[0127] The contents of the tubes are then read by
flow cytometry.
[0128] The results of two screenings of certain
monoclonal antibodies analyzed 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

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cripto-positive cell lines. The results are given in
units of mean fluorescent index (MFI).
Table 1:
Anti-Cripto Monoclonal Antibody Characterization
Hybridoma ATCC EL1SA ELISA DU4475 NCCIT GEO HT3
Subclone deposit Cripto Cripto MFI MFI MFI MFI
no. delC EGFlike
Sups domain
Sups
Control-
ELISA 0.06 0.07
Control-
MouseIg 14 9 37 18
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

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Table 2:
Anti-Cripto Monoclonal Antibody Characterization
Hybridoma ATCC ELISA ELISA DU4475 NCCIT GEO HT3
Subclone deposit Cripto Cripto MFI MFI MFI MFI
no. delC EGFlike
domain
Control-
ELISA 0.05 0.05
Control-
MouseIg 10 6 4 6
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
[0129] We also
employed numerous other immunization
protocols using the CR(delC)-Fc protein as well as
region-specific Cripto peptides as described below. In
these cases, we also generated hybridomas as described.
The Cripto peptides were used in addition to or in
place of the CR(delC)-Fc protein. These protocols
identified some of the antibodies of the invention. As
will be appreciated by one of skill in the art, these
experiments indicate that multiple, diverse

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immunization protocols may be used to generate the
antibodies of the invention.
[0130] In one exemplary immunization protocol,
eight-week-old female RBF mice (Jackson Labs, Bar
Harbor, ME) were immunized intraperitoneally (IP) with
an emulsion containing either 50 pg soluble CR(delC)-Fc
recombinant protein or a region-specific Cripto peptide
conjugated to keyhole limpet hemocyanin (KLH) and
Freund's complete adjuvant (FCA, Sigma Chemical Co.,
St. Louis, MO). Specifically, CR(delC)-Fc protein or
Cripto peptide CR40 (aa36-42; SEQ ID NO: 3)-KLH was
diluted into phosphate buffered saline, pH 7.2 at an
estimated concentration of 2 mg/ml. To this protein,
an equal volume of FCA was added prior to
emulsification and immunization. =Fifty pl containing
50 pg of emulsified CR(delC)-Fc or Cripto peptide CR40-
KLH was IP administered into each mouse for the primary
immunization. All subsequent immunizations were
similarly dosed using either Freund's Incomplete
Adjuvant (FIA) or RIBI adjuvant1,2 (Sigma Chemical Co.,
St. Louis, MO), as described below. Booster
immunizations were administered every two to three
weeks. Serum samples from immunized mice were
collected before the first immunization, 7 days after
the booster immunizations, and again prior to
lymphocyte cell fusions. Serum titers were measured
using both an ELISA and flow cytometry assay described
below.
[0131] Various exemplary immunization protocols with
different Cripto antigens, as well as antibodies
identified thereby, are as follows:

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ANTIGEN 1 - (CR (del C) -Fe) :
Protocol A:
50 pg CR (del C) -Fc (IP) + FCA
2 50 pg CR (del C) -Fc (IP) + FIA
30 50 pg CR (del C) -Fc (IP) + FIA
4 25 pg CR (del C) -Fc (IV) in PBS
Table 3:
5 Anti-Cripto Monoclonal Antibody Characterization
Purified ELISA ELISA LS174T NCCIT GEO H727 HT3
mAbs Cripto Cripto MFI MFI
MFI MFI MFI
delC CFC
domain
1-1A4C.2 0.197 0.682 141 371 441 503 596
2-2C9.2 0.567 0.796 305 407 309 820 205
2-3H9.2 0.637 0.354 127 84 123 191 59
2-4E5.6 0.626 0.328 90 71 127 183 47
2-4D1.3, 0.866 0.946 31 18 67 197 104
Control 0.05 0.05 33 16 47 74 19
IgG
Protocol B:
1 50 pg CR (del C) -Fc (IP) + FCA
2 50 pg CR (del C) -Fc (IP) + FIA
30 50 pg CR (del C) -Fc (IP) + FIA
40 50 pg CR (del C) -Fc (IP) + FIA
50 25 pg CR (del C) -Fc (IV) in PBS

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ANTIGEN 2 - (CR(delC)-Fc + CR40 peptide) :
Protocol C:
50 lag CR(delC)-Fc (IP) + FCA
2 50 pg CR(delC)-Fc (IP) + FIA
30 50 pg CR(delC)-Fc (IP) + FIA
40 50 pg A10B2-KLH (IP) + FIA
50 50 pg A10B2-KLH (IP) + FIA
Table 4:
5 Anti-Cripto Monoclonal Antibody Characterization
Hybridoina ELISA ELISA LS174T NCCIT GEO H727 HT3
Subc lone Cripto Cripto MFI MFI MFI MFI MFI
delC CFC
domain
3-4E8.3 0.6 0.3 ND ND ND ND ND
3-3G1.1 0.5139 0.9206 298 97 1099 677 1538
Control 0.05 0.05 33 17 88 19 10
IgG
ND = not determined
10 ANTIGEN 3 - (CR(delC)-Fc + LS174 T tumor membrane
preparation) :
Protocol D:
1 50 pg CR(delC)-Fc (IP) + FCA
2 50 pg CR(delC)-Fc (IP) + FIA
30 50 pg CR(delC)-Fc (IP) + FIA
40 -50 pg LS174T Cripto (IP) + RIBI

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Table 5:
Anti-Cripto Monoclonal Antibody Characterization
Hybridoma ELISA ELISA H727 HT3
Subclone Cripto Cripto MFI MFI
delC CFC
domain
4-2F6 1.915 0.1145 19 5
4-3A7 0.1286 0.1186 49 5
4-1E2 0.13 0.3 53 24
Control IgG 0.05 0.05 8 5
ANTIGEN 4 - (x=number of peptide conjugated to KLH; we
also inject peptide conjugated to KLH into mice that
have been pre-immunized with CR(delC)-Fc as described,
e.g., in Protocol C):
Sequences of exemplary CRx peptides that we used and
their positions in the full length Cripto protein are
as follows:
CR40 = FRDDSIWPQEEPAIRPR (aa46 - 42, Al0.B2; SEQ ID NO:
3)
CR41 = CPPSFYGRNCEHDVRKE (aa97-113; SEQ ID NO: 4)
CR43 = GSVPHDTWLPKKC (aa 116-128; SEQ ID NO: 5)
CR44 = SLCKSWHGQLRCFPQ (aa 129-143; SEQ ID NO: 6)
CR49 = acetylated N-SFYGRNCEHDVRRENCGSVPHDTWLPKK-000-
(aa100-aa127; SEQ ID NO: 7)
CR50 = acetylated N-LNEGTCMLGSFCACPPSFYGRNCEHDVRK-000-
(aa84-aa112, includes the fucosylation region; SEQ ID
NO: 8)
CR51 = acetylated N- PHNTWLPKKCSLCKCWHGQLRCFPQAFLPGCD-
000- (aa119-aa150; SEQ ID NO: 9)

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Protocol E:
1 50 pg CRx-KLH (IP) + FCA
2 50 pg CRx-KLH (IP) + FIA
30 50 pg CRx-KLH (IP) + FIA
4 25 pg CR(delC)-Fc (IV) in PBS
Protocol F:
50 pg CR(delC)-Fc (IP) + FCA
2 50 pg CR(delC)-Fc (IP) + FIA
30 50 pg CR(delC)-Fc (IP) + FIA
40 25 pg CRx-KLH (IP) + FIA
Example 3: Null Cell Assay for Inhibition of Cripto
5 Activity
[0132] The following describes an F9 Cripto null
cell signaling assay used to assess inhibition of
Cripto activity.
[0133] Day 0 Coat 6 welled plates with 0.1%
10 gelatin 2m1/well at 37 C for 15 min.
Seed cells at 6x105 F9 CRIPTO NULL cells per well.
[0134] Day / Transfection:
Each of the following samples is added to 300
pl OptiMeml to yield Solution A for each sample:
Sample 1: 0.5pg (N2)7 luciferase FAST reporter cDNA
plus 1.5 pg empty vector cDNA.
Sample 2: 0.5pg (N2)7 luciferase, 0.5pg FAST, and 1 pg
empty vector cDNAs.
Sample 3: 0.5pg (N2)7 luciferase, 0.5pg Cripto ADD 0.5
FAST, and 0.5pg empty vector cDNAs.

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Sample 4: 0.5pg (N2)7 luciferase, 0.5pg Cripto, 0.5
FAST, and 0.5 pg empty vector cDNAs
Sample 5: 0.5pg (N2)7 luciferase, 0.5pg Cripto, 0.5
FAST, and 0.5 pg empty vector cDNAs.
Sample 6: 0.5pg (N2)7 luciferase, 0.5pg Cripto, 0.5
FAST, and 0.5 pg empty vector cDNAs.
Sample 7: 0.5pg (N2)7 luciferase, 0.5pg Cripto, 0.5
FAST, and 0.5 pg empty vector cDNAs.
Sample 8: 0.5pg (N2)7 luciferase, 0.5pg Cripto, 0.5
FAST, and 0.5 pg empty vector cDNAs.
Sample 9: 0.5pg (N2)7 luciferase, 0.5pg Cripto, 0.5
FAST, and 0.5 pg empty vector cDNAs.
Solution B comprises 30 pl of Lipofectamine plus 270 pl
of OptiMem1.
[0135] For each sample, mix solution A and solution
B together. Incubate 45 minutes at room temperature.
Rinse wells with 2 ml/well of OptiMeml. Aspirate just
before next step.
[0136] Add 2.4 ml of OptiMeml to each mixture of
solutions A+B, mix, add 1.5 ml/well to duplicate wells.
Incubate 5 hours at 37 C. Add 1.5 ml/well of DMEM+20%
FCS, 2mM Gin, P/S to wells which received samples 1-3.
Add anti-Cripto antibodies as follows: Sample 4 wells:
A27F6.1, 10 pg/ml; Sample 5 wells: A27F6.1, 2pg/m1;
Sample 6 wells: A40G12.8; 10pg/ml, Sample 7 wells:
A40G12.8 2pg/m1; Sample 8 wells: A10B2.18, 10pg/m1;
Sample 9 wells: A10B2.18, 2pg/ml.
[0137] Day 2 Remove media, wash cells with PBS,
2ml/well. Add DMEM+0.5% FCS, 2mM Gin, P/S with the
same amounts of Cripto antibodies as the previous day,
to the same wells.
[0138] Day 3 Develop luciferase signal. Wash
wells with PBS+Ca2+ and Mg2+, 2 ml/well. Use LucLite

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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 +
Ca2+ and Mg2+. Aspirate wells. Quickly add 250 pl of
diluted substrate per well using a repeat pipettor.
Swirl solution and transfer 200 pl to wells of a 96
welled white opaque bottom plate, Falcon 35-3296. Read
plate on luminometer using Winglow, exporting data to
Excel.
[0139] The results of this assay with certain of the
antibodies of the invention are summarized below in
Table 3.
Table 6:
Cripto Activity Assay: Inhibition
with Anti-Cripto Monoclonal Antibodies
cDNAs transfected Anti-Cripto Relative
Antibody Luminescent
Units
(N2) 7 luc None 123
(N2)7 luc, FAST None 259
(N2)7 luc, FAST, Cripto None 3091
(N2)7 luc, FAST, Cripto A27F6.1 10pg/m1 1507
(N2)7 luc, FAST, Cripto A27F6.1 2pg/m1 2297
(N2)7 luc, FAST, Cripto A40G12.8 10pg/m1 1213
(N2)7 luc, FAST, Cripto A40G12.8 2pg/m1 2626
(N2)7 luc, FAST, Cripto A10132.18 10pg/m1 3466
(N2)7 luc, FAST, Cripto A10132.18 2pg/m1 3103
[0140] We also tested other concentrations of
A27F6.1 antibody. We observed that addition of 0.5-20
mg/ml mab A27F6.1 to the media of these cells inhibited
the Cripto-induced luciferase signal by 34-95% (Figure
1). We also tested other antibodies of the invention
and observed that mabs A6C12.11 and A8G3.5 also
inhibited signal production.

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[0 14 1] As as alternative assay system, we tested the
Cripto specific mabs of the invention for inhibition of
FAST/(N2)7-luc activity in NCCIT cells. These assays
were similar to those for the F9 cells, except ectopic
expression of Nodal and ALK4 is required in these cells
for reporter activation. We observed that mab A27F6.1
decreased luciferase activity in these cells by 90% at
both 2 and 20 g/ml (Figure 2).
Example 4: Assay for In Vitro Inhibition of Tumor Cell
Growth
[0142] Inhibition of Cripto activity may also be
assayed by measuring the growth of GEO cells in soft
agar. See, e.g., Ciardiello et al., 1994, Oncogene
9:291-98; Ciardiello et al., 1991, Cancer Res. 51:1051-
54.
[0143] We melted 3% bactoagar and kept it at 42 C in
a water bath. We mixed the 3% bactoagar solution with
prewarmed complete media to make a solution of 0.6%
bactoagar and kept it at 42 C. We plated 4 ml of the
solution in a 6 cm dish and let it cool for at least 30
minutes to allow the bottom agar layer to form. We
trypsinized GEO cells and resuspend to 105 cells/ml in
complete media. We added antibodies to be assayed, or
controls, to the cell suspensions, titrating antibodies
from 20 pg to 1 pg. We mixed equal volumes of the GEO
cell suspensions and 0.6% bactoagar and overlaid 2 ml
on top of the bottom agar layer. We allowed the plates
to cool for at least 1 hour. We incubated for 14 days
at 37 C in CO2 incubator. We counted colonies visible
without the use of a microscope. The absence of
colonies, as compared to negative controls, indicated

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that the antibody tested inhibited in vitro tumor cell
growth.
[0144] 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 7:
Results of growth in soft agar assay
Antibody Average number
of colonies
none 109.0
none 104.3
A27.F6 20pg/m1 82.0
A27F6.1 10pg/m1 78.3
A27F6.1 5pg/m1 79.0
A27F6.1 lpg/ml 108.7
B6G7.10 20pg ml 102.3
B6G7.10 10pg/m1 71.7
[0145] We also performed growth inhibition assays
with T-47D cells (ATCC) which is a non-tumorigenic
human breast carcinoma as described in Example 9.
Example 5: Assay for In Vivo Inhibition of Tumor Cell
Growth
[0146] 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.

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[0147] 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 described
below.
[0148] 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:
Group # Treatment # of
Mice
1. Saline Control, 0.2 ml/mouse, i.p. 20
three times weekly (M,W,F)
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 10
(M,W,F)
6. Cisplatin, 2 mg/kg/inj, s.c., 10
3 Rx/wk (M,W,F)
7. Adriamycin, 1.6 mg/kg/inj, i.p., 10
3 Rx/wk (M,W,F)
8. Irinotecan, 10 mg/kg/inj., i.p., 10
5 Rx/wk ( M-F)
9. MAID, low dose, i.p. + 5-FU 10
(intermediate dose)
10. MAID, middle dose, i.p. + 5-FU 10
(intermediate dose)
11. MAID, high dose, i.p. + 5-FU 10
(intermediate dose)
12. MAID, low dose, i.p. + Cisplatin 10

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Group # Treatment # of
Mice
(intermediate dose)
13. MAb, middle dose, i.p. + Cisplatin 10
(intermediate dose)
14. MAID, high dose, i.p. + Cisplatin 10
(intermediate dose)
15. MAID, low dose, i.p. + Adriamycin 10
(intermediate dose)
16. MAb, middle dose, i.p. + 10
Adriamycin (intermediate dose)
17. MAID, high dose, i.p. + Adriamycin 10
(intermediate dose)
18.
MAID, low dose, i.p. + Irinotecan 10
(intermediate dose)
19. MAb, middle dose, i.p. + 10
Irinotecan ( intermediate dose)
20. MAID, high dose, i.p. + Irinotecan 10
(intermediate dose)
Day 0: Implant tumor, record initial body weight
of animals.
Day 1: Initiate treatments as indicated above.
Day 5: Begin tumor size and body weight
measurements and continue two times weekly until
termination of experiment.
[0149] Initial body weight, tumor size and body
weight measurements, histology at sacrifice, and
immunohistochemistry analysis on tumors are examined
for Cripto expression, tumor growth, and inhibition
thereof.
Example 6: In Vivo Xenograft Tumor Model - Cys-rich
blocking anti-Cripto antibody
[0150] To assess the response of an NCCIT, a human
testicular carcinoma cell line was implanted

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subcutaneously with an antibody which binds to a cys-
rich domain of Cripto. The experimental methods are
listed below. The results are shown in Figure 3.
Methods and Materials
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 x 106 cells
/ 0.2 ml matrigel on the animals right
flank.
Group # Treatment # of
Mice
1. Vehicle Control (25 mM sodium 20
phosphate, 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., 10
3x / wk (M, W, F) for 6 treatments
Treatments began on day 1.
Testing schedule
Day -1: Randomized mice into control and treatments
groups. Recorded initial body weight of

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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 2x / week. Monitored the study daily
and made notations of any unusual
observation on animals.
Endpoints: Initial body weight
Tumor size and body weight measurements
Example 7: In Vivo Xenograft Tumor Model - EGF-like
domain blocking anti-Cripto antibody
[0151] 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 Figure 4.
Methods and Materials
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

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originally obtained Trom 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 x 106 cells
/ 0.2 ml matrigel on the animals right
flank.
Group # Treatment # of
Mice
1. Vehicle Control, (25 mM sodium 18
phosphate, 100 mM sodium chloride,
pH 7.2), 0.2 ml/mouse, i.p., Q14D
Treatments begin on day -1
2. A27F6.1, 1 mg/kg/ii, 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/ii, 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., 10
3x / wk (M, W, F) for 6 treatments
Treatments began on day 1.
Testing schedule
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.

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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 2x / week. Monitored the study daily
and made notations of any unusual
observation on animals.
Endpoints: Initial body weight
Tumor size and body weight measurements
Example 8: Cripto mabs Modulate ALK4 Binding
[0152] 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 puromycin at a
10:1 ratio. The transfected cells were then selected
in puromycin until colonies formed. Colonies were then
picked, expanded and 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.
[0153] 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 (CR(delC)-
Fc) was employed. Approximately 5 pg/ml of CR(delC)-Fc
or control Fc protein was incubated with 3x105 293-A1k4-
21 cells on ice for 30 minutes in 50 pl total volume of

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FACS buffer ( PBS with 0 . 1% BSA) . For samples
containing anti-Cripto antibodies, 5pg/m1 Crde1C-Fc was
preincubated with 50pg/m1 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 Figure 5.
Example 9: Cripto Disrupts Activin B-Induced Growth
Suppression of Breast Cancer Cells
[0154] T47D cells, maintained in RPMI/10%FCS/10pg/m1
insulin, at passage #2 from the ATCC were transfected
with an expression plasmid for the Ecotropic Receptor
(EcoR; B. Elenbaas) and selected in media containing
100 pg/ml hygromycin. Colonies of T-47D-EcoR that
permitted infection of pBABE-GFP murine leukemia virus
(MLV) were grown out, infected with pBABE-hCr-PURO-MLV
and selected in puromycin media. This oligoclonal line
(T-47D-hCr) was analyzed by FACS for hCr (human Cripto)
expression with specific anti-Cripto antibodies.
Approximately 4000 cells/well of T-47D-EcoR or T-47D-
hCr were plated in a 96 well plate in media containing
2% serum with/without 25 ng/ml Activin B (R&D) or
25ng/m1 Activin B plus 0.1-50 pg/ml A8G3.5. Media with
factors was replaced daily for 7-8 days. The plate was
harvested by adding 20 p1/well CellTiter AQueous One

CA 02480119 2004-09-22
WO 03/083041 PCT/US02/31462
- 63 -
solution (Promega), incubating 2 hours 37 C, and reading
at 490 nm.
[0155] We grew T-47D and T-47D-EcoR cells in low
serum conditions, with or without Activin A or B and
assayed for proliferating cells using an MTT
calorimetric assay. We observed that proliferation of
T-47D cells was inhibited by Activin A or B by
approximately 40% compared to untreated cells (Figure
6). We also observed that T-47D-CR cells were
inhibited by Activin A, but discovered that the T-47D-
CR cells were not responsive to Activin B (Figure 6).
This result indicates that Cripto's effect on
proliferation of these cells is specific to Activin B.
We observed in control experiments that untreated T-47D
and T-47D-CR cells did not differ in proliferation
rates in either normal media or in low serum
conditions.
[0156] We then tested whether antibodies of the
invention can inhibit the Cripto-Activin B interaction.
We treated T-47D-CR cells with Activin B in the
presence of various antibodies of the invention. We
observed that the growth suppressive activity of
Activin B was restored in the presence of 10 or 20
g/ml mab A8G3.5 (Figure 7). We observed that the
A27F6.1 mab was unable to restore Activin B growth
suppression of these cells.
Example 10: Cripto Binds Directly to Activin B
[0157] We discovered that Cripto binds directly to
Activin B. We coated ELISA plates with purified
Activin B, Activin A, TGFP1, GDNF or BMP2 and incubated
with CR-Fc. We then added anti-Fc antibody conjugated
to alkaline phosphatase and monitored binding by adding

CA 02480119 2013-05-07
- 64 -
an alkaline phosphatase substrate and developing. We
observed that CR-Fc bound to wells containing Activin
B, but not to Activin A or the other ligands (Figure
8). In addition, we pre-incubated CR-Fc with Activin A
or B in solution before adding to an Activin B coated
plate and observed that only Activin B inhibited
binding (Figure 9). These results confirmed that
Cripto specifically binds Activin B.
[0158] We also analyzed the Cripto-Activin B
interaction using Biacore. We discovered that Activin
B directly binds CR-Fc immobilized on a Biacore chip
with a high intensity, but not to a control LTPR-Fc
protein (Figure 10). We also observed that Activin A
binding to CR-Fc was negligible. These data indicate
that Activin B binds Cripto with a fast on rate and an
apparent affinity in solution measured a competition
format assay of about 1 nM.
[0159] Cripto anti-CFC monoclonal antibodies 2-2C9.2
and A8G3 modulated the interaction of Cripto with
Activin B. This was shown by coimmunoprecipitation
experiments in which Cripto binding to Activin B was
inhibited by addition of A8G3 or 2-2C9.2. The
inhibition was measured using conventional western
blotting techniques with anti-Activin B antibodies.
[0160] Some of the embodiments of the invention
described above are outlined below and include, but are
not limited to, the following embodiments.
The scope of the claims should not be limited by the
preferred embodiments set forth in the examples, but
should be given the broadest interpretation consistent
with the description as a whole.

A CA 02480119 2005-11-07 ,
,
,
,
SEQUENCE LISTING
<110> Biogen Idec MA Inc.
<120> Cripto-Specific Antibodies
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<150> PCT/0S02/11950
<151> 2002-04-17
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CA 02480119 2005-11-07 .
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3
,

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date 2014-04-08
(86) PCT Filing Date 2002-10-01
(87) PCT Publication Date 2003-10-09
(85) National Entry 2004-09-22
Examination Requested 2007-10-01
(45) Issued 2014-04-08
Deemed Expired 2017-10-02

Abandonment History

Abandonment Date Reason Reinstatement Date
2012-05-07 R30(2) - Failure to Respond 2013-05-07
2012-10-01 FAILURE TO PAY APPLICATION MAINTENANCE FEE 2013-06-05

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $100.00 2004-09-22
Application Fee $400.00 2004-09-22
Maintenance Fee - Application - New Act 2 2004-10-01 $100.00 2004-09-22
Registration of a document - section 124 $100.00 2005-07-07
Registration of a document - section 124 $100.00 2005-07-07
Registration of a document - section 124 $100.00 2005-07-07
Maintenance Fee - Application - New Act 3 2005-10-03 $100.00 2005-09-26
Maintenance Fee - Application - New Act 4 2006-10-02 $100.00 2006-09-25
Maintenance Fee - Application - New Act 5 2007-10-01 $200.00 2007-09-18
Request for Examination $800.00 2007-10-01
Maintenance Fee - Application - New Act 6 2008-10-01 $200.00 2008-09-18
Maintenance Fee - Application - New Act 7 2009-10-01 $200.00 2009-09-17
Maintenance Fee - Application - New Act 8 2010-10-01 $200.00 2010-09-20
Maintenance Fee - Application - New Act 9 2011-10-03 $200.00 2011-09-22
Reinstatement - failure to respond to examiners report $200.00 2013-05-07
Reinstatement: Failure to Pay Application Maintenance Fees $200.00 2013-06-05
Maintenance Fee - Application - New Act 10 2012-10-01 $250.00 2013-06-05
Maintenance Fee - Application - New Act 11 2013-10-01 $250.00 2013-09-24
Final Fee $300.00 2014-01-23
Maintenance Fee - Patent - New Act 12 2014-10-01 $250.00 2014-09-29
Maintenance Fee - Patent - New Act 13 2015-10-01 $250.00 2015-09-28
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
BIOGEN IDEC MA INC.
Past Owners on Record
ADKINS, HEATHER
BIOGEN IDEC MA, INC.
BIOGEN, INC.
MIKLASZ, STEVEN DONALD
RAYHORN, PAUL
SANICOLA-NADEL, MICHELE
SCHIFFER, SUSAN GAIL
WILLIAMS, KEVIN P.
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Abstract 2004-09-22 1 64
Claims 2004-09-22 12 256
Drawings 2004-09-22 10 121
Description 2004-09-22 65 2,415
Cover Page 2005-01-05 1 33
Description 2005-11-07 68 2,536
Claims 2007-06-14 3 92
Claims 2009-12-14 2 63
Description 2009-12-14 69 2,553
Claims 2010-09-10 2 62
Description 2010-09-10 70 2,564
Claims 2013-05-07 2 57
Description 2013-05-07 69 2,554
Cover Page 2014-03-06 1 34
Correspondence 2005-06-21 2 31
Assignment 2005-10-07 1 27
Correspondence 2005-06-17 1 57
Assignment 2004-09-22 3 89
PCT 2004-09-22 4 103
Prosecution-Amendment 2010-03-10 4 189
Prosecution-Amendment 2010-01-11 5 227
PCT 2004-09-22 1 71
Correspondence 2004-12-24 1 25
PCT 2004-09-23 5 230
Assignment 2005-07-07 25 830
Assignment 2005-10-25 2 59
Prosecution-Amendment 2005-11-07 5 115
PCT 2006-06-07 1 67
Prosecution-Amendment 2007-06-14 5 130
Prosecution-Amendment 2007-10-01 1 32
Prosecution-Amendment 2007-10-01 2 51
Correspondence 2008-03-13 1 34
Correspondence 2008-04-24 1 15
Correspondence 2008-04-24 1 15
Prosecution-Amendment 2009-12-14 6 176
Correspondence 2010-01-15 1 13
Prosecution-Amendment 2010-09-10 15 568
Prosecution-Amendment 2011-11-07 5 249
Prosecution-Amendment 2013-05-07 15 604
Fees 2013-06-05 1 163
Correspondence 2014-01-23 2 53

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