WO2015075710A1 - Compositions comprenant des anticorps anti-ceacam1, agents d'activation des lymphocytes et lymphocytes activés pour une cancérothérapie - Google Patents

Compositions comprenant des anticorps anti-ceacam1, agents d'activation des lymphocytes et lymphocytes activés pour une cancérothérapie Download PDF

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WO2015075710A1
WO2015075710A1 PCT/IL2014/050996 IL2014050996W WO2015075710A1 WO 2015075710 A1 WO2015075710 A1 WO 2015075710A1 IL 2014050996 W IL2014050996 W IL 2014050996W WO 2015075710 A1 WO2015075710 A1 WO 2015075710A1
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human
cancer
pharmaceutical composition
cell
antigen
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PCT/IL2014/050996
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Tehila Ben-Moshe
Yair SAPIR
Ilana MANDEL
Edna MEILIN
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Ccam Biotherapeutics Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464482—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 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 A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas
    • 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 A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • 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 A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma

Definitions

  • the present invention relates to cancer immunotherapy, in particular to combinations of anti-CEACAMl antibodies, lymphocyte activating agent and activated lymphocytes, and their use in treating cancer.
  • CEACAMl The transmembrane protein carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMl, also known as biliary glycoprotein (BGP), CD66a and C-CAM1), is a member of the carcinoembryonic antigen family (CEA) that also belongs to the immunoglobulin superfamily.
  • Human CEACAMl has been assigned the SwissProt accession number P13688.
  • CEACAMl interacts with itself and with other known CEACAM proteins, including 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 protein Many different functions have been attributed to the CEACAMl protein. It was shown that the CEACAMl protein is over expressed in some carcinomas of colon, prostate, as well as other types of cancer, such as melanoma. 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 (WO 99/52552 and Morales et al. J. Immunol. 1999, 163, 1363-1370).
  • 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.
  • monoclonal antibodies against the CEACAMl protein are already known, such as 26H7, 5F4, TEC-11, 12-140-4, 4/3/17, COL-4, F36-54, 34B 1, YG-C28F2, D14HD11, bl8.7.7, D11-AD11, HEA81, B l.l, CLB-gran-10, F34-187, T84.1, B6.2, B 1.13, YG-C94G7, 12-140-5, TET-2 and scFv-DIATHISl.
  • Interleukin 2 is an interleukin, a type of cytokine signaling molecule in the immune system. It is a protein that regulates the activities of white blood cells (leukocytes, often lymphocytes) that are responsible for immunity. IL-2 is part of the body's natural response to microbial infection, and in discriminating between foreign ("non-self") and "self". IL-2 mediates its effects by binding to IL-2 receptors. Human IL-2 has been assigned the SwissProt accession number P60568.
  • IL-2 has been approved for human therapy in several European countries on 1990, and later on 1992 by the United States Food and Drug Administration (FDA) for the treatment of cancers (malignant melanoma, metastatic renal cell cancer) in large intermittent doses, and has been extensively used in continuous doses. Since then, dozens of clinical trials had been conducted with recombinant or purified IL-2. In addition to testing IL-2 as the sole active ingredient, it was also tested in combination with other drugs, or using cell therapies, in which cells were taken from patients, activated with IL-2, and then infused back into the patients.
  • FDA United States Food and Drug Administration
  • LAK lymphokine-activated killer
  • rlL-2 cultured cells from all patients demonstrated cytotoxic activity, indicating in vitro generation of LAK activity. Results from these studies demonstrated that all patients were capable of generating a cytotoxic response.
  • the long time role of IL-2 in the therapy of cancer is elaborately reviewed by J. Bubenok (Folia Biologica, 2004, Vol. 50, No. 3-4, pp. 120-130), inter alia focusing on IL-2's ability to produce LAK cells when administered systemically. It is thus well established that IL-2 is capable of activating lymphocytes to become cytotoxic LAK cells both in vitro and in vivo.
  • the present invention stems from the surprising finding that combinations of anti- human-carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) antibodies and IL-2 activated human peripheral blood mononucleated cells (PBMC), known as lymphokine-activated killer (LAK) cells, significantly elevated cytotoxicity toward a variety of different types of human cancer in a dose-dependent manner.
  • CEACAM1 human-carcinoembryonic antigen-related cell adhesion molecule 1
  • LAK lymphokine-activated killer
  • the present invention thus provides, in one aspect, a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and an activated, cytotoxic lymphocyte cell.
  • the activated, cytotoxic lymphocyte cell is selected from the group consisting of a LAK cell, a CIK cell, and any combination thereof. Each possibility represents a separate embodiment of the invention.
  • the activated, cytotoxic lymphocyte cell is a lymphokine-activated killer (LAK) cell.
  • the present invention provides, in another aspect, a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • the lymphocyte activating agent is selected from the group consisting of IL-2, IFNy, an anti-CD3 antibody and fragments, analogs or fusion proteins thereof.
  • the lymphocyte activating agent is IL-2 or a fragment, analog or fusion protein thereof.
  • the present invention provides, in another aspect, a pharmaceutical composition as described above, for use in treating cancer.
  • the present invention provides, in another aspect, the use of a pharmaceutical composition as described above, in preparing a medicament for use in treating cancer.
  • the present invention provides, in another aspect, a method for treating a patient having cancer, comprising administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising an activated, cytotoxic lymphocyte cell, thereby treating the cancer.
  • the activated, cytotoxic lymphocyte cell is a lymphokine-activated killer (LAK) cell.
  • the present invention provides, in another aspect, a method for treating a patient having cancer, comprising administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a lymphocyte activating agent or a fragment, analog or fusion protein thereof, thereby treating the cancer.
  • the lymphocyte activating agent is IL-2 or a fragment, analog or fusion protein thereof.
  • kits comprising a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • the lymphocyte activating agent is IL-2 or a fragment, analog or fusion protein thereof.
  • the kits described above are for use in treating cancer.
  • the pharmaceutical composition described above comprises an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, an activated, cytotoxic lymphocyte cell, and a lymphocyte activating agent.
  • the pharmaceutical composition described above comprises an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a lymphokine-activated killer (LAK) cell, and a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • LAK lymphokine-activated killer
  • the pharmaceutical composition described above comprises an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, an activated, cytotoxic lymphocyte cell, and IL-2 or a fragment, analog or fusion protein thereof.
  • LAK lymphokine-activated killer
  • the pharmaceutical composition described above comprises an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a lymphokine- activated killer (LAK) cell, and IL-2 or a fragment, analog or fusion protein thereof.
  • LAK lymphokine- activated killer
  • the anti-CEACAMl monoclonal antibody or an antigen- binding fragment thereof is capable of binding with an affinity of at least about 10 "8 M to a human CEACAMl protein. Each possibility represents a separate embodiment of the invention. In some embodiments, the anti-CEACAMl monoclonal antibody or an antigen- binding fragment thereof is capable of binding with an affinity of at least about 5xlO "7 M to at least one of a human CEACAM3 and human CEACAM5 protein. Each possibility represents a separate embodiment of the invention.
  • the anti-CEACAMl monoclonal antibody is selected from the group consisting of CM-24, CM 10, 26H7, 5F4, TEC-11, 12-140-4, 4/3/17, COL-4, F36-54, 34B1, YG-C28F2, D14HD11, M8.7.7, Dl l- AD11, HEA81, Bl. l, CLB-gran-10, F34-187, T84.1, B6.2, B 1.13, YG-C94G7, 12-140-5, TET-2, scFv-DIATHISl , antigen-binding fragments thereof, and any combination thereof.
  • the anti-CEACAMl monoclonal antibody is CM-24, antigen-binding fragments thereof, and any combination thereof.
  • each possibility represents a separate embodiment of the invention.
  • the monoclonal antibody or fragment thereof which recognizes CEACAMl 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 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.
  • the monoclonal antibody or fragment thereof which recognizes CEACAMl 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.
  • SEQ ID NO: 1 SEQ ID NO: 1
  • SEQ ID NO: 2 and SEQ ID NO: 3 amino acid sequence
  • 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.
  • the monoclonal antibody or fragment thereof which recognizes CEACAMl comprises at least one heavy-chain CDR sequence of at least five consecutive 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.
  • the binding site of the monoclonal antibody or fragment thereof which recognizes CEACAMl consists of the six CDRs of SEQ ID NOs: 1, 2, 3, 4, 5, and 6. In some embodiments, the binding site of the monoclonal antibody or fragment thereof which recognizes CEACAMl consists of the six CDRs of SEQ ID NOs: 7, 8, 9, 10, 11, and 12. In some embodiments, the binding site of the monoclonal antibody or fragment thereof which recognizes CEACAMl consists of the six CDRs of SEQ ID NOs: 13, 14, 15, 16, 17, and 18. In some embodiments, the binding site of the monoclonal antibody or fragment thereof which recognizes CEACAMl consists of the six CDRs of SEQ ID NOs: 19, 20, 21, 22, 23, and 24.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy-chain CDRl 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 CDRl 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.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain CDRl having the sequence set forth in SEQ ID NO: 7, heavy chain CDR2 having the sequence set forth in SEQ ID NO: 8 and heavy chain CDR3 having the sequence set forth in SEQ ID NO: 9.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain CDRl having the sequence set forth in SEQ ID NO: 13, heavy chain CDR2 having the sequence set forth in SEQ ID NO: 14 and heavy chain CDR3 having the sequence set forth in SEQ ID NO: 15.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a light chain CDRl having the sequence set forth in SEQ ID NO: 10, light chain CDR2 having the sequence set forth in SEQ ID NO: 11 and light chain CDR3 having the sequence set forth in SEQ ID NO: 12.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a light chain CDRl having the sequence set forth in SEQ ID NO: 16, light chain CDR2 having the sequence set forth in SEQ ID NO: 17, and light chain CDR3 having the sequence set forth in SEQ ID NO: 18.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises CDR sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17, and 18. In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, 11, and 12.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain variable domain sequence having a sequence set forth in SEQ ID NO: 25, or an analog or derivative thereof having at least 97% sequence identity with said heavy chain sequence.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a light chain variable domain sequence having a sequence set forth in SEQ ID NO: 26, or an analog or derivative thereof having at least 97% sequence identity with said light chain sequence.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain variable domain having a sequence set forth in SEQ ID NO: 25 and a light chain variable domain having a sequence set forth in SEQ ID NO: 26, or an analog or derivative thereof having at least 97% sequence identity with the antibody or fragment sequence.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises:
  • 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 an antigen-binding fragment thereof which recognizes human CEACAMl comprises human derived constant regions selected from the group consisting of: human IgGl, human IgG2, and human IgG3.
  • the human or humanized monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAM1 comprises a constant region subclass of human IgGl subtype.
  • the human or humanized monoclonal antibody or an antigen- binding fragment thereof which recognizes human CEACAM1 comprises 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, wherein the monoclonal antibody binds with an affinity of at least about 10 "8 M to CEACAM1.
  • the human or humanized monoclonal antibody or an antigen- binding fragment thereof which recognizes human CEACAM1 comprises a heavy chain sequence set forth in SEQ ID NO: 27.
  • the human or humanized monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAM1 comprises a light chain sequence set forth in SEQ ID NO: 28. In certain embodiments, the human or humanized monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAM1 comprises a heavy chain sequence set forth in SEQ ID NO: 27, and light chain sequence set forth in SEQ ID NO: 28.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAM1 comprises at least the antigen-binding portion, which is capable of binding the same epitope on the CEACAM1 molecule to which a monoclonal antibody having the six CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, 11 and 12, or the six CDR sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17 and 18, binds.
  • the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope within residues 17-29 and 68-79 of human CEACAM1 having the sequences VLLLVHNLPQQLF (SEQ ID NO:32) and YPNASLLIQNVT (SEQ ID NO:33) respectively.
  • the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope comprising at least four amino acids of the sequence VLLLVHNLPQQLF (SEQ ID NO: 29). In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope comprising amino acid residues within the sequences VLLLVHNLPQQLF (SEQ ID NO: 29) and YPNASLLIQNVT (SEQ ID NO: 30). In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope within sequences VLLLVHNLPQQLF (SEQ ID NO: 29) and PNASLLI (SEQ ID NO: 31).
  • the anti-CEACAMl monoclonal antibody is a human or humanized monoclonal antibody.
  • a human antibody is an isolated human antibody, i.e. isolated from a human donor.
  • a human antibody is a human antibody isolated from a hybridoma cell line.
  • a human antibody refers to a recombinant human antibody, i.e. produced by recombinant DNA technology.
  • the lymphocyte activating agent fragment retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the corresponding full lymphocyte activating agent lymphocyte- activating capability.
  • the lymphocyte activating agent fragment retains at least 50% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the lymphocyte activating agent fragment retains at least 90% of the corresponding full lymphocyte activating agent lymphocyte-activating capability. Holte and coworkers describe an SPR-based fragment mapping of IL-2 (Holte et al., 2011, FASEB, Vol. 25, Ibl48).
  • the lymphocyte activating agent analog retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the corresponding original lymphocyte activating agent lymphocyte- activating capability.
  • the lymphocyte activating agent analog retains at least 50% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the lymphocyte activating agent analog retains at least 90% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the analog is isolated or derived from a non-mammalian organism. In certain embodiments, the analog is isolated or derived from a non-primate organism.
  • the analog is isolated. In certain embodiments, the analog is produced by recombinant DNA technology.
  • IL-2 mutants R38A and F42K are examples of lymphocyte activating agent analogs (Heaton et al., 1993, Cancer Research, Vol. 53, pages 2597-2602).
  • the lymphocyte activating agent fusion protein retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the corresponding original lymphocyte activating agent lymphocyte- activating capability. Each possibility represents a separate embodiment of the invention.
  • the lymphocyte activating agent fusion protein retains at least 50% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the lymphocyte activating agent fusion protein retains at least 90% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • Hu and coworkers describe the generation of low-toxicity IL-2 fusion proteins (Hu et al., 2003, Blood, Vol. 101(12), pages 4853-4861).
  • the activated cytotoxic lymphocyte cell expresses CEACAM1. In some embodiments, the LAK cell expresses CEACAM1.
  • the activated cytotoxic lymphocyte cell is derived from a peripheral blood mononucleated (PBMC) cell.
  • the LAK cell is derived from a PBMC cell.
  • the activated cytotoxic lymphocyte cell is derived from a PBMC cell by incubation with a lymphocyte activating agent.
  • the activated cytotoxic lymphocyte cell is derived from a cancer patient, optionally expanded or further activated ex-vivo.
  • the LAK cell is derived from a PBMC cell by incubation with a lymphocyte activating agent.
  • the LAK cell is derived from a cancer patient, optionally expanded or further activated ex-vivo.
  • the activated cytotoxic lymphocyte cell is cytotoxic to a cancer cell line.
  • the LAK cell is cytotoxic to a cancer cell line.
  • the cancer cell line is selected from the group consisting of SKMEL28, MEL14, T3M4, SU8686, PANC2, H358 and H460. Each possibility represents a separate embodiment of the invention.
  • the cancer cell line expresses CEACAM1.
  • the lymphocyte activating agent is capable of activating lymphocytes in-vivo.
  • the lymphocyte activating agent is selected from the group consisting of Interleukin (IL) 2, IL- ⁇ , IL-6, IL-12, Tumor necrosis factor alpha (TNF-a), Interferon gamma (IFN- ⁇ ), Granulocyte-macrophage colony-stimulating factor (GM-CSF), complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IF A), an anti-CD3 antibody, pokeweed mitogen (PWM), Phytohaemagglutinin (PHA), phorbol myristate acetate (PMA), Concavalin A, Ionomycin, Lipopolysaccharides (LPS), Toll like receptor (TLR) activators, Ca+2 ionophores, Staphylococcal Enterotoxin B (SEB), mitogen stimulators, a fragment thereof, an analog thereof, a fusion
  • IL Interleuk
  • the lymphocyte activating agent is a recombinant lymphocyte activating agent. In certain embodiments, the lymphocyte activating agent is a human lymphocyte activating agent. In certain embodiments, the lymphocyte activating agent is an isolated human lymphocyte activating agent. In certain embodiments, the lymphocyte activating agent is IL-2 or a fragment, analog or fusion protein thereof. Each possibility represents a separate embodiment of the invention. In certain embodiments, the lymphocyte activating agent is a recombinant IL-2 (rIL-2) or a fragment, analog or fusion protein thereof. Each possibility represents a separate embodiment of the invention. In certain embodiments, the lymphocyte activating agent is a human IL-2 (hIL-2) or a fragment, analog or fusion protein thereof. Each possibility represents a separate embodiment of the invention. In certain embodiments, the lymphocyte activating agent is an isolated human hIL-2 or a fragment, analog or fusion protein thereof. Each possibility represents a separate embodiment of the invention.
  • the cancer is selected from the group consisting of a melanoma, pancreatic, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, and endometrial cells cancer.
  • the cancer is selected from the group consisting of melanoma cancer, pancreatic cancer and lung cancer.
  • the cancer is melanoma cancer.
  • the cancer is pancreatic cancer.
  • the cancer is lung cancer.
  • the method described above comprises administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising an activated cytotoxic lymphocyte cell, and a pharmaceutical composition comprising a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • the method described above comprises administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising a LAK cell, and a pharmaceutical composition comprising a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • the method described above comprises administrating to the patient a pharmaceutical composition comprising an anti- human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising an activated cytotoxic lymphocyte cell, and a pharmaceutical composition comprising IL-2 or a fragment, analog or fusion protein thereof.
  • the method described above comprises administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising a LAK cell, and a pharmaceutical composition comprising IL-2 or a fragment, analog or fusion protein thereof.
  • the activated cytotoxic lymphocyte cell is derived from a peripheral blood mononucleated (PBMC) cell of the cancer patient. In certain embodiments, the activated cytotoxic lymphocyte cell is derived from an HLA-matching donor. In certain embodiments, the LAK cell is derived from a peripheral blood mononucleated (PBMC) cell of the cancer patient. In certain embodiments, the LAK cell is derived from an HLA-matching donor.
  • PBMC peripheral blood mononucleated
  • Human LAK cells express CEACAMl upon IL-2 activation.
  • PBMC cells were isolated from a healthy donor followed by activation with IL-2 (500 units/ml) for 7 days to generate a population of LAK cells, followed by FACS analysis with PE-conjugated CM-24 (black line). For comparison, a similar FACS analysis was performed to non-activated PBMC cells (gray line).
  • Figure 2 Human cancer cells express CEACAMl.
  • Human cancer cell lines SU8686 (A), T3M4 (B), H358 (C) and SKMEL28 (D) were analyzed by FACS with a PE-conjugated or biotinylated anti-CEACAMl antibody (CM-24) followed by staining with Strepavidin-APC (black lines). A similar assay was conducted for the appropriate isotype controls (gray lines).
  • FIG. 3A-B Cytotoxicity of human LAK cells against a variety of human melanoma cells.
  • PBMC cells were isolated from a healthy donor followed by activation with IL-2 (0, 300, 1000 or 3000 units/ml) for 3 days to generate a population of human LAK cells.
  • IL-2 0.01, 300, 1000 or 3000 units/ml
  • Two different human melanoma cell lines, SKMEL28 (A) and MEL 14 (B) were added for an incubation of 24 hours at the indicated effector-to-target ratios (2.5: 1 and 10:1).
  • Results represent an average of % cytotoxicity +SE as determined by classical LDH release assay from triplicate wells per treatment.
  • Anti-CEACAMl antibodies increase the cytotoxicity of human LAK cells against human melanoma cells.
  • Human LAK cells were incubated with an anti-CEACAMl antibody (CM-24) in different concentrations for 30 minutes at 37°C.
  • Human melanoma cancer cells (SKMEL28) were added for an incubation of 24 hours.
  • Results represent an average of % cytotoxicity +SE as determined by classical LDH release assay from triplicate wells per treatment. * P ⁇ 0.05 paired T-test compared to effectors + target cells with medium only.
  • FIG. 5A-E Anti-CEACAMl antibodies increase the cytotoxicity of human LAK cells against a variety of human pancreatic and lung cancer cells.
  • Human LAK cells were incubated with an anti-CEACAMl antibody (CM-24) in different concentrations for 30 minutes at 37°C.
  • CM-24 anti-CEACAMl antibody
  • Three different human pancreatic cancer cells, T3M4 (A), SU8686 (B) and PANC2 (C), and two different human lung cancer cells, H358 (D) and H460 (E) were added for an incubation of 24 hours.
  • Results represent an average of % cytotoxicity +SE as determined by classical LDH release assay from triplicate wells per treatment. * P ⁇ 0.05 paired T-test compared to effectors + target cells with medium only.
  • FIG. 6A-B Anti-CEACAMl antibodies enhance granzyme B secretion of human LAK cells in the presence of human pancreatic and lung cancer cells.
  • Human LAK cells were incubated with an anti-CEACAM-1 antibody (CM-24) in different concentrations for 30 minutes at 37°C.
  • Human pancreatic cancer cells T3M4 (A) or human lung cancer cells H358 (B) were added for an incubation of 24 hours.
  • Granzyme B secretion was measured by ELISA. Results represent the mean + S.E of Granzyme B release values from 3 repeats per treatment. *P ⁇ 0.05 paired T-test, compared to effectors + target cells with medium only.
  • FIG. 7A-B Anti-CEACAMl antibodies enhance IFN- ⁇ secretion of human LAK cells in the presence of human cancer cells.
  • Human LAK cells were incubated with an anti- CEACAM-1 antibody (CM-24) in different concentrations for 30 minutes at 37°C.
  • Human lung cancer cells H358 (A) or H460 (B) were added for an incubation of 24 hours.
  • IFN- ⁇ secretion was measured by ELISA. Results represent the mean + S.E of Granzyme B release values from 3 repeats per treatment. *P ⁇ 0.05 paired T-test, compared to effectors + target cells with medium only. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention stems from the surprising finding that a combination of anti- CEACAM1 antibodies and IL-2-activated human peripheral blood mononucleated (PBMC) cells, known as lymphokine-activated killer (LAK) cells, significantly elevates cytotoxicity toward a variety of different types of human cancer in a dose-dependent manner. Further, it was found that the interaction of anti-CEACAMl antibodies and human LAK cells with human cancer cells significantly elevates Granzyme B and IFN- ⁇ levels in the vicinity of these cancer cells.
  • PBMC peripheral blood mononucleated
  • LAK lymphokine-activated killer
  • activated lymphocytes expressing CEACAM1 can be substantially protected or shielded from heterotypic immuno-suppressive interactions with other CEACAM1 molecules presented e.g. by cancer cells, so these lymphocytes may become and remain cytotoxic toward these cancer cells upon activation.
  • anti-CEACAMl antibodies obstruct the major immuno-suppressive homotypic interaction CEACAMl 1ym P hoc 5*7CEACAMl cancer cdl , and increase the level of anti-cancer cytotoxic cells within the body of a patient diagnosed with cancer.
  • lymphocyte activating agents such as IL-2, Interferon- ⁇ (IFN- ⁇ ) and/or anti-CD3 monoclonal antibodies
  • IL-2 Interferon- ⁇
  • IFN- ⁇ Interferon- ⁇
  • PBMC cells a cell population termed "activated, cytotoxic lymphocyte cells”, “activated lymphocyte cells”, “cytotoxic lymphocyte cells” or “activated lymphocytes”, both in vitro and in vivo.
  • activated lymphocyte cells activated lymphocyte cells
  • cytotoxic lymphocyte cells cytotoxic lymphocyte cells
  • activated lymphocytes both in vitro and in vivo.
  • the present invention provides ample guidance for a person of average skill in the field as to how to activate PBMCs to produce diverse populations of activated, cytotoxic lymphocyte cells without adverse side effects. For example, Stephen E.
  • Cytokine Induced Killer (CIK) cells are in-vitro activated human CD8 T cells which have acquired non-specific anti tumoral cytotoxicity, thus representing a cell population with double T cell and NK cell phenotype. Due to their in-vivo intratumoral homing and lack of Graft versus Host (GVH) reactivity, CIK cells have been extensively used in cancer patients either in autologous or allogeneic contexts.
  • M. Introna et al. (Immunology Letters, 2013, Vol. 155, pages 27- 30) provides a summary of CIK cells' main biological features as well as their most prominent clinical results.
  • the present invention thus provides pharmaceutical compositions directed to obstruct the major immuno-suppressive homotypic interaction CEACAMl 1 y m P hoc y t 7CEACAMl cancer cdl , and to increase the level of anti-cancer cytotoxic cells within the body of a patient diagnosed with cancer.
  • the present invention is based on the surprising finding that anti-CEACAMl antibodies significantly elevate the cytotoxic effect of cytotoxic cells against cancer cells in a dose-dependent manner.
  • compositions of the present invention generate a "two-punch" combination, in which the level of anti-cancer cytotoxic lymphocytes within the cancer patient is raised, while their cytotoxicity is maintained by protective interaction with anti-CEACAMl antibodies, bound to CEACAM1 molecules presented by the lymphocyte themselves, the CEACAM1 molecules presented by the cancer cells, or both.
  • the present invention provides, in one aspect, a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and an activated, cytotoxic lymphocyte cell.
  • the activated, cytotoxic lymphocyte cell is selected from the group consisting of a LAK cell, a CIK cell, and any combination thereof. Each possibility represents a separate embodiment of the invention.
  • the activated, cytotoxic lymphocyte cell is a lymphokine-activated killer (LAK) cell.
  • the present invention provides, in another aspect, a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • the lymphocyte activating agent is selected from the group consisting of IL-2, IFNy, an anti-CD3 antibody and fragments, analogs or fusion proteins thereof.
  • the lymphocyte activating agent is IL-2 or a fragment, analog or fusion protein thereof.
  • the present invention provides, in another aspect, a pharmaceutical composition as described above, for use in treating cancer.
  • the present invention provides, in another aspect, the use of a pharmaceutical composition as described above, in preparing a medicament for use in treating cancer.
  • the present invention provides, in another aspect, a method for treating a patient having cancer, comprising administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising an activated, cytotoxic lymphocyte cell, thereby treating the cancer.
  • the present invention further provides, in another aspect, a method for treating a patient having cancer, comprising administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a LAK cell, thereby treating the cancer.
  • the present invention provides, in another aspect, a method for treating a patient having cancer, comprising administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a lymphocyte activating agent or a fragment, analog or fusion protein thereof, thereby treating the cancer.
  • the present invention further provides, in another aspect, a method for treating a patient having cancer, comprising administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising IL-2 or a fragment, analog or fusion protein thereof, thereby treating the cancer.
  • the present invention provides, in yet another aspect, a kit comprising a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • the present invention further provides, in yet another aspect, a kit comprising a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, and a pharmaceutical composition comprising IL-2 or a fragment, analog or fusion protein thereof.
  • CEACAMl is used to refer to the protein product of the CEACAMl gene e.g., NP_001020083.1, NP_001703.2. In humans, 11 different CEACAMl splice variants have been detected so far. Individual CEACAMl isoforms differ with respect to the number of extracellular immunoglobulin-like domains (for example, CEACAMl with four extracellular immunoglobulin-like domains is known as CEACAMl -4), membrane anchorage and/or the length of their cytoplasmic tail (for example, CEACAMl -4 with a long cytoplasmic tail is known as CEACAMl -4L and CEACAMl -4 with a short cytoplasmic tail is known as CEACAM1-4S).
  • the N-terminal domain of CEACAMl 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 "CEACAMl".
  • composition refers to a composition comprising at least one biologically active ingredient.
  • Antibodies, antigen-binding fragments thereof, lymphocyte cells, lymphocyte activating agents and fragments, analogs and fusion proteins thereof are non-limiting examples of biologically active ingredients.
  • antibody is used in the broadest sense and includes monoclonal antibodies
  • polyclonal antibodies including full length or intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multi-specific antibodies (e.g., bi-specific 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.
  • an “anti-CEACAMl antibody”, “an antibody which recognizes CEACAM1”, “an antibody against CEACAM1”, or “an antibody to CEACAM1” is an antibody that binds to the CEACAM1 protein with sufficient affinity and specificity.
  • an anti-CEACAMl antibody is capable of binding CEACAM1 with a minimal affinity of about 10 "8 or 10 "9 M.
  • Some of the monoclonal anti-CEACAMl antibodies are capable of binding CEACAM3, 5 and/or 8 with a minimal affinity of about 5xl0 "7 M.
  • the anti- CEACAMl antibody is capable of preventing, interfering or dissociating an interaction between CEACAM1 presented by lymphocytes and CEACAM1 presented by cancer cells.
  • neutralizing antibody refers to a molecule having an antigenic) 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.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising
  • 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 is 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.
  • 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).
  • 25 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 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 30 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.
  • antigenic determinant or “epitope” refers to the region of an antigen molecule that specifically reacts with particular antibody.
  • 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.
  • An antigen according to the present invention is a CEACAM1 protein or a fragment thereof.
  • 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 (CL) 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).
  • VL variable domain
  • CL constant domain
  • CHI first constant domain of the heavy chain
  • 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.
  • anti-CEACAMl antibody refers to full immunoglobulin molecules, e.g. IgMs, IgDs, IgEs, IgAs or IgGs, antigen-binding-domains of such immunoglobulin molecules, e.g. Fab-fragments, Fab'-fragments, F(ab)2-fragements, chimeric F(ab)2 or chimeric Fab' fragments, chimeric Fab-fragments or isolated VH- or CDR-regions, and known isoforms and modifications of immunoglobulins, e.g. single-chain antibodies or single chain Fv fragments (scAB/scFv) or bispecific antibody constructs, capable of binding to their indicated targets.
  • 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.
  • an antigen-binding fragment of an antibody refers to one or more fragments of an antibody that retains the ability to bind specifically to the disclosed antigen.
  • the antigen-binding fragment may include, but not limited to, Fab fragment, F(ab')2 fragment, scFv fragment, dAb fragment, CDR-containing fragment or isolated CDR. Therefore, an antigen-binding fragment of an anti-CEACAMl antibody may be e.g. an Fab fragment of an anti-CEACAMl antibody, or any molecule which mimics the sequences and structure of such an Fab fragment, without being directly obtained from an anti-CEACAMl antibody, e.g. by chemical or enzymatic cleavage.
  • 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
  • single chain variable fragment scFv
  • 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 (linked VH-VL or single chain Fv (scFv)).
  • VH and VL 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.
  • 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).
  • molecule having the antigen-binding portion of an antibody 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, WO 96/13583, WO 96/37621, 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 a receptor, or molecules developed to deliver therapeutic moieties by means of a portion of the molecule containing such a reactive
  • Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.
  • the term "antigen" as used herein refers to a molecule or a portion of a molecule capable of eliciting antibody formation and/or being bound by an antibody.
  • An antigen may have one or more than one epitope.
  • the protein CEACAM1 is considered an antigen by the present invention.
  • the antigens are human antigens.
  • treating cancer refers to administering therapeutic effective amounts of agents such as antibodies and/or lymphocyte cells and/or lymphocyte activating agents to a patient diagnosed with cancer, to inhibit the further growth of malignant cells in the patient, to inhibit the spread of the malignant cells in the patient, and/or to cause the death of malignant cells in the patient.
  • treating cancer means attenuating tumor progression, inhibiting the spread of the malignant cells in the patient, causing the death of malignant cells in the patient and any combination thereof.
  • treating cancer means attenuating tumor progression, causing the death of malignant cells in the patient, or both.
  • 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.
  • 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.
  • 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 use in 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.
  • kit refers to a combination of reagents and other materials.
  • the kit may include reagents such as antibodies, antibody mixtures, buffers, diluents and other aqueous solutions, and/or one or more storage vials or other containers. It is not intended that the term “kit” be limited to a particular combination of reagents and/or other materials.
  • the pharmaceutical composition described above comprises an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, an activated, cytotoxic lymphocyte cell, and a lymphocyte activating agent.
  • the pharmaceutical composition described above comprises an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a lymphokine-activated killer (LAK) cell, and a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • LAK lymphokine-activated killer
  • the pharmaceutical composition described above comprises an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, an activated, cytotoxic lymphocyte cell, and IL-2 or a fragment, analog or fusion protein thereof.
  • an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof an activated, cytotoxic lymphocyte cell, and IL-2 or a fragment, analog or fusion protein thereof.
  • the pharmaceutical composition described above comprises an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a lymphokine-activated killer (LAK) cell, and IL-2 or a fragment, analog or fusion protein thereof.
  • LAK lymphokine-activated killer
  • the anti-CEACAMl monoclonal antibody or an antigen- binding fragment thereof is capable of binding with an affinity of at least about 10 "8 M to a human CEACAM1 protein.
  • the anti-CEACAMl monoclonal antibody or an antigen- binding fragment thereof is capable of binding with an affinity of at least about 5xlO "7 M to at least one of a human CEACAM3 and human CEACAM5 protein.
  • Many monoclonal antibodies against the CEACAM1 protein are already known, all of which considered appropriate for use in the compositions and methods of the present inventions.
  • the anti-CEACAMl monoclonal antibody is selected from the group consisting of CM-24, CM10, 26H7, 5F4, TEC-11, 12-140-4, 4/3/17, COL-4, F36-54, 34B 1, YG-C28F2, D14HD11, M8.7.7, D11-AD11, HEA81, B l. l, CLB- gran-10, F34-187, T84.1, B6.2, B 1.13, YG-C94G7, 12-140-5, TET-2, scFv-DIATHISl, antigen-binding fragments thereof, and any combination thereof.
  • Each possibility represents a separate embodiment of the invention.
  • the anti-CEACAMl monoclonal antibody is CM-24, antigen-binding fragments thereof, and any combination thereof.
  • CM-24 antigen-binding fragments thereof, and any combination thereof.
  • Each possibility represents a separate embodiment of the invention.
  • the CDR segments of CM-24 were identified using two different algorithm methods:
  • Table 1 summarizes the determined CDR sequences using the two methods as well as the minimal consensus sequence and combined sequence of sequences identified using both methods.
  • the monoclonal antibody or fragment thereof which recognizes CEACAMl 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 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.
  • the monoclonal antibody or fragment thereof which recognizes CEACAMl 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.
  • SEQ ID NO: 1 SEQ ID NO: 1
  • SEQ ID NO: 2 and SEQ ID NO: 3 amino acid sequence
  • 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.
  • the monoclonal antibody or fragment thereof which recognizes CEACAMl comprises at least one heavy-chain CDR sequence of at least five consecutive 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.
  • the binding site of the monoclonal antibody or fragment thereof which recognizes CEACAMl consists of the six CDRs of SEQ ID NOs: 1, 2, 3, 4, 5, and 6. In some embodiments, the binding site of the monoclonal antibody or fragment thereof which recognizes CEACAMl consists of the six CDRs of SEQ ID NOs: 7, 8, 9, 10, 11, and 12. In some embodiments, the binding site of the monoclonal antibody or fragment thereof which recognizes CEACAMl consists of the six CDRs of SEQ ID NOs: 13, 14, 15, 16, 17, and 18. In some embodiments, the binding site of the monoclonal antibody or fragment thereof which recognizes CEACAMl consists of the six CDRs of SEQ ID NOs: 19, 20, 21, 22, 23, and 24.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises 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.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain CDR1 having the sequence set forth in SEQ ID NO: 7, heavy chain CDR2 having the sequence set forth in SEQ ID NO: 8 and heavy chain CDR3 having the sequence set forth in SEQ ID NO: 9.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain CDRl having the sequence set forth in SEQ ID NO: 13, heavy chain CDR2 having the sequence set forth in SEQ ID NO: 14 and heavy chain CDR3 having the sequence set forth in SEQ ID NO: 15.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a light chain CDRl having the sequence set forth in SEQ ID NO: 10, light chain CDR2 having the sequence set forth in SEQ ID NO: 11 and light chain CDR3 having the sequence set forth in SEQ ID NO: 12.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a light chain CDRl having the sequence set forth in SEQ ID NO: 16, light chain CDR2 having the sequence set forth in SEQ ID NO: 17, and light chain CDR3 having the sequence set forth in SEQ ID NO: 18.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises CDR sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17, and 18. In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, 11, and 12.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain variable domain sequence having a sequence set forth in SEQ ID NO: 25, or an analog or derivative thereof having at least 97% sequence identity with said heavy chain sequence.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a light chain variable domain sequence having a sequence set forth in SEQ ID NO: 26, or an analog or derivative thereof having at least 97% sequence identity with said light chain sequence.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain variable domain having a sequence set forth in SEQ ID NO: 25 and a light chain variable domain having a sequence set forth in SEQ ID NO: 26, or an analog or derivative thereof having at least 97% sequence identity with the antibody or fragment sequence.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises:
  • 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 an antigen-binding fragment thereof which recognizes human CEACAMl comprises human derived constant regions selected from the group consisting of: human IgGl, human IgG2, and human IgG3.
  • the human or humanized monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a constant region subclass of human IgGl subtype.
  • the human or humanized monoclonal antibody or an antigen- binding fragment thereof which recognizes human CEACAMl comprises 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, wherein the monoclonal antibody binds with an affinity of at least about 10 "8 M to CEACAMl.
  • the human or humanized monoclonal antibody or an antigen- binding fragment thereof which recognizes human CEACAMl comprises a heavy chain sequence set forth in SEQ ID NO: 27. In certain embodiments, the human or humanized monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a light chain sequence set forth in SEQ ID NO: 28. In certain embodiments, the human or humanized monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises a heavy chain sequence set forth in SEQ ID NO: 27, and light chain sequence set forth in SEQ ID NO: 28.
  • the monoclonal antibody or an antigen-binding fragment thereof which recognizes human CEACAMl comprises at least the antigen-binding portion, which is capable of binding the same epitope on the CEACAMl molecule to which a monoclonal antibody having the six CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, 11 and 12, or the six CDR sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17 and 18, binds.
  • the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope within residues 17-29 and 68-79 of human CEACAMl having the sequences VLLLVHNLPQQLF (SEQ ID NO:32) and YPNASLLIQNVT (SEQ ID NO:33) respectively.
  • the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope comprising at least four amino acids of the sequence VLLLVHNLPQQLF (SEQ ID NO: 29). In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope comprising amino acid residues within the sequences VLLLVHNLPQQLF (SEQ ID NO: 29) and YPNASLLIQNVT (SEQ ID NO: 30). In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope within sequences VLLLVHNLPQQLF (SEQ ID NO: 29) and PNASLLI (SEQ ID NO: 31).
  • the protein sequences of the antibodies produced are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients.
  • the anti- CEACAMl monoclonal antibody is a human or humanized monoclonal antibody.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In certain embodiments, the term “human antibody” refers to an isolated human antibody, i.e. isolated from a human donor.
  • human antibody refers to a human antibody isolated from a hybridoma cell line.
  • human antibody refers to a recombinant human antibody, i.e. produced by recombinant DNA technology.
  • a "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.
  • 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. Biol, 1991, 222, 581).
  • 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.
  • 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.
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • humanized antibody refers is intended to include antibodies that have their CDRs (complementarity determining regions) derived from a non-human species immunoglobulin and the remainder of the antibody molecule derived mainly from a human immunoglobulin.
  • "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.
  • 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 comprises 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 also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • lymphocyte cell refers to any one of a natural killer (NK) cell (usually involved in cell-mediated, cytotoxic innate immunity), a T cell (usually involved in cell-mediated, cytotoxic adaptive immunity), a B cell (usually involved in humoral, antibody-driven adaptive immunity), a plurality thereof and any combination thereof.
  • NK natural killer
  • TIL tumor-infiltrating- lymphocyte
  • LAK lymphokine-activated killer
  • lymphocytes include cytotoxic lymphocytes (CTLs, CD8 + or CD4 + ), NK cells (CD2 + ), and T helper cells (CD4 + ).
  • LAK cell lymphocyte cell that has been stimulated or activated to kill tumor cells, i.e. to become more cytotoxic toward tumor cells.
  • LAK cells may be produced from homogenous or heterogeneous cell populations, the terms above further refer to homogenous or heterogeneous cell populations, stimulated or activated to become more cytotoxic toward tumor cells.
  • LAK cells are produced from PBMC cells by IL-2.
  • Interleukin 2 T cell growth factor
  • TCGF transforming growth factor
  • IL2 interleukin-2 receptor
  • IL-2 interleukin-2 receptor
  • IL-2R interleukin-2 receptor
  • IL-2R lymphocyte-activating analogs thereof
  • IL-2R agonists lymphocyte-activating fusion protein thereof.
  • IL-2 further refers to mouse, rat, rabbit, primate, pig and bovine IL-2.
  • lymphocyte activating agent refers to any chemical or biological molecule, capable of inducing a lymphocyte cell to gain cytotoxic activity, or increasing its native cytotoxic activity, toward at least one cancer cell type, in vitro and/or in vivo.
  • An example of a lymphocyte activating agent is the human IL-2 (UniProt No. P60568), a cytokine signaling molecule in the immune system, and fragments, analogs or fusion proteins thereof, that retain the biological activity of human IL-2.
  • fragment of a lymphocyte activating agent refers to any part of a lymphocyte activating agent, retaining a substantial lymphocyte-activating capability.
  • the lymphocyte activating agent fragment retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the lymphocyte activating agent fragment retains at least 50% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the lymphocyte activating agent fragment retains at least 90% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • analog of a lymphocyte activating agent refers to any chemical or biological molecule having sequence similarity or structural similarity to a lymphocyte activating agent, retaining a substantial lymphocyte-activating capability.
  • the lymphocyte activating agent analog retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the corresponding original lymphocyte activating agent lymphocyte-activating capability.
  • the lymphocyte activating agent analog retains at least 50% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the lymphocyte activating agent analog retains at least 90% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the analog is isolated or derived from a non-primate organism. In certain embodiments, the analog is isolated or derived from a non-mammalian organism. In certain embodiments, the analog is isolated. In certain embodiments, the analog is produced by recombinant DNA technology.
  • fusion protein of a lymphocyte activating agent refers to any chemical or biological molecule covalently linked, either directly or indirectly, to a lymphocyte activating agent, retaining a substantial lymphocyte-activating capability.
  • the lymphocyte activating agent fusion protein retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the corresponding original lymphocyte activating agent lymphocyte- activating capability.
  • the lymphocyte activating agent fusion protein retains at least 50% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the lymphocyte activating agent fusion protein retains at least 90% of the corresponding full lymphocyte activating agent lymphocyte-activating capability.
  • the activated cytotoxic lymphocyte cell expresses CEACAM1. In some embodiments, the LAK cell expresses CEACAM1.
  • PBMC cells activated by IL-2 are often referred to as "LAK" cells.
  • LAK cells activated cytotoxic lymphocyte cell is derived from a peripheral blood mononucleated (PBMC) cell.
  • PBMC peripheral blood mononucleated
  • LAK cell is derived from a PBMC cell.
  • the term "derived from” is used herein to identify the original source of a cell or cell population but is not meant to limit the agent or method by which the cell or cell population is made which can be, for example, by natural or recombinant means.
  • the activated cytotoxic lymphocyte cell is derived from a PBMC cell by incubation with a lymphocyte activating agent.
  • the activated cytotoxic lymphocyte cell is derived from a cancer patient, optionally expanded or further activated ex-vivo.
  • the LAK cell is derived from a PBMC cell by incubation with a lymphocyte activating agent.
  • the LAK cell is derived from a cancer patient, optionally expanded or further activated ex-vivo.
  • ex-vivo may be replaced by the term "in-vitro", and generally refers to processes taking place outside a body of an organism.
  • the main functional difference between an original PBMC cell population and a LAK cell population derived from the original PBMC cell population relevant to the present invention is the increased cytotoxicity of the LAK cell population compared to the original PBMC cell population toward cancer cells.
  • Many standard commercial cell lines where suggested by others, and also disclosed by the present invention, as convenient means to test the cytotoxicity of LAK cell populations, PBMC cell populations, or both, for determining their relative cytotoxicity.
  • the activated cytotoxic lymphocyte cell is cytotoxic to a cancer cell line.
  • the LAK cell is cytotoxic to a cancer cell line.
  • cytotoxic refers to an immune cell which is harmful to a cancer cell structure and/or function, e.g. which damages or affects the cancer cell's membrane and/or DNA integrity, and may ultimately cause cell death.
  • the cancer cell line is selected from the group consisting of SKMEL28, MEL14, T3M4, SU8686, PANC2, H358 and H460. Each possibility represents a separate embodiment of the invention.
  • the cancer cell line expresses CEACAM1.
  • a lymphocyte activating agent may be administered systemically, as described in the literature, e.g. by Joseph H. Phillips et al. and coworkers (J. Clin. Oncol., 1987, Vol. 5, pp.1933-1941).
  • the lymphocyte activating agent is capable of activating lymphocytes in-vivo.
  • lymphocyte activating lymphocytes in-vivo refers to the ability of a lymphocyte activating agent to increase the number of lymphocytes, to increase the cytotoxicity of lymphocytes, or both increase the number and cytotoxicity of lymphocytes in the human body, e.g. in a cancer patient's body.
  • lymphocyte activating agent capable of inducing lymphocyte to gain cytotoxic activity or increase their native cytotoxic activity, all of which are considered lymphocyte activating agent by the present invention.
  • the lymphocyte activating agent is selected from the group consisting of Interleukin (IL) 2, IL- ⁇ , IL-6, IL-12, Tumor necrosis factor alpha (TNF-a), Interferon gamma (IFN- ⁇ ), Granulocyte-macrophage colony-stimulating factor (GM-CSF), complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), an anti-CD3 antibody, poke weed mitogen (PWM), Phytohaemagglutinin (PHA), phorbol myristate acetate (PMA), Concavalin A, Ionomycin, Lipopolysaccharides (LPS), Toll like receptor (TLR) activators, Ca+2 ionophores, Staphylococcal Enterotoxin B (SEB), mitogen stimulators, a fragment thereof, an analog thereof, a fusion protein thereof, and any combination thereof.
  • IL Interleukin
  • TNF-a Tumor necrosis
  • the lymphocyte activating agent is a recombinant lymphocyte activating agent.
  • the lymphocyte activating agent is a human lymphocyte activating agent.
  • the lymphocyte activating agent is an isolated human lymphocyte activating agent.
  • the lymphocyte activating agent is IL-2 or a fragment, analog or fusion protein thereof. Each possibility represents a separate embodiment of the invention.
  • the lymphocyte activating agent is a recombinant IL-2 (rIL-2) or a fragment, analog or fusion protein thereof. Each possibility represents a separate embodiment of the invention.
  • the lymphocyte activating agent is a human IL-2 (hIL-2) or a fragment, analog or fusion protein thereof.
  • the lymphocyte activating agent is an isolated human hIL-2 or a fragment, analog or fusion protein thereof.
  • the cancer is selected from the group consisting of a melanoma, pancreatic, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, and endometrial cells cancer.
  • the cancer is selected from the group consisting of melanoma cancer, pancreatic cancer and lung cancer.
  • the cancer is melanoma cancer.
  • the cancer is pancreatic cancer.
  • the cancer is lung cancer.
  • the method described above comprises administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising an activated cytotoxic lymphocyte cell, and a pharmaceutical composition comprising a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • the method described above comprises administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising a LAK cell, and a pharmaceutical composition comprising a lymphocyte activating agent or a fragment, analog or fusion protein thereof.
  • the method described above comprises administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising an activated cytotoxic lymphocyte cell, and a pharmaceutical composition comprising IL-2 or a fragment, analog or fusion protein thereof.
  • the method described above comprises administrating to the patient a pharmaceutical composition comprising an anti-human-CEACAMl monoclonal antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising a LAK cell, and a pharmaceutical composition comprising IL-2 or a fragment, analog or fusion protein thereof.
  • the human leukocyte antigen (HLA) system is the locus of genes that encode for proteins on the surface of cells that are responsible for regulation of the immune system in humans.
  • the HLA genes are the human versions of the major histocompatibility complex (MHC) genes that are found in most vertebrates. HLAs are routinely used to match a patient of a disease with a healthy donor for cell or organ transplant.
  • MHC major histocompatibility complex
  • the activated cytotoxic lymphocyte cell is derived from a peripheral blood mononucleated (PBMC) cell of the cancer patient.
  • the activated cytotoxic lymphocyte cell is derived from an HLA-matching donor.
  • the LAK cell is derived from a peripheral blood mononucleated (PBMC) cell of the cancer patient.
  • the LAK cell is derived from an HLA-matching donor.
  • Example 1 Human LAK cells express CEACAM1 upon IL-2 activation.
  • PBMC cells Human peripheral blood mononucleated cells (PBMC cells) were isolated from a healthy donor, and activated with IL-2 (500 units/ml) for 7 days to generate a population of human lymphokine-activated killer cells (LAK cells).
  • LAK cells human lymphokine-activated killer cells
  • Figure 1 demonstrates that incubation of human PBMC cells with IL-2 produced CEACAM1 positive human LAK cells.
  • Example 2 Human cancer cells express CEACAM1.
  • CEACAM1 The expression of CEACAM1 on four different human cancer cell lines, SU8686 (Figure 2A), T3M4 (Figure 2B), H358 (Figure 2C) and SKMEL28 (Figure 2D), was confirmed by FACS analysis with a PE-conjugated antibody (CM-24, black lines). For comparison, a similar FACS analysis was performed to appropriate isotype controls (gray lines).
  • Example 3 Cytotoxicity of human LAK cells against a variety of human melanoma cells.
  • PBMC cells were isolated from a healthy donor followed by activation with IL-2 (0, 300, 1000 or 3000 units/ml) for 3 days to generate a population of human LAK cells.
  • Melanoma cancer cell lines ( Figure 3A, SKMEL28; Figure 3B, MEL14) were added for an incubation of 24 hours at effector-to-target ratio of 2.5: 1 or 10: 1, as indicated.
  • Figure 3 demonstrates that while human PBMC cells were not cytotoxic (incubated without IL-2), their incubation with IL-2 produced human LAK cells, cytotoxic to a variety of human melanoma cells.
  • Figure 3 also demonstrates that the cytotoxicity of LAK cells is influenced in a dose-dependent manner by the IL-2 concentration in which they were activated. Results represent an average of % cytotoxicity +SE as determined by classical LDH release assay from triplicate wells per treatment.
  • Example 4 Anti-CEACAMl antibodies increase the cytotoxicity of human LAK cells against human melanoma cells.
  • PBMC cells were isolated from a healthy donor followed by activation with IL-2 (500 or 1000 units/ml) for 3 days to generate a population of human LAK cells. Then, the human LAK cells were incubated with 0 ⁇ g/m ⁇ , 0 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml or 10 ⁇ g/ml of an anti- CEACAM1 antibody (CM-24) for 30 minutes at 37°C. Human melanoma cells (SKMEL28) were added for an incubation of 24 hours, after which cytotoxicity was determined.
  • IL-2 500 or 1000 units/ml
  • Figure 4 demonstrates that while human LAK cells are cytotoxic to human melanoma cancer cells on their own (compare e.g. two left bars), the addition of anti-CEACAMl antibodies significantly increases cytotoxicity to these human melanoma cancer cells in a dose- dependent manner. Results represent an average of % cytotoxicity +SE as determined by classical LDH release assay from triplicate wells per treatment. * P ⁇ 0.05 paired T-test compared to effectors + target cells with medium only.
  • Example 5 - Anti-CEACAMl antibodies increase the cytotoxicity of human LAK cells against a variety of human pancreatic and lung cancer cells.
  • PBMC cells were isolated from a healthy donor followed by activation with IL-2 (500 units/ml) for 7 days to generate a population of human LAK cells. Then, the human LAK cells were incubated with 0. ⁇ g/ml to 10 ⁇ g/ml of an anti-CEACAMl antibody (CM-24) as indicated, for 30 minutes at 37°C.
  • CM-24 an anti-CEACAMl antibody
  • Figure 5 demonstrates that while human LAK cells are cytotoxic to human pancreatic and lung cancer cells on their own, the addition of anti-CEACAMl antibodies significantly increases cytotoxicity to these human cancer cells in a dose-dependent manner.
  • Results represent an average of % cytotoxicity +SE as determined by classical LDH release assay from triplicate wells per treatment. * P ⁇ 0.05 paired T-test compared to effectors + target cells with medium only.
  • Example 6 Anti-CEACAMl antibodies enhance granzyme B secretion of human LAK cells in the presence of human pancreatic and lung cancer cells.
  • Human LAK cells were incubated with an anti-CEACAM-1 antibody (CM-24) in different concentrations for 30 minutes at 37°C.
  • Human pancreatic cancer cells T3M4 (A) or human lung cancer cells H358 (B) were then added for an incubation of 24 hours.
  • Granzyme B secretion was measured by ELISA.
  • Figure 6 demonstrates that while human LAK cells produce high levels of Granzyme B (a serine protease most commonly found in the granules of cytotoxic lymphocytes (CTLs), natural killer cells (NK cells) and cytotoxic T cells.
  • CTLs cytotoxic lymphocytes
  • NK cells natural killer cells
  • Example 7 Anti-CEACAMl antibodies enhance enhances IFN- ⁇ secretion of human LAK cells in the presence of human cancer cells.
  • Human LAK cells were incubated with an anti-CEACAM-1 antibody (CM-24) in different concentrations for 30 minutes at 37°C.
  • Human lung cancer cells H358 ( Figure 7A) or H460 ( Figure 7B) were then added for an incubation of 24 hours. IFN- ⁇ secretion was measured by ELISA.
  • Figure 7 demonstrates that while human LAK cells produce high levels of IFN- ⁇ (a dimerized soluble cytokine, produced predominantly by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, and by CD4 Thl and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops) on their own, the addition of anti-CEACAMl antibodies significantly increases IFN- ⁇ levels in a dose- dependent manner. Results represent the mean + S.E of Granzyme B release values from 3 repeats per treatment. *P ⁇ 0.05 paired T-test, compared to effectors + target cells with medium only.
  • Example 8 Treatment of cancer xenograft model with a combination of an anti-CEACAMl antibody and activated, cytotoxic lymphocyte cells or lymphocyte activating agent.
  • Tumors are xenografted into SCID NOD mice by sub-cutaneous (SC) injection of a cancer cell line (e.g. melanoma cell line such as SKMEL5).
  • SC sub-cutaneous
  • an anti-CEACAMl antibody e.g. CM-24
  • activated, cytotoxic lymphocyte cells and/or lymphocyte activating agent is examined by tumor eradication, tumor growth inhibition and/or mice mortality rate.
  • SCID-NOD mice are randomized into the groups disclosed in Table 2. Table 2. SCID-NOD mice groups.
  • Tumor initiation Each mouse is administrated with a cancer cells (e.g. melanoma) in PBS by SC injection to the mice right flank.
  • Treatment starts at the tumor inoculation day.
  • Tumors growth measurements - Measurements are done twice a week, using a caliper by a person blind to the experiment procedure. The measurements starts from tumor initiation day. End point— 60-80 days from tumor initiation day. At assay termination day, all mice are sacrificed after total body bleeding and sera separation. Tumors are transferred to fixation in separate tube per mouse.
  • Example 9 Human lymphocyte activating agent activates human PBMC cells to become activated, cytotoxic lymphocyte cells in immune-compromised mice.
  • Human PBMC cells are isolated from healthy donor blood samples and are xenografted into SCID /NOD or NOG mice by a single IV injection, followed by daily administration of human lymphocyte activating agent.
  • the effect of human lymphocyte activating agent on activated, cytotoxic lymphocyte generation is evaluated by flow cytometry staining of human PBMC cells from mouse blood, spleens and draining lymph nodes (DLN), and examination of human PBMC activation markers such as CD25, CD69 and CD62L on the human PBMC subpopulations.
  • Various human PBMC donors, lymphocyte activating agent concentrations and time lines are evaluated.
  • Assay setup SCID-NOD mice are randomized the groups disclosed in Table 3.
  • Example 10 Murine lymphocyte activating agent activates murine PBMC cells to become activated, cytotoxic lymphocyte cells in immune-competent mice.
  • mice strains such as C57/b or Balb/C are administrated with mouse lymphocyte activating agent daily, and generation of mouse activated, cytotoxic lymphocyte is evaluate by flow cytometry staining of mouse PBMC isolated from blood, spleens and DLN using murine activation markers such as CD25, CD69, CD62L and etc.
  • mice are randomized into the groups disclosed in Table 4.
  • Human tumors are xenografted into SCID/NOD NOG mice by sub-cutaneous injection of a cancer cell line (e.g. melanoma cell line such as SKMEL5).
  • Human PBMC are isolate from healthy donor blood samples and are administrated into the tumor bearing mice by intravenous (IV) or intrathecal (IT) injection followed by daily administration of human lymphocyte activating agent.
  • IV intravenous
  • IT intrathecal
  • the effect of human lymphocyte activating agent on human activated, cytotoxic lymphocyte generation is evaluated by flow cytometry staining of human PBMC from mouse blood, spleens and DLN and examination of human PBMC activation markers such as CD25, CD69 and CD62L on the human PBMC subpopulations.
  • the cytotoxic effect of human activated, cytotoxic lymphocyte is examined by tumor eradication, tumor growth inhibition and/or mice mortality rate.
  • Tumor initiation Each mouse (of groups 1 to 4) is administrated with cancer cells (e.g. melanoma) in PBS by SC injection to the mice right flank.
  • Treatment - human lymphocyte activating agent and/or human PBMC administration starts one day after the tumor inoculation day.
  • Tumors growth measurements - Measurements are done twice a week, using a caliper by a person blind to the experiment procedure. The measurements start from tumor initiation day. End point - At assay termination day, all mice are sacrificed after total body bleeding and sera separation. Tumors are transferred to fixation in separate tube per mouse.
  • Example 12- murine activated, cytotoxic lymphocyte cells produced in immune-competent mice are cytotoxic to tumor cells.
  • mice strains such as C57/b are engrafted by sub-cutaneous or IV injections of cancer cells (e.g. a melanoma cell line such as B-16-F10).
  • cancer cells e.g. a melanoma cell line such as B-16-F10
  • the mice are then administrated with mouse lymphocyte activating agent in various regimens.
  • the effect of murine activated, cytotoxic lymphocyte is examined by tumor eradication, tumor growth inhibition and/or mice mortality rate.
  • Assay setup mice are engrafted with melanoma cells and randomized into the groups disclosed in Table 6.
  • Tumor initiation Each mouse (of groups 1 to 6) is administrated with cancer cells (e.g. melanoma) in PBS by SC injection to the mice right flank.
  • Treatment - murine lymphocyte activating agent administration starts one day after the tumor inoculation day.
  • Tumors growth measurements - Measurements are done twice a week, using a caliper by a person blind to the experiment procedure. The measurements start from tumor initiation day. End point - At assay termination day, all mice are sacrificed after total body bleeding and sera separation. Tumors are transferred to fixation in separate tube per mouse.
  • Example 13 Synergism between human lymphocyte activating agent and CM-24 against melanoma tumor cells in immune -compromised mice.
  • Human tumors are xenografted into SCID/NOD or NOG mice by sub-cutaneous injection of cancer cells (e.g. a melanoma cell line such as SKMEL5).
  • cancer cells e.g. a melanoma cell line such as SKMEL5
  • Human PBMC are isolated from healthy donor blood samples and are administrated into the tumor bearing mice by IV or IT injection followed by daily administration of human lymphocyte activating agent.
  • the effect of human lymphocyte activating agent on human activated, cytotoxic lymphocyte generation is evaluated by flow cytometry staining of human PBMC from mouse blood, spleens and DLN, and examination of human PBMC activation markers such as CD25, CD69 and CD62L on the human PBMC subpopulations.
  • Tumor initiation Each mouse (of groups 1 to 8) is administrated with cancer cells (e.g. melanoma) in PBS by SC injection to the mice right flank.
  • Treatment antibodies, human lymphocyte activating agent and human PBMC administration starts one day after the tumor inoculation day.
  • Tumors growth measurements - Measurements are done twice a week, using a caliper by a person blind to the experiment procedure. The measurements start from tumor initiation day. End point - At assay termination day, all mice are sacrificed after total body bleeding and sera separation. Tumors are transferred to fixation in separate tube per mouse.
  • Example 14 Synergism between murine lymphocyte activating agent and CM-24 against melanoma tumor cells in immune -competent mice.
  • mice strains such as C57/b or Balb/C are engrafted by sub-cutaneous or IV injections of cancer cells (e.g. melanoma cell line such as B-16-F10).
  • the mice are administrated with mouse lymphocyte activating agent in order to generate activated murine activated, cytotoxic lymphocyte cells in vivo.
  • the combined effect of anti-murine CEACAM1 antibody and lymphocyte activating agent is examined by tumor eradication, tumor growth inhibition and/or mice mortality rate.
  • Assay setup mice are engrafted with melanoma cells and randomized into the groups disclosed in Table 8.
  • Tumor initiation Each mouse is administrated with cancer cells (e.g. melanoma) in PBS by SC injection to the mice right flank.
  • Treatment - murine lymphocyte activating agent and antibodies administration starts one day after the tumor inoculation day.
  • Tumors growth measurements - Measurements are done twice a week, using a caliper by a person blind to the experiment procedure. The measurements start from tumor initiation day. End point - At assay termination day, all mice are sacrificed after total body bleeding and sera separation. Tumors are transferred to fixation in separate tube per mouse.
  • Example 15 Synergism between human lymphocyte activating agent and CM-24 against pancreatic tumor cells in immune-compromised mice.
  • Human tumors are xenografted into SCID/NOD or NOG mice by sub-cutaneous injection of cancer cells (e.g. a pancreatic cell line such as T3A4).
  • Human PBMC are isolated from healthy donor blood samples and are administrated into the tumor bearing mice by IV or IT injection followed by daily administration of human lymphocyte activating agent.
  • human lymphocyte activating agent on human activated, cytotoxic lymphocyte generation is evaluated by flow cytometry staining of human PBMC from mouse blood, spleens and DLN, and examination of human PBMC activation markers such as CD25, CD69 and CD62L on the human PBMC subpopulations.
  • human PBMC activation markers such as CD25, CD69 and CD62L on the human PBMC subpopulations.
  • the anti-cancer effect of the combination of human activated, cytotoxic lymphocyte and CM-24 is examined by tumor eradication, tumor growth inhibition and/or mice mortality rate.
  • SCID-NOD mice are xenografted with human melanoma cells and randomized into the groups disclosed in Table 9. Table 9. SCID/NOD or NOG mice groups.
  • Tumor initiation Each mouse (of groups 1 to 8) is administrated with cancer cells (e.g. melanoma) in PBS by SC injection to the mice right flank.
  • Treatment antibodies, human lymphocyte activating agent and human PBMC administration starts one day after the tumor inoculation day.
  • Tumors growth measurements - Measurements are done twice a week, using a caliper by a person blind to the experiment procedure. The measurements start from tumor initiation day. End point - At assay termination day, all mice are sacrificed after total body bleeding and sera separation. Tumors are transferred to fixation in separate tube per mouse.
  • Example 16 Synergism between murine lymphocyte activating agent and CM-24 against pancreatic tumor cells in immune-competent mice.
  • mice strains such as C57/b or Balb/C are engrafted by sub-cutaneous or IV injections of cancer cells (e.g. pancreatic cell line such as LTPA).
  • the mice are administrated with mouse lymphocyte activating agent in order to generate activated murine activated, cytotoxic lymphocyte cells in vivo.
  • the combined effect of anti-murine CEACAM1 antibody and lymphocyte activating agent is examined by tumor eradication, tumor growth inhibition and/or mice mortality rate.
  • Assay setup mice are engrafted with melanoma cells and randomized into the groups disclosed in Table 10.
  • Tumor initiation Each mouse is administrated with cancer cells (e.g. melanoma) in PBS by SC injection to the mice right flank.
  • Treatment - murine lymphocyte activating agent and antibodies administration starts one day after the tumor inoculation day.
  • Tumors growth measurements - Measurements are done twice a week, using a caliper by a person blind to the experiment procedure. The measurements start from tumor initiation day. End point - At assay termination day, all mice are sacrificed after total body bleeding and sera separation. Tumors are transferred to fixation in separate tube per mouse.

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Abstract

La présente invention concerne des compositions comprenant des anticorps anti-CEACAM1, des agents d'activation des lymphocytes et des lymphocytes activés, ainsi que des méthodes pour leur utilisation combinée dans le traitement du cancer.
PCT/IL2014/050996 2013-11-25 2014-11-18 Compositions comprenant des anticorps anti-ceacam1, agents d'activation des lymphocytes et lymphocytes activés pour une cancérothérapie WO2015075710A1 (fr)

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WO2015166484A1 (fr) * 2014-04-27 2015-11-05 Ccam Therapeutics Ltd. Anticorps humanisés dirigés contre ceacam1
JP2018017723A (ja) * 2016-07-25 2018-02-01 静岡県 肝外胆管癌、肝内胆管癌、又は胆嚢癌の診断用バイオマーカー
US11427647B2 (en) 2014-04-27 2022-08-30 Famewave Ltd. Polynucleotides encoding humanized antibodies against CEACAM1

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WO2013054331A1 (fr) * 2011-10-11 2013-04-18 Tel Hashomer Medical Research Infrastructure And Services Ltd. Anticorps dirigés contre la molécule d'adhésion cellulaire associée à l'antigène carcinoembryonnaire (ceacam)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015166484A1 (fr) * 2014-04-27 2015-11-05 Ccam Therapeutics Ltd. Anticorps humanisés dirigés contre ceacam1
US10550196B2 (en) 2014-04-27 2020-02-04 Famewave Ltd. Humanized antibodies against CEACAM1
EA037613B1 (ru) * 2014-04-27 2021-04-21 Фэймуэйв Лтд. Гуманизированные антитела против ceacam1
US11427647B2 (en) 2014-04-27 2022-08-30 Famewave Ltd. Polynucleotides encoding humanized antibodies against CEACAM1
US11866509B2 (en) 2014-04-27 2024-01-09 Famewave Ltd. Humanized antibodies against CEACAM1
JP2018017723A (ja) * 2016-07-25 2018-02-01 静岡県 肝外胆管癌、肝内胆管癌、又は胆嚢癌の診断用バイオマーカー
WO2018020530A1 (fr) * 2016-07-25 2018-02-01 静岡県 Biomarqueur diagnostique pour cancer des voies biliaires extra-hépatique ou cancer de la vésicule biliaire
US10641773B2 (en) 2016-07-25 2020-05-05 Shizuoka Prefecture Biomarker for diagnosis of extrahepatic bile duct carcinoma, intrahepatic bile duct carcinoma, or gallbladder carcinoma

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