JP2009161539A - Cripto-specific antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a Cripto-specific antibody or biologically functional fragments thereof, and uses thereof. <P>SOLUTION: An antibody that combines with Cripto and inhibits Cripto activity is provided. An antibody that combines with Cripto, inhibits interaction between Cripto and ALK4 and/or inhibits interaction between Cripto and activin B is provided. An antibody that combines with Cripto and inhibits tumor proliferation is also provided. An antibody that combines with Cripto, inhibits Cripto activity and inhibits tumor proliferation is also provided. Additionally, a method for using these antibodies in cure application, diagnosis application and research application is provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  No. 60 / 367,002 filed on Mar. 22, 2002; U.S. Application No. 60 / 301,091 filed on Jun. 26, 2001; U.S. Application Number filed on May 17, 2001. No. 60 / 293,020; priority to International Patent Application No. PCT / US02 / 11950, filed Apr. 24, 2002, claiming priority to U.S. Application No. 60 / 286,782, filed Apr. 26, 2001. Insist on the right. The entire contents of which are incorporated herein by reference.

  In general, the present invention relates to genetics and cell biology as well as molecular biology. More particularly, the present invention relates to anti-Cripto antibodies.

  Cripto is a 188 amino acid cell surface protein. Cripto was unexpectedly isolated on the cDNA screen of a human embryonic cancer library (Ciccocola et al., 1989, EMBO J. 8: 1987-91). The Cripto protein has at least two notable domains: the first characterized domain as being similar to the cysteine-rich (cys-rich) domain and the domain found in the epidermal growth factor (EGF) family . Cripto was originally classified as a member of the EGF family (Ciccocola et al., Supra); however, subsequent analysis showed that Cripto does not bind to any known EGF receptor and that It has been shown that the domain actually differs from the EGF family (Bianco et al., 1999, J. Biol. Chem. 274: 8624-29).

  The Cripto signaling pathway remains ununderstood despite continued work. The literature supports the activation of a variety of different pathways. Various pathways include the MAP kinase pathway (DeSantis et al., 1997, Cell Growth Differ. 8: 1257-66; Kannan et al., 1997, J. Biol. Chem. 272: 3330-35); TGF-β 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 crosstalk with the EGF pathway (Bianco et al., 1999, J. Biol. Chem. 274: 8624-29).

    US Pat. No. 5,256,643 and two related patents (US Pat. No. 5,654,140, US Pat. No. 5,792,616) disclose antibodies to human Cripto gene, Cripto protein and Cripto is doing.

  US Pat. No. 5,264,557 and three related patents (US Pat. No. 5,620,866, US Pat. No. 5,650,285 and US Pat. No. 5,854,399) are listed in Human Cripto. And genes related to human Cripto. Also disclosed are antibodies that bind to the Cripto-related protein but do not cross-react by binding of the Cripto protein itself.

  Overexpression of Cripto protein is associated with cancer in many tissues (brain, breast, testis, colon, as shown by immunostaining of human tissues with rabbit polyclonal antibodies raised against small Cripto peptides. , Lung, ovary, bladder, uterus, cervix, pancreas, and stomach). Panico et al., 1996, Int. J. et al. Cancer 65: 51-56; Byrne et al., 1998, J. MoI. Pathology. 185: 108-11; De Angelis et al., 1999, Int. J. et al. Oncology 14: 437-40. Accordingly, the art needs methods for regulating, limiting, and / or preventing such overexpression, inhibiting Cripto activity, and inhibiting Cripto expression results (ie, promoting cell transformation and / or Maintenance).

(Summary of the Invention)
The present invention provides novel antibodies that specifically bind to Cripto, as well as methods for making and using such antibodies. The invention also includes antibodies that bind to Cripto as well as antibodies that inhibit Cripto activity or protein interaction (eg, the signal produced by Cripto-protein interaction is downregulated to bind to Cripto). provide. The present invention also provides antibodies that bind to Cripto and block the interaction between Cripto and ALK4. The invention also provides antibodies that bind to Cripto and block the interaction between Cripto and activin B. The invention also provides antibodies that bind to Cripto and inhibit tumor growth. The present invention also provides antibodies that bind to Cripto, inhibit Cripto activity, and inhibit tumor growth. The invention also provides antibodies that bind to Cripto, block the interaction between Cripto and ALK4 and / or the interaction between Cripto and activin B, and inhibit tumor growth. Accordingly, the present invention also provides the following.
(1)
An antibody that specifically binds to an epitope in a Cripto ligand / receptor binding domain.
(2)
The antibody according to item 1, wherein Cripto is selected from the group consisting of SEQ ID NO: 1 or 2.
(3)
The antibody according to Item 2, wherein the epitope is present in an EGF-like domain.
(4)
Item 3. The antibody according to Item 2, wherein the epitope is present in a cys-rich domain.
(5)
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-lA4C. 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 The antibody according to item 2, which is selected from the group consisting of:
(6)
4. The antibody according to item 3, selected from the group consisting of A40G12.8, A8H3.1, A27F6.1, B6G7.10, A17G12.1 and A18B3.11.
(7)
A10A10.30, A19A10.30, A8G3.5, A6F8.6, A6C12.11, 1-lA4C. Item 5. The antibody according to item 4, which is selected from the group consisting of 2 and 2-2C9.2.
(8)
An antibody that specifically binds to an epitope comprising a domain spanning amino acid residues 46-62 of Cripto.
(9)
Item 9. The antibody according to item 8, selected from the group consisting of A10B2.18, B3F6.17, 2-3H9.2, 2-4E5.6, 2-4D1.2 and 3-4E8.3.
(10)
Antibodies 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-lA4C. 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 An antibody that specifically binds to an epitope selected from the group consisting of binding epitopes.
(11)
An antibody that specifically binds to Cripto and inhibits Cripto activity.
(12)
Item 12. The antibody according to Item 11, which specifically binds to an epitope present in Cripto's EGF-like domain.
(13)
A40G12.8, A8H3.1, A27F6.1, 1-lA4C. Item 13. The antibody according to item 12, selected from the group consisting of 2, 2-2C9.2 and 2-4D1.3.
(14)
Item 12. The antibody according to Item 11, which specifically binds to an epitope present in a Cypto cys-rich domain.
(15)
A6C12.11, 1-1A4C. Item 15. The antibody according to item 14, selected from the group consisting of 2 and 2-2C9.2.
(16)
Item 12. The antibody according to Item 11, selected from the group consisting of A40G12.8, A8H3.1, A27F6.1, and A6C12.11.
(17)
An antibody that specifically binds to Cripto and inhibits tumor growth.
(18)
Item 18. The antibody according to item 17, which specifically binds to an epitope present in Cripto's EGF-like domain.
(19)
Item 18. The antibody according to Item 17, which specifically binds to an epitope present in a Cypto cys-rich domain.
(20)
Item 18. The antibody according to Item 17, selected from the group consisting of A27F6.1, A8G3.5 and B6G7.10.
(21)
An antibody that specifically binds to Cripto, inhibits Cripto activity, and inhibits tumor growth
(22)
Item 22. The antibody according to Item 21, which specifically binds to an epitope present in Cripto's EGF-like domain.
(23)
Item 22. The antibody according to Item 21, which specifically binds to an epitope present in a Cypto cys-rich domain.
(24)
A27F6.1, A8G3 and 1-1A4C. Item 22. The antibody according to Item 21, selected from the group consisting of two.
(25)
A6F8.6 (ATCC accession number PTA-3318), A8G3.5 (ATCC accession number PTA-3317), A8H3.1 (ATCC accession number PTA-3315), A10B2.18 (ATCC accession number PTA-3331), A27F6. 1 (ATCC Deposit Number PTA-3310), A40G12.8 (ATCC Deposit Number PTA-3316), A17G12.1 (ATCC Deposit Number PTA-3314), A18B3.11 (ATCC Deposit Number PTA-3312), B3F6.17 ( ATCC deposit number PTA-3319), B6G7.10 (ATCC deposit number PTA-3313), 1-lA4C. 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 An antibody produced by a hybridoma selected from the group consisting of:
(26)
An antibody that specifically binds to Cripto and inhibits the interaction between Cripto and ALK4.
(27)
27. The antibody according to item 26, which specifically binds to an epitope present in a Cypto cys-rich domain.
(28)
27. The antibody according to item 26, which is selected from the group consisting of A8G3.5, A6F8.6 and A6C12.11.
(29)
An antibody that specifically binds to Cripto, inhibits the interaction between Cripto and ALK4, and inhibits tumor growth.
(30)
30. The antibody of item 29, which specifically binds to an epitope present in a Cypto cys-rich domain.
(31)
A8G3.5 or 1-lA4C. 30. The antibody according to item 29, which is 2.
(32)
A Cripto antibody capable of internalizing Cripto.
(33)
The antibody of item 32, wherein the antibody is conjugated to a chemotherapeutic agent.
(34)
A27F6.1, B3F6.17, A8G3, 1-lA4C. The antibody of item 32, selected from the group consisting of 2 and 3-4E8.3.
(35)
35. A composition comprising at least one of the antibodies of any of items 1-34 for administration to a mammal having a tumor that expresses Cripto.
(36)
36. The composition of item 35, wherein the mammal is a human.
(37)
36. The composition of item 35, further comprising a pharmaceutically acceptable excipient.
(38)
36. The composition of item 35, wherein the antibody is conjugated to a chemotherapeutic agent.
(39)
36. The composition of item 35, further comprising an unconjugated chemotherapeutic agent.
(40)
36. A method of inhibiting tumor cell growth in vitro in a sample, the method comprising adding to the sample a composition according to item 35.
(41)
36. A method of inhibiting tumor cell growth in vitro in a mammal, the method comprising administering to the mammal the composition of item 35.
(42)
42. The method of item 41, wherein the mammal is a human.
(43)
36. A method of treating a mammal having a tumor that overexpresses Cripto, said method comprising administering to said mammal a composition according to item 35.
(44)
38. A method of treating a patient having a tumor that overexpresses Cripto, said method comprising administering to said patient the composition of item 37.
(45)
40. A method of treating a patient having a tumor that overexpresses Cripto, said method comprising administering to said patient the composition of item 38.
(46)
40. A method of treating a patient having a tumor that overexpresses Cripto, said method comprising administering to said patient the composition of item 39.
(47)
41. The method of item 40, wherein the tumor cells are selected from the group consisting of brain, breast, testis, large intestine, lung, ovary, bladder, uterus, cervix, pancreas and stomach tumor cells.
(48)
42. The method of item 41, wherein the tumor is selected from the group consisting of brain, breast, testis, large intestine, lung, ovary, bladder, uterus, cervix, pancreas and stomach tumor.
(49)
43. The method of item 42, wherein the tumor is selected from the group consisting of brain, breast, testis, large intestine, lung, ovary, bladder, uterus, cervix, pancreas and stomach tumor.
(50)
44. The method of item 43, wherein the tumor is selected from the group consisting of brain, breast, testis, large intestine, lung, ovary, bladder, uterus, cervix, pancreas and stomach tumor.
(51)
45. The method of item 44, wherein the tumor is selected from the group consisting of brain, breast, testis, large intestine, lung, ovary, bladder, uterus, cervix, pancreas and stomach tumor.
(52)
46. The method of item 45, wherein the tumor is selected from the group consisting of brain, breast, testis, large intestine, lung, ovary, bladder, uterus, cervix, pancreas and stomach tumor.
(53)
49. The method of item 46, wherein the tumor is selected from the group consisting of brain, breast, testis, large intestine, lung, ovary, bladder, uterus, cervix, pancreas and stomach tumor.
(54)
A method for determining whether a tissue expresses Cripto, comprising the step of analyzing a tissue derived from a mammal in an immunoassay using the antibody according to any of items 1 to 34. how to.
(55)
A method for determining whether a cell line overexpresses Cripto, comprising the step of analyzing the cell line in an immunoassay using the antibody according to any of items 1-34. how to.
(56)
2. The antibody according to item 1, wherein the antibody is a monoclonal antibody.
(57)
2. The antibody according to item 1, wherein the antibody is a humanized antibody.
(58)
2. The antibody according to item 1, wherein the antibody is a human antibody.
(59)
36. A method of treating a mammal for a condition associated with unwanted cell proliferation, the method comprising administering to the mammal the composition of item 35.
(60)
Item 5. The antibody according to Item 4, wherein the antibody inhibits a binding interaction between Cripto and activin B.
(61)
The antibody may be A8G3.5 or 1-lA4C. 62. The antibody according to item 60, which is 2.
(62)
61. A composition comprising the antibody of item 60 and a pharmaceutically acceptable excipient.
(63)
61. A method of inhibiting the binding interaction between Cripto and activin B, comprising contacting Cripto with the antibody of item 60.
(64)
64. The method of item 63, wherein the contacting is performed in a mammal.
(65) A method according to item 64, wherein the mammal is a human.
(66)
68. The method of item 65, wherein the Cripto is present on the surface of a cell.
(67)
70. The method of item 66, wherein the cell is a tumor cell overexpressing Cripto.
(68)
64. A method of inhibiting the growth of tumor cells in a mammal, the method comprising administering to the mammal the composition of item 62.
(69)
70. The method of item 68, wherein the tumor cell overexpresses Cripto.
(70)
70. The method of item 68, wherein the mammal is a human.
(71)
71. The method of item 70, wherein the tumor cell is a human breast cancer cell.
(72)
64. A method of treating a mammal having a tumor, the method comprising administering to the mammal the composition of item 62.
(73)
73. The method of item 72, wherein the mammal is a human.
(74)
73. The method of item 72, wherein the tumor overexpresses Cripto.
(75)
A method of identifying a compound that inhibits the binding interaction between Cripto and activin B, the method comprising:
Obtaining a candidate compound;
Contacting Cripto with activin B in the presence of the candidate compound; and
Detecting a change in binding interaction between Cripto and Activin B;
Wherein the detection of the change indicates that the candidate compound inhibits the binding interaction between Cripto and activin B.
(76)
76. The method of item 75, wherein the compound is an antibody.

  In one aspect of the invention, the antibody of the invention comprises antibodies A6C12.11, A6F8.6 (ATCC Accession No. PTA-3318), A7H1.19, A8F1.30, A8G3.5 (ATCC Accession No. PTA-3317). A8H3.1 (ATCC accession number PTA-3315), A8H3.2, A10A10.30, A19A10.30, A10B2.17, A10B2.18 (ATCC accession number PTA-3331), A27F6.1 (ATCC accession number PTA- 3310), A40G12.8 (ATCC accession number PTA-3316), A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14, A17A2.16, A17C12.28, A17G12.1 (ATCC accession number) PTA-3314), A17H .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 It specifically binds to an epitope selected from the group of epitopes that bind.

In another aspect of the invention, the antibodies of the invention specifically bind 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 more detailed embodiments, antibodies that specifically bind to an epitope in a ligand / receptor binding domain include, for example, A6C12.11, A6F8.6 (ATCC Accession No. PTA-3318), A7H1.19, A8F1.30, A8G3.5 (ATCC accession number PTA-3317), A8H3.1 (ATCC accession number PTA-3315), A8H3.2, A10A10.30, A19A10.30, A10B2.17, A10B2.18 (ATCC accession number PTA-3)
311), A27F6.1 (ATCC accession number PTA-3310), A40G12.8 (ATCC accession number PTA-3316), A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14, A17A2.16 A17C12.28, A17G12.1 (ATCC Deposit Number PTA-3314), A17H6.1, A18B3.11 (ATCC Deposit Number PTA-3312), A19E2.7, B3F6.17 (ATCC Deposit Number PTA-3319), B6G7 .10 (ATCC accession number 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 Can be mentioned.

  In some embodiments, the epitope to which an antibody of the invention binds is an EGF-like domain. Antibodies that specifically bind to an epitope in the EGF-like domain include A40G12.8 (ATCC accession number PTA-3316), A8H3.1 (ATCC accession number PTA-3315), A27F6.1 (ATCC accession number PTA-3310). ), B6G7.10 (ATCC accession number PTA-3313), A17G12.1 (ATCC accession number PTA-3314), A18B3.11 (ATCC accession number PTA-3312), 1-1A4C. 2, 2-2C9.2, 2-4D1.3, but are not limited thereto.

  In another embodiment, the epitope to which an antibody of the invention binds is a cys-rich domain. Antibodies that specifically bind to an epitope in the cys-rich domain are A19A10.30, A8G3.5 (ATCC accession number PTA-3317), A6F8.6 (ATCC accession number PTA-3318), A6C12.11, 1- 1A4C. 2, 2-2C9.2, but are not limited thereto.

  In yet another embodiment, the epitope to which an antibody of the invention binds is in the amino terminus. Antibodies that specifically bind to an epitope in the amino terminus include, but are not limited to, A10B2.17.

  In yet another embodiment, the epitope to which an antibody of the invention binds is amino acid residues 46-62 spanning the Cripto domain. Antibodies that specifically bind to an epitope in amino acid residues 46-62 spanning the Cripto domain include A10B2.18 (ATCC accession number PTA-3331), B3F6.17 (ATCC accession number PTA-3319), A17A2. 16, 2-3H9.2, 2-4E5.6, 2-4D1.3, 3-4E8.3, 3-1E7.2, 3-3G1.1, but are not limited thereto.

  In another embodiment, the epitope to which the antibody of the present invention binds is 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), in CR50 (SEQ ID NO: 8) or CR51 (SEQ ID NO: 9) polypeptide. Antibodies that specifically bind to an epitope in one of these peptides include A6C12.11, A68.6, 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 For example, but not limited to.

  The present invention also includes antibodies that can specifically bind to Cripto and inhibit the activity of Cripto. Antibodies that can specifically bind to Cripto and inhibit the activity of Cripto include A40G12.8 (ATCC accession number PTA-3316), A8H3.1 (ATCC accession number PTA-3315), A27F6.1 (ATCC accession number) PTA-3310), A6C12.11, 1-1A4C. 2 and 2-2C9.2, but are not limited thereto. In some embodiments, an antibody of the invention that specifically binds Cripto and inhibits Cripto activity binds to an epitope in Cripto's EGF-like or cys-rich domain.

  The invention also includes antibodies that specifically bind to Cripto and block the interaction between Cripto and ALK4. Antibodies that specifically bind Cripto and block the interaction between Cripto and ALK4 include A8G3.5 (ATCC Accession No. PTA-3317), A6F8.6 (ATCC Accession No. PTA-3318), A6C12. 11, 1-1A4C. 2, 2-2C9.2, but are not limited thereto. In some embodiments, an antibody of the invention that specifically binds to Cripto and blocks the interaction between Cripto and ALK4 binds to an epitope in Cripto's EGF-like domain or cys-rich domain.

  The invention also includes antibodies that specifically bind to Cripto and block the interaction between Cripto and activin B. Antibodies that specifically bind to Cripto and block the interaction between Cripto and Activin B include A8G3.5 (ATCC Accession No. PTA-3317), 1-1A4C. 2 is not limited thereto. In some embodiments, an antibody of the invention that specifically binds to Cripto and blocks the interaction between Cripto and activin B binds to an epitope in a Cypto cys-rich domain.

  In another embodiment, the invention includes an antibody that can specifically bind to Cripto and inhibit tumor growth. Antibodies that can specifically bind to Cripto and inhibit tumor growth include A27F6.1 (ATCC accession number PTA-3310), B6G7.10 (ATCC accession number PTA-3313), A8G3.5 (ATCC accession number) PTA-3317), 1-1A4C. 2, 2-2C9.2, but are not limited thereto.

  In some embodiments, an antibody of the invention that specifically binds Cripto and can inhibit tumor growth binds to an epitope in Cripto's EGF-like or cys-rich domain.

  In yet another aspect, the present invention includes an antibody that specifically binds to Cripto, can inhibit Cripto activity, and can inhibit tumor growth. Antibodies that can specifically bind to Cripto, inhibit Cripto activity, and inhibit tumor growth include A27F6.1 (ATCC Accession No. PTA-3310), A8G3.5, 1-1A4C. 2, 2-2C9.2, but are not limited thereto.

  In some embodiments, an antibody of the invention capable of specifically binding to Cripto, inhibiting Cripto activity, and inhibiting tumor growth, binds to an epitope in Cripto's EGF-like domain or cys-rich domain. Join.

  In yet another aspect, the invention includes an antibody that can specifically bind to Cripto, block the interaction between Cripto and ALK4, and inhibit tumor growth. Antibodies that can specifically bind to Cripto, block the interaction between Cripto and ALK4, and inhibit tumor growth include A8G3.5 (ATCC Accession No. PTA-3317), 1-1A4C. 2, 2-2C9.2, but are not limited thereto.

  In yet another aspect, the invention includes an antibody that can specifically bind to Cripto, block the interaction between Cripto and activin B, and inhibit tumor growth. Antibodies that can specifically bind to Cripto, block the interaction between Cripto and activin B, and inhibit tumor growth include A8G3.5 (ATCC Accession No. PTA-3317), 1-1A4C. 2 is not limited thereto.

  In another aspect, the present invention provides a method of inhibiting binding between Cripto and activin B in a sample. This method involves adding to the sample an antibody that specifically binds to Cripto and is capable of blocking the interaction between Cripto and activin B. In a related aspect, the invention includes a method of inhibiting the binding of Cripto and activin B in a mammal. This method involves administering to a mammal an antibody that specifically binds to Cripto and is capable of blocking the interaction between Cripto and activin B.

  In another embodiment, the present invention provides A6F8.6 (ATCC Deposit No. PTA-3318), A8G3.5 (ATCC Deposit No. PTA-3317), A8H3.1 (ATCC Registry Kumogo PTA-3315) A10B2.18. (ATCC deposit number PTA-3331), A27F6.1 (ATCC deposit number PTA-3310), A40G12.8 (ATCC deposit number PTA-3316), A17G12.1 (ATCC deposit number PTA-3314), A18B3.11 (ATCC) Accession number PTA-3312), B3F6.17 (ATCC accession number PTA-3319), B6G7.10 (ATCC accession number 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 An antibody produced by a hybridoma selected from the group is provided. Some of the antibodies have other names such as: 1-1A4C. 2 is the same as 1P1A4-C2.1 (ATCC accession number is specified); 2-2C9.2 is the same as 2P2C9.2 (ATCC accession number is specified). 2-4E5.6 is the same as 2P4E5.6 (ATCC deposit number is specified); 3-3G1.1 is the same as 3P3G1.1 (ATCC deposit number is specified); As well as 3-4E8.3 is the same as 3P4E8.3 (ATCC deposit number specified).

  Antibodies of the present invention include, but are not limited to, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimerized antibodies, and human antibodies.

  The present 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 a human. The composition can include pharmaceutically acceptable excipients. The antibodies described above can be conjugated with chemotherapeutic agents or provided in combination with unconjugated chemotherapy.

  Another aspect of the invention includes a method of inhibiting the growth of tumor cells in vitro in a sample. The method includes adding a composition as described above to a sample.

  Also included are methods of inhibiting growth of mammalian in vivo tumor cells. The method includes the step of administering to the mammal an effective amount of the composition described above. In some embodiments, the mammal is a human.

  Another embodiment of the invention is a method of treating a mammal having a tumor that overexpresses Cripto, the treatment comprising administering to the mammal an effective amount of the composition described above. To do. The composition for administration may comprise a pharmaceutically acceptable excipient, an antibody conjugated to a chemotherapeutic agent and an antibody administered in combination with an unconjugated chemotherapeutic agent.

The method of the present invention comprises inhibiting tumor cell proliferation and / or tumor (where tumor cells are brain tumor cells, breast tumor cells, testicular tumor cells, colon tumor cells, lung tumor cells, ovarian tumor cells, bladder tumor cells, It is particularly useful in the treatment of mammals (eg, humans) having uterine tumor cells, cervical tumor cells, pancreatic tumor cells, and gastric tumor cells.

  In yet another embodiment, the present invention includes a method for determining whether a tissue expresses Cripto or copper. This method involves analyzing a tumor from a mammal in an immunoassay using any of the antibodies described above. Also included is a method of determining whether a cell line overexpresses Cripto. This method involves analyzing cell lines in an immunoassay using any of the antibodies described above.

  In a further aspect, the present invention provides a method of maintaining or sustaining activin B-induced tumor cell inhibition. This method involves exposing tumor cells to an antibody of the invention. In certain embodiments, the tumor cell is a human tumor cell. In some embodiments, the tumor cells are brain tumor cells, breast tumor cells, testicular tumor cells, colon tumor cells, embryonic tumor cells, ovarian tumor cells, bladder tumor cells, uterine tumor cells, cervical tumor cells, pancreatic tumor cells, And gastric tumor cells.

  In yet another embodiment, the present invention includes a method for determining whether a tissue expresses Cripto. This method includes analyzing tissue from a mammal in an immunoassay using any of the antibodies described above. Also included is a method of determining whether a cell line overexpresses Cripto. This method involves analyzing cell lines in an immunoassay using any of the antibodies described above.

  In a further aspect, the present invention provides a method of maintaining or sustaining activin B-induced tumor cell inhibition. This method involves exposing tumor cells to an antibody of the invention. In certain embodiments, the tumor cell is a human tumor cell. In some embodiments, the tumor cells are brain tumor cells, breast tumor cells, testicular tumor cells, colon tumor cells, lung tumor cells, ovarian tumor cells, bladder tumor cells, uterine tumor cells, cervical tumor cells, pancreatic tumor cells, And gastric tumor cells.

  In yet another aspect, the present invention provides a method for identifying a compound capable of blocking the interaction between Cripto and activin B. The method includes contacting Cripto with activin B in the presence of the candidate compound and detecting a change in the interaction between Cripto and activin B. In some embodiments, the compound is an antibody.

  These and other aspects of the invention are described in further detail in the detailed description of the invention below.

FIG. 1 represents the inhibition of Cripto-induced luciferase signal by A27F6.1. FIG. 2 represents the inhibition of Cripto-induced luciferase signal by A27F6.1. FIG. 3 represents the change in tumor weight after subcutaneous implantation of a human testicular carcinoma cell line with an antibody that binds to the Cypto cys-rich domain. FIG. 4 depicts the change in tumor weight after a human testicular carcinoma cell line is implanted subcutaneously with an antibody that binds to Cripto's EGF-like domain. FIG. 5 shows the results of the FACS assay. FIG. 6 is a graph showing that T-47D cell proliferation was inhibited by activin A or activin B by approximately 40% compared to untreated cells. FIG. 7 is a graph showing that the anti-proliferative activity of activin B was restored in the presence of 10 μg / ml or 20 μg / ml of mab A8G3.5. FIG. 8 is a graph showing that CR-Fc bound to wells containing activin B but not to activin A or other ligands. FIG. 9 is a graph showing that CR-Fc was preincubated with activin A or activin B in solution prior to addition to the activin B coated plate, and only activin B inhibited binding. FIG. 10 is a graph showing that activin B binds directly to CR-Fc immobilized on a Biacore chip with high strength but not to the control LTβR-Fc protein.

(Detailed description of the present invention)
The present invention finds that certain Cripto-specific antibodies can affect the activity of Cripto, for example by inhibiting the interaction between Cripto and ALK4 and / or inhibiting the interaction between Cripto and Activin B As well as the discovery that certain Cripto-specific antibodies 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 the native Cripto protein or a modified version of Cripto. For example, these antibodies may bind to an EGF-like domain, a cysteine-rich domain, or a peptide derived from a region of Cripto amino acids 46-150 (eg, about 3 amino acids to about 20 amino acids).

  The antibodies of the present invention are useful in the treatment of mammalian malignant or benign tumors whose tumor growth is at least partially dependent on Cripto. Typically, this growth has an abnormal growth rate that exceeds that required for normal homeostasis and exceeds the growth rate of normal tissue of the same origin.

(I. Definition)
Various definitions are made throughout this document. Most words have the meaning attributed to them by those skilled in the art. Terms defined in detail below or elsewhere in this document have the meaning provided in the context of the present invention and are typically understood by those skilled in the art.

  As used herein, “region” means a physically adjacent portion of the primary structure of a biomolecule. In the case of a protein, a region is defined by adjacent portions in the amino acid sequence of the protein.

  A “domain” as used herein refers to a structural portion of a biomolecule that contributes to a known or potential function of the biomolecule. A domain can occupy a wide area in common with a region or part of a region; a domain can also incorporate a portion of a biomolecule that is distinct from that particular region in addition to all or part of that particular region. Examples of protein domains include extracellular domains (length of about 31 to about 188 residues of Cripto, including CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO: 2)) and transmembrane domains (Length of about 169 to about 188 residues of Cripto including CR-1 and CR-3), but is not limited to these. The ligand / receptor binding domain of the Cripto protein ranges from about 75 to about 150 residues of Cripto, including CR-1 and CR-3; this domain includes, for example, Cripto's, including CR-1 and CR-3 It includes an EGF-like domain that spans from about 75 to about 112, and a cysteine-rich domain that spans from about 114 to about 150, including, for example, CR-1 and CR-3. Many monoclonal antibodies of the invention have been identified as binding to an EGF-like domain or a cysteine-rich domain. Furthermore, monoclonal antibodies A10B2.18 (ATCC accession number PTA-3331), B3F6.17 (ATCC accession number PTA-3312), and A17A2.16 are the lengths of Cripto amino acid residues 46-62 upstream of the EGF-like domain. Identified as binding to an epitope formed in a domain in the region. See Example 3 below.

  The epitope to which the anti-Cripto antibody of the present invention binds can be present in a conformationally native Cripto protein or a modified Cripto protein. In addition, epitopes can be formed by non-adjacent sequences in Cripto polypeptides.

  As used herein, an antibody of the invention can be, for example, a murine antibody, a humanized antibody, a fully human antibody, or a chimeric antibody. The antibody can be an entire antibody of any isotype or subtype (eg comprising IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1 and IgA2; either κ light chain or λ light chain) (ie 2 One full length light chain and two full length heavy chains). Alternatively, an antibody of the invention refers to an antigen-binding fragment of the entire antibody (eg, Fab, F (ab ') 2 and single chain Fv).

  Any of the antibodies of the invention can be combined with chemotherapy as needed, as described below.

As used herein, “an antibody capable of internalizing Cripto” means an antibody that enters a cell during removal of Cripto from the cell surface. For example, by using a fluorescently labeled Cripto monoclonal antibody, it is possible to screen for a Cripto antibody capable of internalizing Cripto. To determine which antibodies can internalize into Cripto positive cells, the uptake of the fluorescent signal of the labeled antibody into the cells can be analyzed by looking at the cells under a fluorescence microscope and / or confocal microscope. These internalized antibodies are detected as fluorescent signals in the cytoplasm or cell vesicles. Non-limiting examples of Cripto antibodies capable of internalizing Cripto include A27F6.1, B3F6.17 and 1-1A4C. 2 is mentioned.

  As used herein, “compound” means any identifiable chemical or molecule. Compounds include, but are not limited to ions, atoms, small molecules, peptides, proteins, sugars, nucleotides and nucleic acids. Such compounds can be natural or synthetic.

  “Modulate” or “modify” as used herein means an increase or decrease in the amount, quality or effect of a particular activity or a particular protein.

  As used herein, “inhibit” means a decrease in the amount, quality or effect of a particular activity or a particular protein.

  As used herein, “modulate Cripto activity” means an increase or decrease in the amount, quality or effect of Cripto activity. An increase or decrease in the amount, quality or effect of Cripto activity is, for example, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. An increase of greater than about 100% is also envisaged. For example, at least about 3 times, 4 times, 5 times, 10 times, 20 times, or even larger. Activity can be measured by assays known in the art, such as the null cell assay shown in Example 3. In some embodiments, protein interactions between Cripto and other proteins are inhibited through the binding of antibodies of the invention.

  As used herein, “block of interaction between Cripto and ALK4” or “modulation of interaction between Cripto and ALK4” refers to interaction (ie, binding between Cripto and ALK4). ) Means increase or decrease. The increase or decrease in interaction can be, for example, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. An increase of greater than about 100% is also envisaged. For example, at least about 3 times, 4 times, 5 times, 10 times, 20 times, or even larger. The activity can be measured by assays known in the art, such as the binding assay shown in Example 8.

  “Blocking the interaction between Cripto and Activin B” or “Modulating the interaction between Cripto and Activin B” used in the present invention refers to the interaction (ie, between Cripto and Activin B). Means an increase or decrease in binding). The increase or decrease in interaction can be, for example, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. An increase of greater than about 100% is also envisaged. For example, at least about 3 times, 4 times, 5 times, 10 times, 20 times, or even larger. Activity can be measured by assays known in the art, such as the binding assay shown in Example 11.

  As used herein, “modulation of tumor cell proliferation 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 is, for example, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% obtain. An increase of greater than about 100% is also envisaged. For example, at least about 3 times, 4 times, 5 times, 10 times, 20 times, or even larger. Modulation of tumor cell growth in vitro can be measured by assays known in the art, such as the GEO cell soft agar assay shown in Example 4.

  As used herein, “modulation of tumor cell growth in vivo” means a decrease in the number of tumor cells, angiogenesis, and / or metastasis in vivo. The decrease in the number of tumor cells can be, for example, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Modulation of tumor cell growth in vivo can be measured by assays known in the art, such as those shown in Example 5.

  As used herein, “therapeutic effect” means inhibition of an abnormal condition. The therapeutic effect somewhat alleviates one or more symptoms of the abnormal condition. With respect to the treatment of an abnormal condition, a therapeutic effect refers to one or more of the following: (a) an increase or decrease in cell proliferation, growth and / or differentiation; (b) inhibition of cell death (ie, delay or arrest). ) Or promotion (ie, elevation or initiation); (c) inhibition of degeneration; (d) some reduction of one or more symptoms associated with the abnormal condition; and (e) enhancement of the function of the cell population. Compounds that show efficacy against abnormal conditions can be identified as described herein.

  “Administration” as used herein refers to a method of incorporating a compound into cells or tissues of an organism. An abnormal condition can be prevented or treated when a cell or tissue of an organism is present in the organism (in vivo) or outside the organism (ex vivo). Cells present outside the organism can be maintained or grown in cell culture dishes or other organisms. For cells present in an organism, there are many techniques for administering compounds in the art. This technique includes, but is not limited to, oral, parenteral, transdermal, injection, and aerosol applications. For cells outside an organism, there are many techniques in the art for administering a compound. This technology includes, but is not limited to, cell microinjection technology, transformation technology and carrier technology. Administration can be accomplished in a number of ways known in the art (eg, oral, intravenous, intraperitoneal, intramuscular, etc.). When used for in vivo therapy, the antibodies of the invention are administered to the patient in an effective amount. As used herein, an “effective amount” is an amount sufficient to produce beneficial or desired clinical results (ie, an amount that eliminates or reduces the burden on the patient's tumor). ). An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of an antibody of the present invention improves, stabilizes, prevents, or prevents the occurrence of a Cripto or activin B related disease state (particularly a tumor related to Cripto or activin B), or An amount sufficient to delay.

  An example of a typical treatment regime includes administration of a dose of about 2-5 mg / kg by intravenous infusion of an antibody of the invention on a weekly schedule. The antibody is administered in outpatient chemical infusion units unless the patient requires hospitalization. Other dosing regimens known in the art are also included.

  By administering the antibody of the present invention to a group of cells having an abnormality in a signal transduction pathway to an organism, the abnormal state can be prevented or treated. The effect of compound administration on its biological function can then be monitored. Preferably, the organism is a human.

  As used herein, “Cripto overexpression” is expressed in a statistically significant amount higher than Cripto expression in normal tissues or cells (eg, at least about 5%, 10%, 20 %, 30%, 40%, 50%, or at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold) means Cripto expression by tissue or cells.

A “chemotherapeutic agent” as used herein is any of those identified in the art as having a therapeutic effect in inhibiting tumor growth, maintaining inhibited tumor growth and / or inducing remission. By drugs (eg natural compounds, synthetic compounds, proteins, modified proteins and radioactive compounds) is meant. Chemotherapeutic drugs include drugs that can be bound to the antibodies of the present invention, or drugs that can be used in combination with the antibodies of the present invention without being bound to the antibodies. Representative chemotherapeutic agents that can be coupled to an antibody of the present invention, radioactive conjugates ^(such as ^{90 Y, 131 I, 99 mTC} , 111 In, 186 Rh), tumor-activated prodrugs (maytansinoids (Maytansinoid) , CC-1065 analogs, Clicheamicin derivatives, anthracyclines, vinca alkaloids, etc.), ricin, diphtheria toxin and Pseudomonas exotoxin.

  A chemotherapeutic agent can be used in combination with an antibody of the invention (ie, a non-conjugated chemotherapeutic agent) rather than conjugated to an antibody of the invention. This includes, but is not limited to: platinum (ie, cis platinum), anthracyclines, nucleoside analogs (purines and pyrimidines), taxanes, camptothecins, epipodophyllotoxins, DNA alkylation Factor, folate antagonist, vinca alkaloid, ribonucleotide reductase inhibitor, estrogen inhibitor, progesterone inhibitor, androgen inhibitor, aromatase inhibitor, interferon, interleukin, monoclonal antibody, taxol, camptosar, adriamycin (dox), 5-FU and Gemcitabine. Such chemotherapeutic agents can be used in the practice of the invention in combination with the antibodies of the invention by concomitant administration of antibodies and non-conjugated chemotherapeutic agents.

  As used herein, “pharmaceutically acceptable carrier or excipient” refers to a biologically inert compound known in the art and is used in the administration of the antibodies of the invention. Acceptable carriers are well known in the art and are described, for example, in Gennaro, Editing, Remington's Pharmaceutical Sciences, Mack Publishing CO. , 1990. Acceptable carriers include biocompatible salts, inert salts or bioabsorbable salts, buffering factors, oligosaccharides or polysaccharides, polymers, viscoelastic compounds such as hyaluronic acid, thickeners, preservatives, etc. Is mentioned.

(II. Antibody of the present invention)
The antibody of the present invention specifically binds to Cripto. As used herein, Cripto includes CR-1 Cripto protein, CR-3 Cripto protein and fragments thereof. Such a fragment can be an entire domain, such as an extracellular or intracellular domain, an EGF-like domain, a cysteine-rich domain, a receptor binding domain. Such fragments can also include adjacent and non-adjacent epitopes in any domain of the Cripto protein. Examples of antigens used to produce antibodies specific to Cripto include 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 CR51 (SEQ ID NO: 9), the amino acid sequences of which are provided in Example 2). It is not limited.

The 188 amino acid sequence of CR-1 is as follows (SEQ ID NO: 1):
MDCRKMARFSYSVIWIMAISKVFELGLVAGLGGHQEFARPSRGYLAFRDDSIWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCCLNGQTCMLGSFCCPPSFYGNRCGVCVCLPHDLCWLPK
The 188 amino acid sequence of CR-3 is as follows (SEQ ID NO: 2):
MDCRKMVRFSYSVIWIMAISKAFELGLVGALGGHQEFARPSRGDLAFRDDDSIWPQEEAIRPRSQRVLPGMGIQHSKENLTGTCTLNGTCLGQTPLGCLPGLCPGLFTCLFCG
In some embodiments, the antibodies of the invention bind to an epitope in the EGF-like domain of Cripto. This EGF-like domain extends from approximately 75 amino acid residues to approximately 112 amino acid residues of the mature Cripto protein. The epitope in this EGF-like domain may include a linear or non-linear span of amino acid residues. Examples of linear epitopes include, but are not limited to, residues approximately 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 a Cripto protein that is configurationally native to a denatured Cripto protein.

  In other embodiments, the antibodies of the invention bind to an epitope in a cysteine-rich domain of Cripto. This cysteine-rich domain ranges from approximately 114 amino acid residues to approximately 150 amino acid residues of the mature Cripto protein. The epitope in this cysteine-rich domain can include a linear or non-linear spread of amino acid residues. Examples of linear epitopes include, but are not limited to, residues approximately 114-125, 120-130, 125-135, 130-140, 135-145, or 140-150. In certain embodiments, the epitope in the cysteine-rich domain is an epitope formed in a Cripto protein that is conformationally native to a denatured Cripto protein.

  Once the antibody is generated, its binding to Cripto can be assayed using standard techniques known in the art (eg, ELISA), while the presence of Cripto on the cell surface is As shown in Example 2, it can be assayed using flow cytometry (FACS). Any other technique for measuring such binding can be used separately.

  The present invention relates to antibodies specific for Cripto or fragments thereof (eg, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional / bispecific antibodies, humanized antibodies, human antibodies and complements). Sex-determining region (CDR) grafted antibodies (including compounds comprising CDR sequences that specifically recognize a polypeptide of the invention) are provided.The terms “specific” and “selective” refer to the antibodies of the invention When used to describe binding, it shows that the variable region of an antibody of the invention recognizes and binds to a Cripto polypeptide. For example, S. aureus protein A or other antibodies in the ELISA technique) to sequences outside the variable regions of those antibodies (in particular, Through interactions with sequences in the constant region of the molecule), it is understood that may interact.

  Screening assays for determining the binding specificity of antibodies of the invention (eg, antibodies that specifically bind to an epitope of a ligand / receptor binding domain or domain spanning amino acid residues 46-62) are well known in the art. Yes, it is done routinely. For a comprehensive discussion of such assays, see Harlow et al., Edited by Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. Also included are antibodies that recognize and bind to a fragment of a Cripto protein, provided that the antibody is specific for a Cripto polypeptide. The antibodies of the invention can be generated and routinely practiced using any method well known in the art.

  In some embodiments, the present invention provides antibodies that specifically bind to an epitope in a Cripto ligand / receptor binding domain. Antibody specificity is described in more detail below. However, it can be generated from other polypeptides previously described in the literature and can accidentally cross-react with Cripto (eg, due to the accidental presence of similar epitopes in both polypeptides). It should be emphasized that antibodies 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 a Cripto epitope is made using any of a number of assays well known in the art (eg, Western blotting assays). To specifically identify cells that express Cripto, and also to inhibit Cripto ligand / receptor binding activity, specifically binds to an extracellular epitope of Cripto protein (ie, the portion of Cripto protein found outside that cell). Antibodies are particularly useful.

  The invention also provides a cell-free composition comprising a polyclonal antibody, at least one of which is an antibody of the invention. Antisera isolated from animals is an exemplary composition, and also a composition comprising an antibody fraction of antisera resuspended in water or another diluent, excipient, or carrier. , An exemplary composition.

  In other embodiments, the present invention provides monoclonal antibodies. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to polyclonal preparations that typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single determinant on that antigen. Monoclonal antibodies are useful for improving the selectivity and specificity of diagnostic and analytical assays that use antigen-antibody binding. Another advantage of monoclonal antibodies is that monoclonal antibodies can be synthesized by cultured cells (eg, hybridomas) and other immunoglobulins can be uncontaminated. Recombinant cells and hybridomas that produce such antibodies are also contemplated as aspects of the invention. See also below.

  In still other related embodiments, the invention provides anti-idiotypic antibodies specific for antibodies that are specific for Cripto. See, eg, US Pat. Nos. 6,063,379 and 5,780,029 for a more detailed discussion of anti-idiotype antibodies.

  Antibodies contain relatively small antigen binding domains that can be isolated either chemically or by recombinant techniques. Such domains are useful Cripto binding molecules per se and can also be reintroduced into human antibodies or fused to chemotherapeutic agents or polypeptides. Thus, in yet another embodiment, the invention provides a polypeptide comprising a fragment of a Cripto-specific antibody, and the fragment and related molecule (if present) binds to this Cripto. By way of non-limiting example, the present invention provides polypeptides that are single chain antibodies and CDR-grafted antibodies. For a more detailed discussion of CDR-grafted antibodies, see, for example, US Pat. No. 5,859,205 and the discussion below.

  In other embodiments, non-human antibodies can be humanized by any method known in the art. Humanized antibodies are useful for in vivo therapeutic applications. In addition, recombinant “humanized” antibodies can be synthesized. A humanized antibody is a non-human in which some or all of the amino acids that are not required for antigen binding are replaced using amino acids from the corresponding region of a human immunoglobulin light or heavy chain using recombinant DNA technology. The first antibody derived from an animal. That is, humanized antibodies are chimeras that mostly contain human immunoglobulin sequences, with the region responsible for specific antigen binding being replaced.

  Various forms of antibodies can be generated using standard recombinant DNA techniques (Winter and Milstein, 1991, Nature 349: 293-99). For example, the monoclonal antibodies of the present invention can be generated by well-known hybridoma technology. For example, an animal (eg, mouse, rat, or rabbit) can be a purified or crude Cripto preparation, a cell transfected with a cDNA construct encoding Cripto, a cell that constitutively expresses Cripto, etc. Can be used to immunize. Furthermore, the antigen can be delivered as a purified protein, a protein expressed on a cell, a protein fragment or peptide thereof, or as a naked DNA or viral vector encoding the protein, protein fragment or peptide. The immunized animal serum is then tested for the presence of anti-Cripto antibodies. B cells are isolated from test positive animals and hybridomas are generated using these B cells.

  The antibody secreted by the hybridoma for its ability to specifically bind to Cripto (eg, binds to Cripto-transfected cells but not to non-transfected parental cells) and any other desirable Screened for characteristics (eg, having the desired CDR consensus sequence, inhibiting binding between Cripto and ALK4 or binding between Cripto and Activin B (or not in the case of non-blockers)) The

  Hybridoma cells that test positive in this screening assay are cultured in nutrient medium under conditions that allow the cells to secrete monoclonal antibodies into the culture medium. Thereafter, the conditioned hybridoma culture supernatant is collected and the antibody contained in the supernatant is purified. Alternatively, the desired antibody can be generated by injecting the hybridoma cells into the peritoneal cavity of a non-immunized animal (eg, a mouse). The hybridoma cells grow in the peritoneal cavity and secrete antibodies that accumulate as ascites. The antibody can then be collected by collecting ascites from the abdominal cavity with a syringe.

  The monoclonal antibody also isolates the antibody-encoding cDNA from the desired hybridoma, transfects a mammalian host cell (eg, a CHO cell or NSO cell) with the cDNA, cultures the transfected host cell, It can then be produced by removing the antibody from the culture medium.

The monoclonal antibodies of the invention can also be generated by manipulating cognate hybridoma (eg, mouse, rat, or rabbit) antibodies. For example, a cognate antibody can be produced by recombinant DNA technology in which the heavy and / or light chain hinge and / or constant region part or all of the corresponding component of an antibody from another species (eg, human). Can be modified to be substituted. In general, the engineered antibody variable domain remains the same or substantially the same as the variable domain of the cognate antibody. Such engineered antibodies are referred to as chimeric antibodies and are less antigenic than their cognate antibodies when administered to individuals of species (eg, humans) originating from their hinge and / or constant regions. Methods for producing chimeric antibodies are well known in the art. The human constant region includes a region derived from IgG1 and a region derived from IgG4.

  The monoclonal antibodies of the present invention also include fully human antibodies. The fully human antibody is described in Boerner et al., 1991, J. MoI. Immunol. 147: 86-95 using in vitro primed human splenocytes or using a phage display antibody library as described for example in US Pat. No. 6,300,064. Can be prepared.

  Alternatively, fully human antibodies are described in Persson et al., 1991, Proc. Natl. Acad. Sci. USA 88: 2432-36; and Huang and Stollar, 1991, J. MoI. Immunol. Methods 141: 227-36 and can be prepared by repertoire cloning. In addition, US Pat. No. 5,798,230 describes the preparation of human monoclonal antibodies from human B cells, in which human antibody-producing B cells are Epstein--a protein required for immortalization. Immortalized by infection with Epstein-Barr virus or its derivatives expressing barvirus nuclear antigen 2 (EBNA2). This EBNA2 function is then blocked, resulting in increased antibody production.

Some other methods for generating fully human antibodies are non-human, which have inactivated endogenous Ig loci and are transgenic for unrearranged human antibody heavy and light chain genes. Includes animal use. Such transgenic animals can be immunized with Cripto and hybridomas are generated from B cells derived from that animal. These methods include, for example, various GenPharm / Medarex (Palo Alto, CA) publications / patents (eg, US Pat. No. 5,789,650) relating to transgenic mice containing the human Ig minilocus; XENOMICE ^™ Various Abgenix (Fremont, CA) publications / patents (eg, US Pat. Nos. 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 "Various Kirin (Japan) publications / patents on transsonic mice (eg, EP 843 961). And Tomizuka et al., 1997, Nature Genetics 16: 1433-43) and also described in, for example, US Patent No. 5,569,825, WO00076310, WO00058499, and WO00037504, which are incorporated herein by reference in their entirety. See incorporated by reference).

  The monoclonal antibodies of the invention also include humanized versions of cognate anti-Cripto antibodies from other species. A humanized antibody is an antibody produced by recombinant DNA technology in which amino acids of a human immunoglobulin light or heavy chain (eg, variable domain constant and framework regions) are not required for antigen binding. Some or all of these are used to substitute with the corresponding amino acid from the light chain or heavy chain of the cognate non-human antibody. By way of example, a humanized version of a murine antibody against a given antigen comprises (1) a constant region of a human antibody; (2) a framework region from the variable domain of a human antibody; 3) It has a CDR derived from a mouse antibody. If necessary, one or more residues in the human framework regions can be changed to corresponding residues in the mouse antibody to preserve the binding affinity of the humanized antibody for the antigen. This change is sometimes referred to as “back mutation”. Humanized antibodies are generally less likely to elicit an immune response in humans compared to chimeric human antibodies. This is because the former contains relatively few non-human components.

  Methods for generating humanized antibodies are described, for example, in 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; US Pat. No. 6,180,370; and Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA 86: 3833-37. See also, for example, PCT Patent Application No. 94/04679. Primatized antibodies can be generated using primate (eg, rhesus monkey, baboon, and chimpanzee) antibody genes as well. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity. See, for example, U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370.

  In general, transplantation of mouse (or other non-human) CDRs onto human antibodies is accomplished as follows. CDNA encoding the heavy chain variable domain and the light chain variable domain is isolated from the hybridoma. The DNA sequence of the variable domain (including CDRs) is determined by sequencing. The DNA encoding the CDRs is transferred to the corresponding region of the variable domain coding sequence of the human antibody heavy or light chain by site-directed mutagenesis. Thereafter, human constant region gene segments of the desired isotype (eg, γ1 for CH, κ for CL) are added. The humanized heavy and light chain genes are coexpressed in mammalian host cells (eg, CHO cells or NSO cells) to produce soluble humanized antibodies. To facilitate large-scale production of antibodies, generating such humanized antibodies in a bioreactor containing cells that express the antibodies, or transgenic animals expressing the antibodies in milk (e.g., It is often desirable to produce goats, cows, or sheep) (see, eg, US Pat. No. 5,827,690).

  Occasionally, direct transfer of CDRs to the human framework results in a loss of antigen binding affinity of the resulting antibody. This is because in some cognate antibodies, certain amino acids in the framework regions interact with the CDRs, thereby affecting the antibody's overall antigen binding affinity. In such cases, a “back mutation” (supra) should be introduced into the framework region of the acceptor antibody to retain the antigen binding activity of the cognate antibody.

  General approaches for generating back mutations are known in the art. See, eg, 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 WO90 / 07861 (Protein Design Labs Inc.) describe an approach involving two important steps. First, human V framework regions are selected by computer analysis for optimal protein sequence homology with the V region framework of cognate mouse antibodies. The tertiary structure of the mouse V region is then computer modeled to visualize framework amino acid residues that may interact with mouse CDRs, after which these mouse amino acid residues are homologous. Layered on the human framework.

  Under this two-step approach, there are several criteria for designing humanized antibodies. The first criterion is to use a framework derived from a specific human immunoglobulin (which is usually homologous to a non-human donor immunoglobulin) as a human acceptor, or a consensus framework from many human antibodies. Is to use. The second criterion is to use a donor amino acid rather than an acceptor if the human acceptor residue is unusual and the donor residue is typical of a human sequence at a particular residue in the framework. The third criterion is to use donor framework amino acid residues rather than acceptors at positions immediately adjacent to the CDRs.

  One skilled in the art may use different approaches as described, for example, in Tempest, 1991, Biotechnology 9: 266-71. Under this approach, the NEWM-derived V region framework, and REI heavy and light chains, respectively, are used for CDR grafting without radial introduction of mouse residues. The advantage of using this approach is that the three-dimensional structure of the NEWM and REI variable regions can be seen from X-ray crystallography, so that specific interactions between CDRs and V region framework residues can be easily modeled. That is.

  The humanized antibodies of the invention have mutations (eg, deletions, substitutions or additions) at specific positions in one or more (eg, 2, 3, 4, 5, 6, 7 or 8) heavy chains. As a result, the effector function of the antibody (eg, the ability of the antibody to bind to an Fc receptor or a complementary factor) is altered without affecting the ability of the antibody to bind to Cripto (US Pat. No. 5, 648, 260). These heavy chain positions include, but are not limited to residues 234, 235, 236, 237, 297, 318, 320 and 322 (EU numbering system). For example, a humanized antibody may include mutations L234A (ie, replacement of leucine with alanine at position 234 of the unmodified antibody) and L235A (EU numbering system) in its heavy chain.

  Furthermore, the humanized antibodies of the present invention may contain mutations (eg, deletions or substitutions) at amino acid residues that are sites for glucosylation, so that this glucosylation site is eliminated. Such antibodies may be clinically beneficial because they have reduced effector functions or other undesired functions while maintaining their Cripto binding affinity. Glycosylation site mutations can also be beneficial for process development (eg, protein expression and purification). For example, the heavy chain of the antibody can contain the mutation N297Q (EU numbering system) so that the heavy chain can no longer be glycosylated at this site.

In yet another embodiment, the heavy and / or light chain of the antibody of the invention is Cript.
Includes mutations that increase affinity for binding to o, thereby increasing efficacy for treating Cripto-mediated disorders.

The monoclonal antibodies of the invention may further comprise other moieties that perform or increase the desired function. For example, the antibody can include a toxic moiety (eg, tetanus toxin or ricin) or a radionuclide (eg, ¹¹¹ In or ⁹⁰ Y) to kill cells targeted by the antibody (eg, US Pat. No. 6, , 307,026). The antibody can include a moiety (eg, biotin, fluorescent moiety, radioactive moiety, histidine tag or other peptide tag) to facilitate isolation or detection. The antibody also has a moiety that can increase its serum half-life (eg, a polyethylene glycol (PEG) moiety), and a member of an immunoglobulin superfamily or fragment thereof (eg, a portion of a human IgG1 heavy chain constant region (eg, Hinge region, CH2 region and CH3 region)).

Antibody fragments and monovalent antibodies can also be used in the methods and compositions of the invention. A monovalent antibody comprises a heavy / light chain dimer linked to the Fc (or stem) region of a second heavy chain. “Fab region” refers to an antibody that is approximately equivalent or similar to the sequence containing the Y branch of the heavy chain and to the entire light chain, and together (in an aggregate) The portion of the chain that has been shown to exhibit activity. The Fab protein comprises an aggregate of one heavy chain and one light chain (commonly known as Fab ′), as well as a tetramer, which is covalently bound by any of the above The two branched segments of antibody Y (generally F (ab)), so long as this aggregation can react specifically with a particular antigen or antigen family 2).

(III. Signal regulation)
The antibodies of the present invention may inhibit Cripto activity and / or interaction between Cripto and its ligand. Overexpression of Cripto activity results in dedifferentiation-promoting mesenchymal cell properties, increased proliferation, and cell migration (Salomon et al., 1999, BioEssays 21: 61-70; Ciadiello et al., 1994, Oncogene 9: 291-98; And Baldassarre et al., 1996, Int. J. Cancer 66: 538-43), may result in a phenotype associated with cell transformation seen in neoplasia.

  One way to test the activity of Cripto antibodies and their ability to inhibit Cripto activity is to use the F9-Cripto knockout (KO) cell line (Mnchiotti et al., 2000, Mech. Dev. 90: 133-42). ). Cripto stimulates Smad2 phosphorylation and transcription factor FAST in Xenopus embryos, and the activity of this transcription factor FAST can be monitored by measuring luciferase activity from the FAST regulatory element-luciferase reporter gene (Saijoh et al. 2000, Mol.Cell 5: 35-47). F9-Cripto KO cells have a null mutation in the Cripto gene and cannot transmit Cripto-dependent signaling (Minchiotti et al., Supra). Cripto activity can be assessed in F9 Cripto KO cells by transfecting F9 Cripto KO cells with Cripto, FAST, and FAST regulatory element-luciferase gene constructs. Cripto-dependent FAST luciferase activity is not found in these cells unless Cripto and FAST cDNAs are transfected into these cells. An antibody that blocks Cripto-dependent Nodal signaling and an antibody that blocks Cripto activity.

  Other assays that can measure Cripto activity (eg, soft agar assay) can be used by those skilled in the art (see, eg, Example 4 below). The ability of cells to grow on soft agar is related to cell transformation, and the assay is a classic in vitro assay to measure inhibition of tumor cell growth. Other assays useful for determining inhibition of activity include in vitro assays on plastics.

  In certain embodiments, the antibodies of the invention bind to Cripto and inhibit Cripto-activin B interaction. The inventors have discovered that Cripto can bind to activin A and inhibit the activin B signaling pathway. Activin B can inhibit tumor cell growth (Ribbridgeger et al., 2001, Endocr. Rev. 222: 836-58). Cripto binding to activin B can also disrupt activin B-induced inhibition of proliferation.

  One way to test the activity of an anti-Cripto antibody and its ability to inhibit Cripto-activin B interaction is to measure the ability of the antibody to prevent Cripto-mediated destruction of activin B-induced inhibition of proliferation It is. One such method is described in Example 9. A method for directly testing the ability of anti-Cripto antibodies to inhibit Cripto-activin B interaction is described in Example 11.

(IV. Therapeutic use)
The antibodies of the present invention are also useful for therapeutic purposes (eg, inhibition of tumor cell growth), diagnostic purposes for detecting or quantifying Cripto, and purification of Cripto.

  In some embodiments of the invention, an antibody that can specifically bind to Cripto and inhibits the growth of a patient's tumor cells, particularly when tumor growth is mediated by deletion or reduction of activin B signaling Is provided. In certain embodiments, the tumor cells are brain, heart, neck, prostate, breast, testis, colon, lung, ovary, bladder, uterus, cervix, pancreas and stomach tumor cells.

  In other embodiments, antibodies are provided that can specifically bind to Cripto and inhibit tumor cells that overexpress Cripto. In one embodiment, the tumor cell is a cell line that overexpresses Cripto (eg, a cell line derived from brain, breast, testis, colon, lung, ovary, bladder, uterus, cervix, pancreatic and gastric cancer). It is.

  Anti-Cripto antibodies can be screened for in vivo activity as potent anti-cancer agents according to standard protocols used by those skilled in the art, as illustrated in Example 4 below. An example of such a protocol is reviewed by the National Cancer Institute (NCI) in its “in vivo cancer model screening” protocol, NIH Publication No. 84-2635 (Feb 1984).

  In other embodiments of the invention, the antibodies of the invention are used to treat patients with cancerous tumors.

  The antibodies of the invention are combined with pharmaceutically acceptable excipients and administered to a patient at a therapeutically effective dose. See, for example, US Pat. No. 6,165,464 for a discussion of methods for inhibiting tumor growth.

  Also encompassed is a method of treating a mammal suffering from a disorder associated with elevated Cripto levels or reduced activin B levels, wherein the method comprises treating the mammal with an effective amount of an antibody (which is a Cripto ligand / receptor). Administering an epitope (which specifically binds to, but not limited to) an epitope in the binding domain, wherein the epitope is a Cripto EGF-like domain or a cys-rich domain.

  Also included is a method of treating a mammal suffering from a disorder associated with elevated Cripto levels, wherein the method comprises administering to the mammal an effective amount of an antibody, wherein the antibody comprises Cripto and An antibody that specifically forms a complex and is directed to an antibody directed from the group consisting of: A6C12.11, A6F8.6 (ATCC Accession No. PTA-3318), A7H1.19, A8F1 .30, A8G3.5 (ATCC accession number PTA-3317), A8H3.1 (ATCC accession number PTA-3315), A8H3.2, A10A10.30, A19A10.30, A10B2.17, A10B2.18 (ATCC accession number) PTA-3331), A27F6.1 (ATCC accession number PTA-3310), A40G12 8 (ATCC deposit number PTA-3316), A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14, A17A2.16, A17C12.28, A17G12.1 (ATCC deposit number PTA-3314), A17H6.1, A18B3.11 (ATCC accession number PTA-3312), A19E2.7, B3F6.17 (ATCC accession number PTA-3319), B6G7.10 (ATCC accession number PTA-3313), 1-1A4C. 2, 2-2C9.2, 2-3H9.2, 2-4E5.6, 2-4E1.3, 3-4E8.3, 3-3G1.1, 4-2F6, 4-3A7 and 4-1E2.

  Diagnosis by detection of Cripto is easily accomplished by standard assays using the novel antibodies of the present invention, and those skilled in the art can detect the presence of Cripto, particularly in various samples, cultures, etc. .

  Kits containing the antibodies of the invention are also contemplated for any purpose described in the written description. In general, the kit of the invention comprises a control antigen in which the antibody is immunospecific. Embodiments include all reagents and instructions for their use.

  Further features of the present invention will become apparent from the following examples. It is understood that the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any manner.

Example 1: Cripto expression and purification
An expression plasmid named pSGS480 was subcloned into a cDNA encoding amino acid residues 1-169 (amino acids 1-169 (SEQ ID NO: 1)) of the human Cripto protein, and the human IgG ₁ Fc domain (ie, “ CR (del C) -Fc "). For a more detailed description of this vector, see US patent application Ser. No. 60 / 233,148 (filed Sep. 18, 2000). This vector pEAG1100 is a derivative of GIBCO-BRL Life Technologies plasmid pCMV-Sport-betagal and its use in CHO transient transfection was described by Schifferli et al., 1999, Focus 21:16. This vector was made by removing the reporter gene β-galactosidase NotI fragment from the plasmid pCMV-Sport-Betagal (Cat. No. 10586-014) as follows: the plasmid was digested with NotI and EcoRV. The 38 kb NotI vector backbone was gel purified and ligated. The ligated DNA is competent E. coli. E. coli DH5α was transformed. From a single isolated colony, pEAG1100 was isolated as a plasmid containing the desired recombinant. The sequence of pEAG1100 including the promoter, polylinker, and transcription termination signal was confirmed.

  Plasmid pSGS480 was transiently transfected into CHO cells and the cells were grown at 28 ° C. for 7 days. The presence of CR (del C) -Fc protein in these cells and 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 a 4-20% gradient gel under reducing conditions, electrophoretically transferred to nitrocellulose, and Cripto The fusion protein was detected with a rabbit polyclonal antiserum raised against the Cripto17 mer peptide (containing residues 97 to 113 of SEQ ID NO: 1) -keyhole limpet hemocyanin (KLH) conjugate. After centrifugation to remove the cells, Western blot analysis showed that CR (del C) -Fc protein was efficiently secreted into the conditioned medium (supernatant). The supernatant was applied to Protein A-Sepharose® (Pharmacia) and the 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 (pH 8.6) and analyzed for total protein content from absorbance measurements at 240-340 nm and analyzed for purity by SDS-PAGE. The eluted protein was filtered through a 0.2 micron filter and stored at -70 ° C.

  Example 2: Antibody Production and Screening Mice are injected with eluted CR (del C) -Fc protein and other Cripto polypeptides, and hybridoma and monoclonal antibodies are obtained using standard techniques known to those skilled in the art. Produce.

(A. Production of antibodies)
Female Robertsonian mice (Jackson Labs) were immunized intraperitoneally with 25 μg of purified human CR (del C) -Fc emulsified with Freund's complete adjuvant (“FCA”; GibcoBRL # 15721-012). They were emulsified with Freund's incomplete adjuvant (“FIA”; GibcoBRL # 15720-014) twice with 25 μg CR (del C) -Fc and once with protein A beads intraperitoneally (IP) booster. . Serum was screened and 3 weeks after the last booster administration, mice with the highest titer were boosted intraperitoneally with 50 μg soluble CR (del C) -Fc 3 days prior to fusion. The mice were intravenously (IV) boosted with 50 μg CR (del C) -Fc the day before fusion.

  Mouse spleen cells were fused with FL653 myeloma cells at a ratio of spleen 1: myeloma 6 and plated at 100,000, 33,000 and 11,000 cells per well in 96-well tissue culture plates in selective medium. did. One week later, wells that were positive for growth were screened by FACS and ELISA. Two fusions were performed.

(B. Screening for antibodies)
Supernatants generated from the first or second fusion were initially screened on ELISA plates for Cripto (del C) recognition and / or Cripto EGF-like domain proteins. A control fusion protein (LT-β receptor-Fc) was coated on an ELISA plate to exclude monoclonal antibodies that recognize human Fc epitopes. The ELISA was performed as described in Section C below. In the first fusion, the first supernatant was also screened by FACS for its ability to recognize the cell surface Cripto protein on the testicular tumor cell line NCCIT. In the second fusion, the ability of the supernatant to recognize Cripto on two tumor cell lines, NCCIT and breast cancer line DU4475 was analyzed by FACS. The secondary screening involved testing the following: the ability of the monoclonal antibody supernatant to recognize cell surface Cripto on a group of tumor cell lines (see Tables 1 and 2 for results), The ability of a monoclonal antibody to recognize human Cripto immunohistochemically in tissue sections of human breast and colon tumors, the ability of a monoclonal antibody to block signaling in a Cripto-Nodal signaling assay, on plastic or in a soft agar assay The ability to block the growth of tumor cell lines and the ability to internalize cell surface Cripto.

(C. ELISA)
The ELISA assay was performed as follows:
material:
Plate: Costar high-binding Easy-wash 96W plate (07-200-642)
Secondary antibody: Pierce Gt anti-Ms IgG (H + L) -HRP (P131430)
Substrate: Pierce TMB Substrate Kit (34021)
Stop solution: 1N H ₂ SO ₄
Buffer: Binding buffer: 0.1 M NaHPO ₄ (pH 9.0)
Blocking buffer: PBS + 10% Donor calf serum Wash buffer: PBS + 0.1% Tween®20.

  Antigen CR (del C) -Fc and CR-EGF-Fc, control hu IgG1 fusion protein were diluted with binding buffer to 500 ng / ml. 100 μl was added per well and incubated for 1 hour at 37 ° C. or overnight at 4 ° C. The liquid was decanted and the plate was inverted and blotted to dryness. Then 250 μl of blocking buffer was added per well, followed by incubation at 37 ° C. for 30 minutes. Again, the liquid was decanted and the plate was inverted and blotted to dryness. The supernatant was diluted 1:50 with wash buffer and plated at 50 μl per well followed by 1 hour incubation at room temperature. The plate was washed vigorously 3 times with 250 μl wash buffer per well. Then secondary antibody diluted 1: 10,000 with wash buffer was added at 100 μl per well. Subsequently, it was incubated at room temperature for 30 minutes. The plate was then washed vigorously 3 times with 250 μl wash buffer per well and then the substrate was added at 100 μl per well. The color was developed until dark enough, then 100 μl of stop buffer was added per well and the plate was read for absorbance at 450 nm.

In certain experiments, we added 96 μl of 0.5 μg / ml Cripto protein in 0.1 M NaHPO ₄ (pH 9.0) and incubated for 1 hour at 37 ° C. Was coated with Cripto protein. We blocked the plate with PBS / 10% DCS. We added antibodies diluted in PBS / 0.05% Tween® 20 at 50 μl per well and incubated at 37 ° C. for 1 hour. We washed with PBS / 0.05% Tween® 20 and probed with anti-mouse-HRP (Pierce). We detected the bound antibody by adding TMB, stopping the reaction with 1N H ₂ SO ₄ and reading at 450 nm.

In yet other experiments, we coated 96-well plates by adding 100 μl of 3-5 μg / ml activin B or other ligand in 50 mM carbonate (pH 9.5) to each well. We incubated the plates for 1 hour at room temperature and blocked with TBS / 1% BSA overnight at 4 ° C. We incubated the plates with CR-Fc or ALK4-Fc in blocking buffer at room temperature, washed with TBST, and probed with anti-human-AP (Jackson). We washed the plate with TBST, washed once with 10 × substrate buffer (200 mM Tris-HCl, 10 mM MgCl ₂ , pH 9.8), and added CSPD and Sapphire (Applied Biosystems).

(D. Flow cytometry)
Cripto positive cell lines can be used to assay monoclonal antibodies for binding to Cripto using cell surface staining and flow cytometry as follows:
Cells are released from in a T162 flask with 2 ml PBS using 5 mM EDTA for 10 minutes at 37 ° C. Increase the volume to 20 ml with serum-containing medium and pipette up and down several times to break up cell clumps. Rotate at 1200 rpm for 5 minutes. Wash cells with 5-10 ml of PBS (4 ° C.) (wash buffer) containing 0.1% BSA. Rotate at 1200 rpm for 5 minutes. Resuspend at 4 × 10 ⁶ to 10 ⁷ / ml in wash buffer. Keep on ice.

Prepare antibody for staining. The purified antibody is diluted with wash buffer to 1-10 μg / ml. Add 50 μl of cells to 96-well Linbro V bottomed plate (ICN 7632105). Plate cells for each control for each cell line to be analyzed in one well. Such control cells include no antibody, secondary antibody only, hybridoma media, positive control antibody supernatant (if available or purified), and IgG subclass control (purification Cells for the use of antibodies).

Cells for each experimental sample of each cell line to be analyzed were plated in one well. Using a tabletop centrifuge at 4 ° C., rotate the plate at 1200 rpm for 5 minutes. Remove the buffer by inverting the plate and shaking until the liquid is substantially removed. Add 40-50 μl of antibody to the well (or wash buffer for control wells without antibody and secondary antibody only). Incubate at 4 ° C. for at least 30 minutes to 1 hour. Spin the plate at 1200 rpm for 5 minutes. Remove antibody solution. Wash wells twice with 200 μl wash buffer per well (rotate after each wash). Remove the buffer.

  Cells in each well are resuspended in 50 μl (in wash buffer) 1: 200 dilution of R-PE tagged goat anti-mouse IgG (Fc specific) (Jackson Immunoresearch Laboratories catalog number 115-116-071). Incubate at 4 ° C. for 20 minutes in the dark. Add 150 μl wash buffer to cells in each well. Spin the plate at 1200 rpm for 5 minutes. Wash once with 200 μl wash buffer per well. Resuspend cells in 150 μl of 1% PFA in PBS. Transfer the contents of each well to a separate tube (5 ml Falcon polystyrene round bottom tube-352052). Wrap the tube with a thin foil.

  The contents of the tube are then read by flow cytometry.

  The results of two screenings of specific monoclonal antibodies analyzed by this method resulted in the following results summarized in Tables 1 and 2 below. Here, the first column provides the name given for the hybridoma subclone, the next two columns show the results of the ELISA screening, and the remaining columns are the flow sites for the four clipto positive cell lines. The result of a measurement analysis is shown. This result is shown in units of mean fluorescence index (MFI).

We also used a number of alternative immunization protocols using CR (del C) -Fc protein and region specific Cripto peptides, as shown below. In these cases, we also. Hybridomas were produced as described. Cripto peptide was used in addition to or in place of CR (del C) -Fc protein. By these protocols, some of the antibodies of the present invention were identified. As will be appreciated by those skilled in the art, these experiments have shown that multiple diverse immunization protocols can be used to produce the antibodies of the present invention.

In one exemplary immunization protocol, 50 μg of either soluble CR (del C) -Fc recombinant protein or region-specific Cripto peptide conjugated to keyhole limpet hemocyanin (KLH), and Freund's complete adjuvant (FCA, Eight week old female RBF mice (Jackson Labs, Bar Harbor, ME) were immunized intraperitoneally (IP) with an emulsion containing Sigma Chemical Co., St, Louis, MO. Specifically, CR (del C) -Fc protein-KLH or Cripto peptide CR40 (amino acids 36-42; SEQ ID NO: 3) -KLH to phosphate buffered saline (pH 7.2) to an estimated concentration of 2 mg / ml Diluted. An equal volume of FCA was added to the protein prior to emulsification and immunization. 50 μl containing 50 μg of emulsified CR (del C) -Fc protein-KLH or Cripto peptide CR40-KLH was administered IP to each mouse for the first immunization. As shown below, all subsequent immunizations were similarly administered using either Freund's incomplete adjuvant (FIA) or RIBI adjuvants _{1, 2} (Sigma Chemical Co., St, Louis, MO). Booster immunization was administered every 2-3 weeks. Serum samples of immunized mice were collected before the first immunization, 7 days after booster immunization, and also prior to fusion with lymphocyte cells. Serum titers were measured using both ELISA and flow cytometry assays as shown below.

  Various exemplary immunization protocols with different Cripto antigens and the antibodies identified thereby are as follows:

Antigen 4- (x = number of peptides conjugated to KLH; we also conjugated KLH to mice previously immunized with CR (delC) -Fc, eg, as described in Protocol C Infused peptide):
The sequences of exemplary CRx peptides used by the inventors and their positions in the full-length Cripto protein are as follows:
CR40 = FRDDSIWPQEEPAIRPR (aa46-42, A10.B2; SEQ ID NO: 3)
CR41 = CPPSFYGRNCEHDVRKE (aa 97-113; SEQ ID NO: 4)
CR43 = GSVPHDTWLPKKKC (aa 116-128; SEQ ID NO: 5)
CR44 = SLCKSWHGQLRCFQ (aa 129-143; SEQ ID NO: 6)
CR49 = acetylated ^{N-SFYGRNCEHDVRRENCGSVPHDTWLPKK-COO - (aa100} -aa127; SEQ ID NO: 7)
CR50 = acetylated ^{N-LNEGTCMLGSFCACPPSFYGRNCEHDVRK-COO - (aa84} -aa112, includes the fucosylation region; SEQ ID NO: 8)
CR51 = acetylated ^{N-PHNTWLPKKCSLCKCWHGQLRCFPQAFLPGCD-COO - (aa119} -aa150; SEQ ID NO: 9)

Example 3: Null cell assay for inhibition of Cripto activity
The following describes the F9 Cripto null cell signaling assay that was used to assess inhibition of Cripto activity.

Day 0 A 6-well plate is coated with 2 ml / well of 0.1% gelatin for 15 minutes at 37 ° C. Seeding cells with 6 × 10 ⁵ F9 CRIPTO NULL cells per well Day 1 Transfection: Add each of the following samples to 300 μl of OptiMem1 to obtain Solution A for each sample:
Sample 1: 0.5 μg (N ₂ ) ₇ luciferase FAST reporter cDNA
And 1.5 μg empty vector cDNA.
Sample 2: 0.5 μg (N ₂ ) ₇ luciferase, 0.5 μg FAST, and 1 μg empty vector cDNA.
Sample 3: 0.5 μg (N ₂ ) ₇ luciferase, 0.5 μg Cripto ADD 0.5 FAST, and 0.5 μg empty vector cDNA.
Sample 4: 0.5 μg (N ₂ ) ₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNA.
Sample 5: 0.5 μg (N ₂ ) ₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNA.
Sample 6: 0.5 μg (N ₂ ) ₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNA.
Sample 7: 0.5 μg (N ₂ ) ₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNA.
Sample 8: 0.5 μg (N ₂ ) ₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNA.
Sample 9: 0.5 μg (N ₂ ) ₇ luciferase, 0.5 μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNA.

  Solution B contains 30 μl Lipofectamine and 270 μl OptiMem1.

  For each sample, solution A and solution B are mixed together. Incubate at room temperature for 45 minutes. Wash wells with 2 ml / well OptiMem1. Aspirate immediately before next step.

  Add 2.4 ml of OptiMem1 to each mixture of solution A + B, mix and add 1.5 ml / well to replicate wells. Incubate for 5 hours at 37 ° C. 1.5 ml / well DMEM and 20% FCS, 2 mM Gln, P / S are added to the wells and these wells receive samples 1-3. Add anti-Cripto antibody as follows: Sample 4 well: A27F6.1, 10 μg / ml; Sample 5 well: A27F6.1, 2 μg / ml; Sample 6 well: A40G12.8; 10 μg / ml, Sample 7 well: A40G12.8 2 μg / ml; Sample 8 well: A10B2.18, 10 μg / ml; Sample 9 well: A10B2.18, 2 μg / ml.

  Day 2 Remove media and wash cells with PBS (2 ml / well). DMEM and 0.5% FCS, 2 mM Gln, P / S are added to the same well with the same amount of Cripto antibody as the previous day.

Day 3 Develop luciferase signal. Wash wells (2 ml / well) with PBS + Ca ²⁺ and Mg ²⁺ . The LucLite kit (Packard catalog number; 6016911) is used. Bring buffer and substrate to room temperature. Dim the light. Reconstitute the substrate with 10 ml of buffer. Dilute 1: 1 with PBS and Ca ²⁺ and Mg ²⁺ . Aspirate well. Using a repeat pipettor, quickly add 250 μl of diluted substrate per well. Stir the solution and transfer 200 μl to the wells of a 96 well white opaque bottom plate (Falcon 35-3296). Read the plate on a luminometer using Winglow and export the data to Excel®.

  The results of this assay with specific antibodies of the invention are summarized in Table 3 below.

We also tested other concentrations of A27F6.1 antibody. We observed that adding 0.5-20 mg / ml of mab A27F6.1 to the media of these cells inhibited Cripto-induced luciferase signal by 34-95% (FIG. 1). . The inventors also tested other antibodies of the invention and observed that mab A6C12.11 and mab A8G3.5 also inhibited signal generation.

As an 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 F9 cells, except that ectopic expression of Nodal and ALK4 was required in these cells for reporter activation. We observed that mab A27F6.1 reduced luciferase activity in these cells by 90% at both 2 μg / ml and 20 μg / ml (FIG. 2).
Example 4: Assay for in vitro inhibition of tumor cell proliferation
Inhibition of Cripto activity can also be assayed by measuring proliferation of GEO cells in soft agar. For example, Cialdiello et al., 1994, Oncogene.
9: 291-98; Cialdiello et al., 1991, Cancer Res. 51: 1051-54.

We thawed 3% bactogar and maintained at 42 ° C. in a water bath. We mixed a 3% Bactogar solution with a pre-warmed complete medium to make a 0.6% Bactogar solution, which was maintained at 42 ° C. We plated 4 ml of the solution on a 6 cm dish and cooled it for at least 30 minutes to form an agar layer on the bottom. We trypsinized GEO cells and resuspended them at 10 ⁵ cells / ml in complete medium. We added the antibody or control to be assayed to this cell suspension and titrated the antibody from 20 μg to 1 μg. We mixed an equal volume of GEO cell suspension with 0.6% bactagar and placed 2 ml on top of the agar layer at the bottom. We cooled the plate for at least 1 hour. We incubated for 14 days at 37 ° C. in a CO ₂ incubator. The inventors counted colonies that could be confirmed without using a microscope. The absence of colonies compared to the negative control indicated that the tested antibodies inhibited tumor cell growth in vitro.

  This assay was used to obtain the results shown in Table 4 for antibodies A27F6.1 and B6G7.10, both of which demonstrate the ability to reduce the growth of GEO cell colonies.

We performed a growth inhibition assay with T-47D cells (ATCC), which are non-tumorigenic human breast carcinomas as described in Example 9.
Example 5: Assay for in vivo inhibition of tumor cell growth
To assess inhibition of tumor cell growth, human tumor cell lines are implanted subcutaneously in athymic nude mice, with or without additional chemotherapeutic treatment that can provide a synergistic or additive effect on tumor inhibition, The effect of the antibody of the present invention is observed.

  This assay can alternatively be performed using the following different tumor cell lines: For example, GEO (a fully differentiated human colon cancer in vitro cell line, obtained from the American Tissue Type Collection (ATCC)), DU -4475 (a breast cancer in vitro cell line obtained from ATCC), NCCIT (a testicular tumor cell line obtained from ATCC), or others known in the art. One example of such an assay is described below.

Animals are individually marked by punching their ears. GEO cell lines are passaged 1 to 4 times in vitro or in vivo. Animals are implanted with GEO cells subcutaneously in the right flank region. The following groups of animals may be used:
Group number Treatment Mouse number Saline control, 0.2 ml / mouse, 20
i. p. 3 times a week (M, W, F)
2. Mab, low dose, i. p. 10
3. Mab, medium dose, i. p. 104. Mab, high dose, i. p. 10
5. 5-FU, 30 mg / kg / infusion, i. p. , 10
3 Rx / wk (M, W, F)
6). Cisplatin, 2 mg / kg / infusion, s. c. , 10
Rx / wk (M, W, F)
7). Adriamycin, 1.6 mg / kg / infusion, i. p. , 10
3 Rx / wk (M, W, F)
8). Irinotecan, 10 mg / kg / infusion. , I. p. , 10
5 Rx / wk (MF)
9. MAb, low dose, i. p. + 5-FU 10
(Intermediate dose)
10. MAb, medium dose, i. p. + 5-FU 10
(Intermediate dose)
11. MAb, high dose, i. p. + 5-FU 10
(Intermediate dose)
12 MAb, low dose, i. p. + Cisplatin 10
Group number Action Mouse number
(Intermediate dose)
13. MAb, medium dose, i. p. + Cisplatin 10
(Intermediate dose)
14 MAb, high dose, i. p. + Cisplatin 10
(Intermediate dose)
15. MAb, low dose, i. p. + Adriamycin 10
(Intermediate dose)
16. MAb, medium dose, i. p. +10
Adriamycin (intermediate dose)
17. MAb, high dose, i. p. + Adriamycin 10
(Intermediate dose)
18. MAb, low dose, i. p. + Irinotecan 10
(Intermediate dose)
19 MAb, medium dose, i. p. +10
Irinotecan (intermediate dose)
20. MAb, high dose, i. p. + Irinotecan 10
(Intermediate dose)
Day 0: Implant the tumor and record the initial body weight of the animal.
Day 1: Start the above procedure.
Day 5: Start tumor sizing and body weight measurement and continue twice a week until the end of the experiment.

  Initial body weight, tumor size and weight measurement, histology at sacrifice, and immunohistochemical analysis for tumors are tested for Cripto expression, tumor growth, and their inhibition.

Example 6: In vivo xenograft tumor model-Cys rich blocking anti-Cripto antibody
To assess NCCIT response, a human testicular carcinoma cell line was implanted subcutaneously with an antibody that binds to Cripto's cys-rich domain. The experimental methods are listed below. The results are shown in FIG.

(Method and material)
Animals: Athymic nude mice were used. Animals were individually numbered by piercing the ears.
Tumor: NCCIT, mediastinal mixed germ cell human testicular carcinoma in vitro cell line (American
Originally obtained from Tissue Type Collection). Cell lines were passaged in vitro for 6 passages in RPMI-1640 / 10% FBS without antibiotics. The animals were implanted subcutaneously in the right flank of the animals with 5 × 10 ⁶ cells / 0.2 ml matrigel.

Treatment started on day 1.

Test schedule-Day 1: Mice were randomized into control and treatment groups. The initial weight of the animal was recorded. The first treatment was given to the antibody group. A dosing solution was made. The treatment was blinded to the technician until the assay was completed.
Day 0: Tumors were transplanted. Bacterial cultures were performed on tumors implanted in mice.
Day 1: The first treatment was given to the positive chemotherapy group.
Day 4: Initial tumor size measurements were recorded for tumor baseline in Matrigel. Tumor size and body weight continued to be recorded at 2x / week for mice. The study was monitored daily and any abnormal observations were recorded on the animals.
Endpoint: initial body weight Tumor size and body weight measurement.

Example 7: In vivo xenograft tumor model-EGF-like domain blocking anti-Cripto antibody
To assess the response of NCCIT, a human testicular carcinoma cell line was implanted subcutaneously with an antibody that binds to Cripto's EGF-like domain. The experimental methods are listed below. The results are shown in FIG.

(Method and material)
Animals: Athymic nude mice were used. Animals were individually numbered by piercing the ears.
Tumor: NCCIT, mediastinal mixed germinoma human testicular cancer in vitro cell line (American)
Originally obtained from Tissue Type Collection). Cell lines were passaged in vitro for 8 passages in RPMI-1640 / 10% FBS without antibiotics. The animals were implanted subcutaneously in the right flank of the animals with 5 × 10 ⁶ cells / 0.2 ml matrigel.

Treatment started on day 1.

Test schedule-Day 1: Mice were randomized into control and treatment groups. The initial weight of the animal was recorded. The first treatment was given to the antibody group. A dosing solution was made. The treatment was blind to the technician until the assay was completed.
Day 0: Tumors were transplanted. Bacterial cultures were performed on tumors implanted in mice. Bacterial cultures were negative for contamination at 24 and 48 hours after sampling.
Day 1: The first treatment was given to the positive chemotherapy group.
Day 4: Initial tumor size measurements were recorded for tumor baseline in Matrigel. Tumor size and body weight continued to be recorded at 2x / week for mice. The study was monitored daily and any abnormal observations were recorded on the animals.
Endpoint: initial body weight Tumor size and body weight measurement.

(Example 8: Cripto mab regulates ALK4 binding)
To assess whether Cripto-specific monoclonal antibodies could interfere with ALK4 (the ability of Cripto to bind to activin type I receptor), we used a 293 cell line that stably expresses ALK4. In order to create this cell line, 293 cells were treated with a plasmid expressing ALK4 tagged with the HA epitope at the C-terminus and a plasmid expressing puromycin with a ratio of 10: 1. The transfected cells were then selected in puromycin until colonies were formed, which were then picked, expanded, and using Western blot analysis for HA, ALK4 expression was analyzed, clone 21 (293 Alk4-21), compared with untransfected 293 cells in the control, it was found to express ALK4 high levels.

To analyze Cripto-ALK4 binding by flow cytometry, a purified soluble form of human Cripto (aa1-169) fused to the Fc portion of human IgG (CR (delC) -Fc) was used. Approximately 5 μg / ml CR (delC) -Fc protein or control Fc protein in 3 × 10 ⁵ 293 on ice for 30 minutes in 50 μl total volume of FACS buffer (PBS containing 0.1% BSA). Incubated with Alk4-21 cells. For samples containing anti-Cripto antibodies, 5 μg / ml CrdelC-FC was added to 50 μg / ml of each Cripto antibody (A10.B2.18, A40.G12.8, A27. F6.1, A8.H3.1, A19.A10.30, A6.F8.6, A8.G3.5, A6.C12.11). The cells were then washed in FACS buffer and bound Fc protein was detected by incubating the cells with R-phycoerythrin-conjugated goat anti-human IgG (Fc fragment specific) from Jackson Immunologics. Samples were then washed again, fixed in 1% paraformaldehyde in PBS, and analyzed using standard flow cytometry procedures. The results of the FACS assay are shown in FIG.

(Example 9: Cripto disrupts activin B-induced growth suppression of breast cancer cells)
Passage number 2 T47D cells from ATCC (maintained in RPMI / 10% FCS / 10 μg / ml insulin) were transfected with the expression plasmid for Ecotropic Receptor (EcoR; B. Ellenbaas) and 100 μg / ml Selection was made in medium containing hygromycin. Colonies of T-47D-EcoR that allowed infection with pBABE-GFP murine leukemia virus (MLV) were grown, infected with pBABE-hCr-PURO-MLV, and selected in puromycin medium. This oligoclonal strain (T-47D-hCr) was analyzed by FACS for hCr (human Cripto) expression using a specific anti-Cripto antibody. Approximately 4000 cells / well of T-47D-EcoR or T-47D-hCr, 25 ng activin B (R & D), or 2% serum with / without 25 ng activin B and 0.1-50 μg / ml A8G3.5 Plate in 96-well plates in medium containing. Medium with factors was changed daily for 7-8 days. 20 μl / well CellTiter AQ _ueous One solution (Promega) was added and the plates were collected, incubated for 2 hours at 37 ° C. and read at 490 nm.

  To grow T-47D and T-47D-EcoR cells at low serum concentrations with or without activin A or activin B and grow the cells using the MTT colorimetric assay Assayed. We observed that T-47D cell proliferation was inhibited by activin A or activin B by approximately 40% compared to untreated cells (FIG. 6). The inventors also observed that T-47D-CR cells were inhibited by activin A, but found that T-47D-CR cells were not responsive to activin B ( FIG. 6). This result indicates that the effect of Cripto on the proliferation of these cells is specific for activin B. We observed in control experiments that untreated T-47D and T-47D-CR cells did not differ in growth rate in either normal medium or low serum conditions.

  The inventors then tested whether the antibodies of the invention could inhibit Cripto-activin B interaction. We treated T-47D-CR with activin B in the presence of various antibodies of the invention. The present inventors observed that activin B growth inhibitory activity was restored in the presence of 10 μg / ml or 20 μg / ml of mab A8G3.5 (FIG. 7). The inventors observed that A27F6.1 mab was unable to restore the activin B growth inhibition of these cells.

  (Example 10: Cripto binds directly to activin B).

  The inventors have discovered that Cripto binds directly to activin B. We coated ELISA plates with purified activin B, activin A, TGFβ1, GDNF or BMP2 and incubated with CR-Fc. We then monitored binding by adding anti-Fc antibody conjugated to alkaline phosphatase, and adding alkaline phosphatase substrate and causing color development. We observed that CR-Fc bound to wells containing activin B, but not to activin A or other ligands (FIG. 8). Furthermore, we preincubated CR-Fc with activin A or activin B in solution before adding to activin B-coated plates and observed that only activin B inhibited binding (FIG. 9). ). These results confirmed that Cripto specifically binds to activin B.

  We also analyzed the Cripto-Activin B interaction using Biacore. The inventors have discovered that activin B binds directly to CR-Fc immobilized on Biacore chips with high strength but not to the control LTβR-Fc protein (FIG. 10). The inventors have also observed that activin A binding to CR-Fc can be ignored. These data indicate that activin B bound to Cripto with a rapid rate and apparent affinity in a solution measured in a competitive format assay of approximately 1 nM.

  Cripto anti-CFC monoclonal antibodies 2-2C9.2 and A8G3 modulated the interaction between Cripto and activin B. This was shown by co-immunoprecipitation experiments where Cripto binding to activin B was inhibited by the addition of A8G3 or 2-2C9.2. Inhibition was measured using a conventional Western blot technique with anti-activin B antibody.

  Some of the above-described embodiments of the present invention are outlined below, which include but are not limited to the following embodiments. As those skilled in the art will appreciate, many changes and modifications may be made to the various embodiments of the present invention without departing from the spirit of the invention. All such modifications are intended to be within the scope of the present invention.

  The entire disclosure of each publication cited herein is hereby incorporated by reference.

Claims (1)

Invention described in the specification.