WO2002088170A2 - Cripto blocking antibodies and uses thereof - Google Patents

Cripto blocking antibodies and uses thereof Download PDF

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Publication number
WO2002088170A2
WO2002088170A2 PCT/US2002/011950 US0211950W WO02088170A2 WO 2002088170 A2 WO2002088170 A2 WO 2002088170A2 US 0211950 W US0211950 W US 0211950W WO 02088170 A2 WO02088170 A2 WO 02088170A2
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Prior art keywords
cripto
antibody
antibodies
tumor
epitope
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PCT/US2002/011950
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French (fr)
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WO2002088170A3 (en
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Michele Sanicola-Nadel
Kevin Williams
Susan Schiffer
Paul Rayhorn
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Biogen, Inc.
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Priority to JP2002585468A priority Critical patent/JP4307845B2/en
Priority to DE60230868T priority patent/DE60230868D1/en
Priority to KR1020037014078A priority patent/KR100592357B1/en
Priority to HU0501113A priority patent/HUP0501113A3/en
Priority to EP02731384A priority patent/EP1390389B1/en
Priority to CA2443840A priority patent/CA2443840C/en
Priority to BRPI0209254-9A priority patent/BR0209254A/en
Application filed by Biogen, Inc. filed Critical Biogen, Inc.
Priority to EEP200300528A priority patent/EE200300528A/en
Priority to SI200230804T priority patent/SI1390389T1/en
Priority to EA200301158A priority patent/EA007469B1/en
Priority to IL15857302A priority patent/IL158573A0/en
Priority to YU84903A priority patent/RS51635B/en
Priority to SK1443-2003A priority patent/SK14432003A3/en
Priority to DK02731384T priority patent/DK1390389T3/en
Priority to MXPA03009797A priority patent/MXPA03009797A/en
Priority to JP2003580477A priority patent/JP2005520566A/en
Priority to PT02807155T priority patent/PT1494693E/en
Priority to PCT/US2002/031462 priority patent/WO2003083041A2/en
Priority to DE60238559T priority patent/DE60238559D1/en
Priority to AT02807155T priority patent/ATE490981T1/en
Priority to DK02807155.3T priority patent/DK1494693T3/en
Priority to EP02807155A priority patent/EP1494693B1/en
Priority to CA2480119A priority patent/CA2480119C/en
Priority to AU2002334799A priority patent/AU2002334799B2/en
Publication of WO2002088170A2 publication Critical patent/WO2002088170A2/en
Priority to US10/693,538 priority patent/US7531174B2/en
Priority to IS6999A priority patent/IS2662B/en
Priority to NO20034805A priority patent/NO20034805L/en
Priority to BG108363A priority patent/BG108363A/en
Publication of WO2002088170A3 publication Critical patent/WO2002088170A3/en
Priority to HK04101788.0A priority patent/HK1058935A1/en
Priority to US10/945,853 priority patent/US7582299B2/en
Priority to HK05105887.0A priority patent/HK1072381A1/en
Priority to US11/799,361 priority patent/US7674462B2/en
Priority to US12/317,476 priority patent/US8003763B2/en
Priority to US12/353,913 priority patent/US20090285818A1/en
Priority to JP2009020949A priority patent/JP2009161539A/en
Priority to US12/415,659 priority patent/US7888052B2/en
Priority to US13/213,499 priority patent/US8673303B2/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Cripto is a cell surface protein of 188 amino acid residues serendipitously isolated in a cDNA screen of a human embryonic carcinoma library (Ciccodicola et al., 1989, EMBO J., vol. 8, no. 7, pp. 1987-1991).
  • the Cripto protein has at least two notable domains: a cysteine-rich domain, and a domain first characterized as similar to the domain found in the epidermal growth factor (EGF) family.
  • EGF epidermal growth factor
  • the Cripto signaling pathway has remained elusive despite continued investigation, with the literature supporting activation of several different pathways, including a MAP kinase pathway (DeSantis et al., 1997, Cell Growth Differ., 8:1257- 1266; Kannan et al., 1997, J. Biol. Chem., 272:3330-3335), the TGF- ⁇ pathway (Gritsman et al., 1999, Development, 127:921-932; Schier et al., 2000, Nature, 403:385-389), possible interactions with the Wnt pathway (Salomon et al., Endocr Relat Cancer. 2000 Dec;7(4): 199-226; and cross talk with the EGF pathway (Bianco et al., 1999, J. Biol. Chem., 274:8624-8629).
  • Cripto protein overexpression is associated with many tumor types (including but not limited to breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach), as demonstrated by immunostaining of human tissue with rabbit polyclonal antibodies raised against small cripto peptides. Panico et al ., 1996, Int. J. Cancer, 65: 51-56; Byrne et al., 1998, J Pathology, 185:108-111; De Angelis et al., 1999, Int J Oncology, 14:437-440. The art is therefore in need of means of controlling, restricting, and or preventing such overexpression, modulating Cripto signaling, and modulating the consequences of Cripto expression (i.e., promotion and/or maintenance of cell transformation).
  • the antibody of the present invention specifically binds to an epitope in the ligand/receptor binding domain of Cripto.
  • Cripto can be selected from CR-1 (SEQ ID NO:l) or CR-3 (SEQ ID NO:2).
  • antibodies that specifically binds to the epitope in the ligand/receptor binding domain include for example A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318), A8G3.5 (ATCC ACCESSION NO. PTA-3317), A19A10.30, A8H3.1 (ATCC ACCESSION NO. PTA-3315), A27F6.1 (ATCC ACCESSION NO.
  • the epitope to which the antibodies of the present invention bind is in an EGF-like domain.
  • Antibodies that specifically bind to the epitope in the EGF-like domain include but are not limited to A40G12.8 (ATCC ACCESSION NO. PTA-3316), A8H3.1 (ATCC ACCESSION NO. PTA-3315), A27F6.1 (ATCC ACCESSION NO. PTA-3310), B6G7.10 (ATCC ACCESSION NO. PTA-3313), A17G12.1 (ATCC ACCESSION NO. PTA-3314) and A18B3.11 (ATCC ACCESSION NO. PTA-3312).
  • the epitope to which the antibodies of the present invention bind is in a cys-rich domain.
  • Antibodies that specifically bind to the epitope in the cys-rich domain include but are not limited to A19A10.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A6F8.6 (ATCC ACCESSION NO. PTA-3318) and A6C12.11.
  • the epitope to which the antibodies of the present invention bind is in the domain spanning amino acid residues 46-62 of Cripto.
  • Antibodies that specifically bind to the epitope in the domain spanning amino acid residues 46-62 of Cripto include but are not limited to A10B2.18 (ATCC ACCESSION NO. PTA-3311), B3F6.17 (ATCC ACCESSION NO. PTA-3319) and A17A2.16.
  • the present inventions also comtemplate antibodies which binds specifically to
  • Antibodies that bind specifically to Cripto and are capable of modulating Cripto signaling include but are not limited to, A40G12.8 (ATCC ACCESSION NO. PTA-3316), A8H3.1 (ATCC ACCESSION NO. PTA-3315), A27F6.1 (ATCC ACCESSION NO. PTA-3310), and A6C12.11.
  • the antibodies of the present invention which binds specifically to Cripto and are capable of modulating Cripto signaling bind to an epitope in an EGF-like domain or a cys-rich domain of Cripto.
  • the present inventions also comtemplate antibodies which binds specifically to Cripto and blocks the interaction between Cripto and ALK4.
  • Antibodies that bind specifically to Cripto and are capable of blocking the interaction between Cripto and AL 4 include but are not limited to, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A6F8.6 (ATCC ACCESSION NO. PTA-3318) and A6C12.11.
  • the antibodies of the present invention which binds specifically to Cripto and are capable of blocking the interaction between Cripto and ALK4 bind to an epitope in an EGF-like domain or a cys-rich domain of Cripto.
  • the present invention comtemplates antibodies which bind specifically to Cripto and are capable of modulating tumor growth.
  • Antibodies that specifically bind to Cripto and are capable of modulating tumor growth include but are not limited to, A27F6.1 (ATCC ACCESSION NO. PTA-3310), B6G7.10 (ATCC ACCESSION NO. PTA-3313) and A8G3.5 (ATCC ACCESSION NO. PTA-3317).
  • the antibodies of the present invention which bind specifically to Cripto and are capable of modulating tumor growth bind to an epitope in an EGF-like domain or a cys-rich domain of Cripto.
  • the antibodies of the present invention which bind specifically to Cripto, which are capable of modulating Cripto signaling, and which are capable of modulating tumor growth bind to an epitope in an EGF-like domain or a cys-rich domain of Cripto.
  • the present invention comtemplates antibodies which bind specifically to Cripto, which are capable of blocking the interaction between Cripto and ALK4, and which are capable of modulating tumor growth.
  • Antibodies that specifically bind to Cripto, which are capable of blocking the interaction between Cripto and AL 4, and which are capable of modulating tumor growth include but are not limited to A8G3.5 (ATCC ACCESSION NO. PTA-3317).
  • the antibodies of the present invention include but are not limited to monoclonal, polyclonal, humanized, chimeric and human antibodies.
  • the present invention also provides for a composition for administration to a subject having a tumor that expresses Cripto comprising at least one of the antibodies described above.
  • the subject is human.
  • the composition may include a pharmaceutically acceptable excipient.
  • the antibodies described above can be conjugated to a chemotherapeutic agent or be provided in combination with a nonconjugated chemotherapeutic.
  • Contemplated in another aspect of the invention are methods of modulating growth of tumor cells in vitro in a sample comprising the step of adding to the sample the compositions described above.
  • the methods of the present invention are particularly useful in modulating growth of tumor cells and/or treating a subject (i.e. a human) having a tumor where the tumor cell is selected from breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumor cells.
  • the present invention contemplates methods of determining whether a tissue expresses Cripto, comprising the step of analyzing tissue from the subject in an immunoassay using any of the antibodies described above. Also contemplated are methods of determining whether a cell line overexpresses Cripto, comprising the step of analyzing the cell line in an immunoassay using any of the antibodies described above.
  • antibodies that specifically bind to Cripto include, for example, antibodies that specifically bind to an epitope in the ligand/receptor binding domain of either a native Cripto protein or a denatured form of Cripto; antibodies that bind an EGF-like domain, a cys-rich domain, or a peptide (e.g., from about 3 to about 20 amino acids) from the region comprising amino acid residues 46 to 150; antibodies that bind Cripto and modulate Cripto signalling; antibodies that bind Cripto and modulate tumor cell growth; and antibodies that bind to Cripto, modulate Cripto signaling, and modulate tumor cell growth. These antibodies are selected using conventional in vitro assays for selecting antibodies which bind the ligand/receptor binding domain, modulate Cripto signaling, or modulate tumor cell growth.
  • chimeric antibodies may be constructed, in which the antigen binding domain from an animal antibody is linked to a human constant domain (an antibody derived initially from a nonhuman mammal in which recombinant DNA technology has been used to replace all or part of the hinge and constant regions of the heavy chain and/or the constant region of the light chain, with corresponding regions from a human immunoglobulin light chain or heavy chain) (see, e.g., Cabilly et al., United States patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. 81: 6851-55, 1984). Chimeric antibodies reduce the immunogenic responses elicited by animal antibodies when used in human clinical treatments.
  • Humanized antibodies minimize the use of heterologous (inter-species) sequences in antibodies for use in human therapies, and are less likely to elicit unwanted immune responses. Primatized antibodies can be produced similarly.
  • the term “compound” means any identifiable chemical or molecule, including, but not limited to, ion, atom, small molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and such compound can be natural or synthetic.
  • the terms “modulates” or “modifies” means an increase or decreas'e in the amount, quality, or effect of a particular activity or protein.
  • blocking the interaction between Cripto and ALK 4" means an increase or decrease in the interaction, i.e. binding, between Cripto and ALK4, by about 5%, preferably 10%, more preferably 20%, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 100%,.
  • Activity may be measured by assays known in the art, such as the binding assay shown in Example 8.
  • treating refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism. Treating includes maintenance of inhibited tumor growth, and induction of remission.
  • therapeutic effect refers to the inhibition of an abnormal condition.
  • administering relates to a method of incorporating a compound into cells or tissues of an organism.
  • the abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.
  • Cells existing outside the organism can be maintained or grown in cell culture dishes, or in another organism.
  • many techniques exist in the art to administer compounds including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
  • multiple techniques exist in the art to administer the compounds including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques.
  • Administration may be accomplished by the many modes known in the art, e.g., oral, intravenous, intraperitoneal, intramuscular, and the like.
  • an "effective amount" is an amount sufficient to effect beneficial or desired clinical results (i.e., amounts that eliminate or reduce the patient's tumor burden).
  • An effective amount can be administered in one or more administrations.
  • an effective amount of the antibodies of the present invention is an amount of the antibodies that is sufficient to ameliorate, stabilize, or delay the development of the Cripto-associated disease state, particularly Cripto-associated tumors. Detection and measurement of these indicators of efficacy are discussed below.
  • An example of a typical treatment regime includes administering by intravenous infusion to the subject antibodies of the invention on a weekly schedule, at a dose of about 2-5 mg/kg.
  • the antibodies are administered in an outpatient chemoinfusion unit, unless the patient requires hospitalization.
  • Other administration regimes known in the art are also contemplated.
  • “Cripto overexpression” is intended to mean the expression of Cripto by a tissue which expression is greater than the Cripto expression of adjacent normal tissue in a statistically significant amount.
  • “Chemotherapeutics” refers to any agents identified in the art as having therapeutic effect on the inhibition of tumor growth, maintenace of inhibited tumor growth, and/or induction of remission, such as natural compounds, synthetic compounds, proteins, modified proteins, and radioactive compounds. Chemotherapeutic agents contemplated herewith include agents that can be conjugated to the antibodies of the present invention or alternatively agents that can be used in combination with the antibodies of the present invention without being conjugated to the antibody.
  • chemotherapeutics that can be conjugated to the antibodies of the present invention include, but are not limited to radioconjugates (90 Y, 1311, 99mTc, lllln, 186Rh, et al.), tumor-activated prodrugs (maytansinoids, CC-1065 analogs, clicheamicin derivatives, anthracyclines, vinca alkaloids, et al.), ricin, diptheria toxin, pseudomonas exotoxin.
  • radioconjugates 90 Y, 1311, 99mTc, lllln, 186Rh, et al.
  • tumor-activated prodrugs maytansinoids, CC-1065 analogs, clicheamicin derivatives, anthracyclines, vinca alkaloids, et al.
  • ricin diptheria toxin
  • pseudomonas exotoxin pseudomonas exotoxin.
  • a “subject” refers to vertebrates, particularly members of a mammalian species, and includes but is not limited to domestic animals, sports animals, and primates, including humans.
  • Antibodies of the Invention The antibodies of the invention specifically bind to Cripto: As used herein,
  • Cripto includes the CR-1 Cripto protein, the CR-3 Cripto protein, and fragments thereof. Such fragments may be entire domains, such as the extracellular or intracellular domains, the EGF-like domain, the cys-rich domain, the receptor binding domain, and the like. Such fragments may also include contiguous and noncontiguous epitopes in any domain of the Cripto protein.
  • the 188 amino acid sequence for CR-1 is as follows [SEQ ID NO: 1]: DCRK ARFSYSVI IMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS IWPQEEPAIRPRSSQ VPPMGIQHSKELNRTCCLNGGTCM GSFCACPPS FYGRNCEHDVRK ⁇ NCGSVPHDT LPKKCSLCKC HGQLRCFPQAF PGCD GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYYY
  • the 188 amino acid sequence for CR-3 is as follows [SEQ ID NO: 2]:
  • the antibodies of the invention bind to an epitope in the EGF-like domain of Cripto.
  • the EGF-like domain spans from about amino acid residue 75 to about amino acid residue 112 of the mature Cripto protein.
  • Epitopes in the EGF-like domain may comprise linear or nonlinear spans of amino acid residues.
  • Example of linear epitopes contemplated include but are not limited to about residues 75-85, 80-90, 85-95, 90-100, 95-105, 100-110, or 105-112.
  • the epitope in the EGF domain is an epitope formed in the conformational native Cripto protein versus a denatured Cripto protein.
  • the antibodies of the invention bind to an epitope in the cys-rich domain of Cripto.
  • the cys-rich domain spans from about amino acid residue 114 to about amino acid residue 150 of the mature Cripto protein.
  • Epitopes in the cys- rich domain may comprise linear or nonlinear spans of amino acid residues.
  • Example of linear epitopes contemplated include but are not limited to about residues 114-125, 120-130, 125-135, 130-140, 135-145, or 140-150.
  • the epitope in the cys-rich domain is an epitope formed in the conformational native Cripto protein versus a denatured Cripto protein
  • the present invention provides antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for Cripto or fragments thereof.
  • Antibody fragments including Fab, Fab', F(ab') 2 , and F v , are also provided by the invention.
  • the terms "specific " and "selective,” when used to describe binding of the antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind Cripto polypeptides.
  • Hybridomas that produce such antibodies are also intended as aspects of the invention.
  • the invention provides an anti-idiotypic antibody specific for an antibody that is specific for Cripto.
  • anti-idiotypic antibodies see, e.g., U.S. Patents 6,063,379 and 5,780,029.
  • antibodies contain relatively small antigen binding domains that can be isolated chemically or by recombinant techniques. Such domains are useful Cripto binding molecules themselves, and also may be reintroduced into human antibodies, or fused to a chemotherapeutic or polypeptide.
  • the invention provides a polypeptide comprising a fragment of a Cripto- specific antibody, wherein the fragment and associated molecule, if any, bind to the Cripto.
  • the invention provides polypeptides that are single chain antibodies and CDR-grafted antibodies. For a more detailed discussion of CDR-grafted antibodies, see, e.g., U.S. Patent5,859,205.
  • non-human antibodies may be humanized by any of the methods known in the art. Humanized antibodies are useful for in vivo therapeutic applications.
  • recombinant "humanized" antibodies may be synthesized. Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobulin light or heavy chain. That is, they are chimeras comprising mostly human immunoglobulin sequences into which the regions responsible for specific antigen-binding have been inserted (see, e.g., PCT patent application WO 94/04679).
  • Another embodiment of the invention includes the use of human antibodies, which can be produced in nonhuman animals, such as transgenic animals harboring one or more human immunoglobulin transgenes. Such animals may be used as a source for splenocytes for producing hybridomas, as is described in United States patent 5,569,825, WO00076310, WO00058499 and WO00037504 and incorporated by reference herein.
  • Signal Modulation is described in United States patent 5,569,825, WO00076310, WO00058499 and WO00037504 and incorporated by reference herein.
  • Also contemplated are methods of treating a subject suffering from a disorder associated with abnormal levels (i.e. elevated or depleted) of Cripto wherein the method comprises administering to the subject an effective amount of an antibody which specifically forms a complex with Cripto and is directed to the epitope to which an antibody selected from the group consisting of A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318), A7H1.19, A8F1.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A8H3.1 (ATCC ACCESSION NO. PTA-3315), A8H3.2, A19A10.30, A10B2.18 (ATCC ACCESSION NO.
  • the vector pEAGl 100 is a derivative of GEBCO-BRL Life Technologies plasmid pCMV-Sport- betagal, the use of which in CHO transient transfections was described by Schifferli et al., 1999, Focus 21: 16. It was made by removing the reporter gene beta-galactosidase Notl fragment from the plasmid pCMV-Sport-Betagal (catalog number 10586-014) as follows: The plasmid was digested with Notl and EcoRV, the 4.38 kb Notl vector backbone fragment was gel-purified and ligated. Ligated DNA was transformed into competent E. coli DH5alpha.
  • the eluted CR(del C)-Fc protein is injected into mice, and standard hybridoma techniques known to those of skill in the art are used to generate monoclonal antibodies.
  • the mouse was boosted intravenously with 50 ⁇ g CR del C-Fc the day before fusion.
  • the mouse spleen cells were fused with FL653 myeloma cell at a 1 spleen :6 myeloma ratio and were plated at 100,000, 33,000and 11,000 cells per well into 96 well tissue culture plates in selection media. Wells positive for growth were screened by FACS and ELIS A a week later. Two fusions were performed.
  • B. Screening of Antibodies Supernatants resulting from the first or second fusion were screened first on
  • Cripto positive cell lines may be used to assay the monoclonal antibodies for binding to Cripto using cell surface staining and flow cytometry as follows: Release cells from T162 flasks with 2 ml PBS " with 5 mM EDTA, 10 min., 37°C. Bring up to 20 ml with media with serum, pipetting up and down several times to unclump cells. Spin at 1200 rpm for 5 minutes. Wash cells with 5-10 ml 4° C PBS with 0.1% BSA (wash buffer). Spin at 1200 rpm for 5 minutes. Resuspend at 4xl0 6 - 10 7 /ml in wash buffer. Keep on ice.
  • Purified antibodies are diluted to 1-10 ⁇ g/ml in wash buffer. Add 50 ⁇ l of cells to a 96-well Linbro V bottomed plate (ICN 7632105). Plate one well of cells for each control for each cell line to be analyzed, including cells for no antibody, 2° antibody only, hybridoma media, positive control antibody supernatant, if available, or purified, and an IgG subclass control (if using purified antibodies).
  • the following describes an F9 Cripto null cell signaling assay used to assess inhibition of Cripto signaling.
  • Sample 1 0.5 ⁇ g (N 2 ) 7 luciferase FAST reporter cDNA plus 1.5 ⁇ g empty vector cDNA.
  • Sample 2 0.5 ⁇ g (N ) 7 luciferase, 0.5 ⁇ g FAST, and 1 ⁇ g empty vector cDNAs.
  • Sample 3 0.5 ⁇ g (N 2 ) 7 luciferase, 0.5 ⁇ g Cripto ADD 0.5 FAST, and -0.5 ⁇ g empty vector cDNAs.
  • Sample 4 0.5 ⁇ g (N 2 ) 7 luciferase, 0.5 ⁇ g Cripto, 0.5 FAST, and 0.5 ⁇ g empty vector cDNAs
  • Sample 5 0.5 ⁇ g (N 2 ) luciferase, 0.5 ⁇ g Cripto, 0.5 FAST, and 0.5 ⁇ g empty vector cDNAs.
  • Sample 6 0.5 ⁇ g (N 2 ) 7 luciferase, 0.5 ⁇ g Cripto, 0.5 FAST, and 0.5 ⁇ g empty vector cDNAs.
  • Sample 7 0.5 ⁇ g (N 2 ) 7 luciferase, 0.5 ⁇ g Cripto, 0.5 FAST, and 0.5 ⁇ g empty vector cDNAs.
  • Sample 8 0.5 ⁇ g (N 2 ) 7 luciferase, 0.5 ⁇ g Cripto, 0.5 FAST, and 0.5 ⁇ g empty vector cDNAs.
  • Sample 9 0.5 ⁇ g (N 2 ) 7 luciferase, 0.5 ⁇ g Cripto, 0.5 FAST, and 0.5 ⁇ g empty vector cDNAs.
  • Solution B comprises 30 ⁇ l of Lipofectamine plus 270 ⁇ l of OptiMeml.
  • Sample 6 wells A40G12.8; lO ⁇ g/ml, Sample 7 wells: A40G12.8 2 ⁇ g/ml; Sample 8 wells: A10B2.18, lO ⁇ g/ml; Sample 9 wells: A10B2.18, 2 ⁇ g/ml.
  • Day 2 Remove media, wash cells with PBS, 2ml/well. Add DMEM+0.5% FCS, 2mM Gin, P/S with the same amounts of Cripto antibodies as the previous day, to the same wells.
  • Day 0 Implant tumor, record initial body weight of animals.
  • a human testicular carcinoma cell line was implanted subcutaneously with an antibody which binds to a cys-rich domain of Cripto.
  • Day -1 Randomized mice into control and treatments groups. Recorded initial body weight of animals. Administered first treatments to antibody groups. Dosing solutions were made. Treatments were blinded to the technicians until the assay was terminated.

Abstract

The invention provides Cripto blocking antibodies, or biologically functional fragments thereof, and uses thereof. Antibodies which bind Cripto and modulate Cripto signaling are provided. Antibodies which bind Cripto and block the interaction between Cripto and ALK4 are provided. Antibodies which bind Cripto and modulate tumor growth are also provided. Antibodies which bind Cripto, modulate signaling, and modulate tumor growth are also provided. Antibodies which bind Cripto, block the interaction between Cripto and ALK4 and modulate tumor growth are provided. The invention also provides methods of using these antibodies in therapeutic, diagnostic, and research applications.

Description

CRIPTO BLOCKING ANTIBODIES AND USES THEREOF Related Applications
This is a continuation of U.S.S.N. 60/367,002, filed March 22, 2002, which is a continuation-in-part of U.S.S.N. 60/301,091, filed June 26, 2001, which is a continuation-in-part of U.S.S.N. 60/293,020, filed on May 17, 2001, which is a continuation-in-part of U.S.S.N. 60/286,782, filed on April 26, 2001. The entire disclosure of each of the aforesaid patent applications are incorporated herein by reference.
Technical Field of the Invention The present invention relates generally to the fields of genetics and cellular and molecular biology. More particularly, the invention relates to antibodies which bind to and modulate the signaling of Cripto, kits comprising such antibodies, and methods which use the antibodies.
Background of the Invention Cripto is a cell surface protein of 188 amino acid residues serendipitously isolated in a cDNA screen of a human embryonic carcinoma library (Ciccodicola et al., 1989, EMBO J., vol. 8, no. 7, pp. 1987-1991). The Cripto protein has at least two notable domains: a cysteine-rich domain, and a domain first characterized as similar to the domain found in the epidermal growth factor (EGF) family. Cripto was originally classified as a member of the EGF family (Ciccodicola et al., supra); however, subsequent analysis showed that Cripto did not bind any of the known EGF receptors and its EGF-like domain was actually divergent from the EGF family (Bianco et al, 1999, J. Biol. Chem., 274:8624-8629).
The Cripto signaling pathway has remained elusive despite continued investigation, with the literature supporting activation of several different pathways, including a MAP kinase pathway (DeSantis et al., 1997, Cell Growth Differ., 8:1257- 1266; Kannan et al., 1997, J. Biol. Chem., 272:3330-3335), the TGF-β pathway (Gritsman et al., 1999, Development, 127:921-932; Schier et al., 2000, Nature, 403:385-389), possible interactions with the Wnt pathway (Salomon et al., Endocr Relat Cancer. 2000 Dec;7(4): 199-226; and cross talk with the EGF pathway (Bianco et al., 1999, J. Biol. Chem., 274:8624-8629).
U.S. Patent 5,256,643 and two divisional applications related thereto (US. Patents 5,654,140 and 5,792,616), disclose a human Cripto gene, Cripto protein, and antibodies to Cripto.
U.S. Patent 5,264,557 and three divisional applications related thereto (U.S. Patents 5,620,866, 5,650,285, and 5,854,399), disclose a human Cripto-related gene and protein. Also disclosed are antibodies which bind to the Cripto-related protein but do not cross react by binding to the Cripto protein itself.
Cripto protein overexpression is associated with many tumor types (including but not limited to breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach), as demonstrated by immunostaining of human tissue with rabbit polyclonal antibodies raised against small cripto peptides. Panico et al ., 1996, Int. J. Cancer, 65: 51-56; Byrne et al., 1998, J Pathology, 185:108-111; De Angelis et al., 1999, Int J Oncology, 14:437-440. The art is therefore in need of means of controlling, restricting, and or preventing such overexpression, modulating Cripto signaling, and modulating the consequences of Cripto expression (i.e., promotion and/or maintenance of cell transformation).
Summary of the Invention
The present invention provides novel antibodies which specifically bind to
Cripto, and methods of making and using such antibodies. The invention also provides antibodies which bind to Cripto, and modulate Cripto signaling or protein interaction, e.g., an antibody which binds to Cripto such that the signal resulting from a protein interaction with Cripto is modulated downward. The invention also provides antibodies which bind to Cripto and block the interaction between Cripto and ALK4. The invention also provides antibodies which bind to Cripto and modulate tumor growth. The invention also provides antibodies which bind to Cripto, modulate Cripto signaling and modulate tumor growth. The invention also provides antibodies which bind to Cripto, block the interaction between Cripto and ALK4 and modulate tumor growth
In one aspect of the invention, the antibody of the present invention specifically binds to an epitope selected from the group of epitopes to which antibodies A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318), A7H1.19, A8F1.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A8H3.1 (ATCC ACCESSION NO. PTA-3315), A8H3.2, A19A10.30, A10B2.18 (ATCC ACCESSION NO. PTA-3311), A27F6.1 (ATCC ACCESSION NO. PTA-3310), A40G12.8 (ATCC ACCESSION NO. PTA- 3316), A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14, A17A2.16, A17C12.28, A17G12.1 (ATCC ACCESSION NO. PTA-3314), A17H6.1, A18B3.11 (ATCC ACCESSION NO. PTA-3312), A19E2.7, B3F6.17 (ATCC ACCESSION NO. PTA- 3319), B6G7.10 (ATCC ACCESSION NO. PTA-3313), B11H8.4 bind.
In another aspect of the invention, the antibody of the present invention specifically binds to an epitope in the ligand/receptor binding domain of Cripto. Cripto can be selected from CR-1 (SEQ ID NO:l) or CR-3 (SEQ ID NO:2). In a more particular embodiment, antibodies that specifically binds to the epitope in the ligand/receptor binding domain include for example A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318), A8G3.5 (ATCC ACCESSION NO. PTA-3317), A19A10.30, A8H3.1 (ATCC ACCESSION NO. PTA-3315), A27F6.1 (ATCC ACCESSION NO. PTA-3310), A40G12.8 (ATCC ACCESSION NO. PTA-3316), A17G12.1 (ATCC ACCESSION NO. PTA-3314), A18B3.11 (ATCC ACCESSION NO. PTA-3312) and B6G7.10 (ATCC ACCESSION NO. PTA-3313).
In one embodiment the epitope to which the antibodies of the present invention bind is in an EGF-like domain. Antibodies that specifically bind to the epitope in the EGF-like domain include but are not limited to A40G12.8 (ATCC ACCESSION NO. PTA-3316), A8H3.1 (ATCC ACCESSION NO. PTA-3315), A27F6.1 (ATCC ACCESSION NO. PTA-3310), B6G7.10 (ATCC ACCESSION NO. PTA-3313), A17G12.1 (ATCC ACCESSION NO. PTA-3314) and A18B3.11 (ATCC ACCESSION NO. PTA-3312). In another embodiment the epitope to which the antibodies of the present invention bind is in a cys-rich domain. Antibodies that specifically bind to the epitope in the cys-rich domain include but are not limited to A19A10.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A6F8.6 (ATCC ACCESSION NO. PTA-3318) and A6C12.11. In another embodiment the epitope to which the antibodies of the present invention bind is in the domain spanning amino acid residues 46-62 of Cripto. Antibodies that specifically bind to the epitope in the domain spanning amino acid residues 46-62 of Cripto include but are not limited to A10B2.18 (ATCC ACCESSION NO. PTA-3311), B3F6.17 (ATCC ACCESSION NO. PTA-3319) and A17A2.16. The present inventions also comtemplate antibodies which binds specifically to
Cripto and are capable of modulating Cripto signaling. Antibodies that bind specifically to Cripto and are capable of modulating Cripto signaling, include but are not limited to, A40G12.8 (ATCC ACCESSION NO. PTA-3316), A8H3.1 (ATCC ACCESSION NO. PTA-3315), A27F6.1 (ATCC ACCESSION NO. PTA-3310), and A6C12.11. In one embodiment the antibodies of the present invention which binds specifically to Cripto and are capable of modulating Cripto signaling bind to an epitope in an EGF-like domain or a cys-rich domain of Cripto.
The present inventions also comtemplate antibodies which binds specifically to Cripto and blocks the interaction between Cripto and ALK4. Antibodies that bind specifically to Cripto and are capable of blocking the interaction between Cripto and AL 4, include but are not limited to, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A6F8.6 (ATCC ACCESSION NO. PTA-3318) and A6C12.11. In one embodiment the antibodies of the present invention which binds specifically to Cripto and are capable of blocking the interaction between Cripto and ALK4 bind to an epitope in an EGF-like domain or a cys-rich domain of Cripto.
In another aspect, the present invention comtemplates antibodies which bind specifically to Cripto and are capable of modulating tumor growth. Antibodies that specifically bind to Cripto and are capable of modulating tumor growth include but are not limited to, A27F6.1 (ATCC ACCESSION NO. PTA-3310), B6G7.10 (ATCC ACCESSION NO. PTA-3313) and A8G3.5 (ATCC ACCESSION NO. PTA-3317).
In one embodiment the antibodies of the present invention which bind specifically to Cripto and are capable of modulating tumor growth bind to an epitope in an EGF-like domain or a cys-rich domain of Cripto.
In yet another aspect, the present invention comtemplates antibodies which bind specifically to Cripto, which are capable of modulating Cripto signaling, and which are capable of modulating tumor growth. Antibodies that specifically bind to Cripto, which are capable of modulating Cripto signaling, and which are capable of modulating tumor growth, include but are not limited to A27F6.1 (ATCC ACCESSION NO. PTA-3310).
In one embodiment the antibodies of the present invention which bind specifically to Cripto, which are capable of modulating Cripto signaling, and which are capable of modulating tumor growth bind to an epitope in an EGF-like domain or a cys-rich domain of Cripto.
In yet another aspect, the present invention comtemplates antibodies which bind specifically to Cripto, which are capable of blocking the interaction between Cripto and ALK4, and which are capable of modulating tumor growth. Antibodies that specifically bind to Cripto, which are capable of blocking the interaction between Cripto and AL 4, and which are capable of modulating tumor growth, include but are not limited to A8G3.5 (ATCC ACCESSION NO. PTA-3317).
In another embodiment, the present invention provides an antibody produced by a hybridoma selected from the group consisting of A6F8.6 (ATCC Accession No. PTA- 3318), A8G3.5 (ATCC Accession No. PTA-3317), A8H3.1 (ATCC Accession No. PTA-3315), A10B2.18 (ATCC Accession No. PTA-3311), A27F6.1 (ATCC Accession No. PTA-3310), A40G12.8 (ATCC Accession No. PTA-3316), A17G12.1 (ATCC Accession No. PTA-3314), A18B3.11 (ATCC Accession No. PTA-3312), B3F6.17 (ATCC Accession No. PTA-3319), and B6G7.10 (ATCC Accession No. PTA-3313).
The antibodies of the present invention include but are not limited to monoclonal, polyclonal, humanized, chimeric and human antibodies.
The present invention also provides for a composition for administration to a subject having a tumor that expresses Cripto comprising at least one of the antibodies described above. In a more particular embodiment the subject is human. The composition may include a pharmaceutically acceptable excipient. The antibodies described above can be conjugated to a chemotherapeutic agent or be provided in combination with a nonconjugated chemotherapeutic. Contemplated in another aspect of the invention are methods of modulating growth of tumor cells in vitro in a sample comprising the step of adding to the sample the compositions described above.
Also comtemplated are methods of modulating growth of tumor cells in vivo in a subject comprising the step of administering to the subject an effective amount of the compositions described above. In a particular embodiment the subject is human.
Another aspect of the invention are methods of treating subjects having a tumor that over-expresses Cripto comprising administering to the subject the compositions described above in an effective amount. Compositions for administration may include pharmaceutically acceptable excipients, antibodies conjugated to chemotherapeutic agents and antibodies administered in combination with nonconjugated chemotherapeutic agents
The methods of the present invention are particularly useful in modulating growth of tumor cells and/or treating a subject (i.e. a human) having a tumor where the tumor cell is selected from breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumor cells.
In yet another embodiment, the present invention contemplates methods of determining whether a tissue expresses Cripto, comprising the step of analyzing tissue from the subject in an immunoassay using any of the antibodies described above. Also contemplated are methods of determining whether a cell line overexpresses Cripto, comprising the step of analyzing the cell line in an immunoassay using any of the antibodies described above.
These and other aspects of the invention are set forth in greater detail below in the Detailed Description of the Invention. Detailed Description of the Invention Antibodies that specifically bind to Cripto and their uses for modulating Cripto signaling or protein interaction, and/or block the interaction between Cripto and ALK4, and/or modulate the growth of tumor cells have been discovered. Various classes of antibodies that specifically bind to Cripto have been discovered, including, for example, antibodies that specifically bind to an epitope in the ligand/receptor binding domain of either a native Cripto protein or a denatured form of Cripto; antibodies that bind an EGF-like domain, a cys-rich domain, or a peptide (e.g., from about 3 to about 20 amino acids) from the region comprising amino acid residues 46 to 150; antibodies that bind Cripto and modulate Cripto signalling; antibodies that bind Cripto and modulate tumor cell growth; and antibodies that bind to Cripto, modulate Cripto signaling, and modulate tumor cell growth. These antibodies are selected using conventional in vitro assays for selecting antibodies which bind the ligand/receptor binding domain, modulate Cripto signaling, or modulate tumor cell growth.
The methods of this invention are useful in the therapy of malignant or benign tumors of mammals where the growth rate of the tumor (which is an abnormal rate for the normal tissue) is at least partially dependent upon Cripto. Abnormal growth rate is a rate of growth which is in excess of that required for normal homeostasis and is in excess of that for normal tissues of the same origin.
Definitions Various definitions are made throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art. As used herein, the term "region" means a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.
As used herein, the term "domain" refers to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region. Examples of protein domains include, but are not limited to the extracellular domain (spans from about residue 31 to about residue 188 of Cripto, including Cripto, CR-1 (SEQ ED NO: 1) and CR-3 (SEQ ED NO:2)) and transmembrane domain (spans from about residue 169 to about residue 188 of Cripto, including Cripto, CR-1 (SEQ ED NO: 1) and CR-3 (SEQ ED NO: 2)). A ligand/receptor binding domain of the Cripto protein spans from about residue 75 to about residue 150 of Cripto, including Cripto, CR-1 (SEQ ED NO: 1) and CR-3 (SEQ ID NO:2) and includes the EGF-like domain of Cripto, which spans, for example, from about residue 75 to about residue 112 of Cripto, including Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ED NO:2) and the cysteine- rich domain of Cripto, which spans, for example, from about residue 114 to about residue 150 of Cripto, including Cripto, CR-1 (SEQ ID NO: 1) and CR-3 (SEQ ID NO:2). For example, many monoclonal antibodies of the present invention have been identified as binding to the EGF-like or cys-rich domains. Additionally monoclonal antibody A10B2.18 (ATCC ACCESSION NO. PTA-3311), B3F6.17 (ATCC ACCESSION NO. PTA-3319) and A17A2.16 have been identified as binding to an epitope formed in a domain in the region spanning amino acid residues 46-62, upstream of the EGF-like domain. See Example 3 below. An epitope in the ligand/receptor binding domain is an epitope, whether formed in the conformational native Cripto protein, or the denatured Cripto protein, to which antibodies may bind.
As used herein, the term "antibody" is meant to refer to complete, intact antibodies, and Fab, Fab', F(ab)2, and other fragments thereof. Complete, intact antibodies include, but are not limited to, monoclonal antibodies such as murine monoclonal antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, and humanized antibodies. Various forms of antibodies may be produced using standard recombinant DNA techniques (Winter and Milstein, Nature 349: 293-99, 1991). For example, "chimeric" antibodies may be constructed, in which the antigen binding domain from an animal antibody is linked to a human constant domain (an antibody derived initially from a nonhuman mammal in which recombinant DNA technology has been used to replace all or part of the hinge and constant regions of the heavy chain and/or the constant region of the light chain, with corresponding regions from a human immunoglobulin light chain or heavy chain) (see, e.g., Cabilly et al., United States patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. 81: 6851-55, 1984). Chimeric antibodies reduce the immunogenic responses elicited by animal antibodies when used in human clinical treatments.
In addition, recombinant "humanized" antibodies may be synthesized. Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobulin light or heavy chain. That is, they are chimeras comprising mostly human immunoglobulin sequences into which the regions responsible for specific antigen-binding have been inserted (see, e.g., PCT patent application WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated and the portion of the variable region sequences responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of the human antibody genes in which the antigen binding regions have been deleted. Humanized antibodies minimize the use of heterologous (inter-species) sequences in antibodies for use in human therapies, and are less likely to elicit unwanted immune responses. Primatized antibodies can be produced similarly.
Another embodiment of the invention includes the use of human antibodies, which can be produced in nonhuman animals, such as transgenic animals harboring one or more human immunoglobulin transgenes. Such animals may be used as a source for splenocytes for producing hybridomas, as is described in US Patent 5,569,825.
Antibody fragments and univalent antibodies may also be used in the methods and compositions of this invention. Univalent antibodies comprise a heavy chain/light chain dimer bound to the Fc (or stem) region of a second heavy chain. "Fab region" refers to those portions of the chains which are roughly equivalent, or analogous, to the sequences which comprise the Y branch portions of the heavy chain and to the light chain in its entirety, and which collectively (in aggregates) have been shown to exhibit antibody activity. A Fab protein includes aggregates of one heavy and one light chain (commonly known as Fab7), as well as tetramers which correspond to the two branch segments of the antibody Y, (commonly known as F(ab)2), whether any of the above are covalently or non-covalently aggregated, so long as the aggregation is capable of specifically reacting with a particular antigen or antigen family.
Any of the antibodies of the invention may optionally be conjugated to a chemotherapeutic, as defined below. As used herein, the term "binding" means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof, including the interaction between an antibody and a protein. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc. The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates. Binding may be detected in many different manners. Methods of detecting binding are well-known to those of skill in the art.
As used herein, "an antibody capable of internalizing Cripto" means an antibody which enters the cell while removing Cripto from the cell surface. One can screen for Cripto antibodies which are capable of internalizing Cripto by using fluorescent labeled Cripto monoclonal antibodies. In order to determine which antibodies internalize into the Cripto positive cells one can assay for the uptake of the fluorescent signal of the antibodies into the cells by viewing the cells under a fluorescent and/or confocal microscope. Those antibodies that get internalized will be seen as fluorescent signals in the cytoplasmic and or cellular vesicles. Non-limiting examples of Cripto antibodies capable of internalizing Cripto include A27F6.1 and B3F6.17.
As used herein, the term "compound" means any identifiable chemical or molecule, including, but not limited to, ion, atom, small molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and such compound can be natural or synthetic. As used herein, the terms "modulates" or "modifies" means an increase or decreas'e in the amount, quality, or effect of a particular activity or protein. As used herein, the term "modulate Cripto signaling" means an increase or decrease in the amount, quality, or effect of Cripto activity, by about 5%, preferably 10%, more preferably 20%, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 100%,. Activity may be measured by assays known in the art, such as the null cell assay shown in Example 3. In another embodiment, protein interaction between Cripto and another protein is similarly modulated downward via binding of the antibodies of the invention.
As used herein, the term "blocking the interaction between Cripto and ALK 4" means an increase or decrease in the interaction, i.e. binding, between Cripto and ALK4, by about 5%, preferably 10%, more preferably 20%, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 100%,. Activity may be measured by assays known in the art, such as the binding assay shown in Example 8.
As used herein, the term "modulate growth of tumor cells in vitro" means an increase or decrease in the number of tumor cells, in vitro, by about 5%, preferably 10%, more preferably 20%, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 100%. In vitro modulation of tumor cell growth may be measured by assays known in the art, such as the GEO cell soft agar assay shown in Example 4.
As used herein, the term "modulate growth of tumor cells in vivo" means an increase or decrease in the number of tumor cells, in vivo, by about 5%, preferably 10%, more preferably 20%, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 100%. In vivo modulation of tumor cell growth may be measured by assays known in the art, such as the one shown in Example 5. The term "preventing" refers to decreasing the probability that an organism contracts or develops an abnormal condition.
The term "treating" refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism. Treating includes maintenance of inhibited tumor growth, and induction of remission. The term "therapeutic effect" refers to the inhibition of an abnormal condition.
A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase or decrease in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) or promotion of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of a population of cells. Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
The term "administering" relates to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes, or in another organism. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications. For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques. Administration may be accomplished by the many modes known in the art, e.g., oral, intravenous, intraperitoneal, intramuscular, and the like. When used in in vivo therapy, the antibodies of the subject invention are administered to a patient in effective amounts. As used herein an "effective amount" is an amount sufficient to effect beneficial or desired clinical results (i.e., amounts that eliminate or reduce the patient's tumor burden). An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of the antibodies of the present invention is an amount of the antibodies that is sufficient to ameliorate, stabilize, or delay the development of the Cripto-associated disease state, particularly Cripto-associated tumors. Detection and measurement of these indicators of efficacy are discussed below. An example of a typical treatment regime includes administering by intravenous infusion to the subject antibodies of the invention on a weekly schedule, at a dose of about 2-5 mg/kg. The antibodies are administered in an outpatient chemoinfusion unit, unless the patient requires hospitalization. Other administration regimes known in the art are also contemplated.
The abnormal condition can also be prevented or treated by administering an antibody of the invention to a group of cells having an aberration in a signal transduction pathway to an organism. The effect of administering a compound on organism function can then be monitored. The organism is preferably a human.
"Cripto overexpression" is intended to mean the expression of Cripto by a tissue which expression is greater than the Cripto expression of adjacent normal tissue in a statistically significant amount. "Chemotherapeutics" refers to any agents identified in the art as having therapeutic effect on the inhibition of tumor growth, maintenace of inhibited tumor growth, and/or induction of remission, such as natural compounds, synthetic compounds, proteins, modified proteins, and radioactive compounds. Chemotherapeutic agents contemplated herewith include agents that can be conjugated to the antibodies of the present invention or alternatively agents that can be used in combination with the antibodies of the present invention without being conjugated to the antibody. Exemplary chemotherapeutics that can be conjugated to the antibodies of the present invention include, but are not limited to radioconjugates (90 Y, 1311, 99mTc, lllln, 186Rh, et al.), tumor-activated prodrugs (maytansinoids, CC-1065 analogs, clicheamicin derivatives, anthracyclines, vinca alkaloids, et al.), ricin, diptheria toxin, pseudomonas exotoxin..
Chemotherapeutic agents may be used in combination with the antibodies of the invention, rather than being conjugated thereto (i.e. nonconjugated chemotherapeutics), include, but are not limited to the following: platinums (i.e. cis platinum), anthracyclines, nucleoside analogs (purine and pyrimidine), taxanes, camptothecins, epipodophyllotoxins, DNA alkylating agents, folate antagonists, vinca alkaloids, ribonucleotide reductase inhibitors, estrogen inhibitors, progesterone inhibitors, androgen inhibitors, aromatase inhibitors, interferons, interleukins, monoclonal antibodies, taxol, camptosar, adriamycin (dox), 5-FU and gemcitabine. Such chemotherapeutics may be employed in the practice of the invention in combination with the antibodies of the invention by coadministration of the antibody and the nonconjugated chemotherapeutic.
"Pharmaceutically acceptable carrier or excipient" refers to biologically inert compounds known in the art and employed in the administration of the antibodies of the invention. Acceptable carriers are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., 1990. Acceptable carriers can include biocompatible, inert or bioabsorbable salts, buffering agents, oligo- or polysaccharides, polymers, viscoelastic compound such as hyaluronic acid, viscosity-improving agents, preservatives, and the like.
A "subject" refers to vertebrates, particularly members of a mammalian species, and includes but is not limited to domestic animals, sports animals, and primates, including humans.
Antibodies of the Invention The antibodies of the invention specifically bind to Cripto: As used herein,
Cripto includes the CR-1 Cripto protein, the CR-3 Cripto protein, and fragments thereof. Such fragments may be entire domains, such as the extracellular or intracellular domains, the EGF-like domain, the cys-rich domain, the receptor binding domain, and the like. Such fragments may also include contiguous and noncontiguous epitopes in any domain of the Cripto protein.
The 188 amino acid sequence for CR-1 is as follows [SEQ ID NO: 1]: DCRK ARFSYSVI IMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS IWPQEEPAIRPRSSQ VPPMGIQHSKELNRTCCLNGGTCM GSFCACPPS FYGRNCEHDVRKΞNCGSVPHDT LPKKCSLCKC HGQLRCFPQAF PGCD GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY
The 188 amino acid sequence for CR-3 is as follows [SEQ ID NO: 2]:
IvfDCRB-MVRFSYSVIWIMAISKAFELG VAGLGHQEFARPSRGDLAFRDDS IWPQEEPAIRPRSSQRVLPMGIQHSKELNRTCC NGGTCM ESFCACPPSF YGR CEHDVRKENCGSVPHDTW PKKCS CKCWHGQLRCFPQAFLPGCDGL
V DEHLVASRTPELPPSARTTTFMLAGICLSIQSYY
In a one embodiment, the antibodies of the invention bind to an epitope in the EGF-like domain of Cripto. The EGF-like domain spans from about amino acid residue 75 to about amino acid residue 112 of the mature Cripto protein. Epitopes in the EGF-like domain may comprise linear or nonlinear spans of amino acid residues. Example of linear epitopes contemplated include but are not limited to about residues 75-85, 80-90, 85-95, 90-100, 95-105, 100-110, or 105-112. In one embodiment, the epitope in the EGF domain is an epitope formed in the conformational native Cripto protein versus a denatured Cripto protein. In another embodiment, the antibodies of the invention bind to an epitope in the cys-rich domain of Cripto. The cys-rich domain spans from about amino acid residue 114 to about amino acid residue 150 of the mature Cripto protein. Epitopes in the cys- rich domain may comprise linear or nonlinear spans of amino acid residues. Example of linear epitopes contemplated include but are not limited to about residues 114-125, 120-130, 125-135, 130-140, 135-145, or 140-150. In one embodiment, the epitope in the cys-rich domain is an epitope formed in the conformational native Cripto protein versus a denatured Cripto protein
Once antibodies are generated, binding of the antibodies to Cripto may be assayed using standard techniques known in the art, such as ELISA, while the presence of Cripto on a cell surface may be assayed using flow cytometry (FACS), as shown in Example 2. Any other techniques of measuring such binding may alternatively be used.
The present invention provides antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for Cripto or fragments thereof. Antibody fragments, including Fab, Fab', F(ab')2, and Fv, are also provided by the invention. The terms "specific " and "selective," when used to describe binding of the antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind Cripto polypeptides. It will be understood that specific antibodies of the invention may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention (i.e. antibodies that specifically bind to an epitope the ligand/receptor binding domain and the domain spanning amino acid residues 46-62) are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6.
Antibodies that recognize and bind fragments of Cripto protein are also contemplated, provided that the antibodies are specific for Cripto polypeptides. Antibodies of the invention can be produced using any method well known and routinely practiced in the art. In one embodiment, the invention provides an antibody that specifically binds to an epitope in the ligand/receptor binding domain of Cripto. Antibody specificity is described in greater detail below. However, it should be emphasized that antibodies that can be generated from other polypeptides that have previously been described in the literature and that are capable of fortuitously cross-reacting with Cripto (e.g., due to the fortuitous existence of a similar epitope in both polypeptides) are considered "cross- reactive" antibodies. Such cross-reactive antibodies are not antibodies that are "specific" for Cripto. The determination of whether an antibody specifically binds to an epitope of Cripto is made using any of several assays, such as Western blotting assays, that are well known in the art. For identifying cells that express Cripto and also for modulating Cripto ligand/receptor binding activity, antibodies that specifically bind to an extracellular epitope of the Cripto protein (i.e., portions of the Cripto protein found outside the cell) are particularly useful.
In one embodiment, the invention provides a cell-free composition comprising polyclonal antibodies, wherein at least one of the antibodies is an antibody of the invention specific for Cripto. Antisera isolated from an animal is an exemplary composition, as is a composition comprising an antibody fraction of an antisera that has been resuspended in water or in another diluent, excipient, or carrier.
In another embodiment, the invention provides monoclonal antibodies. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Further in contrast to polyclonal preparations which typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies are useful to improve selectivity and specificity of diagnostic and analytical assay methods using antigen-antibody binding. Another advantage of monoclonal antibodies is that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins.
Hybridomas that produce such antibodies are also intended as aspects of the invention.
In still another related embodiment, the invention provides an anti-idiotypic antibody specific for an antibody that is specific for Cripto. For a more detailed discussion of anti-idiotypic antibodies, see, e.g., U.S. Patents 6,063,379 and 5,780,029. It is well known that antibodies contain relatively small antigen binding domains that can be isolated chemically or by recombinant techniques. Such domains are useful Cripto binding molecules themselves, and also may be reintroduced into human antibodies, or fused to a chemotherapeutic or polypeptide. Thus, in still another embodiment, the invention provides a polypeptide comprising a fragment of a Cripto- specific antibody, wherein the fragment and associated molecule, if any, bind to the Cripto. By way of non-limiting example, the invention provides polypeptides that are single chain antibodies and CDR-grafted antibodies. For a more detailed discussion of CDR-grafted antibodies, see, e.g., U.S. Patent5,859,205.
In another embodiment, non-human antibodies may be humanized by any of the methods known in the art. Humanized antibodies are useful for in vivo therapeutic applications. In addition, recombinant "humanized" antibodies may be synthesized. Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobulin light or heavy chain. That is, they are chimeras comprising mostly human immunoglobulin sequences into which the regions responsible for specific antigen-binding have been inserted (see, e.g., PCT patent application WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated and the portion of the variable region sequences responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of the human antibody genes in which the antigen binding regions have been deleted. Humanized antibodies minimize the use of heterologous (inter-species) sequences in antibodies for use in human therapies, and are less likely to elicit unwanted immune responses. Primatized antibodies can be produced similarly using primate (e.g., rhesus, baboon and chimpanzee) antibody genes. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity. See, e.g., U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370.
Another embodiment of the invention includes the use of human antibodies, which can be produced in nonhuman animals, such as transgenic animals harboring one or more human immunoglobulin transgenes. Such animals may be used as a source for splenocytes for producing hybridomas, as is described in United States patent 5,569,825, WO00076310, WO00058499 and WO00037504 and incorporated by reference herein. Signal Modulation
In another embodiment, the antibodies of the invention bind to Cripto, and modulate Cripto signaling or Cripto-protein interactions. Over-expression of Cripto activity can lead to a de-differentiated state promoting mesenchymal cell characteristics, increased proliferation, and cell migration (Salomon et al., BioEssays 21: 61-70, 1999; Ciardiello et al., Oncogene 9: 291-298, 1994; and Baldassarre et al., Int. J. Cancer 66:538-543, 1996), phenotypes associated with cell transformation seen in neoplasia.
One method of testing the activity of anti-Cripto antibodies and their ability to modulate Cripto signaling is with an F9-Cripto knock-out (KO) cell line (Minchiotti at al., Mech. Dev. 90: 133-142, 2000). Cripto stimulates smad2 phosphorylation and the transcription factor FAST in Xenopus embryos, and the activity of the transcription factor FAST can be monitored by measuring the luciferase activity from a FAST regulatory element-luciferase reporter gene (Saijoh et al., Mol. Cell 5:35-47, 2000). F9-Cripto KO cells are deleted for the Cripto gene and are thus null for Cripto and Cripto-dependent signaling (Minchiotti at al., Mech. Dev. 90: 133-142, 2000). Cripto signaling can be assessed in the F9 Cripto KO cells by transfecting in Cripto, FAST, and the FAST regulatory element-luciferase gene construct. No Cripto dependent FAST luciferase activity will be seen in these cell lines unless Cripto cDNA, and FAST cDNA is transfected into them. Antibodies capable of blocking Cripto-dependent Nodal signaling are antibodies that block Cripto signaling function. Other assays capable of measuring the activity of Cripto can be employed by those of skill in the art, such as a growth in soft agar assay (see Example 4 below). The ability of cells to grow in soft agar is associated with cell transformation and the assay is a classical in vitro assay for measuring inhibition of tumor cell growth. Other assays useful in determining inhibition of activity include in vitro assays on plastic, and the like.
Therapeutic Uses
Antibodies of the invention are also useful for, therapeutic purposes, such as modulation of tumor cell growth, diagnostic purposes to detect or quantitate Cripto, and purification of Cripto.
In one embodiment of the invention, antibodies are provided which are capable of binding specifically to Cripto and which modulate growth of tumor cells in a patient. In one embodiment, the tumor cells are testicular, breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumor cells. In another embodiment, antibodies are provided which are capable of binding specifically to Cripto and which modulate growth of tumor cells which overexpress Cripto. In one embodiment, the tumor cells are cell lines which overexpress Cripto, such as cell lines derived from breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach cancer. Anti-Cripto antibodies may be screened for in vivo activity as potential anticancer agents following standard protocols used by those of skill in the art, as illustrated in Example 4 below. Example of such protocols are outlined by the National Cancer institute (NCI) in their "in vivo cancer models screening" protocols, NEH publication number 84-2635 (Feb 1984). In another embodiment of the invention, the antibodies of the invention are used to treat a patient having a cancerous tumor.
The antibodies of the present invention can be combined with a pharmaceutically acceptable excipient and administered in a therapeutically effective dose to the patient. For a discussion of methods of inhibiting growth of tumors, see, e.g., U.S. Patent 6,165,464.
Also contemplated are methods of treating a subject suffering from a disorder associated with abnormal levels (i.e. elevated or depleted) of Cripto wherein the method comprises administering to the subject an effective amount of an antibody that specifically binds to an epitope in the ligand/receptor binding domain of Cripto, including but not limited to where the epitope is in an EGF-like domain or a cys-rich domain of Cripto.
Also contemplated are methods of treating a subject suffering from a disorder associated with abnormal levels (i.e. elevated or depleted) of Cripto wherein the method comprises administering to the subject an effective amount of an antibody which specifically forms a complex with Cripto and is directed to the epitope to which an antibody selected from the group consisting of A6C12.11, A6F8.6 (ATCC ACCESSION NO. PTA-3318), A7H1.19, A8F1.30, A8G3.5 (ATCC ACCESSION NO. PTA-3317), A8H3.1 (ATCC ACCESSION NO. PTA-3315), A8H3.2, A19A10.30, A10B2.18 (ATCC ACCESSION NO. PTA-3311), A27F6.1 (ATCC ACCESSION NO. PTA-3310), A40G12.8 (ATCC ACCESSION NO. PTA-3316), A2D3.23, A7A10.29, A9G9.9, A15C12.10, A15E4.14, A17A2.16, A17C12.28, A17G12.1 (ATCC ACCESSION NO. PTA-3314), A17H6.1, A18B3.11 (ATCC ACCESSION NO. PTA- 3312), A19E2.7, B3F6.17 (ATCC ACCESSION NO. PTA-3319), and B6G7.10 (ATCC ACCESSION NO. PTA-3313) is directed. Diagnosis via detection of Cripto is readily accomplished through standard binding assays using the novel antibodies of the invention, allowing those of skill in the art to detect the presence of Cripto specifically in a wide variety of samples, cultures, and the like. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific. Embodiments include kits comprising all reagents and instructions for the use thereof.
Additional features of the invention will be apparent from the following illustrative Examples.
Examples Example 1: Expression and Purification of Cripto
An expression plasmid designated pSGS480 was constructed by sub-cloning a cDNA encoding human Cripto amino acids residues 1 to 169 of Cripto [amino acids 1- 169 of SEQ ED NO: 1], fused to human IgGj Fc domain (i.e., "CR(del C)-Fc") into vector pEAGl 100. For a more detailed description of this vector, see copending U.S. Patent Application Serial No. 60/233,148, filed September 18, 2000. The vector pEAGl 100 is a derivative of GEBCO-BRL Life Technologies plasmid pCMV-Sport- betagal, the use of which in CHO transient transfections was described by Schifferli et al., 1999, Focus 21: 16. It was made by removing the reporter gene beta-galactosidase Notl fragment from the plasmid pCMV-Sport-Betagal (catalog number 10586-014) as follows: The plasmid was digested with Notl and EcoRV, the 4.38 kb Notl vector backbone fragment was gel-purified and ligated. Ligated DNA was transformed into competent E. coli DH5alpha. pEAGHOO was isolated as a plasmid containing the desired recombinant from an isolated single colony. The sequence of pEAGl 100 spanning 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 the conditioned media was examined by Western blot analysis. For Western blot analysis, conditioned media and cells from Cripto transfected cells were subjected to SDS-PAGE on 4-20% gradient gels under reducing conditions, transferred electrophoretically to nitrocellulose, and the Cripto fusion protein was detected with a rabbit polyclonal antiserum raised against a Cripto 17-mer peptide (comprising residues 97-113 of SEQ ED NO: l)-keyhole limpet hemocyanin conjugate. After centrifugation to remove the cells, Western blot analysis showed that the CR( del C)-Fc protein was efficiently secreted into the conditioned media (supernatant). The supernatant was applied to a Protein A-Sepaharose (Pharmacia), and bound protein was eluted with 25 mM sodium phosphate pH 2.8, 100 mM NaCl. The eluted protein was neutralized with 0.5 M sodium phosphate at pH 8.6, and analyzed for total protein content from absorbance measurements at 240-340 nm, and for purity by SDS-PAGE. The eluted protein was filtered through a 0.2 micron filter, and stored at -70°C.
Example 2: Generation and Screening of Antibodies
The eluted CR(del C)-Fc protein is injected into mice, and standard hybridoma techniques known to those of skill in the art are used to generate monoclonal antibodies.
A. Generation of Antibodies
Particularly, female Robertsonian mice (Jackson Labs) were immunized intraperitoneally with 25 μg of purified human CR del C-Fc emulsified with complete fruend's adjuvant (GibcoBRL #15721-012). They were boosted two times intraperitoneally with 25 μg of CR del C -Fc emulsified with incomplete freunds's adjuvant (GibcoBRL #15720-014) and once on Protein A beads. The sera were screened and 3 weeks after the last boost, the mouse with the best titer was boosted intraperitoneally with 50 μg soluble CR del C-Fc three days before fusion. The mouse was boosted intravenously with 50 μg CR del C-Fc the day before fusion. The mouse spleen cells were fused with FL653 myeloma cell at a 1 spleen :6 myeloma ratio and were plated at 100,000, 33,000and 11,000 cells per well into 96 well tissue culture plates in selection media. Wells positive for growth were screened by FACS and ELIS A a week later. Two fusions were performed. B. Screening of Antibodies Supernatants resulting from the first or second fusion were screened first on
ELISA plates for recognition of Cripto del C and/or Cripto EGF-like domain proteins. A control fusion protein (LT-beta receptor-Fc) was coated on ELISA plates to discard monoclonal antibodies that recognized the human Fc epitope. The ELISA was performed as described below in section C. In the first fusion, primary supernatants were also screened for their ability to recognize cell surface Cripto protein on the testicular tumor cell line, NCCIT by FACS. In the case of the second fusion, the ability of supernatants to recognize Cripto on two tumor cell lines, NCCIT and the breast cancer line, DU4475 by FACs was analyzed. Secondary screens included testing the monoclonal antibody supernatant's ability to recognize cell surface Cripto on a panel of tumor cell lines (see Tables 1 and 2 for results), ability of monoclonal antibodies to recognize human Cripto immunohistochemically on human breast and colon tumor tissue sections, ability of monoclonal antibodies to block in Cripto-Nodal signalling assay, ability to block growth of tumor cell lines on plastic or in soft agar assays, and ability to internalize cell surface Cripto. C. ELISA The ELISA assays were performed as follows: Materials:
Plates: Costar high-binding Easy-wash 96W plates (07-200-642) 2' antibody: Pierce Gt anti- Ms IgG (H+L)- HRP (P131430) Substrate: Pierce TMB Substrate Kit (34021)
Stop solution: IN H2SO4 Buffers:
Binding buffer: 0.1 M NaHPO4 pH 9.0 Blocking buffer: PBS + 10% Donor Calf Serum Wash buffer: PBS + 0.1 % tween-20
Antigens CR-del-C-Fc and CR-EGF-fc, control hu IgGl fusion protein were diluted in binding buffer to 500ng/ml. 100 μl were added per well and incubated for 1 hr at 37° C or overnight at 4° C. The liquid was decanted and the plate inverted and blotted until dry. 250 μl/well blocking buffer was then added, followed by incubation for 30 min. at 37° C. Again, the liquid was decanted and the plate inverted and blotted until dry. Supernatants were diluted 1:50 in wash buffer, and plated at 50 μl/well, followed by incubation for 1 hour at room temperature. Plates were washed 3X vigorously with 250 μl/well wash buffer. Then 100 μl/well 2' antibody diluted in wash buffer at 1:10,000 was added, followed by incubation for 30 min. at room temperature. Plates were then washed 3X vigorously with 250 μl/well wash buffer, then substrate added at lOOμl/well. Color was permitted to develop until sufficiently dark, then 100 μl/well stop solution was added and the plates read for absorbance at 450nm. D. Flow Cytometry
Cripto positive cell lines may be used to assay the monoclonal antibodies for binding to Cripto using cell surface staining and flow cytometry as follows: Release cells from T162 flasks with 2 ml PBS" with 5 mM EDTA, 10 min., 37°C. Bring up to 20 ml with media with serum, pipetting up and down several times to unclump cells. Spin at 1200 rpm for 5 minutes. Wash cells with 5-10 ml 4° C PBS with 0.1% BSA (wash buffer). Spin at 1200 rpm for 5 minutes. Resuspend at 4xl06- 107/ml in wash buffer. Keep on ice.
Prepare antibodies for staining. Purified antibodies are diluted to 1-10 μg/ml in wash buffer. Add 50 μl of cells to a 96-well Linbro V bottomed plate (ICN 7632105). Plate one well of cells for each control for each cell line to be analyzed, including cells for no antibody, 2° antibody only, hybridoma media, positive control antibody supernatant, if available, or purified, and an IgG subclass control (if using purified antibodies).
Plate one well of cells for each experimental sample for each cell line to be analyzed. Spin plate, 1200 rpm for 5 minutes, using a table top centrifuge at 4° C. Flick out buffer by inverting the plate and shaking until the liquid is substantially discarded. Add 40-50 μl of antibodies (or wash buffer for the no-antibody and 2° antibody-only control wells) to wells. Incubate at least 30 min.-l hour at 4° C. Spin plate, 1200 rpm for 5 minutes. Flick out antibody solutions. Wash wells twice with 200 μl wash buffer per well, spinning after each wash. Flick out buffer.
Resuspend cells in each well in 50 μl of 1 :200 dilution (in wash buffer) of R-PE tagged goat anti-mouse IgG, Fc Specific (Jackson Immunoresearch Laboratories Cat# 115-116-071). Incubate 20 min, 4°C, in the dark. Add 150 μl wash buffer to cells in each well. Spin plate at 1200 rpm for 5 minutes. Wash once with 200 μl wash buffer per well. Resuspend cells in 150 μl 1% PFA in PBS. Transfer contents of each well to separate tubes (5 ml Falcon polystyrene round bottomed tube-352052). Wrap tubes in tin foil. The contents of the tubes are then read by flow cytometry.
The results of a two screenings of monoclonal antibodies produced by this method yielded the following results, summarized in Tables 1 and 2 below, wherein the first column provides the designated names for the hybridoma subclones, the next two columns show the results of ELISA screens, and the remaining columns show flow cytometry analysis results on four cripto-positive cell lines. The results are given in units of mean fluorescent index (MFI).
Table 1: Anti-Cripto Monoclonal Antibody Characterization
Figure imgf000032_0001
Table 2: Anti-Cripto Monoclonal Antibody Characterization
Figure imgf000033_0001
Example 3: Null Cell Assay for Inhibition of Cripto Signaling
The following describes an F9 Cripto null cell signaling assay used to assess inhibition of Cripto signaling.
Day 0 Coat 6 welled plates with 0.1% gelatin 2ml/well at 37°C for 15 min. Seed cells at 6xl05 F9 CRIPTO NULL cells per well. Day 1 Transfection:
Each of the following samples is added to 300 μl OptiMeml to yield Solution A for each sample:
Sample 1: 0.5μg (N2)7 luciferase FAST reporter cDNA plus 1.5 μg empty vector cDNA. Sample 2: 0.5μg (N )7 luciferase, 0.5μg FAST, and 1 μg empty vector cDNAs. Sample 3: 0.5μg (N2)7 luciferase, 0.5μg Cripto ADD 0.5 FAST, and -0.5μg empty vector cDNAs.
Sample 4: 0.5μg (N2)7 luciferase, 0.5μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNAs Sample 5: 0.5μg (N2) luciferase, 0.5μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNAs.
Sample 6: 0.5μg (N2)7 luciferase, 0.5μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNAs.
Sample 7: 0.5μg (N2)7 luciferase, 0.5μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNAs.
Sample 8: 0.5μg (N2)7 luciferase, 0.5μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNAs.
Sample 9: 0.5μg (N2)7 luciferase, 0.5μg Cripto, 0.5 FAST, and 0.5 μg empty vector cDNAs. Solution B comprises 30 μl of Lipofectamine plus 270 μl of OptiMeml.
For each sample, mix solution A and solution B together. Incubate 45 minutes at room temperature. Rinse wells with 2 ml/well of OptiMeml. Aspirate just before next step.
Add 2.4 ml of OptiMeml to each mixture of solutions A+B, mix, add 1.5 ml/well to duplicate wells. Incubate 5 hours at 37°C. Add 1.5 ml/well of DMEM+20% FCS,
2mM Gin, P/S to wells which received samples 1-3. Add anti-Cripto antibodies as follows: Sample 4 wells: A27F6.1, 10 μg/ml; Sample 5 wells: A27F6.1, 2μg/ml;
Sample 6 wells: A40G12.8; lOμg/ml, Sample 7 wells: A40G12.8 2μg/ml; Sample 8 wells: A10B2.18, lOμg/ml; Sample 9 wells: A10B2.18, 2μg/ml. Day 2 Remove media, wash cells with PBS, 2ml/well. Add DMEM+0.5% FCS, 2mM Gin, P/S with the same amounts of Cripto antibodies as the previous day, to the same wells.
Day 3 Develop luciferase signal. Wash wells with PBS+Ca 2+ a„nd Mg ,2+ , 2 ml/well. Use LucLite kit, Packard cat# 6016911. Bring buffer and substrate to room temperature. Dim lights. Reconstitute substrate with 10 ml of buffer. Dilute 1:1 with PBS + Ca2+ and Mg2+. Aspirate wells. Quickly add 250 μl of diluted substrate per well using a repeat pipettor. Swirl solution and transfer 200 μl to wells of a 96 welled white opaque bottom plate, Falcon 35-3296. Read plate on luminometer using Winglow, exporting data to Excel.
The results of this assay are summarized below in Table 3.
Table 3: Cripto Signaling Assay: Inhibition with Anti-Cripto Monoclonal Antibodies
Figure imgf000035_0001
Example 4: Assay for In Vitro Inhibition of Tumor Cell Growth
Inhibition of Cripto Signaling may also be assayed by measuring the growth of
GEO cells in soft agar. See, e.g., Ciardiello et al., Oncogene. 1994 Jan;9(l):291-8; Ciardiello et al., Cancer Res. 1991 Feb l;51(3):1051-4.
First, melt 3% bactoagar. Keep at 42 °C in a water bath. Then, mix 3% bactoagar solution with prewarmed complete media to make a solution of 0.6% bactoagar, keeping at 42 °C. Plate 4 nils of the solution in a 6 cm dish and let cool for at least 30 minutes to form the bottom agar layer. Trypsinize GEO cells and resuspend to 105 cells/ml in complete media. Add antibodies to be assayed, or controls, to the cell suspensions, titrating antibodies from 20μg to 1 μg. Mix equal volumes of the GEO cell suspensions and 0.6% bactoagar and overlay 2 mis on top of the bottom agar layer. Let cool for at least 1 hour. Incubate for 14 days at 37 °C in CO2 incubator. Count colonies visible without the use of a microscope. The absence of colonies, as compared to negative controls, indicates that the antibody tested inhibits in vitro tumor cell growth.
This assay was used to yield the results shown in Table 4, for the antibodies A27F6.1 and B6G7.10, both of which demonstrate the ability to decrease growth of GEO cell colonies.
Table 4: Results of growth in soft agar assay
Figure imgf000036_0001
Example 5: Assay for In Vivo Inhibition of Tumor Cell Growth To assess the inhibition of tumor cell growth, a human tumor cell line is implanted subcutaneously in athymic nude mice and the effects of the antibodies of the invention are observed, with and without additional chemotherapeutic treatments which may provide synergistic or additive effects on tumor inhibition. This assay may be performed alternatively using different tumor cell lines, such as, for example, GEO (a well differentiated human colon cancer in-vitro cell line, is obtained from the American Tissue Type Collection (ATCC)), DU-4475 (a breast cancer in-vitro cell line obtained from the ATCC), NCCIT (a testicular tumor cell line obtained from ATCC), or others known in the art. One example of such assays is as follows:
Animals are individually marked by ear punches. The GEO cell line is passed in-vitro or in- vivo for 1-4 passages. Animals are implanted with GEO cells subcutaneously in the right flank area. The following groups of animals may be used: Group # Treatment # of Mice
1. Saline Control, 0.2 ml/mouse, i.p. three times weekly (M,W,F) 20
2. mAb, low dose, i.p. 10
3. mAb, middle dose, i.p. 10
4. mAb, high dose, i.p. 10 5. 5-FU, 30 mg/kg/inj, i.p., 3 Rx/wk (M,W,F) 10
6. Cisplatin, 2 mg/kg/inj, s.c, 3 Rx/wk (M,W,F) 10
7. Adriamycin, 1.6 mg/kg/inj, i.p., 3 Rx/wk (M,W,F) 10
8. Irinotecan, 10 mg kg/inj., i.p., 5 Rx/wk ( M-F) 10
9. mAb, low dose, i.p. + 5-FU ( intermediate dose) 10 10. mAb, middle dose, i.p. + 5-FU ( intermediate dose) 10
11. mAb, high dose, i.p. + 5-FU ( intermediate dose) 10
12. mAb, low dose, i.p. + Cisplatin ( intermediate dose) 10
13. mAb, middle dose, i.p. + Cisplatin ( intermediate dose) 10
14. mAb, high dose, i.p. + Cisplatin ( intermediate dose) 10 15. mAb, low dose, i.p. + Adriamycin ( intermediate dose) 10 16. mAb, middle dose, i.p. + Adriamycin ( intermediate dose) 10
17. mAb, high dose, i.p. + Adriamycin ( intermediate dose) 10
18. mAb, low dose, i.p. + Irinotecan ( intermediate dose) 10
19. mAb, middle dose, i.p. + Irinotecan ( intermediate dose) 10 20. mAb, high dose, i.p. + Irinotecan ( intermediate dose) 10
Day 0: Implant tumor, record initial body weight of animals.
Day 1: Initiate treatments as indicated above.
Day 5: Begin tumor size and body weight measurements and continue two times weekly until termination of experiment.
Initial body weight, tumor size and body weight measurements, histology at sacrifice, and immunohistochemistry analysis on tumors are examined, analyzing for Cripto expression, tumor growth, and inhibition thereof.
Example 6: In Vivo Xenograft Tumor Model - Cys-rich blocking anti-Cripto antibody
To assess the response of an NCCIT, a human testicular carcinoma cell line was implanted subcutaneously with an antibody which binds to a cys-rich domain of Cripto.
The experimental methods are listed below. The results are shown in Fi ure 1. Methods and Materials
Animals: Athymic nude male mice were used. Animals were individually numbered by ear punches.
Tumor:NCCrT, mediastinal mixed germ cell human testicular carcinoma in-vitro cell line originally obtained from the American Tissue Type Collection. Cell line was passed in-vitro for six passages in RPMI-1640/ 10% FBS without antibiotics. Animals implanted subcutaneously with 5 x 106 cells / 0.2 ml matrigel on the animals right flank. Group # Treatment # of Mice
1 Vehicle Control, (25 mM sodium phosphate, 100 mM sodium 20 chloride, pH 7.2), 0.2 ml/mouse, i.p., Q14D Treatments begin on day -1
2. A8G3.5, 1 mg/kg/inj, i.p., Q14D 10
Treatments begin on day -1 3. A8G3.5, 3 mg/kg/inj, i.p., Q14D 10
Treatments begin on day -1
4. A8G3.5, 10 mg/kg/inj, i.p., Q14D 10 Treatments begin on day -1
5. Cis-platinum, 2 mg kg/inj, s.c, 3 x/wk (M,W, F) for 6 treatments 10
Treatments began on day 1 Testing schedule
Day -1: Randomized mice into control and treatments groups. Recorded initial body weight of animals. Administered first treatments to antibody groups. Dosing solutions were made. Treatments were blinded to the technicians until the assay was terminated.
Day 0: Implanted tumor. Ran bacterial cultures on the tumor implanted into mice.
Day 1: Administered first treatment to the positive chemotherapeutic group.
Day 4: Recorded initial tumor size measurements for tumor baseline on matrigel. Continued to record tumor size and body weights on mice 2x / week. Monitored the study daily and made notations of any unusual observation on animals.
Endpoints: Initial body weight
Tumor size and body weight measurements
Example 7: In Vivo Xenograft Tumor Model - EGF-like domain blocking anti- Cripto antibody
To assess the response of an NCCIT, a human testicular carcinoma cell line was implanted subcutaneously with an antibody which binds to a EGF-like domain of
Cripto. The experimental methods are listed below. The results are shown in Figure 2. Methods and Materials:
Animals: Athymic nude male mice were used. Animals were individually numbered by ear punches.
Tumor: NCCIT, mediastinal mixed germ cell human testicular carcinoma in- vitro cell line originally obtained from the American Tissue Type Collection. Cell line was passed in-vitro for eight passages in RPMI- 1640/ 10% FBS without antibiotics. Animals implanted subcutaneously with 5 x 106 cells / 0.2 ml matrigel on the animals right flank.
Group # Treatment # of Mice
Vehicle Control, (25 mM sodium phosphate, 100 mM sodium 18 chloride, pH 7.2), 0.2 ml/mouse, i.p., Q14D Treatments begin on day -1
A27F6.1, 1 mg/kg/inj, i.p., Q14D 10
Treatments begin on day -1 with a loading dose of 2.6 mg/kg/mouse
3. A27F6.1, 10 mg/kg/inj, i.p., Q14D 10
Treatments begin on day -1 with a loading dose of 21.2 mg kg/mouse
Cis-platinum, 2 mg/kg/inj, s.c, 3 x/wk (M,W, F) for 6 treatments 10
Treatments began on day 1.
Testing schedule
Day -1: Randomized mice into control and treatments groups. Recorded initial body weight of animals. Administered first treatments to antibody groups. Dosing solutions were made. Treatments were blinded to the technicians until the assay was terminated.
Day O: Implant tumor. Ran bacterial cultures on the tumors implanted into mice. Bacterial culture were negative for contamination at 24 and 48 hours post sampling.
Day l: Administered first treatment to the positive chemotherapeutic group. Day 4: Recorded initial tumor size measurements for tumor baseline on matrigel. Continued to record tumor size and body weights on mice 2x / week. Monitored the study daily and made notations of any unusual observation on animals.
Endpoints:
Initial body weight
Tumor size and body weight measurements Example 8: Cripto mabs that block ALK4 binding
In order to assess whether Cripto-specific monoclonal antibodies can interfere with Cripto 's ability to bind to Alk4, the activin type I receptor, we used flow cytometry analysis using a 293 cell line which stably expresses Alk4. To generate this cell line, 293 cells were cotransfected with a plasmid that expresses Alk4 tagged at the C- terminus with a HA epitope and a plasmid that expresses the drug, puromycin, at a 10:1 ratio. The transfected cells were then selected in puromycin until colonies formed. Colonies were then picked, expanded and then analyzed for Alk4 expression using western blotting analysis for HA. Clone 21 (293-Alk4-21) was found to express high levels of Alk4 compared to control, untransfected 293 cells.
To analyze Cripto- Alk4 binding by flow cytometry, a purified, soluble form of human Cripto (aa 1-169) fused to the Fc portion of human IgG (CrdelC-Fc) was employed. Approximately 5μg/ml of CrdelC-Fc or control Fc protein was incubated
with 3xl05 293-Alk4-21 cells on ice for 30 minutes in 50μl total volume of FACS buffer (PBS with 0.1% BSA). For samples containing anti-Cripto antibodies, 5μg/ml CrdelC-Fc was preincubated with 50/ g ml of each Cripto antibody (A10.B2.18, A40.G12.8, A27.F6.1, A8.H3.1, A19.A10.30, A6.F8.6, A8.G3.5, A6.C12.11) on ice prior to addition of the cells. The cells were then washed in FACS buffer and the bound Fc protein was detected by incubating the cells with a R-phycoerytherin-conjugated goat anti-human IgG (Fc fragment specific) from Jackson Immunologies. Samples were then washed again, fixed in 1% paraformaldehyde in PBS, and analyzed using standard flow cytometry procedures. The results of the FACS assay are shown in Figure 3.
Some of the embodiments of the invention described above are outlined below and include, but are not limited to, the following embodiments. As those skilled in the art will appreciate, numerous changes and modifications may be made to the various embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention. •
The entire disclosure of each publication cited herein is hereby incorporated by reference.

Claims

We claim:
1. An antibody that specifically binds to an epitope in the ligand/receptor binding domain of Cripto.
2. The antibody of claim 1 wherein the Cripto is selected from the group consisting of SEQ ID NO: 1 or 2.
3. The antibody of claim 2 wherein the epitope is in an EGF-like domain.
4. The antibody of claim 2 wherein the epitope is in a cys-rich domain.
5. The antibody of claim 2 which is selected from the group consisting of A6C12.11, A6F8.6, A7H1.19, A8F1.30, A8G3.5, A8H3.1, A8H3.2, A19A10.30, 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, and B6G7.10.
6. The antibody of claim 3 which is selected from the group consisting of A40G12.8, A8H3.1, A27F6.1, B6G7.10, A17G12.1 and A18B3.11.
7. The antibody of claim 4 which is selected from the group consisting of
A19A10.30, A8G3.5, A6F8.6 and A6C12.11.
8. An antibody that specifically binds to an epitope comprised in the domain spanning amino acid residues 46-62 of Cripto.
9. The antibody of claim 8 which is A10B2.18 and B3F6.17. 10. An antibody which binds specifically to an epitope selected from the group of epitopes to which antibodies A6C12.11, A6F8.6, A7H1.19, A8F1.30, A8G3.5, A8H3.1, A8H3.2, A19A10.30, 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, and B6G7.
10 bind.
11. An antibody which binds specifically to Cripto and is capable of modulating Cripto signaling.
12. The antibody of claim 11 which specifically binds to an epitope in an EGFlike domain of Cripto.
13. The antibody of claim 12 which is selected from A40G12.8, A8H3.1 and
A27F6.1.
14. The antibody of claim 11 which specifically binds to an epitope in a cys- rich domain of Cripto.
15. The antibody of claim 14 which is A6C12.11.
16. The antibody of claim 11 which is selected from the group consisting of
A40G12.8, A8H3.1, A27F6.1, and A6C12.11.
17. An antibody which binds specifically to Cripto and is capable of modulating tumor growth.
18. The antibody of claim 17 which specifically binds to an epitope in an EGF- like domain of Cripto.
19. The antibody of claim 17 which specifically binds to an epitope in a cys- rich domain of Cripto.
20. The antibody of claim 17 which is selected from the group consisting of A27F6.1, A8G3.5 and B6G7.10.
21. An antibody which binds specifically to Cripto, which is capable of modulating Cripto signaling, and which is capable of modulating tumor growth.
22. The antibody of claim 21 which specifically binds to an epitope in an EGFlike domain of Cripto.
23. The antibody of claim 21 which specifically binds to an epitope in a cys- rich domain of Cripto.
24. The antibody of claim 21 which is A27F6.1.
25. An antibody produced by a hybridoma selected from the group consisting of A6F8.6 (ATCC Accession No. PTA-3318), A8G3.5 (ATCC Accession No. PTA- 3317), A8H3.1 (ATCC Accession No. PTA-3315), A10B2.18 (ATCC Accession No. PTA-3311), A27F6.1 (ATCC Accession No. PTA-3310), A40G12.8 (ATCC Accession No. PTA-3316), A17G12.1 (ATCC Accession No. PTA-3314), A18B3.11 (ATCC Accession No. PTA-3312), B3F6.17 (ATCC Accession No. PTA-3319), and B6G7.10 (ATCC Accession No. PTA-3313).
26. An antibody which binds specifically to Cripto and is capable of blocking the interaction between Cripto and ALK4.
27. The antibody of claim 26 which specifically binds to an epitope in an EGFlike domain of Cripto.
28. The antibody of claim 26 which specifically binds to an epitope in a cys- rich domain of Cripto.
29. The antibody of claim 26 which is selected from the group consisting of
A8G3.5, A6F8.6 and A6C12.11.
30. An antibody which binds specifically to Cripto, which is capable of blocking the interaction between Cripto and ALK4, and which is capable of modulating tumor growth.
31. The antibody of claim 30 which specifically binds to an epitope in an EGFlike domain of Cripto.
32. The antibody of claim 30 which specifically binds to an epitope in a cys- rich domain of Cripto.
33. The antibody of claim 30 which is A8G3.5.
34. A Cripto antibody capable of internalizing Cripto.
35. The antibody of claim 34 wherein the antibody is conjugated to a chemotherapeutic .
36. The antibody of claim 34 which is selected from A27F6.1 and B3F6.17.
37. A composition for administration to a subject having a tumor that expresses Cripto comprising at least one of the antibodies of any of claims 1-35.
38. The composition according to claim 37, wherein the subject is human.
39. The composition according to claim 37, further comprising a pharmaceutically acceptable excipient.
40. The composition according to claim 37, wherein the antibody is conjugated to a chemotherapeutic.
41. The composition according to claim 37, further comprising a nonconjugated chemotherapeutic.
42. A method of modulating growth of tumor cells in vitro in a sample comprising the step of adding to the sample the composition of claim 37.
43. A method of modulating growth of tumor cells in vivo in a subject comprising the step of administering to the subject an effective amount of the composition of claim 37.
44. The method according to claim 43, wherein the subject is human.
45. A method of treating a subject having a tumor that over-expresses Cripto comprising administering to said subject the composition of claim 37 in an effective amount.
46. A method of treating a patient having a tumor that over-expresses Cripto comprising administering to said patient the composition of claim 39 in an effective amount.
47. A method of treating a patient having a tumor that over-expresses Cripto comprising administering to said patient the composition of claim 40 in an effective amount.
48. A method of treating a patient having a tumor that over-expresses Cripto comprising administering to said patient the composition of claim 41 in an effective amount.
49. The method according to claim 42, wherein the tumor cell is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumor cells.
50. The method according to claim 43, wherein the tumor cell is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumor cells.
51. The method according to claim 44, wherein the tumor cell is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumor cells.
52. The method according to claim 45, wherein the tumor is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumors.
53. The method according to claim 46, wherein the tumor is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumors.
54. The method according to claim 47, wherein the tumor is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumors.
55. The method according to claim 48, wherein the tumor is selected from the group consisting of breast, testicular, colon, lung, ovary, bladder, uterine, cervical, pancreatic, and stomach tumors.
56. A method of determining whether a tissue expresses Cripto, comprising the step of analyzing tissue from the subject in an immunoassay using an antibody of any of claims 1-37.
57. A method of determining whether a cell line overexpresses Cripto, comprising the step of analyzing the cell line in an immunoassay using an antibody of any of claims 1-37.
58. The antibody of claim 1 wherein the antibody is monoclonal antibodies.
59. The antibody of claim 1 wherein the antibody is humanized antibodies.
60. The antibody of claim 1 wherein the antibody is human antibodies.
61. A method of treating a subject for a condition associated with undesired cell proliferation, said method comprising administering to said subject the composition of claim 37.
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MXPA03009797A MXPA03009797A (en) 2001-04-26 2002-04-17 Cripto blocking antibodies and uses thereof.
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KR1020037014078A KR100592357B1 (en) 2001-04-26 2002-04-17 Cryptoblocking antibodies and uses thereof
HU0501113A HUP0501113A3 (en) 2001-04-26 2002-04-17 Cripto blocking antibodies and uses thereof
EEP200300528A EE200300528A (en) 2001-04-26 2002-04-17 Cripto protein blocking antibodies and their use
CA2443840A CA2443840C (en) 2001-04-26 2002-04-17 Cripto blocking antibodies and uses thereof
DE60230868T DE60230868D1 (en) 2001-04-26 2002-04-17 CRIPTOBLOCKING ANTIBODIES AND THEIR USE
JP2002585468A JP4307845B2 (en) 2001-04-26 2002-04-17 Antibodies that block Cripto and uses thereof
EP02731384A EP1390389B1 (en) 2001-04-26 2002-04-17 Cripto blocking antibodies and uses thereof
SI200230804T SI1390389T1 (en) 2001-04-26 2002-04-17 Cripto blocking antibodies and uses thereof
EA200301158A EA007469B1 (en) 2001-04-26 2002-04-17 Cripto blocking antibodies and uses thereof
IL15857302A IL158573A0 (en) 2000-03-20 2002-04-17 Cripto blocking antibodies and uses thereof
YU84903A RS51635B (en) 2001-04-26 2002-04-17 Cripto blocking antibodies and uses thereof
SK1443-2003A SK14432003A3 (en) 2001-04-26 2002-04-17 Cripto blocking antibodies and uses thereof
DK02731384T DK1390389T3 (en) 2001-04-26 2002-04-17 Crypto-blocking antibodies and uses thereof
AU2002334799A AU2002334799B2 (en) 2001-04-26 2002-10-01 Cripto-specific antibodies
PCT/US2002/031462 WO2003083041A2 (en) 2002-03-22 2002-10-01 Cripto-specific antibodies
CA2480119A CA2480119C (en) 2002-03-22 2002-10-01 Cripto-specific antibodies
PT02807155T PT1494693E (en) 2002-03-22 2002-10-01 Cripto-specific antibodies
JP2003580477A JP2005520566A (en) 2002-03-22 2002-10-01 Cripto specific antibodies
DE60238559T DE60238559D1 (en) 2002-03-22 2002-10-01 CRIPT-SPECIFIC ANTIBODIES
AT02807155T ATE490981T1 (en) 2002-03-22 2002-10-01 CRIPTO-SPECIFIC ANTIBODIES
DK02807155.3T DK1494693T3 (en) 2002-03-22 2002-10-01 Crypto-specific antibodies
EP02807155A EP1494693B1 (en) 2002-03-22 2002-10-01 Cripto-specific antibodies
US10/693,538 US7531174B2 (en) 2001-04-26 2003-10-23 Cripto blocking antibodies and uses thereof
IS6999A IS2662B (en) 2001-04-26 2003-10-24 Antibodies that block the Cripto protein and their use
NO20034805A NO20034805L (en) 2001-04-26 2003-10-27 CRIPTO-blocking antibody and its use
BG108363A BG108363A (en) 2001-04-26 2003-11-14 Cripto blocking antibodies and use thereof
HK04101788.0A HK1058935A1 (en) 2001-04-26 2004-03-11 Cripto blocking antibodies and uses thereof
US10/945,853 US7582299B2 (en) 2001-04-26 2004-09-20 Cripto-specific antibodies
HK05105887.0A HK1072381A1 (en) 2002-03-22 2005-07-11 Cripto-specific antibodies cripto-specific
US11/799,361 US7674462B2 (en) 2001-04-26 2007-04-30 Cripto blocking antibodies and uses thereof
US12/317,476 US8003763B2 (en) 2001-04-26 2008-12-22 Cripto blocking antibodies and uses thereof
US12/353,913 US20090285818A1 (en) 2001-04-26 2009-01-14 Cripto Blocking Antibodies and Uses Thereof
JP2009020949A JP2009161539A (en) 2002-03-22 2009-01-30 Cripto-specific antibody
US12/415,659 US7888052B2 (en) 2001-04-26 2009-03-31 Cripto blocking antibodies and uses thereof
US13/213,499 US8673303B2 (en) 2001-04-26 2011-08-19 Cripto blocking antibodies and uses thereof

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Cited By (90)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005000899A2 (en) 2003-06-27 2005-01-06 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
EP1494693A2 (en) * 2002-03-22 2005-01-12 Biogen Inc. Cripto-specific antibodies
EP1670511A2 (en) * 2003-09-15 2006-06-21 Research Development Foundation Cripto antagonism of activin and tgf-b signaling
WO2006074397A2 (en) * 2005-01-05 2006-07-13 Biogen Idec Ma Inc. Cripto binding molecules
US7318924B2 (en) 2001-03-26 2008-01-15 The Austin Research Institute Antibodies against cancer
WO2008150530A2 (en) * 2007-06-01 2008-12-11 Biogen Idec Ma Inc. Cripto binding molecules
US7531174B2 (en) 2001-04-26 2009-05-12 Biogen Idec Ma Inc. Cripto blocking antibodies and uses thereof
US7582299B2 (en) 2001-04-26 2009-09-01 Biogen Idec Ma Inc. Cripto-specific antibodies
EP2260858A2 (en) 2003-11-06 2010-12-15 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
EP2286844A2 (en) 2004-06-01 2011-02-23 Genentech, Inc. Antibody-drug conjugates and methods
WO2011031870A1 (en) 2009-09-09 2011-03-17 Centrose, Llc Extracellular targeted drug conjugates
WO2011056983A1 (en) 2009-11-05 2011-05-12 Genentech, Inc. Zirconium-radiolabeled, cysteine engineered antibody conjugates
WO2011130598A1 (en) 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazepines and conjugates thereof
EP2385069A2 (en) 2003-11-12 2011-11-09 Biogen Idec MA Inc. Neonatal Fc rReceptor (FcRn)- binding polypeptide variants, dimeric Fc binding proteins and methods related thereto
WO2011156328A1 (en) 2010-06-08 2011-12-15 Genentech, Inc. Cysteine engineered antibodies and conjugates
US8084026B2 (en) 2005-01-05 2011-12-27 Biogen Idec Ma Inc. Multispecific binding molecules comprising connecting peptides
WO2012074757A1 (en) 2010-11-17 2012-06-07 Genentech, Inc. Alaninyl maytansinol antibody conjugates
WO2012143496A2 (en) 2011-04-21 2012-10-26 Bayer Intellectual Property Gmbh Novel binder-drug conjugates (adcs) and their use
WO2012155019A1 (en) 2011-05-12 2012-11-15 Genentech, Inc. Multiple reaction monitoring lc-ms/ms method to detect therapeutic antibodies in animal samples using framework signature pepides
WO2013012733A1 (en) 2011-07-15 2013-01-24 Biogen Idec Ma Inc. Heterodimeric fc regions, binding molecules comprising same, and methods relating thereto
WO2013130093A1 (en) 2012-03-02 2013-09-06 Genentech, Inc. Biomarkers for treatment with anti-tubulin chemotherapeutic compounds
WO2014057074A1 (en) 2012-10-12 2014-04-17 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
WO2014140862A2 (en) 2013-03-13 2014-09-18 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2014140174A1 (en) 2013-03-13 2014-09-18 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
WO2014159981A2 (en) 2013-03-13 2014-10-02 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2015023355A1 (en) 2013-08-12 2015-02-19 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
US8975037B2 (en) 2006-07-28 2015-03-10 Ann & Robert H. Lurie Children's Hospital of Chicago Methods of inhibiting tumor cell aggressiveness using the microenvironment of human embryonic stem cells
WO2015095212A1 (en) 2013-12-16 2015-06-25 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
WO2015095227A2 (en) 2013-12-16 2015-06-25 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2015095223A2 (en) 2013-12-16 2015-06-25 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2016040856A2 (en) 2014-09-12 2016-03-17 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2016040825A1 (en) 2014-09-12 2016-03-17 Genentech, Inc. Anthracycline disulfide intermediates, antibody-drug conjugates and methods
WO2016037644A1 (en) 2014-09-10 2016-03-17 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2016090050A1 (en) 2014-12-03 2016-06-09 Genentech, Inc. Quaternary amine compounds and antibody-drug conjugates thereof
WO2016161477A1 (en) * 2015-04-07 2016-10-13 Prince Henry's Institute Of Medical Research (Acn 132 025 024) A method of treating neoplasias
EP3088004A1 (en) 2004-09-23 2016-11-02 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2016207089A1 (en) 2015-06-22 2016-12-29 Bayer Pharma Aktiengesellschaft Antibody drug conjugates (adcs) and antibody prodrug conjugates (apdcs) with enzymatically cleavable groups
WO2017059289A1 (en) 2015-10-02 2017-04-06 Genentech, Inc. Pyrrolobenzodiazepine antibody drug conjugates and methods of use
WO2017060322A2 (en) 2015-10-10 2017-04-13 Bayer Pharma Aktiengesellschaft Ptefb-inhibitor-adc
WO2017064675A1 (en) 2015-10-16 2017-04-20 Genentech, Inc. Hindered disulfide drug conjugates
WO2017068511A1 (en) 2015-10-20 2017-04-27 Genentech, Inc. Calicheamicin-antibody-drug conjugates and methods of use
WO2017165734A1 (en) 2016-03-25 2017-09-28 Genentech, Inc. Multiplexed total antibody and antibody-conjugated drug quantification assay
WO2017162663A1 (en) 2016-03-24 2017-09-28 Bayer Pharma Aktiengesellschaft Prodrugs of cytotoxic active agents having enzymatically cleavable groups
EP3235820A1 (en) 2014-09-17 2017-10-25 Genentech, Inc. Pyrrolobenzodiazepines and antibody disulfide conjugates thereof
WO2017201449A1 (en) 2016-05-20 2017-11-23 Genentech, Inc. Protac antibody conjugates and methods of use
WO2017205741A1 (en) 2016-05-27 2017-11-30 Genentech, Inc. Bioanalytical method for the characterization of site-specific antibody-drug conjugates
WO2017214024A1 (en) 2016-06-06 2017-12-14 Genentech, Inc. Silvestrol antibody-drug conjugates and methods of use
WO2018031662A1 (en) 2016-08-11 2018-02-15 Genentech, Inc. Pyrrolobenzodiazepine prodrugs and antibody conjugates thereof
US9919056B2 (en) 2012-10-12 2018-03-20 Adc Therapeutics S.A. Pyrrolobenzodiazepine-anti-CD22 antibody conjugates
US9931415B2 (en) 2012-10-12 2018-04-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US9931414B2 (en) 2012-10-12 2018-04-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
WO2018065501A1 (en) 2016-10-05 2018-04-12 F. Hoffmann-La Roche Ag Methods for preparing antibody drug conjugates
US9950078B2 (en) 2013-10-11 2018-04-24 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US9956299B2 (en) 2013-10-11 2018-05-01 Medimmune Limited Pyrrolobenzodiazepine—antibody conjugates
WO2018114798A1 (en) 2016-12-21 2018-06-28 Bayer Aktiengesellschaft Prodrugs of cytotoxic active agents having enzymatically cleavable groups
WO2018114578A1 (en) 2016-12-21 2018-06-28 Bayer Pharma Aktiengesellschaft Antibody drug conjugates (adcs) having enzymatically cleavable groups
US10010624B2 (en) 2013-10-11 2018-07-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10022453B2 (en) 2013-12-23 2018-07-17 Bayer Pharma Aktiengesellschaft Antibody drug conjugates (ADCs) with kinesin spindel protein (KSP)
US10029018B2 (en) 2013-10-11 2018-07-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2019060398A1 (en) 2017-09-20 2019-03-28 Ph Pharma Co., Ltd. Thailanstatin analogs
US10392393B2 (en) 2016-01-26 2019-08-27 Medimmune Limited Pyrrolobenzodiazepines
US10420777B2 (en) 2014-09-12 2019-09-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2019232449A1 (en) 2018-06-01 2019-12-05 Eisai R&D Management Co., Ltd. Splicing modulator antibody-drug conjugates and methods of use
US10544223B2 (en) 2017-04-20 2020-01-28 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate
US10543279B2 (en) 2016-04-29 2020-01-28 Medimmune Limited Pyrrolobenzodiazepine conjugates and their use for the treatment of cancer
WO2020049286A1 (en) 2018-09-03 2020-03-12 Femtogenix Limited Polycyclic amides as cytotoxic agents
WO2020086858A1 (en) 2018-10-24 2020-04-30 Genentech, Inc. Conjugated chemical inducers of degradation and methods of use
WO2020123836A2 (en) 2018-12-13 2020-06-18 Eisai R&D Management Co., Ltd. Herboxidiene splicing modulator antibody-drug conjugates and methods of use
WO2020123275A1 (en) 2018-12-10 2020-06-18 Genentech, Inc. Photocrosslinking peptides for site specific conjugation to fc-containing proteins
US10695433B2 (en) 2012-10-12 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10695439B2 (en) 2016-02-10 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine conjugates
WO2020157491A1 (en) 2019-01-29 2020-08-06 Femtogenix Limited G-a crosslinking cytotoxic agents
US10736903B2 (en) 2012-10-12 2020-08-11 Medimmune Limited Pyrrolobenzodiazepine-anti-PSMA antibody conjugates
US10751346B2 (en) 2012-10-12 2020-08-25 Medimmune Limited Pyrrolobenzodiazepine—anti-PSMA antibody conjugates
US10780096B2 (en) 2014-11-25 2020-09-22 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US10799595B2 (en) 2016-10-14 2020-10-13 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11001636B2 (en) 2016-06-15 2021-05-11 Bayer Pharma Aktiengesellschaft Specific antibody-drug-conjugates (ADCs) with KSP inhibitors and anti-CD123-antibodies
US11059893B2 (en) 2015-04-15 2021-07-13 Bergenbio Asa Humanized anti-AXL antibodies
US11135303B2 (en) 2011-10-14 2021-10-05 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US11160872B2 (en) 2017-02-08 2021-11-02 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
WO2022023735A1 (en) 2020-07-28 2022-02-03 Femtogenix Limited Cytotoxic agents
US11318211B2 (en) 2017-06-14 2022-05-03 Adc Therapeutics Sa Dosage regimes for the administration of an anti-CD19 ADC
US11352324B2 (en) 2018-03-01 2022-06-07 Medimmune Limited Methods
US11370801B2 (en) 2017-04-18 2022-06-28 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11433140B2 (en) 2016-12-21 2022-09-06 Bayer Pharma Aktiengesellschaft Specific antibody drug conjugates (ADCs) having KSP inhibitors
US11517626B2 (en) 2016-02-10 2022-12-06 Medimmune Limited Pyrrolobenzodiazepine antibody conjugates
US11524969B2 (en) 2018-04-12 2022-12-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof as antitumour agents
US11612665B2 (en) 2017-02-08 2023-03-28 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11649250B2 (en) 2017-08-18 2023-05-16 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11702473B2 (en) 2015-04-15 2023-07-18 Medimmune Limited Site-specific antibody-drug conjugates

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090123470A1 (en) * 2001-03-26 2009-05-14 The Macfarlane Burnet Istitute For Medical Research And Public Health Ltd. Antibodies Against Cancer
EP2279414A4 (en) * 2008-04-21 2011-06-29 Merck Sharp & Dohme Pancreatic beta-cell mass biomarker
JP5805538B2 (en) * 2008-11-07 2015-11-04 リサーチ ディベロップメント ファウンデーション Compositions and methods for inhibiting Cripto / GSP78 complex formation and signaling
EP3138581B1 (en) 2011-03-17 2019-01-02 The University of Birmingham Re-directed immunotherapy
WO2014096365A1 (en) 2012-12-21 2014-06-26 Spirogen Sàrl Unsymmetrical pyrrolobenzodiazepines-dimers for use in the treatment of proliferative and autoimmune diseases
EA031585B1 (en) 2012-12-21 2019-01-31 Медимьюн Лимитед Pyrrolobenzodiazepines and conjugates thereof
AU2019402097A1 (en) 2018-12-17 2021-06-10 Revitope Limited Twin immune cell engager

Family Cites Families (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ207394A (en) * 1983-03-08 1987-03-06 Commw Serum Lab Commission Detecting or determining sequence of amino acids
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1989006692A1 (en) 1988-01-12 1989-07-27 Genentech, Inc. Method of treating tumor cells by inhibiting growth factor receptor function
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US5780029A (en) 1989-11-14 1998-07-14 New York Medical College Antidiotypic monoclonal antibodies for treatment of melanoma
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US5256643A (en) * 1990-05-29 1993-10-26 The Government Of The United States Human cripto protein
ES2246502T3 (en) 1990-08-29 2006-02-16 Genpharm International, Inc. TRANSGENIC NON-HUMAN ANIMALS ABLE TO PRODUCE HETEROLOGICAL ANTIBODIES.
WO1994004679A1 (en) 1991-06-14 1994-03-03 Genentech, Inc. Method for making humanized antibodies
US5264557A (en) 1991-08-23 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Polypeptide of a human cripto-related gene, CR-3
US6063379A (en) 1993-12-09 2000-05-16 Centro De Inmunologia Molecular Anti-idiotypic monoclonal antibodies and compositions including the anti-idiotypic monoclonal antibodies
US5981215A (en) * 1995-06-06 1999-11-09 Human Genome Sciences, Inc. Human criptin growth factor
NZ332566A (en) * 1996-05-22 2000-08-25 Novopharm Biotech Inc Antigen binding fragments that specifically detect cancer cells
IL125608A0 (en) 1998-07-30 1999-03-12 Yeda Res & Dev Tumor associated antigen peptides and use of same as anti-tumor vaccines
UA76936C2 (en) 1998-12-23 2006-10-16 Pfizer Human monoclonal antibody binding the antigen-4 of cytotoxic t-lymphocyte (ctla-4), nucleic acid, cell line, method of obtaining transgenic mammal, method to increase producing il-2 or ifn-? in the t-cells of a patient, method to treat and pharmaceutical composition used in treatment of cancer, inflammation or autoimmune disease, including the antibody
WO2000052204A2 (en) * 1999-02-22 2000-09-08 Orntoft Torben F Gene expression in bladder tumors
WO2000058499A1 (en) 1999-03-30 2000-10-05 Japan Tobacco Inc. Process for producing monoclonal antibody
WO2000063693A1 (en) * 1999-04-20 2000-10-26 Kokolus William J Improved method of identifying and locating immunobiologically-active linear peptides
US6833268B1 (en) 1999-06-10 2004-12-21 Abgenix, Inc. Transgenic animals for producing specific isotypes of human antibodies via non-cognate switch regions
JP2001046066A (en) 1999-08-03 2001-02-20 Kyowa Hakko Kogyo Co Ltd Antibody against human vegf receptor kdr having new complementary determining region
DE60039448D1 (en) 1999-10-29 2008-08-21 Genentech Inc AGAINST PROSTATE-STATE-TEMPORARY (PSCA) ANTIBODIES AND THEIR USE
US6663613B1 (en) 2000-01-25 2003-12-16 Bacchus Vascular, Inc. System and methods for clot dissolution
AU3607301A (en) 2000-03-03 2001-09-12 Kyowa Hakko Kogyo Kk Gene recombinant antibody and its fragment
US6333410B1 (en) * 2000-08-18 2001-12-25 Immunogen, Inc. Process for the preparation and purification of thiol-containing maytansinoids
GB0020953D0 (en) 2000-08-24 2000-10-11 Smithkline Beecham Biolog Vaccine
AU2001292724B2 (en) * 2000-09-18 2005-05-26 Biogen Idec Ma Inc. CRIPTO mutant and uses thereof
JP3821779B2 (en) 2001-01-26 2006-09-13 アメリカ合衆国 Detection and quantification of CRIPTO-1
PL372140A1 (en) 2001-01-29 2005-07-11 Idec Pharmaceuticals Corporation Modified antibodies and methods of use
WO2002096948A2 (en) 2001-01-29 2002-12-05 Idec Pharmaceuticals Corporation Engineered tetravalent antibodies and methods of use
AUPR395801A0 (en) 2001-03-26 2001-04-26 Austin Research Institute, The Antibodies against cancer
CA2443840C (en) 2001-04-26 2010-10-26 Biogen, Inc. Cripto blocking antibodies and uses thereof
US7582299B2 (en) * 2001-04-26 2009-09-01 Biogen Idec Ma Inc. Cripto-specific antibodies
ES2390531T3 (en) * 2001-09-18 2012-11-13 Genentech, Inc. Compositions and procedures for the diagnosis and treatment of tumor
US20040014690A1 (en) * 2002-02-13 2004-01-22 Zhenkun Ma Macrolides with activity against methicillin-resistant staphylococcus aureus
EP1494693B1 (en) * 2002-03-22 2010-12-08 Biogen Idec MA Inc. Cripto-specific antibodies
US7820620B2 (en) * 2003-09-15 2010-10-26 Research Development Foundation Cripto antagonism of activin and TGF-b signaling
EP2311880A3 (en) 2005-01-05 2011-07-27 Biogen Idec MA Inc. Cripto binding molecules
US20100041032A1 (en) * 2006-04-28 2010-02-18 Biogen Idec Ma Inc. Composition and methods for the detection of cripto-3

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BALDASSARRE ET AL., INT. J. CANCER, vol. 66, 1996, pages 538 - 543
CIARDIELLO ET AL., ONCOGENE, vol. 9, 1994, pages 291 - 298
MINCHIOTTI, MECH. DEV., vol. 90, 2000, pages 133 - 142
SAIJOH ET AL., MOL. CELL, vol. 5, 2000, pages 35 - 47
SALOMON ET AL., BIOESSAYS, vol. 21, 1999, pages 61 - 70
SCHIFFERLI ET AL., FOCUS, vol. 21, 1999, pages 16

Cited By (135)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7318924B2 (en) 2001-03-26 2008-01-15 The Austin Research Institute Antibodies against cancer
US7674462B2 (en) 2001-04-26 2010-03-09 Biogen Idec Ma Inc. Cripto blocking antibodies and uses thereof
US7888052B2 (en) 2001-04-26 2011-02-15 Biogen Idec Ma Inc. Cripto blocking antibodies and uses thereof
US8003763B2 (en) 2001-04-26 2011-08-23 Biogen Idec Ma Inc. Cripto blocking antibodies and uses thereof
US8673303B2 (en) 2001-04-26 2014-03-18 Biogen Idec Ma Inc. Cripto blocking antibodies and uses thereof
US7531174B2 (en) 2001-04-26 2009-05-12 Biogen Idec Ma Inc. Cripto blocking antibodies and uses thereof
US7582299B2 (en) 2001-04-26 2009-09-01 Biogen Idec Ma Inc. Cripto-specific antibodies
EP1494693A4 (en) * 2002-03-22 2006-08-02 Biogen Idec Inc Cripto-specific antibodies
EP1494693A2 (en) * 2002-03-22 2005-01-12 Biogen Inc. Cripto-specific antibodies
US7700097B2 (en) 2003-06-27 2010-04-20 Biogen Idec Ma Inc. Purification and preferential synthesis of binding molecules
US8603473B2 (en) 2003-06-27 2013-12-10 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
WO2005000899A2 (en) 2003-06-27 2005-01-06 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
US7820620B2 (en) 2003-09-15 2010-10-26 Research Development Foundation Cripto antagonism of activin and TGF-b signaling
EP1670511A2 (en) * 2003-09-15 2006-06-21 Research Development Foundation Cripto antagonism of activin and tgf-b signaling
US9546210B2 (en) 2003-09-15 2017-01-17 Research Development Foundation Cripto antagonism of activin and TGF-b signaling
AU2004274433B2 (en) * 2003-09-15 2010-08-12 Research Development Foundation Cripto antagonism of activin and TGF-beta signaling
EP1670511A4 (en) * 2003-09-15 2008-03-26 Res Dev Foundation Cripto antagonism of activin and tgf-b signaling
EP3858387A1 (en) 2003-11-06 2021-08-04 Seagen Inc. Monomethylvaline compounds capable of conjugation to ligands
EP2260858A2 (en) 2003-11-06 2010-12-15 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
EP2489364A1 (en) 2003-11-06 2012-08-22 Seattle Genetics, Inc. Monomethylvaline compounds onjugated to antibodies
EP2486933A1 (en) 2003-11-06 2012-08-15 Seattle Genetics, Inc. Monomethylvaline compounds conjugated with antibodies
EP2478912A1 (en) 2003-11-06 2012-07-25 Seattle Genetics, Inc. Auristatin conjugates with anti-HER2 or anti-CD22 antibodies and their use in therapy
EP3434275A1 (en) 2003-11-06 2019-01-30 Seattle Genetics, Inc. Assay for cancer cells based on the use of auristatin conjugates with antibodies
EP2385069A2 (en) 2003-11-12 2011-11-09 Biogen Idec MA Inc. Neonatal Fc rReceptor (FcRn)- binding polypeptide variants, dimeric Fc binding proteins and methods related thereto
EP2286844A2 (en) 2004-06-01 2011-02-23 Genentech, Inc. Antibody-drug conjugates and methods
EP3088004A1 (en) 2004-09-23 2016-11-02 Genentech, Inc. Cysteine engineered antibodies and conjugates
EP2311880A3 (en) * 2005-01-05 2011-07-27 Biogen Idec MA Inc. Cripto binding molecules
CN101137673B (en) * 2005-01-05 2013-12-04 比奥根艾迪克Ma公司 Cripto binding molecules
US8021661B2 (en) 2005-01-05 2011-09-20 Biogen Idec Ma Inc. Cripto binding molecules
WO2006074397A2 (en) * 2005-01-05 2006-07-13 Biogen Idec Ma Inc. Cripto binding molecules
US8084026B2 (en) 2005-01-05 2011-12-27 Biogen Idec Ma Inc. Multispecific binding molecules comprising connecting peptides
WO2006074397A3 (en) * 2005-01-05 2006-12-28 Biogen Idec Inc Cripto binding molecules
EP2311880A2 (en) 2005-01-05 2011-04-20 Biogen Idec MA Inc. Cripto binding molecules
EP2311881A3 (en) * 2005-01-05 2011-07-27 Biogen Idec MA Inc. Cripto binding molecules
EP2311881A2 (en) 2005-01-05 2011-04-20 Biogen Idec MA Inc. Cripto binding molecules
US8975037B2 (en) 2006-07-28 2015-03-10 Ann & Robert H. Lurie Children's Hospital of Chicago Methods of inhibiting tumor cell aggressiveness using the microenvironment of human embryonic stem cells
WO2008150530A2 (en) * 2007-06-01 2008-12-11 Biogen Idec Ma Inc. Cripto binding molecules
WO2008150530A3 (en) * 2007-06-01 2009-02-19 Biogen Idec Inc Cripto binding molecules
WO2011031870A1 (en) 2009-09-09 2011-03-17 Centrose, Llc Extracellular targeted drug conjugates
WO2011056983A1 (en) 2009-11-05 2011-05-12 Genentech, Inc. Zirconium-radiolabeled, cysteine engineered antibody conjugates
WO2011130598A1 (en) 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazepines and conjugates thereof
WO2011156328A1 (en) 2010-06-08 2011-12-15 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2012074757A1 (en) 2010-11-17 2012-06-07 Genentech, Inc. Alaninyl maytansinol antibody conjugates
WO2012143496A2 (en) 2011-04-21 2012-10-26 Bayer Intellectual Property Gmbh Novel binder-drug conjugates (adcs) and their use
WO2012143499A2 (en) 2011-04-21 2012-10-26 Bayer Intellectual Property Gmbh Novel binder-drug conjugates (adcs) and their use
WO2012155019A1 (en) 2011-05-12 2012-11-15 Genentech, Inc. Multiple reaction monitoring lc-ms/ms method to detect therapeutic antibodies in animal samples using framework signature pepides
WO2013012733A1 (en) 2011-07-15 2013-01-24 Biogen Idec Ma Inc. Heterodimeric fc regions, binding molecules comprising same, and methods relating thereto
US11135303B2 (en) 2011-10-14 2021-10-05 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2013130093A1 (en) 2012-03-02 2013-09-06 Genentech, Inc. Biomarkers for treatment with anti-tubulin chemotherapeutic compounds
US10722594B2 (en) 2012-10-12 2020-07-28 Adc Therapeutics S.A. Pyrrolobenzodiazepine-anti-CD22 antibody conjugates
EP2839860A1 (en) 2012-10-12 2015-02-25 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
US10646584B2 (en) 2012-10-12 2020-05-12 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2014057074A1 (en) 2012-10-12 2014-04-17 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
US9889207B2 (en) 2012-10-12 2018-02-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10695433B2 (en) 2012-10-12 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10736903B2 (en) 2012-10-12 2020-08-11 Medimmune Limited Pyrrolobenzodiazepine-anti-PSMA antibody conjugates
US11690918B2 (en) 2012-10-12 2023-07-04 Medimmune Limited Pyrrolobenzodiazepine-anti-CD22 antibody conjugates
US10335497B2 (en) 2012-10-12 2019-07-02 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9931415B2 (en) 2012-10-12 2018-04-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11701430B2 (en) 2012-10-12 2023-07-18 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9919056B2 (en) 2012-10-12 2018-03-20 Adc Therapeutics S.A. Pyrrolobenzodiazepine-anti-CD22 antibody conjugates
US10751346B2 (en) 2012-10-12 2020-08-25 Medimmune Limited Pyrrolobenzodiazepine—anti-PSMA antibody conjugates
US11771775B2 (en) 2012-10-12 2023-10-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11779650B2 (en) 2012-10-12 2023-10-10 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10799596B2 (en) 2012-10-12 2020-10-13 Adc Therapeutics S.A. Pyrrolobenzodiazepine-antibody conjugates
US10994023B2 (en) 2012-10-12 2021-05-04 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10780181B2 (en) 2012-10-12 2020-09-22 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US9931414B2 (en) 2012-10-12 2018-04-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
WO2014140174A1 (en) 2013-03-13 2014-09-18 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
WO2014159981A2 (en) 2013-03-13 2014-10-02 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2014140862A2 (en) 2013-03-13 2014-09-18 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2015023355A1 (en) 2013-08-12 2015-02-19 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
US10029018B2 (en) 2013-10-11 2018-07-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10010624B2 (en) 2013-10-11 2018-07-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US9956299B2 (en) 2013-10-11 2018-05-01 Medimmune Limited Pyrrolobenzodiazepine—antibody conjugates
US9950078B2 (en) 2013-10-11 2018-04-24 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
WO2015095223A2 (en) 2013-12-16 2015-06-25 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2015095212A1 (en) 2013-12-16 2015-06-25 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
WO2015095227A2 (en) 2013-12-16 2015-06-25 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
US10022453B2 (en) 2013-12-23 2018-07-17 Bayer Pharma Aktiengesellschaft Antibody drug conjugates (ADCs) with kinesin spindel protein (KSP)
US10188746B2 (en) 2014-09-10 2019-01-29 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2016037644A1 (en) 2014-09-10 2016-03-17 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2016040856A2 (en) 2014-09-12 2016-03-17 Genentech, Inc. Cysteine engineered antibodies and conjugates
US10420777B2 (en) 2014-09-12 2019-09-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2016040825A1 (en) 2014-09-12 2016-03-17 Genentech, Inc. Anthracycline disulfide intermediates, antibody-drug conjugates and methods
EP3235820A1 (en) 2014-09-17 2017-10-25 Genentech, Inc. Pyrrolobenzodiazepines and antibody disulfide conjugates thereof
US10780096B2 (en) 2014-11-25 2020-09-22 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
WO2016090050A1 (en) 2014-12-03 2016-06-09 Genentech, Inc. Quaternary amine compounds and antibody-drug conjugates thereof
WO2016161477A1 (en) * 2015-04-07 2016-10-13 Prince Henry's Institute Of Medical Research (Acn 132 025 024) A method of treating neoplasias
US11059893B2 (en) 2015-04-15 2021-07-13 Bergenbio Asa Humanized anti-AXL antibodies
US11702473B2 (en) 2015-04-15 2023-07-18 Medimmune Limited Site-specific antibody-drug conjugates
WO2016207089A1 (en) 2015-06-22 2016-12-29 Bayer Pharma Aktiengesellschaft Antibody drug conjugates (adcs) and antibody prodrug conjugates (apdcs) with enzymatically cleavable groups
US11123439B2 (en) 2015-06-22 2021-09-21 Bayer Pharma Aktiengesellschaft Antibody drug conjugates (ADCS) and antibody prodrug conjugates (APDCS) with enzymatically cleavable groups
WO2017059289A1 (en) 2015-10-02 2017-04-06 Genentech, Inc. Pyrrolobenzodiazepine antibody drug conjugates and methods of use
WO2017060322A2 (en) 2015-10-10 2017-04-13 Bayer Pharma Aktiengesellschaft Ptefb-inhibitor-adc
WO2017064675A1 (en) 2015-10-16 2017-04-20 Genentech, Inc. Hindered disulfide drug conjugates
WO2017068511A1 (en) 2015-10-20 2017-04-27 Genentech, Inc. Calicheamicin-antibody-drug conjugates and methods of use
US10392393B2 (en) 2016-01-26 2019-08-27 Medimmune Limited Pyrrolobenzodiazepines
US11517626B2 (en) 2016-02-10 2022-12-06 Medimmune Limited Pyrrolobenzodiazepine antibody conjugates
US10695439B2 (en) 2016-02-10 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11685714B2 (en) 2016-03-24 2023-06-27 Bayer Pharma Aktiengesellschaft Prodrugs of cytotoxic active agents having enzymatically cleavable groups
WO2017162663A1 (en) 2016-03-24 2017-09-28 Bayer Pharma Aktiengesellschaft Prodrugs of cytotoxic active agents having enzymatically cleavable groups
EP4273551A2 (en) 2016-03-25 2023-11-08 F. Hoffmann-La Roche AG Multiplexed total antibody and antibody-conjugated drug quantification assay
WO2017165734A1 (en) 2016-03-25 2017-09-28 Genentech, Inc. Multiplexed total antibody and antibody-conjugated drug quantification assay
US10543279B2 (en) 2016-04-29 2020-01-28 Medimmune Limited Pyrrolobenzodiazepine conjugates and their use for the treatment of cancer
WO2017201449A1 (en) 2016-05-20 2017-11-23 Genentech, Inc. Protac antibody conjugates and methods of use
WO2017205741A1 (en) 2016-05-27 2017-11-30 Genentech, Inc. Bioanalytical method for the characterization of site-specific antibody-drug conjugates
WO2017214024A1 (en) 2016-06-06 2017-12-14 Genentech, Inc. Silvestrol antibody-drug conjugates and methods of use
US11643469B2 (en) 2016-06-15 2023-05-09 Bayer Pharma Aktiengesellschaft Specific antibody-drug-conjugates (ADCs) with KSP inhibitors and anti-CD123-antibodies
US11001636B2 (en) 2016-06-15 2021-05-11 Bayer Pharma Aktiengesellschaft Specific antibody-drug-conjugates (ADCs) with KSP inhibitors and anti-CD123-antibodies
WO2018031662A1 (en) 2016-08-11 2018-02-15 Genentech, Inc. Pyrrolobenzodiazepine prodrugs and antibody conjugates thereof
WO2018065501A1 (en) 2016-10-05 2018-04-12 F. Hoffmann-La Roche Ag Methods for preparing antibody drug conjugates
US10799595B2 (en) 2016-10-14 2020-10-13 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11660351B2 (en) 2016-12-21 2023-05-30 Bayer Aktiengesellschaft Antibody drug conjugates (ADCs) having enzymatically cleavable groups
WO2018114798A1 (en) 2016-12-21 2018-06-28 Bayer Aktiengesellschaft Prodrugs of cytotoxic active agents having enzymatically cleavable groups
US11433140B2 (en) 2016-12-21 2022-09-06 Bayer Pharma Aktiengesellschaft Specific antibody drug conjugates (ADCs) having KSP inhibitors
US11478554B2 (en) 2016-12-21 2022-10-25 Bayer Pharma Aktiengesellschaft Antibody drug conjugates (ADCS) having enzymatically cleavable groups
WO2018114578A1 (en) 2016-12-21 2018-06-28 Bayer Pharma Aktiengesellschaft Antibody drug conjugates (adcs) having enzymatically cleavable groups
US11813335B2 (en) 2017-02-08 2023-11-14 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11160872B2 (en) 2017-02-08 2021-11-02 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11612665B2 (en) 2017-02-08 2023-03-28 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11370801B2 (en) 2017-04-18 2022-06-28 Medimmune Limited Pyrrolobenzodiazepine conjugates
US10544223B2 (en) 2017-04-20 2020-01-28 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate
US11318211B2 (en) 2017-06-14 2022-05-03 Adc Therapeutics Sa Dosage regimes for the administration of an anti-CD19 ADC
US11649250B2 (en) 2017-08-18 2023-05-16 Medimmune Limited Pyrrolobenzodiazepine conjugates
WO2019060398A1 (en) 2017-09-20 2019-03-28 Ph Pharma Co., Ltd. Thailanstatin analogs
US11352324B2 (en) 2018-03-01 2022-06-07 Medimmune Limited Methods
US11524969B2 (en) 2018-04-12 2022-12-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof as antitumour agents
WO2019232449A1 (en) 2018-06-01 2019-12-05 Eisai R&D Management Co., Ltd. Splicing modulator antibody-drug conjugates and methods of use
WO2020049286A1 (en) 2018-09-03 2020-03-12 Femtogenix Limited Polycyclic amides as cytotoxic agents
WO2020086858A1 (en) 2018-10-24 2020-04-30 Genentech, Inc. Conjugated chemical inducers of degradation and methods of use
WO2020123275A1 (en) 2018-12-10 2020-06-18 Genentech, Inc. Photocrosslinking peptides for site specific conjugation to fc-containing proteins
WO2020123836A2 (en) 2018-12-13 2020-06-18 Eisai R&D Management Co., Ltd. Herboxidiene splicing modulator antibody-drug conjugates and methods of use
WO2020157491A1 (en) 2019-01-29 2020-08-06 Femtogenix Limited G-a crosslinking cytotoxic agents
WO2022023735A1 (en) 2020-07-28 2022-02-03 Femtogenix Limited Cytotoxic agents

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