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  • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
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  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39566—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against immunoglobulins, e.g. anti-idiotypic antibodies
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  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [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
  • C07K16/3007—Carcino-embryonic Antigens
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
  • G01N33/686—Anti-idiotype
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/57—Skin; melanoma
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the present invention relates to therapeutic and diagnostic antibodies, useful in diseases involving Carcinoembryonic Antigen-Related Cell Adhesion Molecule (CEACAM), expression, activation or function.
• CEACAM Carcinoembryonic Antigen-Related Cell Adhesion Molecule
• the present invention provides antibodies having specific complementarity determining regions (CDRs) and improved properties over other antibodies which recognize CEACAMl.
• CEACAMl Carcinoembryonic antigen-related cell adhesion molecule 1
• BGP biliary glycoprotein
• CD66a and C-CAM1 are members of the carcinoembryonic antigen family (CEA) that also belongs to the immunoglobulin superfamily.
• CEACAMl interacts with other known CEACAM proteins, including CD66a (CEACAMl), CD66e (CEACAM6) and CD66e (CEACAM5, CEA) proteins. It is expressed on a wide spectrum of cells, ranging from epithelial cells to those of hemopoietic origin (e.g. immune cells).
• CEACAMl has been attributed to many different functions. It was shown that the CEACAMl protein exhibits anti-proliferative properties in carcinomas of colon, prostate, as well as other types of cancer. Additional data support the central involvement of CEACAMl in angiogenesis and metastasis. CEACAMl also plays a role in the modulation of innate and adaptive immune responses. For example, CEACAMl was shown to be an inhibitory receptor for activated T cells contained within the human intestinal epithelium ( W099/52552 and Morales et al. J. Immunol. 1999, 163, 1363-1370). Additional reports have indicated that CEACAMl engagement either by T Cell Receptor cross-linking with Monoclonal antibodies (mAbs) or by Neisseria gonorrhoeae Opa proteins inhibits T cell activation and proliferation.
• mAbs Monoclonal antibodies
• Neisseria gonorrhoeae Opa proteins inhibits T cell activation and proliferation.
• Melanoma is a malignancy of pigment-producing cells (melanocytes), responsible for 75% of skin cancer-related mortality worldwide, mainly due to extensive metastasis. Metastatic melanoma (MM) responds feebly to most anticancer regimens and overall survival mean for patients with MM is 8.5 months.
• CEACAMl is rarely expressed by normal melanocytes, but frequently found on melanoma cells.
• CEACAMl expression on primary cutaneous melanoma lesions strongly predicts the development of metastatic disease with poor prognosis.
• increased CEACAMl expression was observed on NK cells derived from some patients with metastatic melanoma compared with healthy donors.
• CEACAMl can be correlated with poor prognosis and is detected in the majority of metastatic melanoma cases.
• CEACAMl may have an important role in virus infections.
• Markel at el. J. Clinical Investigation 2002, 110, 943-953 demonstrated that lymphocytes isolated from the deciduae of CMV-infected patients express the CEACAMl protein in increased levels.
• the increased CEACAMl expression on the decidual lymphocytes might diminish the local immune response and serve as another mechanism developed by the virus to avoid recognition and clearance primarily by activated decidual lymphocytes.
• WO2007/063424 and U.S. Patent Application No. 20070110668 disclose methods for regulating the immune system, and in particular methods for the regulation of a specific immune response, including the regulation of lymphocyte activity. These methods comprise both the negative and positive modulation of CEACAMl protein function.
• U.S. Patent Application No. 20070071758 teaches methods and compositions for enhancing the efficacy of tumor-infiltrating lymphocyte (TIL) therapy in the treatment of cancer by negatively modulating the activity of the CEACAMl protein, such as for example, by using an immunoglobulin specific for CEACAMl.
• TIL tumor-infiltrating lymphocyte
• U.S. Patent Application No. 20080108140 discloses methods of modulating specific immune responses to create a protective immunity in the treatment of autoimmune diseases and diseases requiring the transplantation of tissue.
• U.S. Patent Application No. 20080108140 relates to the suppression of immune responses in a targeted fashion, by increasing the functional concentration of the CEACAMl protein in the target tissue.
• U.S. Patent Application No. 20040047858 discloses specific antibodies which are capable of modulating T cell activity via CEACAM 1 and uses thereof in treating immune response related diseases (e.g. graft versus host disease, autoimmune diseases, cancers etc.).
• compositions which bind T cell inhibitory receptor molecules and modulate (i.e. enhance or suppress) T cell activity (e.g. cytotoxicity and proliferation), such as biliary glycoprotein binding agents, and methods of using such compositions such as for treatment of diseases (e.g. an autoimmune disease, immunodeficiency, cancer etc.).
• diseases e.g. an autoimmune disease, immunodeficiency, cancer etc.
• WO 2010/125571 discloses a murine monoclonal antibody produced by a specific hybridoma cell.
• the mAb is highly selective to CEACAM 1 and does not cross-react with other members of the CEACAM family.
• the present invention discloses monoclonal antibodies which recognize a specific set of CEACAM subtypes.
• the antibodies of the invention show binding to CEACAM 1 and at least one additional subtype selected from CEACAM5 and CEACAM3.
• the antibodies of the invention are characterized by unique CDR sequences and combinations of frameworks and CDR.
• the unique specificity of the monoclonal antibodies of the present invention broaden their therapeutic utility for treatment and diagnosis of additional types of malignancies and viral infections.
• the present invention also provides methods for obtaining such antibodies, methods for their production, and therapeutic and diagnostic uses thereof.
• the monoclonal antibodies according to the present invention have specific combinations of CDRs and frameworks and possess unique properties and improved specificity and potency over known anti CEACAM 1 antibodies.
• the present invention provides a monoclonal antibody which recognizes CEACAM 1, or an antibody fragment comprising at least an antigen-binding portion thereof, having heavy-chain CDRs comprising sequences set forth in SEQ ID NOs: 1, 2 and 3, and light-chain CDRs comprising sequences set forth in SEQ ID NOs: 4, 5 and 6, and analogs and derivatives thereof.
• a monoclonal antibody or antibody fragment which recognizes CEACAM1 having a heavy-chain CDR1 comprising a sequence set forth in SEQ ID NO: 1 , a heavy-chain CDR2 comprising a sequence set forth in SEQ ID NO: 2 a heavy-chain CDR3 comprising a sequence set forth in SEQ ID NO: 3, a light-chain CDR1 comprising a sequence set forth in SEQ ID NO: 4, a light-chain CDR2 comprising a sequence set forth in SEQ ID NO: 5 and a light-chain CDR3 comprising a sequence set forth in SEQ ID NO: 6, and analogs and derivatives thereof.
• CEACAM1 or a fragment thereof comprising at least an antigen binding portion is provided, comprising heavy chain CDRs having the sequences set forth in SEQ ID NOs: 7, 8 and 9.
• a monoclonal antibody which recognizes CEACAM1 or a fragment thereof comprising at least an antigen binding portion comprising heavy chain CDRs having the sequences set forth in SEQ ID NOs: 13, 14 and 15.
• a monoclonal antibody which recognizes CEACAM1 or a fragment thereof comprising at least an antigen binding portion comprising light chain CDRs having the sequences set forth in SEQ ID NOs: 10, 11 and 12.
• a monoclonal antibody which recognizes CEACAM1 or a fragment thereof comprising at least an antigen binding portion is provided, wherein the light chain CDRs having the sequences set forth in SEQ ID NOs: 16, 17 and 18.
• a monoclonal antibody having CDR sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17, and 18.
• a monoclonal antibody having CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, 11, and 12.
• Analogs and derivatives of the monoclonal antibody or fragment thereof, having at least 90% sequence identity with the antigen-binding portion of the reference sequence are also within the scope of the present invention.
• analogs and derivatives of the monoclonal antibody or fragment thereof having at least 95% sequence identity with the antigen-binding portion of the reference sequence are provided.
• the antibody comprises a heavy chain variable domain sequence having a sequence set forth in SEQ ID NO: 26: QVQLQQSGAELVRPGTSVKVSCKASGYAFTNNLIEWVKQRPGQGLEWIGVINPGSG DTNYNEKFKGKATLTADKSSNTAYMQLSSLTSDDSAVYFCARGDYYGGFAVDYW GQGTSVTVSS, or an analog or derivative thereof having at least 97% sequence identity with the heavy chain sequence.
• the antibody comprises a light chain variable domain sequence having a sequence set forth in SEQ ID NO: 28:
• the antibody or fragment thereof comprises a heavy chain variable domain having a sequence set forth in SEQ ID NO: 26 and a light chain variable domain having a sequence set forth in SEQ ID NO: 28, or an analog or derivative thereof having at least 97% sequence identity with the antibody or fragment sequence.
• the present invention encompasses monoclonal antibodies isolated from hybridoma cells or other biological systems, as well as monoclonal antibodies produced recombinantly or synthetically.
• a monoclonal antibody according to the present invention may contain a constant region from any mammalian species, including but not limited to mouse, rat and human.
• a monoclonal antibody according to the present invention includes a chimeric antibody, a humanized antibody, a fully human antibody, a xenogeneic antibody, and an antibody fragment comprising at least the antigen-binding portion of an antibody.
• the antibody fragment is selected from the group consisting of: Fab, Fab', F(ab') 2 , Fd, Fd', Fv, dAb, isolated CDR region, single chain antibody, "diabodies", and "linear antibodies”.
• the present invention provides a monoclonal antibody, or an antibody fragment comprising:
• a framework sequence selected from the group consisting of: mouse IgG2a, mouse IgG2b, mouse IgG3, human IgGl, human IgG2, human IgG3; and
• the monoclonal antibody or fragment thereof binds with an affinity of at least about 5xl0 "7 M to at least two CEACAM subtypes. According to other embodiments, the monoclonal antibody or fragment thereof binds with an affinity of at least about 10 "8 M to CEAC AM 1.
• the monoclonal antibody is a chimeric monoclonal antibody.
• the chimeric antibody comprises human-derived constant regions.
• the human constant regions of the chimeric antibody are selected from the group consisting of: human IgGl, human IgG2, and human IgG3
• a chimeric or humanized monoclonal antibody which recognizes CEACAMl comprising the six CDRs having sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17, and 18; or the six CDRs having sequences set forth in SEQ ID NOs: 7, 8, 9, 10, 11, and 12; and analogs and derivatives thereof having at least 95% sequence identity with said CDR sequences, and a constant region subclass selected from human IgGl, human IgG2 and human IgG3, wherein the monoclonal antibody binds with an affinity of at least about 5x10 ⁇ 7 M to at least two CEAC AM subtypes.
• the chimeric or humanized monoclonal antibody or fragment thereof comprises a constant region subclass of human IgGl subtype.
• a chimeric monoclonal antibody or a fragment thereof comprising at least the antigen-binding portion comprising a heavy chain sequence set forth in SEQ ID NO: 30.
• a chimeric monoclonal antibody or a fragment thereof comprising at least the antigen-binding portion comprising a light chain sequence set forth in SEQ ID NO: 31.
• a chimeric monoclonal antibody or a fragment thereof comprising at least the antigen-binding portion having a heavy chain sequence set forth in SEQ ID NO: 30, and light chain sequence set forth in SEQ ID NO: 31.
• a monoclonal antibody which recognizes CEACAMl is provided produced from DNA sequences of the heavy and light chains contained in a plasmid deposited on September 28, 2011 under ATCC Accession Number
• Monoclonal antibodies of the present invention exhibit, according to some embodiments, specific binding to more than one CEACAM subtype.
• the monoclonal antibody binds at least two different CEACAM subtypes.
• the monoclonal antibody binds to CEACAMl and at least one of CEACAM3 and CEACAM5.
• the monoclonal antibody binds to CEACAMl and CEACAM5.
• the monoclonal antibody binds to CEACAMl and CEACAM3.
• a monoclonal antibody according to the present invention binds to CEACAM subtypes 1, 3 and 5.
• a monoclonal antibody according to the present invention does not bind to CEACAM4 and CEACAM6.
• the present invention provides a monoclonal antibody which recognizes CEACAMl, or a fragment thereof comprising at least the antigen-binding portion, which is capable of binding the same epitope on the CEACAMl molecule to which a monoclonal antibody to CEACAMl having a heavy chain sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 30 and a light chain sequence set forth as SEQ ID NO: 28 or SEQ ID NO: 31, binds.
• the monoclonal antibody or fragment thereof binds to the same epitope of which an antibody having the six CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, 11 and 12 binds.
• the monoclonal antibody or fragment thereof binds to the same epitope of which an antibody having the CDR sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17 and 18 binds.
• the monoclonal antibody or fragment thereof binds to the same epitope of which an antibody produced from DNA sequences deposited on
• nucleic acid molecules encoding an antibody or antibody fragment according to the invention, having affinity and specificity for CEACAMl .
• an isolated polynucleotide encoding an antibody which recognizes CEACAMl or an antibody fragment thereof is disclosed.
• the isolated polynucleotide sequence comprises a DNA sequence set forth in SEQ ID NO: 25 or analog thereof having at least 90% sequence identity with said DNA sequence. According to other embodiments, the isolated polynucleotide sequence comprises a DNA sequence set forth in SEQ ID NO: 27 or analog thereof having at least 90% sequence identity with said DNA sequence. Plasmids comprising at least one polynucleotide sequence encoding a monoclonal antibody or fragment thereof according to the invention are also disclosed, as well as host cells comprising these plasmids.
• plasmid comprising polynucleotide sequences set forth in SEQ ID NOs: 25 and 27, deposited on September 28, 2011 under ATCC Accession Number , is disclosed.
• the present invention is related to a pharmaceutical composition useful for preventing, attenuating or treating a disease or disorder associated with CEACAMl, CEACAM3 or CEACAM5 expression, activation or function.
• a pharmaceutical composition according to the invention comprises a therapeutically effective amount of a monoclonal antibody which recognizes CEACAMl, CEACAM3 or CEACAM5 or an antibody fragment thereof comprising at least an antigen-binding portion; and a pharmaceutically acceptable carrier.
• the pharmaceutical composition comprises a monoclonal antibody capable of binding to CEAC AM 1.
• the pharmaceutical composition comprises a monoclonal antibody capable of binding with an affinity of at least about 10 " kD to CEACAMl and with affinity of at least about 5xlO "7 M to at least one of CEACAM3 and CEACAM5.
• the pharmaceutical composition comprises a monoclonal antibody capable of binding with an affinity of at least about 5x10 ⁇ 7 kD to
• CEACAMl CEACAM3 and CEACAM5.
• CEACAMl, CEACAM3 and/or CEACAM5 expression, activation or function is a cell proliferative disease or disorder.
• the cell proliferative disease or disorder is cancer.
• CEACAM5 is selected from the group consisting of: gastrointestinal, colorectal (CRC) pancreatic non-small cell lung cancer (NSCL), breast, thyroid, stomach, ovarian and uterine.
• CRC colorectal
• NSC pancreatic non-small cell lung cancer
• CEACAMl are melanoma, pancreatic cancer, lung cancers and myeloma.
• the pharmaceutical composition according to the present invention may be administered as a stand alone treatment or in addition to a treatment with any other therapeutic agent.
• antibodies according to the present invention are administered to a subject in need thereof as part of a treatment regimen in conjunction with at least one anti-cancer agent.
• the pharmaceutical composition according to the present invention may be administered together with the other agent or separately.
• the pharmaceutical composition according to the present invention may be administered together with an anti-neoplastic composition.
• the present invention provides diagnostic compositions useful for detecting CEACAM 1, CEACAM3 and/or CEACAM5 in a subject.
• a diagnostic composition according to the invention comprises a therapeutically effective amount of a monoclonal antibody having affinity of at least about 5x10 ⁇ 7 M to CEACAM 1, CEACAM3 and/or CEACAM5 or an antibody fragment thereof comprising at least an antigen-binding portion; and an optional carrier or excipient.
• the present invention is related to a method of preventing, attenuating or treating a disease or disorder associated with expression, activation or function of CEACAM, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an antibody to CEACAM; and a pharmaceutically acceptable carrier.
• the disease or disorder is a cell proliferative disease or disorder.
• the cell proliferative disease or disorder is cancer.
• the monoclonal antibody, or fragment thereof has an affinity of at least about 10 "8 M to CEACAM 1 and the cancer is melanoma.
• the monoclonal antibody, or fragment thereof has an affinity of at least about 5xl0 "7 M to CEACAM5 and the cancer is selected from the group consisting of: gastrointestinal, colorectal (CRC) pancreatic, non-small cell lung cancer (NSCL), breast, thyroid, stomach, ovarian, myeloma and uterine.
• CRC colorectal
• NSC non-small cell lung cancer
• the disease or disorder associated with over expression of CEACAM 1 is viral infection.
• the viral infection is caused by a virus selected from the group consisting of: DNA viruses, such as but not limited to cytomegalovirus (CMV), adenovirus, hepatitis virus and human papillomavirus (HPV); and RNA viruses such as but not limited to influenza virus and human immuno-deficiency virus (HIV).
• DNA viruses such as but not limited to cytomegalovirus (CMV), adenovirus, hepatitis virus and human papillomavirus (HPV); and RNA viruses such as but not limited to influenza virus and human immuno-deficiency virus (HIV).
• a method of immunomodulation comprising contacting a CEACAM- expressing lymphocyte with the antibody or antibody fragment.
• a method of inhibiting migration of a CEACAM expressing tumor cell comprising contacting the CEACAM expressing tumor cell with the antibody or antibody fragment, thereby inhibiting migration of a CEACAM expressing tumor cell.
• the tumor cell comprises a melanoma tumor cell.
• a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antibody fragment, thereby treating the cancer in the subject.
• a method of inhibiting CEACAM homotypic or heterotypic protein-protein interaction comprising contacting a CEACAM 1 -expressing lymphocyte with the antibody or antibody fragment, thereby inhibiting CEACAM 1 homotypic or heterotypic protein-protein interaction.
• the isolated antibody or antibody fragment is attached to a cytotoxic moiety.
• the cytotoxic moiety comprises a cytotoxin, a chemokine, a chemotherapeutic composition, a pro-apoptotic, an interferon, a radioactive moiety, or combinations thereof.
• the isolated antibody or antibody fragment is attached to an identifiable moiety.
• cells of the cancer are characterized by over expression of CEACAM 1 as compared to unaffected cells.
• the method of treating cancer further comprises administering to the subject lymphocytes.
• the lymphocytes comprise T cells or NK cells. According to some embodiments, the lymphocytes express CEACAM1.
• the CEACAM 1 -expressing lymphocyte is a Tumor Infiltrating Lymphocyte or NK cell. According to other embodiments, the CEACAM1- expressing lymphocyte is a cytotoxic T cell.
• the antibody of the present invention can be used to block CEACAM on either or both immune effector cells (CEACAM expressing lymphocytes e.g., tumor infiltrating cells,
• T cells or NK cells T cells or NK cells
• target cells e.g., CEACAM expressing pathological cells such as cancer cells
• cancer cells which are candidates for this therapy include, but are not limited to, melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, or endometrial cells.
• a method of rendering a CEACAM expressing tumor cell susceptible to immunomodulation comprising contacting the CEACAM expressing tumor cell (e.g., melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary or endometrial cell) with the antibody or antibody fragment described above, thereby rendering the CEACAM expressing tumor cell susceptible to immunomodulation.
• the CEACAM expressing tumor cell e.g., melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary or endometrial cell
• the present invention also envisages a method of immunomodulation (e.g., inhibiting CEACAM 1 homotypic or heterotypic protein-protein interaction), by contacting a CEACAM 1 -expressing lymphocyte with the antibody or antibody fragment described herein.
• a method of immunomodulation e.g., inhibiting CEACAM 1 homotypic or heterotypic protein-protein interaction
• the methods of the present teachings can be effected ex-vivo (e.g., used in T cell based adoptive immunotherapy) or in- vivo.
• Antibodies of some embodiments of the invention can have anti cancer activity which is independent from its immunomodulatory activity described above.
• the present invention provides a method for increasing the duration or progression of response or survival of a subject having cancer, comprising administering to the subject effective amounts of a composition comprising an antibody which recognizes CEACAM and an anti-neoplastic composition, wherein said anti-neoplastic composition comprises at least one chemotherapeutic agent, whereby the co-administration of the antibody and the anti-neoplastic composition effectively increases the duration or progression of response or survival.
• the present invention provides a method for treating a subject having cancer, comprising administering to the subject effective amounts of a composition comprising an antibody to CEACAM and an anti-neoplastic composition whereby co- administration of the antibody to CEACAM and the anti-neoplastic composition effectively increases the response incidence in the group of subjects.
• antibodies of the present invention can also be used in diagnostic applications.
• a method for diagnosing a cancer in a subject in need thereof comprising contacting a biological sample derived from the subject (in- vivo, in vitro or ex- vivo) with the antibody or antibody fragment described herein, wherein a complex formation beyond a predetermined threshold is indicative of the cancer in the subject.
• cells of the cancer are characterized by over expression of CEACAM as compared to unaffected cells.
• the diagnosed cancer is selected from the group consisting of: melanoma, pancreatic cancer, lung cancer and myeloma.
• the measured protein is CEACAM5 and the diagnosed cancer is selected from the group consisting of: gastrointestinal, colorectal (CRC) pancreatic non-small cell lung cancer (NSCL), breast, thyroid, stomach, ovarian and uterine.
• CRC colorectal
• NSC pancreatic non-small cell lung cancer
• the method of the invention is affected under conditions sufficient to form an immunocomplex; such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art, and examples are disclosed herein.
• immunocomplex refers to a complex which comprises the antibody of the invention and the CEACAM. Determining a presence or level of the immunocomplex of the invention may be direct or by detecting an identifiable (detectable) moiety which may be attached to the antibody.
• the level of the immunocomplex in the tested cell (e.g., a cell of a subject in need thereof) is compared to a predetermined threshold.
• a predetermined threshold may be determined based on a known reference level and/or a level in a control cell or serum.
• the control cell can be obtained from a control, healthy subject (e.g., a subject not suffering from the cancer) or from the same subject prior to disease initiation or following treatment.
• the control subject is of the same species e.g. human, preferably matched with the same age, weight, sex etc. as the subject in need thereof.
• a method for detecting or quantifying the presence of CEACAM in is provided.
• the present invention also provides methods for diagnosing conditions associated with CEACAM expression using antibodies which recognizes CEACAM. Diagnostic methods according to the invention may be performed according to specific embodiments, in-vilro or ex-vivo.
• the antibodies according to the present invention may be also used to configure screening methods.
• an ELISA assay can be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art.
• a method for detecting or quantifying the presence of CEACAM comprising the steps of:
• the biological sample is a body fluid.
• the antibodies of the present invention may be also used in screening assays for assessing the CEACAM levels in patients and for prediction of the effectiveness of treatment.
• the screening assays with the antibodies of the present invention may allow determination of the levels of CEACAM and therefore prediction of treatment outcome and planning of an appropriate treatment regimen.
• the level of at least one of CEACAM1, CEACAM3 and CEACAM5 is assessed.
• the level of CEACAM1 is assessed.
• the antibody is attached to an identifiable moiety.
• the identifiable moiety can be a member of a binding pair, which is identifiable via its interaction with an additional member of the binding pair and a label which is directly visualized.
• the member of the binding pair is an antigen which is identified by a corresponding labeled antibody.
• the label is a fluorescent protein or an enzyme producing a colorimetric reaction.
• Another aspect of the present invention relates to the use of an antibody to CEACAM or an antibody fragment thereof, for diagnosis or treatment of a cell proliferative or angiogenesis-related disease or disorder or a viral infection.
• the cell proliferative disease is melanoma.
• the cell proliferative disease or disorder is a cancer selected from the group consisting of: gastrointestinal, colorectal (CRC) pancreatic non-small cell lung cancer (NSCL), breast, thyroid, stomach, ovarian, uterine, and myeloma.
• CRC colorectal
• NSC pancreatic non-small cell lung cancer
• breast thyroid
• stomach ovarian
• uterine myeloma
• the present invention provides use of an antibody to CEACAM or an antibody fragment thereof comprising at least an antigen-binding portion, for preparation of a medicament for treatment of a disorder or disease associated with expression or activation of, including but not limited to cancer and viral infection.
• the invention also relates to use of an antibody to CEACAM or an antibody fragment thereof, for the manufacture of a diagnostic composition for the diagnosis of a cell proliferative or angiogenesis-related disease or disorder or a viral infection.
• CEACAM 1 Essentially all of the uses known or envisioned in the prior art for CEACAM 1, CEACAM3 and CEACAM5 antibodies can be accomplished with the antibodies of the present invention which are shown to posses improved affinity toward these proteins and superior inhibitory and in indirect immunomodulatory effects on CEACAM 1, bearing cells. These uses include diagnostic, prophylactic and therapeutic techniques. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter.
• Figure 1 is an SDS-PAGE image showing light and heavy chains of the chimeric antibody CMIO.
• Figure 2 shows specific binding curve of CMIO to purified hCEACAMl.
• Figure 3 demonstrates specific binding of CMIO to CEACAMl as detected by Flow
• Figure 4 confirms that CMIO blocks CEACAMl -CEACAMl interaction between cells.
• Figure 5 shows CMIO enhancement of the specific killing activity of CEACAMl -positive melanoma cells.
• FIG. 6 demonstrates that CMIO stimulates the killing activity of TILs.
• Figure 7 demonstrates that CMIO enhances the killing activity of NK cells on CEACAMl positive melanoma cell lines.
• CMIO immunomodulatory effect inhibits tumor growth in-vivo. Arrows indicate time of administration (CMIO cyrcles, TIL triangles, CMIO and TIL open squares).
• Figure 9 is a schematic presentation of CMIO immunomodulatory mode of action.
• Figure 10 represents CEACAM binding intensity level in tumors.
• Figure 11 shows quantification of CM- 10 molecules bound per cell.
• Figure 12 confirms that CMIO has no effect on PBMC Proliferation. Results represent average proliferation rates from three donors for each treatment.
• Figure 13 presents FACS analysis of binding between CMIO to CEACAM family proteins.
• CEACAMl, 5, 6 and 8 were expressed by 721.221 cells, and CEACAM3 and 4 by HEK293T cells
• Figure 14 represents results of complement-dependent cytotoxicity (CDC) assay in melanoma cell lines.
• the present invention provides antibodies which recognize CEACAMl comprising specific and unique CDR sequences which possess improved and unique specificity, selectivity, affinity and/or activity.
• Antibodies according to the present invention bind CEACAM1 with higher affinity than other anti-CEACAMl antibodies, they blocks the function of CEACAM1, while not all anti CEACAM antibodies do, and more efficiently than polyclonal anti CEACAM antibodies.
• antibodies according to the present invention are effective against cancer cells, in particular melanoma cells: the antibodies render melanoma cells more susceptible to lymphocytes, inhibit melanoma growth rate in vivo, an effect which is enhanced when the antibody is combined with adoptive T cell transfer in vivo.
• the in vivo anti-melanoma effect of anti- CEACAMl antibodies according to the invention is a combined direct anti-tumor effect as well as immunomodulatory effect rendering the cells more susceptible to reactive lymphocytes.
• An antibody according to the present invention fragments and derivatives can be used as an effective tool for diagnosis, immunomodulation and cancer treatment.
• the antibody inhibits CEACAM 1 homophilic interactions, as determined by co- incubation of immune effector cells and target cells expressing CEACAM 1 and assaying IL-2 secretion and by the in vitro killing assays.
• an antibody according to the invention is effective in inhibiting melanoma cells invasion.
• in vivo administration of an antibody according to the invention either alone or in combination with reactive lymphocytes was shown effective in inhibiting growth of melanoma tumors.
• isolated antibody or antibody fragment having the same binding specificity and selectivity to an antibody defined herein comprising an antigen recognition domain having specific CDR segments described above.
• isolated antibody or antibody fragment is capable of binding the same epitope determinant of the CEACAM 1 protein as does the antibody described above by its specific CDR segments sequences.
• Monoclonal antibodies can be designed to selectively target tumor cells and elicit a variety of responses once bound. These agents can destruct tumor cells in different ways such as blocking tumor cell proliferation or activating the immune system.
• Chimeric monoclonal antibodies according to the present invention were designed to specifically bind and neutralize various functions of the CEACAM 1 protein and other CEACAM subtype proteins, and to induce the specific death of tumor cells. Without wishing to be bound to any theory, it is suggested that monoclonal antibodies according to the present invention act also via activation of the immune system against cancerous cells. Both the clinical and biological evidence highlight CEACAM1 as a promising target for the development of targeted-immunotherapy.
• CEACAM1 is not found on normal melanocytes, but undergoes neo-expression and is widely expressed on the vast majority of metastatic melanoma specimens. It has been previously demonstrated mechanistically that CEACAM1 protects melanoma cells by inhibiting effector functions of NK cells (Markel G et al JI 2002, Markel G et al JI 2004) and T cells (Markel G et al JI 2006, Markel G et al Immunology 2009).
• CM10 is a chimeric monoclonal antibody which binds with high affinity to human CEACAM1.
• CM10 efficiently blocked CEACAMl-homophilic interactions in a dose dependent manner and improves CEACAM1 positive melanoma cells killing by T cells and NK cells.
• CM 10 significantly inhibited the in- vivo growth of melanoma xenografts when administered systemically along with melanoma-reactive human T cells (TIL). Without wishing to be bound to any theory, this is in line with the suggested mechanism of action; abrogation of immune-protective interactions of the tumor cells with the activated lymphocytes.
• CEACAM1 is expressed by a wide variety of epithelial cells, including colon, prostate, breast, kidney etc. Extensive examination of CEACAM1 expression profile on normal and malignant tissues by IHC have been performed. The expression analysis showed a strong staining of melanoma cells, as compared to no staining of the vast majority of the tissues tested in a normal human tissue. Nevertheless, some selective staining was observed in restricted sites of several organs. When more quantitative method was used to quantify the number of CM 10 mAb molecules bound to malignant and normal primary cells, very low CM 10 molecules could be detected in normal cells, which may indicate that CM 10 binds mostly to patient's tumor cells. Furthermore it is shown that CM 10 has no effect on primary cells proliferation and is unable to induce CDC or ADCC indicating the potential safety of the monoclonal antibody in human subjects.
• CM 10 Since CM 10 has an immunomodulation activity, possible immune-related side effects, should be evaluated. Following PBMC activation, CEACAM1 is upregulated on the activated lymphocytes (Gray-Owen and Blumberg 2006, Nat Rev Immunol 6, 433-46). Ex- vivo human PBMC proliferation assay revealed that CM 10 has no effect on naive and activated PBMC proliferative response.
• the main advantage of CEACAM1 blockade over abrogation of generalized inhibitory mechanisms is the expected selectivity to the vicinity of the tumor and therefore fewer adverse events compare to other general immune toxicity agents.
• CM 10 shows encouraging activity and safety profile and is a promising candidate for cancer immunotherapy and can be used as a strategy to selectively enhance the anti-tumor properties of the endogenous immune response in several malignancies, such as melanoma and non-small cell lung cancer (Laack et al., 2002, J Clin Oncol., 20(21), 4279-84 and Sienel et al. 2003, , Clin Cancer Res., 9(6), 2260-6).
• binding to additional CEACAM subtypes increases the therapeutic profile of the antibody, thus it can be used for diagnosis and treatment of other types of malignancies which do not extensively express CEACAM 1 but express CEACAM5, for example.
• CEACAM5 has been found to be over-expressed in a high percentage of many human tumors, including 90% of gastrointestinal, colorectal (CRC) and pancreatic cancers, 70% of non-small cell lung cancer cells and 50% of breast cancers. It is also over-expressed in thyroid, stomach, ovarian and uterine cancers (Thompson, Grunert et al. 1991, J Clin Lab Anal 5, 344-66). CEACAM5 even serves as a clinical marker for liver metastasis in CRC and post-surgical surveillance of colon cancer (Duffy 2001, Clin Chem 47, 624-30).
• CM10 is capable to bind CEACA5 is very important and can expand the possible indications that can be treated by CM10 from 4-5 types of malignancies to above 10.
• the anti-CEACAM5 agents that have entered clinical trials include anti-CEACAM5 antibodies conjugated to toxic substances such as radioactive substances for both diagnostic purposes and for the treatment of various malignancies. It seems that even these toxic conjugated forms don't show safety problems, which can indicate that CEACAM5 is a safe target.
• Murine IgG2a and IgG2b and human IgGl and IgG3 share the ability to fix complement and bind to protein antigens (Hussain et al., 1995, Clinical and Diagnostic Laboratory Immunology 726-732).
• Murine IgGl and human IgG4 are considered to be similar because of their property of binding to mast cells.
• Human IgG4 is the only human IgG subclass which does not activate complement and the subclasses IgGl and 3 are the most effective in activating complements.
• chimeric monoclonal antibodies comprise a human IgGl constant framework.
• the present invention provides a monoclonal antibody which recognizes CEACAM1, or an antibody fragment comprising at least an antigen-binding portion thereof, comprising at least one heavy-chain CDR comprising a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and at least one light-chain CDR comprising a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and analogs and derivatives thereof.
• analogs and derivatives of the monoclonal antibody or fragment thereof, having at least 90% sequence identity with the sequence of the reference sequence are disclosed.
• analogs and derivatives of the monoclonal antibody or fragment thereof having at least 95% sequence identity with the reference sequence are disclosed.
• analogs and derivatives of the monoclonal antibody or fragment thereof having at least 98% sequence identity with the CDR sequence of the reference antibody are disclosed.
• the antibody or antibody fragment comprises at least two heavy-chain CDRs comprising a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and at least one light-chain CDRs comprising a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and analogs and derivatives thereof having at least 97% sequence identity with the sequence of the monoclonal antibody or fragment thereof.
• the antibody or antibody fragment comprises at least one heavy-chain CDR comprising a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and at least two light-chain CDRs comprising a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and analogs and derivatives thereof having at least 97% sequence identity with the sequence of the monoclonal antibody or fragment thereof.
• the antibody or antibody fragment comprises at least two heavy-chain CDRs comprising a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and at least two light-chain CDRs comprising a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and analogs and derivatives thereof having at least 97% sequence identity with the sequence of the monoclonal antibody or fragment thereof.
• the antibody or antibody fragment comprises at least one heavy-chain CDR sequence of at least five amino acids derived from a sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and at least one light-chain CDR sequence of at least five amino acids derived from a sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, and analogs and derivatives thereof having at least 97% sequence identity with the sequence of the monoclonal antibody or fragment thereof.
• the antibody binding site of the antibody or fragment thereof consists of three heavy chain CDRs selected from the group consisting of SEQ ID NOs: 7, 8, 9, 13, 14 and 15 and three light chain CDRs selected from the group consisting of SEQ ID NOs: 10, 11, 12, 16, 17, 18, and analogs and derivatives thereof having at least 97% sequence identity with the antibody binding site.
• the antibody binding site consists of the six CDRs of SEQ ID NOs: 13, 14, 15, 16, 17, and 18.
• the antibody binding site consists of the six CDRs of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.
• the heavy chain CDR1 of the antibody according to the invention or a fragment thereof is selected from NNLIE (SEQ ID NO: 7) and GYAFTNNL (SEQ ID NO: 13).
• the heavy chain CDR2 of the antibody according to the invention or a fragment thereof is selected from VINPGSGDTNYNEKFKG (SEQ ID NO: 8) and INPGSGDT (SEQ ID NO: 14).
• the heavy chain CDR3 of the antibody according to the invention or a fragment thereof is selected from GDYYGGFAVDY (SEQ ID NO: 9) and ARGDYYGGFAVD Y (SEQ ID NO: 15).
• the light chain CDR1 of the antibody according to the invention or a fragment thereof is selected from RTSQDIGNYLN (SEQ ID NO: 10) and QDIGNY (SEQ ID NO: 16).
• the light chain CDR2 of the antibody according to the invention or a fragment thereof is selected from YTSRLHS (SEQ ID NO: 11) and YTS (SEQ ID NO: 17).
• the light chain CDR3 of the antibody according to the invention or a fragment thereof is selected from QQGKSLP (SEQ ID NO: 12) and QQGKSLPRT (SEQ ID NO: 18).
• a monoclonal antibody which recognizes CEACAM1 or a fragment thereof comprising at least an antigen binding portion is provided, wherein the heavy chain CDRs consist of the sequences of SEQ ID NOs: 7, 8 and 9.
• CEACAM1 or a fragment thereof comprising at least an antigen binding portion is provided, wherein the heavy chain CDRs consist of the sequences of SEQ ID NOs: 13, 14 and 15.
• a monoclonal antibody which recognizes CEACAM1 or a fragment thereof comprising at least an antigen binding portion is provided, wherein the light chain CDRs consist of the sequences of SEQ ID NOs: 10, 11 and 12.
• a monoclonal antibody which recognizes CEACAM1 or a fragment thereof comprising at least an antigen binding portion is provided, wherein the light chain CDRs consist of the sequences of SEQ ID NOs: 16, 17 and 18.
• the antibody or fragment thereof comprises a heavy chain variable domain sequence consisting of the of SEQ ID NO: 26 and a light chain variable domain sequence consisting of SEQ ID NO: 28, or an analog or derivative thereof having at least 90% sequence identity with the antibody or fragment sequence.
• the present invention provides a monoclonal antibody, or an antibody fragment comprising a set of si CDRs selected from i. SEQ ID NOs: 13, 14, 15, 16, 17, and 18 and ii. SEQ ID NOs: 7, 8, 9, 10, 11, and 12; and analogs and derivatives thereof having at least 97% sequence identity with said CDR sequences, and a framework sequence selected from mouse IgG2a, mouse IgG2b, mouse IgG3, human IgGl, human IgG2, human IgG3, wherein the monoclonal antibody binds with an affinity of at least about 5xl0 "7 M to at least two CEACAM subtypes.
• a chimeric monoclonal antibody which recognizes CEACAM 1 comprising at least one CDR sequence selected from the group consisting of: SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18; and analogs and derivatives thereof having at least 97% sequence identity with said CDR sequences, and a constant region sequence selected from human IgGl, human IgG2 and human IgG3, wherein the monoclonal antibody binds with an affinity of at least about 5xl0 "7 M to at least two CEACAM subtypes.
• a chimeric or humanized monoclonal antibody which recognizes CEACAM 1 comprising a set of six CDRs selected from i. SEQ ID NOs: 13, 14, 15, 16, 17, and 18 and ii. SEQ ID NOs: 7, 8, 9, 10, 11, and 12; and analogs and derivatives thereof having at least 97% sequence identity with said CDR sequences, and a constant region subclass selected from human IgGl, human IgG2 and human IgG3, wherein the monoclonal antibody binds with an affinity of at least about 5xlO "7 M to at least two CEACAM subtypes.
• a chimeric monoclonal antibody or a fragment thereof comprising at least the antigen-binding portion comprising a heavy chain sequence according to SEQ ID NO: 30.
• a chimeric monoclonal antibody or a fragment thereof comprising at least the antigen-binding portion comprising a light chain sequence according to SEQ ID NO: 31.
• a chimeric monoclonal antibody or a fragment thereof comprising at least the antigen-binding portion comprising a human IgGl heavy chain sequence according to SEQ ID NO: 30, and a human IgGl light chain sequence according to SEQ ID NO: 31.
• CEACAM 1 is used to refer to the protein product of the CEACAM 1 gene e.g., NP_001020083.1, NP_001703.2. In humans, 11 different CEACAM 1 splice variants have been detected so far. Individual CEACAM 1 isoforms differ with respect to the number of extracellular immunoglobulin-like domains (for example, CEACAM 1 with four extracellular immunoglobulin-like domains is known as CEACAM 1-4), membrane anchorage and/or the length of their cytoplasmic tail (for example, CEACAM 1-4 with a long cytoplasmic tail is known as CEACAM 1-4L and CEACAM 1-4 with a short cytoplasmic tail is known as CEACAM1-4S).
• the N-terminal domain of CEACAM1 starts immediately after the signal peptide and its structure is regarded as IgV-type.
• the N-terminal IgV-type domain is comprised of 108 amino acids, from amino acid 35 to 142. This domain was identified as responsible for the homophilic binding activity (Watt et al., 2001, Blood. 98, 1469-79). All variants, including these splice variants are included within the term "CEACAM1".
• an “anti-CEACAMl antibody”, “an antibody which recognizes CEACAM1”, “an antibody against CEACAMl”, or “an antibody to CEACAM1” is an antibody that binds to the CEACAMl protein with sufficient affinity and specificity.
• an antibody according to the present teachings is capable of binding CEACAMl with a minimal affinity of about 10 "8 or 10 "9 M.
• Some of the monoclonal antibodies of the present invention are capable of binding CEACAM3, 5 and/or 8 with a minimal affinity of about 5xl0 "7 M.
• the anti-CEACAMl antibody of the invention can be used as a diagnostic or therapeutic agent in targeting and interfering with diseases or conditions wherein the CEACAMl expression or activity is involved.
• an "antigen” is a molecule or a portion of a molecule capable of eliciting antibody formation and being bound by an antibody.
• An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
• An antigen according to the present invention is a CEACAMl protein or a fragment thereof.
• antigenic determinant or "epitope” according to the invention refers to the region of an antigen molecule that specifically reacts with particular antibody.
• Antibodies, or immunoglobulins comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds in a "Y" shaped configuration.
• Proteolytic digestion of an antibody yields Fv (Fragment variable) and Fc (fragment crystalline) domains.
• the antigen binding domains, Fab include regions where the polypeptide sequence varies.
• F(ab') 2 represents two Fab' arms linked together by disulfide bonds.
• the central axis of the antibody is termed the Fc fragment.
• Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH).
• Each light chain has a variable domain (VL) at one end and a constant domain (C L ) at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CHI).
• the variable domains of each pair of light and heavy chains form the antigen-binding site.
• the domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, joined by three hypervariable domains known as complementarity determining regions (CDRl-3). These domains contribute specificity and affinity of the antigen-binding site.
• the isotype of the heavy chain determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively).
• the light chain is either of two isotypes (kappa, ⁇ or lambda, ⁇ ) found in all antibody classes.
• antibody is used in the broadest sense and includes monoclonal antibodies
• polyclonal antibodies including full length or intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
• the antibody according to the present invention is a molecule comprising at least the antigen-binding portion of an antibody.
• Antibody or antibodies according to the invention include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof such as the Fab or F(ab') 2 fragments. Further included within the scope of the invention are chimeric antibodies; human and humanized antibodies; recombinant and engineered antibodies, and fragments thereof. Furthermore, the DNA encoding the variable region of the antibody can be inserted into the DNA encoding other antibodies to produce chimeric antibodies. Single chain antibodies also fall within the scope of the present invention.
• Antibody fragments comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen.
• Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab') 2 fragments, a
• VL in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448);
• VH-CHl-VH-CHl "linear antibodies” comprising a pair of tandem Fd segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng., 1995, 8, 1057-1062; and U.S. Pat. No. 5,641,870).
• Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain i.e. linked VH-VL or single chain Fv (scFv).
• immunoglobulin light and heavy chain i.e. linked VH-VL or single chain Fv (scFv).
• neutralizing antibody refers to a molecule having an antigen- binding site to a specific receptor or ligand target capable of reducing or inhibiting (blocking) activity or signaling through a receptor, as determined by in vivo or in vitro assays, as per the specification.
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
• the modifier "monoclonal” is not to be construed as requiring production of the antibody by any particular method. mAbs may be obtained by methods known to those skilled in the art.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
• the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581- 597, for example.
• the mAbs of the present invention may be of any immunoglobulin class including IgG, IgM, IgE, IgA.
• a hybridoma producing a mAb may be cultivated in vitro or in vivo.
• High titers of mAbs can be obtained in vivo production where cells from the individual hybridomas are injected intraperitoneally into pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs.
• mAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
• the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984)).
• complementarity determining region (CDR) grafting may be performed to alter certain properties of the antibody molecule including affinity or specificity.
• CDR complementarity determining region
• Chimeric antibodies are molecules, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
• Antibodies which have variable region framework residues substantially from human antibody (termed an acceptor antibody) and complementarity determining regions substantially from a mouse antibody (termed a donor antibody) are also referred to as humanized antibodies.
• Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used.
• Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
• donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
• framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues.
• humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
• the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• human antibody is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
• Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al. Nature Biotechnology 1996 14,309-314; Sheets et al. PNAS (USA), 1998, 95, 6157-6162); Hoogenboom and Winter, J. Mol. Biol., 1991, 227, 381; Marks et al., J. Mol.
• Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
• the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (l):86-95 (1991); and U.S. Pat No. 5,750,373.
• single chain variable fragment scFv
• scFv single chain variable fragment
• Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain
• V H and V L may copy natural monoclonal antibody sequences or one or both of the chains may comprise a CDR-FR construct of the type described in US patent 5,091,513, the entire contents of which are incorporated herein by reference.
• the separate polypeptides analogous to the variable regions of the light and heavy chains are held together by a polypeptide linker.
• a "molecule having the antigen-binding portion of an antibody” as used herein is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab' fragment, the F(ab') 2 fragment, the variable portion of the heavy and/or light chains thereof, Fab mini-antibodies (see WO 93/15210, US patent application 08/256,790, WO 96/13583, US patent application 08/817,788, WO 96/37621, US patent application 08/999,554, the entire contents of which are incorporated herein by reference), dimeric bispecific mini-antibodies (see Muller et al., 1998) and chimeric or single-chain antibodies incorporating such reactive fraction, as well as any other type of molecule or cell in which such antibody reactive fraction has been physically inserted, such as a chimeric T-cell receptor or a T-cell having such
• Antibodies according to the invention can be obtained by administering CEACAM1, or epitope-bearing fragments, analogs, or cells expressing, to an animal, preferably a nonhuman, using routine protocols.
• any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
• antibodies can be generated in vitro using phage display technology.
• Such a production of recombinant antibodies is much faster compared to conventional antibody production and they can be generated against an enormous number of antigens.
• many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals.
• affinity maturation i.e., increasing the affinity and specificity
• large numbers of different antibodies against a specific antigen can be generated in one selection procedure.
• To generate recombinant monoclonal antibodies one can use various methods all based on display libraries to generate a large pool of antibodies with different antigen recognition sites.
• Such a library can be made in several ways: One can generate a synthetic repertoire by cloning synthetic CDR3 regions in a pool of heavy chain germline genes and thus generating a large antibody repertoire, from which recombinant antibody fragments with various specificities can be selected.
• Non-human antibodies may be humanized by any methods known in the art.
• the non-human complementarity determining regions are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
• US Patent 5,585,089 of Queen et al. discloses a humanized immunoglobulin and methods of preparing same, wherein the humanized immunoglobulin comprises complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chains, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin framework outside the Kabat and Chothia CDRs, wherein the donor amino acids replace corresponding amino acids in the acceptor immunoglobulin heavy or light chain frameworks.
• CDRs complementarity determining regions
• US Patent 5,225,539, of Winter also discloses an altered antibody or antigen-binding fragment thereof and methods of preparing same, wherein a variable domain of the antibody or antigen-binding fragment has the framework regions of a first immunoglobulin heavy or light chain variable domain and the complementarity determining regions of a second immunoglobulin heavy or light chain variable domain, wherein said second immunoglobulin heavy or light chain variable domain is different from said first immunoglobulin heavy or light chain variable domain in antigen binding specificity, antigen binding affinity, species, class or subclass.
• Anti-idiotype antibodies specifically immunoreactive with an antibody of the invention are also comprehended.
• phage display technology can be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-CEACAMl or from libraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks, et al, (1992) Biotechnology 10, 779-783).
• the affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al., (1991) Nature 352:628).
• the above-described antibodies can be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by, for example, affinity chromatography.
• the invention also provides conservative amino acid variants of the antibody molecules according to the invention.
• Variants according to the invention also may be made that conserve the overall molecular structure of the encoded proteins. Given the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e. "conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
• a “disorder” is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
• disorders to be treated herein include benign and malignant tumors; leukemias and lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic, immunologic disorders or hyperpermeability states.
• the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
• the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
• the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
• efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
• Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
• cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
• examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, or endometrial cancer.
• the antibody of the present invention is attached to a cytotoxic or therapeutic moiety.
• the cytotoxic or therapeutic moiety can be, for example, a cytotoxic moiety, a toxic moiety, a cytokine moiety, a bi-specific antibody moiety, a cytotoxin, a chemokine, a chemotherapy, a pro-apoptotic, interferon, a radioactive moiety, or combinations thereof, examples of which are provided infra.
• anti-neoplastic composition refers to a composition useful in treating cancer comprising at least one active therapeutic agent capable of inhibiting or preventing tumor growth or function, and/or causing destruction of tumor cells.
• Therapeutic agents suitable in an anti-neoplastic composition for treating cancer include, but not limited to, chemotherapeutic agents, radioactive isotopes, toxins, cytokines such as interferons, and antagonistic agents targeting cytokines, cytokine receptors or antigens associated with tumor cells.
• the therapeutic agent is a chemotherapeutic agent.
• diagnosis refers to determining presence or absence of a pathology, classifying a pathology or a symptom, determining a severity of the pathology, monitoring pathology progression, forecasting an outcome of a pathology and/or prospects of recovery.
• the present invention also contemplates pharmaceutical formulations for human medical use, which comprise as the active agent at least one antibody which recognizes CEACAM1, for the manufacture of a therapeutic or diagnostic composition for the treatment, diagnosis or prophylaxis of the conditions variously described herein.
• the active agent is preferably utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients.
• the carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof.
• the active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.
• the molecules of the present invention comprising the antigen binding portion of an antibody or comprising another polypeptide including a peptidomimetic will be suspended in a sterile saline solution for therapeutic uses.
• the pharmaceutical compositions may alternatively be formulated to control release of active ingredient (molecule comprising the antigen binding portion of an antibody) or to prolong its presence in a patient's system.
• suitable drug delivery systems include, e.g., implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention.
• biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a poly anhydride copolymer of a stearic acid dimer and sebaric acid.
• the rate of release of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles.
• composition of this invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intraarticulary, intralesionally or parenterally. Ordinarily, intravenous (i.v.), intraarticular, topical or parenteral administration will be preferred.
• the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician.
• a "therapeutically effective amount” refers to the amount of a molecule required to alleviate one or more symptoms associated with a disorder being treated over a period of time.
• the daily dosage can generally be between about O.Olmg to about 500 mg, preferably about O.Olmg to about 50 mg, more preferably about 0.1 mg to about 10 mg, per kg body weight.
• the daily dosage can generally be between about O.OOlmg to about 100 mg, preferably about O.OOlmg to about 10 mg, more preferably about O.Olmg to about 1 mg, per kg body weight.
• the daily dosage can be administered, for example in regimens typical of 1- 4 individual administration daily.
• Other preferred methods of administration include intraarticular administration of about O.Olmg to about 100 mg per kg body weight.
• Various considerations in arriving at an effective amount are described, e.g., in Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.
• Suitable dosing regimens of combination chemotherapies are known in the art and described in, for example, Saltz et al. Proc ASCO 1999, 18, 233a and Douillard et al., Lancet 2000, 355, 1041-7.
• the molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known.
• excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof.
• compositions can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.
• the pharmaceutical composition according to the present invention may be administered together with an anti-neoplastic composition.
• the anti-neoplastic composition comprises at least one chemotherapeutic agent.
• the chemotherapy agent which could be administered together with the antibody according to the present invention, or separately, may comprise any such agent known in the art exhibiting anticancer activity, including but not limited to: mitoxantrone, topoisomerase inhibitors, spindle poison vincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabin; podophyllotoxins: etoposide, irinotecan, topotecan, dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, da
• the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L- asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog.
• the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel and doxetaxel.
• 5-fluorouracil 5-FU
• leucovorin LV
• irenotecan oxaliplatin
• capecitabine paclitaxel
• doxetaxel Two or more chemotherapeutic agents can be used in a cocktail to be administered in combination with administration of the anti-CEACAMl antibody.
• Example 1 Generation and characterization of monoclonal antibodies which recognized CEACAM
• Monoclonal antibodies that effectively block the CEACAM 1 homophilic interactions in vitro at nanomolar concentrations were generated by immunizing mice with recombinant human CEACAM 1 protein.
• Hybridomas producing the CEACAM 1 -blocking antibodies were produced and re-cloned several times to yield a stable clone.
• the DNA and amino acid sequence of one exemplary monoclonal antibody which recognizes CEACAMl was determined by Fusion Antibodies Ltd.
• mRNA was extracted from the hybridoma cell pellets and total RNA was extracted from the pellets using RNA extraction protocol.
• RT-PCR-cDNA was created from the RNA by reverse-transcription with an oligo(dT) primer. PCR reactions using variable domain primers were used to amplify both the VH and VL regions of the monoclonal antibody DNA.
• VH Variable heavy chain
• VL Variable light chain
• N-terminal sequence analysis of the light chain was performed by the Edman degradation method to verify the N-terminus sequence of the light chain of one of the monoclonal antibodies.
• the obtained N-terminal sequence was: DIQMTQTTSS (SEQ ID NO: 29), which is in accordance with the N-terminal expected sequence based on the DNA sequence.
• the CDR segments were identified using two different algorithm methods:
• Example 4 Design and production of a chimeric monoclonal antibody
• variable heavy and light chains (SEQ ID NOs 25 and 27) were used to construct a chimeric antibody, comprising the human IgGl isotype constant domains and constant light (CL) human IgKappa domain.
• the parent monoclonal antibody is mouse IgGl and its human equivalent is IgG4, a human IgGl framework was used to construct some of the chimeric antibodies of the present invention.
• the DNA sequences for the light chain and heavy chain were synthesized and cloned into the expression vector pFUSION-DHFRl under separate promoters.
• Suspension CHO cells (Invitrogen, UK) were cultivated at 130rpm, 8% C0 2 , 37°C in Pro CHO 5 serum free medium (Lonza, UK) in 250 and 500ml vented Erlenmeyer flasks (Corning, Netherlands). On the day of transfection, cells were seeded at a density of 2.0 X 106 cells/ml, 2.5g/ml of plasmid DNA (Geneart , Germany) was transfected into the cells using Polyethylenimine (Polysciences Inc, PA, US). Transfected cultures were incubated at 130 rpm, 8% C0 2 , 37°C for 9-10 days. Prior to harvest of the culture supernatants were spinned at 4,000 rpm for 40 minutes.
• CMIO has the following amino acid sequence of the heavy and light chains:
• Variable domain is in bold, CDRs according to IMGT are underlined.
• Variable domain is in bold, CDRs according to IMGT are underlined.
• CM 10 A plasmid containing the DNA sequences of the heavy and light chains of an exemplary chimeric monoclonal antibody denoted CM 10 was deposited on September 28, 2011 under ATCC Accession Number
• Example 5 Affinity characterization of the chimeric monoclonal antibody CM10
• CM 10 The binding specificity of CM 10 to human CEACAM1 was tested in ELISA assay using purified human CEACAM1.
• the chimeric antibody CM 10 was evaluated for CEACAM1 binding by competitive ELISA and by BIAcore analysis.
• the chemically biotinylated CM 10 was used as tracer at a constant concentration and was competed with increasing concentrations of unlabelled CM10. Following incubation and washing, the plate was developed with a StrepAvidin- HRP conjugate and the color reaction was developed with TMB as an HRP substrate.
• Each antibody was immobilized onto a single channel of a CM5 sensor chip by NHS-EDC chemistry.
• CEACAMl was flowed at 50 ⁇ /min over the chip in various concentrations (0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5 and 25nM).
• the running buffer was lOmM p-buffer pH7.4, 150mM NaCl, 3.4mM EDTA and 0.005% tween 20 - PBS-ET.
• the data were analyzed using BIAE valuation software 3.0 and the KD values were as follows: CM10 CEACAMl affinity (KD): 4.07- 5.05 nM in three independent experiments (average 4.56 nM).
• FACS analysis was performed. Several human melanoma cell lines were screened for hCEACAMl expression while 526mel cell line was used as positive control and 003 mel as negative control line. 526 mel, 003 Mel, Malme 3M, Sk mel 5 and A375 cell lines were stained with CM 10. Empty histograms represent mAb staining while darker histograms represent background staining. At least 5000 cells were used to analyze CEACAMl expression in each histogram.
• CM 10 detects membrane-bound endogenous
• CEACAMl .
• Malme 3M and Sk mel5 cell lines showed high expression of CEACAMl while no expression could be detected in A375 melanoma cell-line.
• the chimerization process was carried out successfully.
• the chimeric antibody bind CEACAMl in an affinity of 1.4 nM as validated by two different approaches
• the assay which determines the ability of anti CEACAMl mAb to blocks CEACAMl cell-cell interaction uses murine T cells (BW) that are stably transfected with a chimeric molecule composed of the extracellullar portion of human CEACMA1 fused to mouse z-chain (BW/CEACAMl). Engagement of CEACAMl by co-incubation of BW/CEACAMl.
• Effectors cells BW expressing CEACAM1
• CM10 or PBS for 30 minutes on ice.
• Mouse IL-2 secretion was measured by ELISA.
• the results shown in Figure 4 represent average IL-2 secretion from duplicate wells.
• CEACAM1 -mediated cell-cell interactions between T and B cells were abolished in a dose-depended manner as indicated by blockage of IL-2 secretion.
• TILs Tumor Infiltrating Lymphocytes, derived from melanoma patients
• TILs can destroy melanoma cells with matched HLA.
• TILs were purchased from ELLA Institute at Shiba medical center and were growth according to the clinical lab protocols.
• CFSE-labeled melanoma cells (SKmel5) were pre- incubated with CM10 (10 ⁇ g/ml) for 30 minutes on ice. TIL were added for additional 10 hours incubation at 37°C. Percentage of killing was determined by Pi-staining of the CFSE labeled melanoma cells. Effector-to-target ratio was 5:1.
• CFSE-labeled melanoma cells were pre-incubated with CM 10 for 30 minutes on ice. TIL were added for additional 5 hours incubation at 37°C. Percentage of killing was determined by Pi-staining of the CFSE labeled melanoma cells. Results represent average of % specific killing from triplicate wells ⁇ SE per treatment. Effector-to-target ratio was 5:1.
• NK cells Natural killer cells
• NK cells are a type of cytotoxic lymphocyte that can destroy malignant cells by releasing small proteins called perforin and granzyme that cause the target cell to die by apoptosis.
• NK cells can by activated throw several different pathways among them cytokines, FC receptor, and MHC class 1 absence at the target cells.
• cytokines cytokines, FC receptor, and MHC class 1 absence at the target cells.
• Several activation and inhibition receptors to various ligands on target cells regulate the final cytoxicity activity of NK cells.
• NK 92MI cells are IL-2 independent NK cell line that were purchased from the ATCC.
• NK 92MI were incubated with CM 10 (0 ⁇ g/ml, ⁇ g/ml or 5 ⁇ g/ml) or isotype match control Ab (5 ⁇ g ml) for 30 minutes at 37°C, target cells expressing CEACAMl were added for additional 5 hours. Percentage of killing was determined by classical LDH release assay. Results represent average of % cytotoxicity from triplicate wells ⁇ SE per treatment. Effector-to-target ratio was 2.5:1.
• CM10 strongly enhanced the killing activity of NK cells on two melanoma cell lines (SKMel 5 and G361) expressing CEACAMl compare to PBS or isotype match IgG. Similar results have been demonstrated in two other CEACAM1+ melanoma cell lines and in various E:T (Effector-to-target) ratios.
• E:T Effector-to-target
• ADCC Antibody-Dependent Cell-Mediated Cytotoxicity
• CM 10 The ability of CM 10 to induce ADCC was examined in comparison to a positive control antibody (Polyclonal Ab anti CEACAMl that showed ADCC activity in preliminary experiment). Isotype matched antibody served as negative control (hlgGl K). The results indicate that CM10 do not trigger ADCC in the setting tested.
• Complement proteins are found in the blood, and their action "complements" the work of antibodies.
• Complement-dependent cytotoxicity (CDC) is a mechanism of killing cells in which antibody bound to the target cell surface fixes complement, results in assembly of the membrane attack complex that create pores in the target cell membrane and finally lead to cell lyses.
• CM 10 In order to access the safety profile of CM 10, the ability of CM 10 to induce CDC was examined in two melanoma cell lines (SKmel28 and SKmel5) expressing CEACAMl.
• Commercial pooled human serum was used as complement proteins source.
• the commercial monoclonal antibody Rituximab incubated with Daudi cells was used as the assay positive control and commercial IgGlK as isotype match control to CM10.
• Melanoma cell lines - SKMEL5 and SKMEL28 or positive control Daudi cells were incubated with CM 10 or Rituxiamab respectively for 1 hour in room temperature followed by the addition of normal human serum at a final concentration of 50% for 2 additional hours in a humidified incubator (37°C, 5% C0 2 ).
• the purpose of this experiment is to test the direct effect of CM 10 on melanoma cells in-vivo, as well as to evaluate the immunomodulatory effect, which is missing in the xenograft setting.
• SKMel5 melanoma cells were injected SC (subcutaneous) to SCID-NOD mice and tumor volume was monitored by physical measurements. When the tumor volume reached 100mm A 3, the mice where divided into 5 randomized groups.
• the TILs were injected either IT (Intra Tumoral) at two different concentrations or IV (Intra Venus) at one concentration (20X10 A 6 per mice) while one group received only one injection and the second group received 2 TIL injections.
• TIL injection was followed by 5 days of hlL- 2 administration.
• the calibration experiment demonstrated that TIL IV injection has higher effect on tumor size than IT administration.
• repetitive TIL regime provides better tumor growth inhibition over single injection, as could be predicted from T cells half life. Based on this data, TIL will be administrated every 10 days by IV injections, in future xenograft experiments.
• mice Human CEACAM1 positive S mel5 melanoma cells were injected SC to SCID- NOD mice. When the tumors reached a volume of approximately 100mm , the mice were randomized to one of the following treatment groups: a) Weekly IV injections of PBS; b) Weekly IV injections of 0.45mg CM10; c) Three IV injection of 20xl0 6 anti-tumor reactive human T cells (TIL) and weekly IV injections of PBS; d) Three IV injections of 20xl0 6 anti- tumor reactive human T cells and weekly IV injections of 0.45mg CM 10. A person blind to the experimental setting measured the tumors volume 2-3 times per week. The results of
• Figure 8 represent average tumor volume ⁇ SE from 6-10 mice per group. Arrows indicate time of administration (CM10 cyrcle, TIL triangles, CM10 and TIL open squares). As shown, a moderate inhibition of tumor growth was observed either with CM 10 alone or with TIL only, but the differences did not reach statistical significance when compared to the control treatment. Strikingly, the combination of adoptive human T cell transfer with CM 10 injections exhibited significant synergism and strongly inhibited xenograft growth. This observation concurs with the in-vitro data showing the potentiating effect of CM 10 on T cell killing ( Figures 5 and 6).
• CEACAM1 is known as a regulator of lymphocyte activation.
• CM 10 is an antibody that blocks the interactions between two CEACAM1 molecules (Figure 4) and therefore eliminates the inhibitory signals mediated by CEACAM1, results in stronger cytotoxic lymphocytes activation against tumor cells ( Figures 6 and 7).
• the in-vivo xenograft result ( Figure 8) reinforce the immunomostimulatory nature of CM 10 and demonstrate significant growth inhibition of tumors in mice treated with CM10 in the presence of TIL.
• the scheme presented in Figure 9 demonstrates a non-limitative theory of the mode of action of CM 10 that prevents CEACAM1-CEACAM1 interaction enabling activation of killing signals by immune system cells.
• Example 7 CM10 in-vitro safety assessment
• CEACAM1 In order to evade from the immune system cancer cells alter the expression of many molecules. Several evidences have showed that CEACAM1 expression is increasing during the malignance transformation of melanoma cells. According to the literature CEACAM1 is also expressed on normal cells, therefore it is important to map the possible binding sites of CM10 in the body and to identify if binding of CM10 to normal cells may lead to any undesired outcome.
• Tissue micro array containing 100 cases of malignant melanoma (primary, metastasis) and of benign nevi were analyzed for anti CEACAM1 binding intensity by standard IHC procedure. Each core of tumor was graded from 0 to +3. As shown in Figure 10, binding intensity of anti-CEACAMl mAb was seen in more than 50% of the melanoma samples and in 65% of metastatic melanoma samples.
• the multi normal human organ tissue microarray included 33 types of normal organs, each type taken from 3 normal human individuals. The age ranged from 2 - 67 years, 43 specimens were derived females and 57 specimens from males.
• the following tissues were negative for anti-CEACAMl mAb binding: Cerebrum, cerebellum, ovary, pancreas, parathyroid gland, hypophysis, thyroid gland, tonsil, bone marrow, spleen, thymus, lung, cardiac muscle, stomach, skeletal muscle, skin, peripheral nerves, mesothelium and retina.
• a cell-specific staining was detected in some organs, mainly on the luminal side of epithelial cells forming ducts or glands in hollow visceral organs such as: brush border of small intestine; some apical colonic glands; Breast ductal epithelium; Liver bile canaliuculi; inner surface of renal tubules; few Endometrial glands ;luminal part of Salivary gland.
• some low cellular staining was observed in adrenal gland cortex, apical surface of prostatic glands, Lomme cells of testis and single scattered cells in the pancreas. The only cells of the immune system that found positive were neutrophils within capillaries. No staining of lymphocytes was found in tissues and lymphatic organs.
• weak to moderate positive staining was found in endothelial cells of small blood vessels at selective sites, including: ovary, adrenal gland, kidney, and rarely in pancreas, prostate, hypophysis and endometrium.
• the IHC analysis showed a strong anti CEACAM1 staining of melanoma cells, as compared to no staining of the vast majority of the tissues tested in a normal human tissue. Nevertheless, some selective staining was observed in the luminal aspect of epithelial cells of ducts or glands in hollow viscera. This cellular aspect is generally less accessible to an antibody administered via the peripherally blood.
• QuantiBRITE kit was used. Using the kit the MFI (mean florescence intensity) was directly translated to the number of molecules bound per cell. Three human primary cells of tissues, which were found to be positive for anti-CEACAMl binding, were purchased from ATCC.
• HUVEC cells to represent the positive staining found in endothelial cells; primary prostate epithelial cells, since the apical surface of prostate glands showed positive staining, and primary renal proximal tubule epithelial cells since the inner surface of tubules stained positive.
• SkMel 5, G361, Malme 3M, NK 92MI, HUVEC, Renal primary cells and prostate primary cells were grown according to ATCC protocols.
• CM- 10 was conjugated to a PE molecule (RPE LYNX Rapid Conjugation Kits Serotec) according to the manufacture's protocol and was used ( ⁇ g/ml) with the QuantiBRITE PE beads kit (BD) to determine the ABC (antibodies bound per cell) by Flow cytometry.
• the number of CM- 10 molecules bound per cell was analyzed using flow cytometry in the indicated primary cells in comparison to melanoma cell lines. At least 10000 cells were counted for each cell line.
• HUVEC and primary prostate cells were grown according to the ATCC protocols and were monitored for cell proliferation using XTT standard assay. No effect on cell-proliferation could be detected.
• CEACAM1 is absence on normal melanocytes but undergoes neo- expression and is widely expressed on the vast majority of metastatic melanoma specimens (Figure 10), in other normal organ there is restricted expression of CEACAM1 in specific cells within several tissues.
• Figure 10 A quantitative analysis, measuring the number of CM10 molecules that are bound to cells, revealed a very low numbers of bound-CMlO on normal human primary cell ( Figure 11). These results imply that the majority of CM 10 molecule injected to patients will mainly target cancer cells and not normal tissues due to expression differences.
• CM10 has no effect on cell proliferation, and no ADCC or CDC activity could be observed, which suggests that the binding of CM 10 to non-target cells, would not result in unwanted cell outcome.
• CM 10 is an immunostimulatory antibody that blocks the interaction between two CEACAM1 molecules and by doing so mediates stimulation of lymphocytes against malignant cells. It is important to verify that the antibody will not cause unleashed stimulation of the immune system which can cause severe adverse events. In normal lymphocytes there is a neglect expression of CEACAM1 on the cell membrane, only following cell activation CEACAM1 is mobilized to the membrane, where it rapidly and strongly up-regulated on activated lymphocytes (Gray-Owen and Blumberg 2006, Nat Rev
• PBMC from 3 unrelated donors were isolated and incubated with CM 10 using 3 different concentrations (2 g/ml, 20 ⁇ g/ml or 200 ⁇ g/ml) or with a control mAb IgGlK (200 g/ml) for 60 minutes and with eight replicate wells.
• PHA (1 ⁇ g/ml) was added to 4 of the assay replicate wells and cells were incubated for 96 hours.
• 3H-Thymidine incorporation used to assay cell proliferation. Mock stimulated cells and PHA only stimulated cells used as assay's negative and positive controls respectively. The proliferation was assessed in resting lymphocytes as well as in activated lymphocytes (PHA treated).
• Example 8 CM10 selectivity panel.
• CEACAM1 Characterization of the binding profile was performed using Cell lines over- expressing the different CEACAM family proteins and flow cytometry analysis. .
• the CEA family is encoded by 18 genes and 1 1 pseudogenes on chromosome 19ql3.2.
• Several closely related members belong to the CEACAM family (CEACAM1,3,4,5,6,7,8) and are differentially expressed by various human cell types.
• the CEACAM proteins have been implicated in various adhesion mediated effects that govern the growth and differentiation of normal and cancerous cells (Gray-Owen and Blumberg 2006, Nat Rev Immunol 6, 433-46).
• the closely related proteins in the family share a high amino acid similarity that varies from 45% up to 90% similarity between certain members.
• CMIO conjugated to a Biotin molecule and Strep- Avidin APC as secondary agent.
• 721.221 cells expressing CEACAM 1,5,6,8 or HEK 293 T transient expressing CEACAM 3, 4 were stained with biotinylated CM- 10 (l ⁇ g/m ⁇ ) and Strep- Avidin APC as secondary agent.
• Empty histograms represent mAb staining while red histograms represent background staining. At least 10000 cells were used to analyze CMIO binding in each histogram.
• FAB was calculated by dividing the MFI of the stained cells in the MFI of the background staining. The results demonstrated in Figure 13, clearly indicate that CMIO bind strongly to cells expressing CEACAMl. Moderate staining was observed in cells expressing CEACAM3 and 5. Weak or neglect binding was demonstrated in cells expressing CEACAM 4, 6, 8.
• CM- 10 is a mAb developed to recognize human CEACAMl, a protein that was found to be associated with cancer, in general, and with Melanoma in particular.
• the over- expression of CEACAMl has been identified in a few malignancies among them melanoma, NSCLC, Thyroid cancer and gastric cancer. The evidence indicates that over- expression of CEACAMl can be correlated with poor prognosis in melanoma and NSCLC patients.
• CEACAM5 has been found to be over-expressed in a high percentage of many human tumors, including 90% of gastrointestinal, colorectal (CRC) and pancreatic cancers, 70% of non-small cell lung cancer cells and 50% of breast cancers. It is also over-expressed in thyroid, stomach, ovarian and uterine cancers (Thompson, Grunert et al. 1991, J Clin Lab Anal 5, 344-66). CEACAM5 even serves as a clinical marker for liver metastasis in CRC and post-surgical surveillance of colon cancer (Duffy 2001, Clin Chem 47, 624-30).
• CMIO is capable to bind CEACA5
• the anti-CEACAM5 agents that have entered clinical trials include anti-CEACAM5 antibodies conjugated to toxic substances such as radioactive substances for both diagnostic purposes and for the treatment of various malignancies. It seems that even these toxic conjugated forms don't show safety problems, which can indicate that CEACAM5 is a safe target (Liersch, et al. 2005, J Clin Oncol 23(27), 6763-70; Ychou, et al. 2008, Clin Cancer Res 14(11), 3487-93).
• none of these agents target the immunological regulation of tumors, which can be targeted by an antibody which can bind both CEACAM1 and CEACAM5, such as CM10.
• a humanized antibody typically has a human framework grafted with non human CDRs.
• a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
• Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 32 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 23 :1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• variable domains both light and heavy
• the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity.
• the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
• the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).
• Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
• the same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, ⁇ 9:4285 (1992); Presta et al, J. Immunol., 1 1 :2623 (1993)).
• humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
• Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
• Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
• FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
• the CDR residues are directly and most substantially involved in influencing antigen binding.
• transgenic animals e.g., mice
• transgenic animals e.g., mice
• JH antibody heavy-chain joining region
• Human antibodies can also be derived from phage-display libraries (Hoogenboom et al, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech 74:309 (1996)).
• phage-display libraries Hoogenboom et al, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech 74:309 (1996)).
• these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)).
• these fragments can now be produced directly by recombinant host cells.
• the antibody fragments can be isolated from the antibody phage libraries discussed above.
• Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology 70: 163-167 (1992)).
• F(ab') 2 fragments can be isolated directly from recombinant host cell culture.
• Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
• the antibody of choice is a single chain Fv fragment (scFv).
• the following experiments are used to determine the potential of CM 10 against viral infection.
• the experiments include different target cells, various virus and several in vivo and in vitro models.
• Detection/diagnosis Examination of CEACAM expression level in cell lines and primary cells infected with different virus types, by FACS analysis, RT PCR and Immuno- histochemestry.
• Treatment after viral infection the infected cell are incubated with immune system cell and the killing ability of the effectors cells and the viral load is examined. In addition, the viral load and replication and overall survival are determined in vivo after virus infection and treatment with anti CEACAM antibodies.

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