Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
• • 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
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • 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
  • A61K38/395—Alveolar surfactant peptides; Pulmonary surfactant peptides
• • 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
  • A61K39/12—Viral 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • 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
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present invention in some embodiments thereof, relates to methods of treating cancer and, more particularly, but not exclusively, to the use of a CXCR4 antagonistic peptide and an anti-cancer agent in the treatment of cancer.
• Cancer is the second leading cause of death in the US.
• the estimates for 2014 are that approximately 585,000 people will die of cancer and 1.6 million new cases will be diagnosed (American Cancer Society, Cancer Facts & Figures 2014).
• surgical removal is a very effective treatment.
• standard, non-specific cancer treatments such as chemotherapy and radiotherapy are typically used. These treatments affect many healthy cells and result in elevated toxicity and effective in only a minor percentage of treated individuals.
• individuals that initially respond to therapy are at risk for relapses, and often develop resistance.
• monoclonal antibodies have become part of the therapeutic repertoire for several type of cancers with the anti-CD20 mAb, Rituximab, and the anti- human HER2, Trastuzumab, routinely used for the treatment of breast cancer; and the anti-human EGFR, Cetuximab, routinely used for the treatment of head and neck cancer and colorectal carcinoma [Kirkwood et al. (2012) CA Cancer J Clin. 62(5): 309-335].
• IFNa2 Intron-A, Merck
• Roferon-A Futhogen-A
• IL-2 aldesleukin, Proleukin; Prometheus Inc.
• melanoma; and dendritic cell vaccines have shown safety and efficacy in several solid tumors, for example sipuleucel-T (Provenge; Dendreon Corporation) in prostate cancer and dendritic cells loaded with four melanoma peptides (gplOO, melan-A/MART-1, tyrosinase and MAGE-3), KLH and flu matrix peptide in metastatic melanoma [Kirkwood et al. (2012) CA Cancer J Clin. 62(5): 309-335; and Banchereau (2001) Cancer Research 61: 6451- 58].
• 4F-benzoyl-TN 14003 (also known as BKT140, hereinafter BL-8040), is a 14- residue bio stable synthetic peptide developed as a specific CXCR4 antagonist. It has been shown that BL-8040 binds the CXCR4 receptor with high affinity and long receptor occupancy. Studies in mice demonstrated that a single BL-8040 injection mobilized long term repopulating stem cells sufficient for transplantation.
• a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof; and an anti-cancer agent selected from the group consisting of:
• a vaccine selected from the group consisting of IMCgplOO, Prophage G- 100 & G-200, GV-1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAdl, GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as GI-4000;
• MNBCs anti-cancer reactive mononuclear blood cells
• a cytokine capable of inducing activation and/or proliferation of a T cell
• an immune-check point regulator wherein the immune-check point regulator is not a PDl antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TEVI-3 antagonist, KIR antagonist, IDO antagonist, OX40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS agonist;
• CSF1R colony stimulating factor- 1 receptor
• the administering comprises multiple administrations of the peptide.
• the administering comprises multiple administrations of the agent.
• the administering the peptide and the administering the agent are effected sequentially. According to some embodiments of the invention, the administering the peptide is effected prior to the administering the agent.
• the administering the peptide is effected following the administering the agent.
• the administering the peptide is effected concomitantly with the administering the agent.
• the peptide is administered at a dose of 0.5-1 mg / kg.
• the peptide is administered subcutaneously.
• an article of manufacture identified for use in treating cancer comprising a packaging material packaging a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof and an anti-cancer agent selected from the group consisting of:
• a vaccine selected from the group consisting of IMCgplOO, Prophage G- 100 & G-200, GV-1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAdl, GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as GI-4000;
• MNBCs anti-cancer reactive mononuclear blood cells
• a cytokine capable of inducing activation and/or proliferation of a T cell
• an immune-check point regulator wherein the immune-check point regulator is not a PD1 antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TEVI-3 antagonist, KIR antagonist, IDO antagonist, OX40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS agonist;
• CSF1R colony stimulating factor- 1 receptor
• the peptide and the agent are in separate containers.
• the peptide and the agent are in a co-formulation.
• a pharmaceutical composition comprising as active ingredients a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof and an anti-cancer agent selected from the group consisting of:
• a vaccine selected from the group consisting of IMCgplOO, Prophage G- 100 & G-200, GV-1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAdl, GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as GI-4000;
• MNBCs anti-cancer reactive mononuclear blood cells
• a cytokine capable of inducing activation and/or proliferation of a T cell
• an immune-check point regulator wherein the immune-check point regulator is not a PDl antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TEVI-3 antagonist, KIR antagonist, IDO antagonist, OX40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS agonist;
• CSF1R colony stimulating factor- 1 receptor
• the MNBCs are derived from a subject not treated with a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof. According to some embodiments of the invention, the MNBCs are selected from the group consisting of T cells, NK cells and dendritic cells.
• the MNBCs comprise T cells. According to some embodiments of the invention, the MNBCs comprise dendritic cells.
• the dendritic cells comprise a dendritic cells vaccine.
• the cytokine is selected from the group consisting of IFNa, IFNy, IL-1, IL-2, IL-6, IL-12, IL-15, IL-21 and TNFa.
• the immune-check point regulator targets an immune check-point protein selected from the group consisting of B7-H3, CD 19 and CD70.
• the immune-check point regulator is selected from the group consisting of an antibody, a peptide and a small molecule.
• the binding of the agent to the immune-check point protein expressed on the cancer cell results in at least one of:
• the immune-check point protein expressed on the cancer cell is selected from the group consisting of LAG3, CD 19 and CD70 and CEACAM1.
• the agent capable of binding the immune-check point protein is an antibody or a T cell.
• the T cell comprises a T cell transduced with a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
• TCR T cell receptor
• CAR chimeric antigen receptor
• the analog or derivative has an amino acid sequence as set forth in formula (I) or a salt thereof:
• Ai is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue or a N-a-substituted derivative of these amino acids, or Ai is absent;
• a 2 represents an arginine or glutamic acid residue if Ai is present, or A 2 represents an arginine or glutamic acid residue or a ⁇ - ⁇ -substituted derivative of these amino acids if Ai is absent;
• A3 represents an aromatic amino acid residue
• A4, A5 and A9 each independently represents an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue;
• A6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
• a 7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
• a 10 represents a citrulline, glutamic acid, arginine or lysine residue
• An represents an arginine, glutamic acid, lysine or citrulline residue wherein the C-terminal carboxyl may be derivatized;
• cysteine residue of the 4-position or the 13-position can form a disulfide bond
• amino acids can be of either L or D form.
• the peptide is selected from the group consisting of SEQ ID NOs: 1-72.
• the peptide is as set forth in SEQ ID NO: 1.
• the cancer is a solid tumor cancer.
• the solid tumor is selected from the group consisting of lung cancer, glioma, colon cancer, ovarian cancer, renal cancer, melanoma cancer, hepatocellular cancer, gastric or stomach cancer, glioblastoma, cervical cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, thyroid cancer, head and neck and pancreatic cancer.
• the cancer is a non- solid tumor cancer.
• the non-solid tumor is selected from the group consisting of multiple myeloma and leukemia.
• the present invention in some embodiments thereof, relates to methods of treating cancer and, more particularly, but not exclusively, to the use of a CXCR4 antagonistic peptide and an anti-cancer agent in the treatment of cancer.
• cytotoxic treatments such as chemotherapy and radiotherapy that may at least in part affect many healthy cells and thus result in elevated toxicity.
• Immunotherapy strategies for cancer therapy aiming at harnessing the immune system to fight cancer, include cytokines, monoclonal antibodies against tumor cells or immune regulatory molecules, cancer vaccines as well as cell-based therapies such as adoptive transfer of ex-vivo activated T cells and natural killer (NK) cells.
• 4F-benzoyl-TN 14003 (SEQ ID NO: 1, also known as BKT140, hereinafter BL- 8040) is a CXCR4 peptide antagonist. It has been shown that BL-8040 induces mobilization of CD34+ hematopoietic stem/progenitor cells (HSPC) that can be further used for transplantation. In addition, BL-8040 was found to be toxic against several tumors such as myeloid leukemia, hematopoietic tumors and non- small cell lung cancer.
• HSPC hematopoietic stem/progenitor cells
• BL-8040 induces rapid mobilization of a variety of immune cells including immature stem/progenitor cells as well as fully differentiated T cells and NK cells.
• the present findings therefore can be harnessed to the use of BL- 8040 to induce the mobilization and dissemination of ImDCs and T effector and memory cells into tumor sites and thus can augment the anti-tumor effect of anti-cancer agents such as immunotherapeutics.
• Example 1 suggest the use of a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof in combination with several anti-cancer agents for the treatment of cancer.
• a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof; and an anti-cancer agent selected from the group consisting of:
• a vaccine selected from the group consisting of IMCgplOO, Prophage G- 100 & G-200, GV-1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011,
• MNBCs anti-cancer reactive mononuclear blood cells
• a cytokine capable of inducing activation and/or proliferation of a T cell
• an immune-check point regulator wherein said immune-check point regulator is not a PD1 antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TEVI-3 antagonist, KIR antagonist, IDO antagonist, OX40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS agonist;
• CSF1R colony stimulating factor- 1 receptor
• CXCR2 CXCR2 antagonist
• treating refers to inhibiting, preventing or arresting the development of a pathology (e.g. cancer) and/or causing the reduction, remission, or regression of a pathology.
• pathology e.g. cancer
• Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
• the phrase "subject in need thereof” refers to a mammalian male or female subject (e.g., human being) who is diagnosed with cancer. In a specific embodiment, this term encompasses individuals who are at risk to develop cancer. Veterinary uses are also contemplated. The subject may be of any gender or at any age including neonatal, infant, juvenile, adolescent, adult and elderly adult.
• cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
• Cancers which can be treated by the method of this aspect of some embodiments of the invention can be any solid or non- solid cancer and/or cancer metastasis.
• the cancer is a solid tumor.
• the cancer is a non-solid tumor.
• cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non- small-cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), melanoma cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocy
• the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non- small cell lung cancer, non-Hodgkins lymphoma (NHL), acute lymphoblastic leukemia (ALL); chronic myeloblastic leukemia (CML); acute myeloblastic leukemia (AML); renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma.
• the cancerous conditions amenable for treatment of the invention include metastatic cancers.
• the cancer is selected from the group consisting of lung cancer, glioma, colon cancer, ovarian cancer, renal cancer, melanoma cancer, hepatocellular cancer, gastric or stomach cancer, glioblastoma, cervical cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, thyroid cancer, head and neck and pancreatic cancer.
• the cancer is selected from the group consisting of lung cancer, glioma, colon cancer and pancreatic cancer.
• the cancer is selected from the group consisting of multiple myeloma, Lymphoma and leukemia.
• the cancer is selected from the group consisting of multiple myeloma and leukemia.
• peptide encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
• the peptide is no more than 100 amino acids in length. According to a specific embodiment, the peptide is 5-100 amino acids in length. According to a specific embodiment, the peptide is 5-50 amino acids in length. According to a specific embodiment, the peptide is 5-20 amino acids in length. According to a specific embodiment, the peptide is 5-15 amino acids in length. According to a specific embodiment, the peptide is 10-20 amino acids in length. According to a specific embodiment, the peptide is 10-15 amino acids in length.
• SEQ ID NO: 1 or an analog or derivative thereof refers to 4F-benzoyl-TN 14003 (SEQ ID NO: 1, also known as BKT140, hereinafter BL-8040) peptide and functional analogs or derivatives thereof.
• the peptides of the present invention are structurally and functionally related to the peptides disclosed in patent applications WO2002/020561 and WO2004/020462, also known as "T-140 analogs", as detailed hereinbelow.
• the peptide of the present invention is a CXCR4-antagnoistic peptide i.e. it reduces CXCR- 4 activation by at least 10 % as compared to same in the absence of the peptide antagonist.
• the peptide antagonist is a competitive inhibitor.
• the peptide antagonist is a non- competitive inhibitor.
• a functional CXCR4 antagonistic peptide is capable of inducing mobilization and dissemination of ImDCs, NK cells, B cells, monocytes/macrophages and T effector and memory cells into a tumor of a subject upon administration.
• a functional CXCR4 antagonistic peptide as used herein, is capable of enhancing an immune-response to a tumor.
• the peptide analog or derivative has an amino acid sequence as set forth in the following formula (I) or a salt thereof:
• Ai is an arginine, lysine, ornithine, citruUine, alanine or glutamic acid residue or a N-a-substituted derivative of these amino acids, or Ai is absent;
• a 2 represents an arginine or glutamic acid residue if Ai is present, or A 2 represents an arginine or glutamic acid residue or a ⁇ - ⁇ -substituted derivative of these amino acids if Ai is absent;
• A3 represents an aromatic amino acid residue
• A4, A5 and A9 each independently represents an arginine, lysine, ornithine, citruUine, alanine or glutamic acid residue;
• A6 represents a proline, glycine, ornithine, lysine, alanine, citruUine, arginine or glutamic acid residue;
• a 7 represents a proline, glycine, ornithine, lysine, alanine, citruUine or arginine residue;
• a 10 represents a citruUine, glutamic acid, arginine or lysine residue
• An represents an arginine, glutamic acid, lysine or citruUine residue wherein the C-terminal carboxyl may be derivatized;
• cysteine residue of the 4-position or the 13-position can form a disulfide bond
• amino acids can be of either L or D form.
• Exemplary peptides according to formula (I) are peptides having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-72, as presented in Table 1 hereinbelow.
• each one of SEQ ID NOs: 1-72 two cysteine residues are coupled in a disulfide bond.
• the analog or derivative has an amino acid sequence as set forth in SEQ ID NO: 65 (H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit- Cys-Arg-OH; TC14003).
• the peptide used in the compositions and methods of the invention consists essentially of an amino acid sequence as set forth in SEQ ID NO: 1.
• the peptide used in the compositions and methods of the invention comprises an amino acid sequence as set forth in SEQ ID NO: 1.
• the peptide is at least 60%, at least 70% or at least 80% homologous to SEQ ID NO: 1.
• the peptide is at least 90% homologous to SEQ ID NO: 1.
• the peptide is at least about 95% homologous to SEQ ID NO: 1.
• the peptide is selected from SEQ ID NOs: 1-72, wherein each possibility represents a separate embodiment of the present invention.
• the peptide has an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4, 10, 46, 47, 51-56, 65, 66, 68, 70 and 71. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 10, 46, 47, 68 and 70. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 51, 65 and 66. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOs: 53-56.
• the peptide has an amino acid sequence as set forth in SEQ ID NO: 1. According to a specific embodiment, the peptide is as set forth in SEQ ID NO: 1. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO: 2. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO: 51. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO: 66.
• the peptide is as set forth in SEQ ID NO: 1 and any embodiment described herein should be read as if specifically reading over this peptide.
• the peptides of some embodiments of the invention may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis.
• solid phase peptide synthesis a summary of the many techniques may be found in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973.
• For classical solution synthesis see G. Schroder and K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.
• these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain.
• amino acids or suitably protected amino acids Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group.
• the protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage.
• the protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final peptide compound.
• the CXCR4 antagonistic peptide is administered to the subject in combination with one or more white blood cell mobilizing agents.
• the peptide may be administered in sequential or concomitant combination with one or more other growth factors or cytokines that affect mobilization such as, but not limited to, G-CSF, GM-CSF and SCF.
• the peptide of the present invention is administered in combination with an anti-cancer agent.
• the term "anti-cancer agent” refers to an agent effective in inhibiting, slowing or arresting the growth or metastasis of a cancerous cell or which exhibits a cytotoxic effect on a cancerous cell.
• the anti-cancer agent is an anti-cancer immune modulator agent which is capable of eliciting an immune response (e.g. T cell, NK cell, B cell, complement) against a cancerous cell.
• the anti-cancer agent of the present invention is selected from the group consisting of:
• a vaccine selected from the group consisting of IMCgplOO, Prophage G- 100 & G-200, GV-1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAdl, GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as GI-4000;
• MNBCs anti-cancer reactive mononuclear blood cells
• a cytokine capable of inducing activation and/or proliferation of a T cell;
• an immune-check point regulator wherein said immune-check point regulator is not a PD1 antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TEVI-3 antagonist, KIR antagonist, IDO antagonist, OX40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS agonist;
• CSF1R colony stimulating factor- 1 receptor
• the term "vaccine” refers to a composition used to provoke a specific immune response against a cancerous cell (e.g. the production of anti-cancer antibodies, eliciting a cell mediated immune-response) following administration.
• the vaccine includes: IMC gp 100 [a soluble affinity enhanced T cell receptor (TCR) specific for the melanoma-associated antigen gplOO, fused to an anti-CD3 specific antibody fragment (scFv), e.g., produced by Immunocore]; the antigen based vaccines Prophage G-100 & G-200 (HSPPC-96, e.g., produced by Agenus), GV-1001 (e.g., produced by KAEL- GemVax), IMA-950 (e.g., produced by Immatics Biotechnologies), CV-9201 (e.g., produced by CureVac) and CV-9104 (CV-9103, e.g., produced by CureVac); the viral vectors vaccines Ad-RTS-hIL-12 (INXN-2001, e.g., produced by Ziopharm), ETBX- 011 (Ad5 [E1-, E2b-]-CEA(6D), e
• TCR soluble affinity enhanced
• the vaccine is a human papiloma virus (HPV, e.g., HPV 16 vaccine) typically targeting E6 and/or E7.
• HPV human papiloma virus
• the vaccine may be a preventive vaccine or a therapeutic vaccine.
• HPV vaccines which can be used along with the present teachings can be found in Lin et al. J Formos Med Assoc. 2010 Jan; 109(1): 4-24; and Rice et al. Cancer Gene Therapy 22, 454-462.
• the preventive vaccines utilize the capsid proteins LI and L2 as target antigens, inducing antibodies to neutralize and prevent entry of HPV into cells.
• LI the major component of the capsid
• VLPs virus-like particles
• the vaccines is GardasilTM or CervarixTM Gardasil is a quadrivalent vaccine containing recombinant LI VLPs for HPV genotypes 6, 11, 16 and 18 whereas the bivalent vaccine Cervarix contains LI VLPs for HPV- 16 and 18.
• the vaccine is a monovalent HPV- 16 LI vaccine with an aluminium hydroxyphosphate sulfate adjuvant.
• Exemplary, non-limiting, therapeutic vaccines comprise HPV E6 and E7 antigens. These represent ideal targets for therapeutic vaccines since these are constitutively expressed in HPV-infected cells and not healthy cells. E6 and E7 are essential to the induction and maintenance of cellular transformation, and thus are unlikely to be lost in an attempt to evade the immune system.
• the therapeutic vaccines target E6 and/or
• Therapeutic vaccines typically include:
• Live vector vaccines - Vector-based vaccines can deliver the antigens E6 and E7 to the dendritic cells (DCs), stimulating antigen expression through MHC class I (to CD8+ cytotoxic T cells) and MHC class II (to CD4+ helper T cells).
• DCs dendritic cells
• MHC class I to CD8+ cytotoxic T cells
• MHC class II to CD4+ helper T cells.
• Viral vectors used adenovirus, adeno-associated virus, vaccinia virus and alphaviruses, such as the Venezuelan equine encephalitis (VEE) virus;
• Peptide/protein-based vaccines - Administered peptides and proteins derived from HPV antigens are taken up by DCs, processed and expressed via MHC II and/or I to the appropriate CD4+/CD8+ T cells;
• Cell-based vaccines dendritic cell-based or tumor cell based vaccines; and Nucleic acid-based vaccines e.g., naked DNA based vaccines (e.g., ZYC-101 and ZYC-lOla), naked RNA replicon vaccines.
• Nucleic acid-based vaccines e.g., naked DNA based vaccines (e.g., ZYC-101 and ZYC-lOla), naked RNA replicon vaccines.
• the vaccine may be administered with an adjuvant, such as, but not limited to, incomplete Freund's Adjuvant, aluminum salts, oil-in-water emulsion (MF59), and nontoxic derivatives from Salmonella (MPL), water-in-oil emulsions (e.g. Montanide ISA 51 and ISA 720), saponins (e.g.
• an adjuvant such as, but not limited to, incomplete Freund's Adjuvant, aluminum salts, oil-in-water emulsion (MF59), and nontoxic derivatives from Salmonella (MPL), water-in-oil emulsions (e.g. Montanide ISA 51 and ISA 720), saponins (e.g.
• ISCOM ISCOM
• QS-21 QS-21, AS01 and AS02
• SRL-172 histamine dihydrochloride
• thymocartin Tio- TEPA
• monophosphoryl-lipid A/micobacteria compositions alum, Montanide ISA, Ribi Adjuvant System, TiterMax adjuvant, syntex adjuvant formulations, immune- stimulating complexes (ISCOMs), Gerbu adjuvant, CpG oligodeoxynucleotides, lipopolysaccharide, and polyinosinic:polycytidylic acid.
• MNBCs anti-cancer reactive mononuclear blood cells
• MNBCs include lymphocytes, monocytes and dendritic cells (DCs) which are capable of eliciting an immune response (e.g. T cell, NK cell) against a cancerous cell.
• DCs dendritic cells
• the MNBCs are selected from the group consisting of dendritic cells (DCs), T cells, B cells, NK cells and NKT cells.
• DCs dendritic cells
• T cells refers to a differentiated lymphocyte with a CD3 + , T cell receptor (TCR) + having either CD4 + or CD8 + phenotype.
• TCR T cell receptor
• the T cell may be either an effector or a regulatory T cells.
• effector T cells refers to a T cell that activates or directs other immune cells e.g. by producing cytokines or has a cytotoxic activity e.g., CD4+, Thl/Th2, CD8+ cytotoxic T lymphocyte.
• the term "regulatory T cell” or “Treg” refers to a T cell that negatively regulates the activation of other T cells, including effector T cells, as well as innate immune system cells. Treg cells are characterized by sustained suppression of effector T cell responses. According to a specific embodiment, the Treg is a CD4+CD25+Foxp3+ T cell.
• MNBCs comprise T cells.
• B cell refers to a lymphocyte with a B cell receptor (BCR)+, CD 19+ and or B220+ phenotype. B cells are characterized by their ability to bind a specific antigen and elicit a humoral response.
• BCR B cell receptor
• NK cell refers to a differentiated lymphocyte with a CD 16+ CD56+ and/or CD57+ TCR- phenotype. NKs are characterized by their ability to bind to and kill cells that fail to express "self MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.
• NKT cells refers to a specialized population of T cells that express a semi-invariant ⁇ T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NKl. l.
• NKT cells include NK1.1 + and NKl. l " , as well as CD4 + , CD4 " , CD8 + and CD8 " cells.
• the TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance.
• DC dendritic cell
• hematopoietic cells such as plasmacytoid dendritic cells, myeloid dendritic cells, Langerhans cells and interdigitating cells; and follicular DCs.
• Dendritic cells may be recognized by function, or by phenotype, particularly by cell surface phenotype.
• cell surface phenotype of DCs include CDla+, CD4+, CD86+, or HLA-DR.
• the term DCs encompasses both immature and mature DCs.
• the MNBCs comprise dendritic cells.
• the MNBCs do not comprise CD34+ hematopoietic stem/progenitor cells.
• the MNBCs used according to specific embodiments of the present invention may be autologous or non- autologous; they can be syngeneic or non-synegeneic: allogeneic or xenogeneic. Each possibility represents a separate embodiment of the present invention.
• autologous means that the donor subject is the recipient subject.
• the cells have been removed and re-introduced e.g., re-infused to the subject.
• non- autologous means that the donor subject is not the recipient subject.
• syngeneic means that the donor subject is essentially genetically identical with the recipient subject.
• Examples of syngeneic transplantation include transplantation of cells derived from the subject (also referred to in the art as “autologous”), a clone of the subject, or a homozygotic twin of the subject.
• allogeneic means that the donor is of the same species as the recipient, but which is substantially non-clonal with the recipient. Typically, outbred, non-zygotic twin mammals of the same species are allogeneic with each other. It will be appreciated that an allogeneic donor may be HLA identical or HLA non-identical with respect to the subject.
• xenogeneic means that the donor subject is from a different species relative to the recipient subject.
• Methods of obtaining and/or developing anti-cancer reactive MNBCs that can be used according to specific embodiments of the present invention are well known in the art (see e.g. Hildebrandt et al. Cytotherapy. 2014 16(40): S 120-S 129; Leen et al. Immunol Rev. 2014; 258(1): 12-29; Qian et al. Journal of Immunology Research Volume 2014, Article ID 525913, 9 pages; Martelli et al. Blood. 2014;123(7):967-973; Ophir and Reisner Front Immunol. 2012 3:93; Lask et al. Blood.
• a peripheral blood sample is collected from a subject by methods well known in the art such as drawing whole blood from the subject and collection in a container containing an anti-coagulant (e.g. heparin or citrate); and apheresis.
• an anti-coagulant e.g. heparin or citrate
• apheresis e.g. heparin or citrate
• at least one type of MNBCs is purified from the peripheral blood and enriched for at least one type of MNBCs effective for the treatment of cancer.
• MNBCs e.g. heparin or citrate
• There are several methods and reagents known to those skilled in the art for purifying MNBCs from whole blood such as leukapheresis, sedimentation, density gradient centrifugation (e.g. ficoll), centrifugal elutriation, fractionation, chemical lysis of e.g. red blood cells (e.g.
• Enriching for an anti-cancer reactive MNBCs can be effected by any method known in the art (see e.g. Hildebrandt et al. Cytotherapy. 2014 16(40): S 120-S 129; Leen et al. Immunol Rev. 2014; 258(1): 12-29; Qian et al. Journal of Immunology Research Volume 2014, Article ID 525913, 9 pages; Palucka and Banchereau Immunity. 2013; 39(1): 38-48; and Pizzurro and Barrio Front Immunol.
• the peripheral blood or purified population thereof comprising CD3+, CD4+ or CD8+ T cells or tumor-associated lymphocytes (TALs) selected for T-cell receptor (TCR) specificity (see e.g. Cancer Immunol Immunother. 2009; 58: 553-66) is activated and expanded ex-vivo by incubation with mature DCs preloaded with tumor antigens or transfected with mRNA coding for cancer antigens.
• the peripheral blood or a purified population thereof comprising both APCs (e.g. DCs) and T cells (e.g.
• CD3+, CD4+ or CD8+ T cells or TALs selected for TCR specificity is contacted with tumor antigens or transfected with mRNA coding for cancer antigens under conditions which allow capturing and presentation of the cancer antigen by the APC leading to activation and expansion of anti-cancer antigen-specific T cells.
• a specific non-limiting example of an autologous T cell therapy that can be used according to some embodiments of the present invention is Contego tumor-infiltrating lymphocytes (TILs, produced by Lion Biotechnologies).
• TILs tumor-infiltrating lymphocytes
• T cell specificity by promoting presentation of an anticancer receptor by way of transducing with a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
• TCR T cell receptor
• CAR chimeric antigen receptor
• the MNBCs comprise T cells transduced with a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
• TCR T cell receptor
• CAR chimeric antigen receptor
• transduction with a TCR refers to cloning of variable a- and ⁇ - chains are from T cells with specificity against a cancer antigen presented in the context of MHC.
• Method of transducing with a TCR are known in the art and are disclosed e.g. in Nicholson et al. Adv Hematol. 2012; 2012:404081; Wang and Riviere Cancer Gene Ther. 2015 Mar;22(2): 85-94); and Lamers et al., Cancer Gene Therapy (2002) 9, 613-623.
• transducing with a CAR refers to cloning of a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition moiety and a T-cell activation moiety.
• a chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen binding domain of an antibody (e.g., a single chain variable fragment (scFv)) linked to T-cell signaling or T-cell activation domains.
• scFv single chain variable fragment
• Another non-limiting example is the generation of dendritic cells vaccine that can be used according to some embodiments of the present invention.
• DC-based vaccine DCs are expanded ex vivo and contacted with a cancer antigen or a cancer cell lysate to thereby induce presentation of the cancer antigen (see e.g. Nestle, F. et al. (1998) Nature Medicine 4: 328-332).
• promoting presentation of a cancer antigen by a DC comprises transfecting the DC with a DNA, cDNA or an mRNA coding for a cancer antigen.
• Non-limiting examples of cancer antigens include MAGE-AI, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A7, MAGE-AS, MAGE-A9, MAGE-AIO, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE- 8, BAGE-1, RAGE- 1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE- C1/CT7, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and XAGE, melanocyte differentiation antigens, p53, ras, CEA, MU
• -fetoprotein 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29YBCAA), CA 195, CA 242, CA-50, CAM43, CD68 ⁇ KP1, CO-029, FGF-5, 0250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NBM70K, NYCO-I, RCASI, SDCCAG16, TA-90 (Mac-2 binding protein ⁇ cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, tyrosinase related proteins, TRP-1, or TRP-2 and tumor derived heat shock proteins.
• tumor antigens that may be expressed include out-of-frame peptide-MHC complexes generated by the non-AUG translation initiation mechanisms employed by "stressed" cancer cells (Malarkannan et al. Immunity 1999).
• tumor antigens that may be expressed are well-known in the art (see for example W000/20581; Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverley and Carroll, Cambridge University Press, Cambridge). The sequences of these tumor antigens are readily available from public databases but are also found in WO 1992/020356, WO 1994/005304, WO 1994/023031, WO 1995/020974, WO 1995/023874 & WO 1996/026214.
• EBV-specific antigens can be used as the cancer antigen.
• Other cancer antigens may include the proteins from viruses implicated in human cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV).
• HPV Human Papilloma Viruses
• HBV and HCV Hepatitis Viruses
• KHSV Kaposi's Herpes Sarcoma Virus
• a tumor antigen may be identified using cancer cells obtained from the subject by e.g. biopsy.
• DCs can also be fused directly to tumor cells for the purposes of immunization (see e.g. Kugler, A. et al. (2000) Nature Medicine 6:332-336).
• DC-based vaccines that can be used according to specific embodiments of the present invention include: BPX-201 (produced by Bellicum Pharmaceuticals), AV-0113 (Trivax, produced by Activartis Biotech), ICT-107 (produced by ImmunoCellular Therapeutics) and Ad.p53-DC vaccine (produced by Medvax).
• the anti-cancer reactive MNBCs used according to some embodiments of the present invention can be freshly isolated cells; stored e.g., cryopreserved (i.e. frozen) at e.g. liquid nitrogen temperature cells; and cell lines.
• the MNBCs are derived from a subject not treated with a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof.
• the anti-cancer agent is a cytokine capable of inducing activation and/or proliferation of a T cell.
• cytokines capable of inducing activation and/or proliferation of a T cell include, but are not limited to IL-2, IFNa, IL-12, IFN-gamma, TNF-a, IL-15, IL-6 and IL-1, IL-21.
• the cytokine is selected from the group consisting of IFNa, IFNy, IL-1, IL-2, IL-6, IL-12, IL-15, IL-21 and TNFa.
• the cytokine is selected from the group consisting of IFNa, IL-2, IL-12, IL-21 and IL-15.
• IL-2 (produced by Roche); IL21 (produced by BMY); ALT-803 (IL15 superagonist combined with a soluble IL-15a receptor, produced by Altor Bioscience); Darleukin (L19-IL2, human IL-2 conjugated with an antibody (L19) that is specific to the EDB region of fibronectin, produced by Philogen); Denenicokin [BMS-982470, a recombinant human peptide homologous to IL-21, produced by Bristol-Myers Squibb (ZymoGenetics)]; and Immunopulse (delivery of DNA-based IL-12 leading to localized expression of IL-12 in the tumor microenvironment, produced by Oncosec Medical.
• the anti-cancer agent is an immune-check point regulator.
• immune-check point regulator refers to a molecule that modulates the activity of one or more immune-check point proteins in an agonistic or antagonistic manner resulting in recruitment of an immune cell to elicit an immune activity against a cancer cell.
• the immune-check point regulator may bind directly the immune-check point or through an intermediary molecule, it can modulate the activity of a specific immune-check point protein with no cross reactivity with other immune- check point proteins or modulate the activity of at least 2, at least 3, at least 4 immune- check point proteins; each possibility represents a separate embodiment of the present invention.
• activation refers to the process of stimulating an immune cell (e.g. T cell, NK cell, B cell) that results in cellular proliferation, maturation, cytokine production and/or induction of regulatory or effector functions.
• an immune cell e.g. T cell, NK cell, B cell
• immune-check point protein refers to an antigen independent protein that modulates an immune cell response (i.e. activation or function).
• Immune-check point proteins can be either co-stimulatory proteins [i.e. positively regulating an immune cell activation or function by transmitting a co- stimulatory secondary signal resulting in activation of an immune cell] or inhibitory proteins (i.e. negatively regulating an immune cell activation or function by transmitting an inhibitory signal resulting in suppressing activity of an immune cell).
• the immune-check point protein regulates activation or function of a T cell.
• check-point proteins include, but not limited to, PD1, PDL-1, B7H2, B7H3, B7H4, BTLA-4, HVEM, CTLA-4, CD80, CD86, LAG- 3, TIM-3, KIR, IDO, CD19, OX40, OX40L, 4-1BB (CD137), 4-1BBL, CD27, CD70, CD40, CD40L, GITR, CD28, ICOS (CD278), ICOSL, VISTA and adenosine A2a receptor.
• Methods of determining signaling of a stimulatory or inhibitory signal include, but are not limited to, binding assay using e.g. BiaCore, HPLC or flow cytometry, enzymatic activity assays such as kinase activity assays, and expression of molecules involved in the signaling cascade using e.g. PCR, Western blot, immunoprecipitation and immunohistochemistry. Additionally or alternatively, determining transmission of a signal (co- stimulatory or inhibitory) can be effected by evaluating immune cell activation or function.
• Methods of evaluating immune cell activation or function include, but are not limited to, proliferation assays such as BRDU and thymidine incorporation, cytotoxicity assays such as chromium release, cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA, expression of activation markers such as CD25, CD69 and CD69 using flow cytometry.
• proliferation assays such as BRDU and thymidine incorporation
• cytotoxicity assays such as chromium release
• cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA
• expression of activation markers such as CD25, CD69 and CD69 using flow cytometry.
• determining the signaling activity is effected in-vitro or ex-vivo e.g. in a mixed lymphocyte reaction (MLR).
• MLR mixed lymphocyte reaction
• the signaling activity or the immune cell activation or function are generally expressed in comparison to the signaling, activation or function in a cell of the same species but not contacted with the immune-check point regulator or contacted with a vehicle control, also referred to as control.
• the immune-check point regulator is selected from the group consisting of an antibody, a peptide and a small molecule as further described in details hereinbelow.
• the immune-check point regulator can be an agonist or antagonist.
• the immune-check point regulator is an antagonist.
• an antagonist refers to a molecule that prevents and/or inhibits the biological function and/or expression of an immune-check point protein.
• the antagonist prevents and/or inhibits the suppressive effect of an immune-check point protein on an immune cell (e.g. T cells).
• an immune cell e.g. T cells
• the antagonist prevents and/or inhibits signaling to an immune cell (e.g. T cell) by an immune-check point protein.
• an immune cell e.g. T cell
• an immune-check point protein e.g. T cell
• the molecule may be a reversible or an irreversible antagonist.
• the antagonist completely prevents the biological function (e.g. signal transduction) of the immune-check point protein.
• the antagonist inhibits the biological function (e.g. signal transduction) of the immune-check point protein e.g., as detected by e.g. kinase activity, proliferation assay, cytotoxicity assay or cytokine secretion assay.
• the reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % as compared to same in the absence of the antagonist.
• Preventing and/or inhibiting the biological function of an immune-check point protein can be effected at the protein level (e.g., antibodies, small molecules, inhibitory peptides, enzymes that cleave the polypeptide, aptamers and the like) but may also be effected at the genomic (e.g. homologous recombination and site specific endonucleases) and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents) of an inhibitory immune-check point protein.
• protein level e.g., antibodies, small molecules, inhibitory peptides, enzymes that cleave the polypeptide, aptamers and the like
• genomic e.g. homologous recombination and site specific endonucleases
• transcript level e.g. homologous recombination and site specific endonucleases
• RNA silencing agents e.g., RNA silencing agents
• agents that can function as antagonists are described in details hereinbelow.
• the antagonistic agent is an antibody.
• the antagonistic antibody is capable of specifically binding an inhibitory immune-check point protein. According to specific embodiments, the antagonistic antibody specifically binds at least one epitope of an inhibitory immune-check point protein.
• epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
• Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
• antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VL that are capable of binding to an epitope of an antigen.
• the antibody may be mono-specific (capable of recognizing one epitope or protein), bi-specific (capable of binding two epitopes or proteins) or multi- specific (capable of recognizing multiple epitopes or proteins).
• Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab', and an F(ab')2.
• CDR complementarity-determining region
• light chain referred to herein as "light chain”
• heavy chain a complementarity-determining region of an immunoglobulin heavy chain
• variable region of a light chain a variable region of a heavy chain
• a light chain a variable region of
• CDR complementarity-determining region
• VH VH
• CDR H2 or H2 CDR H3 or H3
• VL VL
• the identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc.
• variable regions and CDRs may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.
• Fv defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;
• scFv single chain Fv
• dsFv disulfide- stabilized Fv
• Fab a fragment of an antibody molecule containing a monovalent antigen- binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CHI domains thereof;
• Fab' a fragment of an antibody molecule containing a monovalent antigen- binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are obtained per antibody molecule);
• F(ab')2 a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two disulfide bonds); and
• Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.
• the antibody may be monoclonal or polyclonal.
• Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
• Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
• antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
• This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
• a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
• an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
• cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
• Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
• sFv single-chain antigen binding proteins
• the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
• the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
• Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97- 105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11: 1271-77 (1993); and U.S. Patent No. 4,946,778, which is hereby incorporated by reference in its entirety.
• CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
• humanized antibodies are preferably used.
• Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary determining region
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
• Fc immunoglobulin constant region
• a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
• the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
• human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
• aptamer refers to double stranded or single stranded RNA molecule that binds to specific molecular target, such as a protein.
• specific molecular target such as a protein.
• SELEX Systematic Evolution of Ligands by Exponential Enrichment
• SELEX Systematic Evolution of Ligands by Exponential Enrichment
• Another agent capable of being an antagonist would be any molecule which interferes with the immune-check point protein function (e.g. catalytic or interaction) by binding to and/or cleaving the immune-check point protein.
• Such molecules can be, but are not limited to, small molecules, inhibitory peptides, enzymes that cleave the immune-check point protein, adnectins, affibodies, avimers, anticalins, tetranectins, DARPins, and engineered Kunitz-type inhibitors wherein each possibility is a separate embodiment of the invention.
• the antagonist is a small molecule.
• the antagonist is a peptide molecule. It will be appreciated that a non-functional analogue of at least a catalytic or binding portion of an inhibitory peptide can be also used as an antagonist.
• Down-regulation at the nucleic acid level is typically effected using a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same.
• the nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se.
• RNA silencing refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co- suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene.
• RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
• RNA silencing agent refers to an RNA which is capable of specifically inhibiting or “silencing" the expression of a target gene.
• the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism.
• RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
• Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
• the RNA silencing agent is capable of inducing RNA interference.
• the RNA silencing agent is capable of mediating translational repression.
• the RNA silencing agent is specific to the target RNA (i.e. an immune-check point protein) and does not cross inhibit or silence other targets or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98 %, 97 %, 96 %, 95 %, 94 %, 93 %, 92 %, 91 %, 90 %, 89 %, 88 %, 87 %, 86 %, 85 %, 84 %, 83 %, 82 %, 81 % global homology to the target gene; as determined by PCR, Western blot, Immunohistochemistry and/or flow cytometry.
• RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
• RNA silencing agents that can be used according to specific embodiments of the present invention.
• DsRNA, siRNA and shRNA - The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer.
• Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs).
• Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes.
• RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single- stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
• RISC RNA-induced silencing complex
• some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.
• dsRNA longer than 30 bp are used.
• dsRNA is provided in cells where the interferon pathway is not activated, see for example Billy et al., PNAS 2001, Vol. 98, pages 14428-14433. and Diallo et al., Oligonucleotides, October 1, 2003, 13(5): 381-392. doi: 10.1089/154545703322617069.
• the long dsRNA are specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression.
• Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.
• siRNAs small inhibitory RNAs
• RNA refers to small inhibitory RNA duplexes (generally between 18-30 base pairs) that induce the RNA interference (RNAi) pathway.
• siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location.
• the observed increased potency obtained using longer RNAs in triggering RNAi is suggested to result from providing Dicer with a substrate (27mer) instead of a product (21mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.
• RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
• RNA agent refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
• the number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11.
• nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop.
• oligonucleotide sequences that can be used to form the loop include 5'-CAAGAGA-3' and 5'-UUACAA-3' (International Patent Application Nos. WO2013126963 and WO2014107763). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem- loop or hairpin structure comprising a double- stranded region capable of interacting with the RNAi machinery.
• RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the inhibitory-check point mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each A A and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245].
• UTRs untranslated regions
• siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www(dot)ambion(dot)com/techlib/tn/91/912(dot)html).
• potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
• sequence alignment software such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/).
• Qualifying target sequences are selected as template for siRNA synthesis.
• Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %.
• Several target sites are preferably selected along the length of the target gene for evaluation.
• a negative control is preferably used in conjunction.
• Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome.
• a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
• RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
• RNA silencing agent may be a miRNA.
• miRNA refers to a collection of non-coding single- stranded RNA molecules of about 19-28 nucleotides in length, which regulate gene expression. miRNAs are found in a wide range of organisms (viruses.fwdarw.humans) and have been shown to play a role in development, homeostasis, and disease etiology.
• pri-miRNA Genes coding for miRNAs are transcribed leading to production of a miRNA precursor known as the pri-miRNA.
• the pri-miRNA may form a hairpin with a stem and loop.
• the hairpin structure of the pri-miRNA is recognized by Drosha, which is a RNase III endonuclease. Drosha typically recognizes terminal loops in the pri-miRNA and cleaves the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a pre-miRNA stem loop with a 5' phosphate and ⁇ 2 nucleotide 3' overhang. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by Ran- GTP and the export receptor Ex-portin-5.
• Drosha is a RNase III endonuclease. Drosha typically recognizes terminal loops in the pri-miRNA and cleaves the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a pre-miRNA stem loop with a 5' phosphate and ⁇ 2 nucleotide 3' overhang.
• the pre-miRNA
• the double- stranded stem or the 5' phosphate and 3' overhang at the base of the stem loop of the pre-miRNA is then recognized by Dicer, which is also an RNase III endonuclease. Dicer then cleaves off the terminal loop two helical turns away from the base of the stem loop leaving an additional 5' phosphate and ⁇ 2 nucleotide 3' overhang.
• the resulting siRNA-like duplex which may comprise mismatches, comprises the mature miRNA and a similar- sized fragment known as the miRNA*. miRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs.
• RISC RNA-induced silencing complex
• the RISC identifies target nucleic acids based on high levels of complementarity between the miRNA and the mRNA, especially by nucleotides 2-7 of the miRNA.
• the target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region.
• miRNAs may direct the RISC to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression.
• the miRNA may specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut is typically between the nucleotides pairing to residues 10 and 11 of the miRNA. Alternatively, the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA.
• contacting cells with a miRNA may be effected by transfecting/loading the cells with e.g. the mature double stranded miRNA, the pre-miRNA or the pri-miRNA.
• the pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
• the pri-miRNA sequence may comprise from 45-30,000, 50-25,000, 100- 20,000, 1,000-1,500 or 80-100 nucleotides.
• Antisense - Antisense is a single stranded RNA designed to prevent or inhibit expression of a gene by specifically hybridizing to its mRNA. Downregulation of an immune-check point can be effected using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the immune-check point protein.
• the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
• the prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example,
• Nucleic acid agents can also operate at the DNA level as summarized infra. Suppressing the biological function of an immune-check point can also be achieved by inactivating the gene via introducing targeted mutations involving loss-of function alterations (e.g. point mutations, deletions and insertions) in the gene structure.
• loss-of function alterations e.g. point mutations, deletions and insertions
• loss-of-function alterations refers to any mutation in the DNA sequence of a gene which results in downregulation of the expression level and/or activity of the expressed product, i.e., the mRNA transcript and/or the translated protein.
• Non-limiting examples of such loss-of-function alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non- functional protein, devoid of the enzymatic activity
• los-of-function alteration of a gene may comprise at least one allele of the gene.
• allele refers to any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
• loss-of-function alteration of a gene comprises both alleles of the gene.
• PB transposases e.g. Sleeping Beauty, piggyBac, Tol2 or Frog Prince
• genome editing by engineered nucleases e.g. meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system
• TALENs transcription-activator like effector nucleases
• rAAV recombinant adeno-associated virus
• Methods for qualifying efficacy and detecting sequence alteration include, but not limited to, DNA sequencing, electrophoresis, an enzyme-based mismatch detection assay and a hybridization assay such as PCR, RT- PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis.
• Sequence alterations in a specific gene can also be determined at the protein level using e.g. chromatography, electrophoretic methods, immunodetection assays such as ELISA and western blot analysis and immunohistochemistry.
• the immune-check point regulator can be an agonist or antagonist.
• the immune-check point regulator is an agonist.
• agonist refers to a molecule that induces and/or increases the biological function and/or expression of an immune-check point protein.
• the agonist induces and/or increases the co- stimulatory effect of an immune-check point protein on an immune cell (e.g. T cells).
• an immune cell e.g. T cells
• the agonist induces and/or increases signaling to an immune cell (e.g. T cell) by an immune-check point protein.
• an immune cell e.g. T cell
• the agonist can be a naturally occurring activator or a functional derivative thereof; or non-naturally occurring activator.
• the agonist is a full agonist, that is, the effect of the agonist is equivalent to the effect of the naturally occurring activator (i.e. ligand).
• the agonist is a partial agonist, that is, the effect of the agonist is lower than the maximal effect of the naturally occurring activator (i.e. ligand).
• the effect of the agonist may be lower by at least 5 %, 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 % as compared to the maximal effect of the naturally occurring activator.
• the agonist is a super agonist, that is, the effect of the agonist is higher than the maximal effect of the naturally occurring activator (i.e. ligand).
• the effect of the agonist may be higher by at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 % at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or at least 2 fold, at least 4 fold, at least 5 fold or at least 10 fold as compared to the maximal effect of the naturally occurring activator.
• the agonist induces complete activation the biological function (e.g. signal transduction) of the immune-check point protein.
• the agonist increases the biological function (e.g. signal transduction) of the immune-check point protein e.g., as detected by e.g. kinase activity, proliferation assay, cytotoxicity assay or cytokine secretion assay.
• the increase may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % as compared to same in the absence of the agonist.
• the agonist binds directly the immune- check point protein.
• the agonist indirectly binds the immune-check point protein by acting through an intermediary molecule, for example the agonist binds to or modulates a molecule that in turn binds to or modulates the immune-check point protein.
• Activating and/or increasing the biological function of an immune-check point protein can be effected at the protein level (e.g., antibodies, small molecules, peptides and the like) but may also be effected at the genomic level (e.g., activation of transcription via promoters, enhancers, regulatory elements) and/or the transcript level using a variety of molecules which promote transcription and/or translation (e.g., correct splicing, polyadenylation, activation of translation) of a co- stimulatory immune- check point protein.
• protein level e.g., antibodies, small molecules, peptides and the like
• genomic level e.g., activation of transcription via promoters, enhancers, regulatory elements
• the transcript level e.g., a variety of molecules which promote transcription and/or translation (e.g., correct splicing, polyadenylation, activation of translation) of a co- stimulatory immune- check point protein.
• agents that can function as agonists are described in details hereinbelow.
• the agonist is the naturally occurring activator or a functional derivative or variant thereof which retain the ability to specifically bind to the immune-check point protein.
• a functional analogue of at least a catalytic or binding portion of a co-stimulatory peptide can be also used as an agonist.
• the agonist is an exogenous polypeptide including at least a functional portion (e.g. catalytic or interaction) of the co-stimulatory immune-check point protein.
• the polypeptide can be a ligand capable of binding and activating the co- stimulatory immune-check point protein receptor.
• the agonist is an antibody.
• the agonistic antibody is capable of specifically binding a co- stimulatory immune-check point protein. According to specific embodiments, the agonistic antibody specifically binds at least one epitope of a co- stimulatory immune-check point protein.
• Another agent capable of being an agonist would be a molecule which promotes and/or increases the co- stimulatory immune-check point protein function (e.g. catalytic or interaction) by binding to the immune-check point protein or an intermediate thereof.
• Such molecules can be, but are not limited to, small molecules, peptides, aptamers, adnectins, affibodies, avimers, anticalins, tetranectins and DARPins, wherein each possibility is a separate embodiment of the invention.
• the agonist is a small molecule.
• the agonist is a peptide.
• An agonist can also be a molecule which is capable of increasing the transcription and/or translation of an endogenous DNA or mRNA encoding the co- stimulatory immune-check point protein and thus increasing endogenous co-stimulatory immune-check point protein activity.
• Another agonistic agent may be an exogenous polynucleotide (DNA or RNA) sequence designed and constructed to express at least a functional portion of the co- stimulatory immune-check point protein.
• DNA or RNA exogenous polynucleotide
• a polynucleotide sequence encoding a specific co- stimulatory immune-check point protein or a homologue thereof which exhibit the desired activity is preferably ligated into a nucleic acid construct suitable for mammalian cell expression.
• a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive [e.g. cytomegalovirus (CMV) and Rous sarcoma virus (RSV)] or inducible (e.g. the tetracycline-inducible promoter) manner.
• CMV cytomegalovirus
• RSV Rous sarcoma virus
• the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in a specific cell population.
• cell type-specific and/or tissue- specific promoters include promoters such as, but not limited to lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins [Banerji et al. (1983) Cell 33729-740].
• the nucleic acid construct (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
• a typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
• such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
• the construct may also include an enhancer element which can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters.
• the vector may or may not include a eukaryotic replicon.
• the nucleic acid construct of some embodiments of the invention can also include a signal sequence for secretion of the peptide from a host cell in which it is placed.
• the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
• Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of a co- stimulatory immune-check point mRNA translation.
• Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
• Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.
• the expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
• IRS internal ribosome entry site
• the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, or yield of the expressed peptide.
• the type of vector used by some embodiments of the invention will depend on the cell type transformed.
• the ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
• Recombinant viral vectors are useful for in vivo expression of an immune-check point protein since they offer advantages such as lateral infection and targeting specificity. Viral vectors can also be produced that are unable to spread laterally.
• nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
• viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
• Useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
• the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
• Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
• the immune-check point regulator targets an immune check-point protein selected from the group consisting of B7-H3, CD 19 and CD70.
• B7-H3 (gene symbol CD276, also known as CD276), is a B7 family protein. The gene is over-expressed in a variety of solid tumors, including prostate, pancreatic, melanoma, renal cell, ovarian, colorectal, gastric, bladder and non-small cell lung cancers has been reported (e.g. Immune Netw. 2014 Dec; 14(6): 265-276).
• the B7-H3 protein refers to the human protein, such as provided in the following GenBank Number NP_001019907.
• CD19 (gene symbol CD19, also known as B-lymphocyte antigen CD19), is a cell surface molecule that assembles with the antigen receptor of B lymphocytes in order to decrease the threshold for antigen receptor-dependent stimulation.
• CD 19 is expressed on follicular dendritic cells and B cells. Typically, it is present on B cells from earliest recognizable B-lineage cells during development to B-cell blasts but is lost on maturation to plasma cells. CD 19 is also expressed on several hematologic e.g. B cell tumors.
• the CD 19 protein refers to the human protein, such as provided in the following GenBank Number NP_001171569.
• CD70 is the ligand for CD27, which is expressed on activated lymphocytes. CD70 expression is found in different types of cancers including renal cell carcinomas, metastatic breast cancers, brain tumours, leukemias, lymphomas and nasopharyngeal carcinomas. According to a specific embodiment, the CD70 protein refers to the human protein, such as provided in the following GenBank Number NP_001243.
• an immune-check point regulator selected from the group consisting of PD1 antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TEVI-3 antagonist, KIR antagonist, IDO antagonist, OX40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist and ICOS agonist.
• PD1 Programmed Death 1
• gene symbol PDCD1 is also known as CD279.
• the PD1 protein refers to the human protein, such as provided in the following GenBank Number NP_005009.
• PDL-1 gene symbol CD274
• CD274 is also known as CD274 and B7-H1.
• the PDL- 1 protein refers to the human protein, such as provided in the following GenBank accession Numbers NP_054862 and NP_054862.
• CTLA4 gene symbol Ctla4 is also known as CD 152.
• CTLA-4 protein refers to the human protein, such as provided in the following GenBank Number NP_001032720.
• LAG-3 (Lymphocyte-activation gene 3), gene symbol LAG3, is also known as
• the LAG-3 protein refers to the human protein, such as provided in the following GenBank Number NP_002277.
• TEVI-3 gene symbol HAVCR2
• HAVCR2 Hepatitis A Virus Cellular
• the TIM-3 protein refers to the human protein, such as provided in the following GenBank Number NP_116171.
• KIRs killer cell Ig-like receptors
• T cells T cells as well as most human NK cells.
• KIR refers to a KIR that delivers an inhibitory signal to the cell. A number of KIRs are well characterized
• GenBank entries of human KIRs have the following accession numbers: KIR2DL1:
• KIR2DL2 Genbank accession number U24075, L76669 or NP_055034
• KIR2DL3 Genbank accession number U24074, L41268 or NP_056952
• KIR2DL4 Genbank accession number X97229
• KIR3DL1 Genbank accession number L41269.
• IDO indoleamine 2,3-dioxygenase
• EC 1.13.11.52 is a heme-containing intracellular enzyme that catalyzes the first and rate-determining step in the degradation of the essential amino acid L-tryptophan to N-formyl-kynurenine.
• the IDO protein refers to the human protein, such as provided in the following GenBank Number NP_002155.
• OX40 gene symbol TNFRSF4
• TNFRSF4 is also known as CD 134, Tumor necrosis factor receptor superfamily, member 4, TNFRSF4, TXGP1L, ACT35 and IMD16.
• the OX40 protein refers to the human protein, such as provided in the following GenBank Number NP_003318.
• CD137 gene symbol TNFRSF9, is also known as 4-1BB and Tumor Necrosis
• CD 137 protein refers to the human protein, such as provided in the following GenBank Number NP_001552.
• CD27 gene symbol CD27, is also known as Tumor Necrosis Factor Receptor Superfamily, Member 7, TNFRSF7 and S 152.
• the CD27 protein refers to the human protein, such as provided in the following GenBank Number NP_001233.
• CD40 gene symbol CD40
• CD40 is also known as Tumor Necrosis Factor Receptor Superfamily, Member 5 and TNFRSF5.
• the CD40 protein refers to the human protein, such as provided in the following GenBank Number NP 001241.
• GITR glucocorticoid-induced tumor necrosis factor receptor
• gene symbol TNFRSF18 is also known as TNF receptor superfamily 18, TNFRSF18, AITR and CD357.
• the GITR protein refers to the human protein, such as provided in the following GenBank Numbers NP_004186, NP_683699, NP_683700.
• CD28 gene symbol CD28
• Tp44 Tp44
• the CD28 protein refers to the human protein, such as provided in the following GenBank Numbers NP_001230006, NP_001230007 and NP_006130.
• ICOS Inducible T-cell co-stimulator
• gene symbol ICOS is also known as
• the ICOS protein refers to the human protein, such as provided in the following GenBank Number NP_036224.
• the anti-cancer agent may be an agent capable of binding an immune-check point protein expressed on a cancer cell.
• the immune-check point protein expressed on said cancer cell is selected from the group consisting of PDL-1, CD27, LAG3, CD 19, CD70 and CEACAM1.
• binding of the anti-cancer agent to the immune-check point protein expressed on the cancer cell results in at least one of:
• agents capable of binding a cancer cell expressing a specific antigen that can be used according to specific embodiments of the present invention are described in details hereinabove.
• the agent capable of binding said immune- check point protein is an antibody or a T cell.
• the T cell comprises a T cell transduced with a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
• TCR T cell receptor
• CAR chimeric antigen receptor
• immune-check point regulators and/or agents capable of binding an immune-check point protein expressed on a cancer cell that can be used according to some embodiments of the invention include:
• EVIP321 a Ig fusion protein comprising a soluble dimeric recombinant form of LAG-3) designed to activate antigen presenting cells, induce maturation and migration of DCs to the lymph nodes and enhanced cross-presentation of antigens to CD8+ T cells [see e.g. Brignone et al., (2007) J. Immunol. 179:4202-4211].
• An agent targeting B7-H3, such as, MGA271 (produced by MacroGenics), a humanized IgGlK monoclonal antibody that recognizes human B7-H3, B7-H3 antagonist such as disclosed e.g. in US Application Publication No. US 20130122021 and International Application Publication Nos. WO 2011109400 and WO 2012004410, or a B7-H3 agonist such as disclosed e.g. in International Application Publication No. WO 2004093894, the contents of each of which are fully incorporated herein by reference.
• An agent capable of binding CD70 such as ARGX-110 (produced by arGEN-X BVBA) a monoclonal antibody that targets CD70 and induces cytotoxic T cell response and ADCC to cells expressing CD70.
• the anti-cancer agent can also be selected from the group consisting of a colony stimulating factor-1 receptor (CSF1R) antagonist, a CXCR2 antagonist, a STAT3 antagonist, PV-10 and Cotara.
• CSF1R colony stimulating factor-1 receptor
• CXCR2 CXCR2
• STAT3 STAT3
• PV-10 Cotara
• CSF1R also known as macrophage colony-stimulating factor receptor, M-CSFR and CD115
• M-CSFR macrophage colony-stimulating factor receptor
• CD115 is a cell single pass type I membrane protein acting as the receptor for colony stimulating factor 1, a cytokine which controls the production, differentiation, and function of macrophages.
• the CSF1R protein refers to the human protein, such as provided in the following GenBank Number NP_001275634.
• CSF1R antagonists that can be used according to some embodiments of the invention include the antibodies FPA008 (produced by Five Prime Therapeutics) and RG7155 (produced by Roche).
• Tumor associated macrophages TAM
• TAMs are myeloid lineage-derived cells that are part of the tumor microenvironmental support system. TAMs are potently angiogenic, remodel the stroma (extracellular matrix and related components) and are immunosuppressive.
• the plethora of critical factors produced by TAM include hypoxia response proteins and growth factors that drive angiogenesis, tissue remodeling and immunosuppression, i.e.
• HIF2a HIF2a, MMP-9, EGF, VEGF and TGF-beta, cytokines that can maintain this response in a chronic state (IL- 10, IL-4) and chemokines that attract myeloid cells and regulatory T cells (CCL22, CCXL8).
• the TAM population can be directly regulated by tumor cell secretion of CSF-1, thus the importance of the CSF1R target.
• CXCR2 (also known as IL-8 receptor, beta) is a G-protein-coupled chemokine receptor which functions as a receptor for e.g. IL-8, CXCL1, CXCL2, CXCL3 and CXCL5.
• the CXCR2 protein refers to the human protein, such as provided in the following GenBank Number NP_001161770.
• a specific non-limiting example of a CXCR2 antagonist that can be used according to some embodiments of the invention include AZD5069 (produced by AstraZeneca). Cancer initiation and progression also depends on escape from host immunosurveillance. In several solid tumors, immune evasion involves a shift of immune responses, including imbalance in Thl/Th2 responses and enhancement of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells. The number of MDSCs in the blood correlates well with clinical cancer stage and metastatic tumor burden in patients (Diaz-Montero et al., 2009; Cancer Immunology, Immunotherapy, January 2009, Volume 58, Issue 1, pp 49-59).
• MDSCs myeloid-derived suppressor cells
• MDSCs contribute to cancer immune evasion via suppressing functions of T and natural killer (NK) cells (Gabrilovich and Nagaraj, 2009 Nature Reviews Immunology 9, 162-174). Accumulation of Myeloid Derived Suppressor Cells (MDSCs) in the tumor bed limits the efficacy of checkpoint blockade in cancer. Expansion of MDSCs is a major mechanism of tumor immune escape. CXCR2 is essential for MDSC infiltration into tumors, which promotes tumor growth and progression via inhibition of CD8+ T cell cytotoxic activity.
• NK natural killer
• STAT3 Signal transducer and activator of transcription 3
• STAT3 is a transcription factor encoded by the STAT3 gene.
• STAT3 is a member of the STAT protein family of signal transducers and activators of transcription, which represent a family of proteins that, when activated by protein tyrosine kinases in the cytoplasm of the cell, migrate to the nucleus and activate gene transcription.
• the STAT3 protein refers to the human protein, such as provided in the following GenBank Number NP_003141.
• STAT3 antagonists that can be used according to some embodiments of the invention include AZD9150 (produced by AstraZeneca), a STAT3 antisense; and STAT3 siRNA such as disclosed e.g. in Herrmann A, et al. J Clin Invest. (2014) 124(7): 2977-87.
• PV-10 (produced by Provectus Biopharmaceuticals Inc.) is a composition containing Rose Bengal shown to eradicate tumors and activate anti-tumor T cell response.
• Cotara (131I-chTNT-l/B Mab, produced by Peregrine Pharmaceuticals Inc.), is an antibody which is combined with radioactive iodine.
• Cotara binds to proteins within the nucleus of necrotic (i.e. dead and dying) cells which are present in most malignant tumors and are primarily found at the center of the tumor.
• necrotic i.e. dead and dying
• the Ab When the Ab is delivered directly to the tumor, it remains within the tumor and the attached radioactive iodine bombards the neighboring living tumor cells with radiation.
• the CXCR4 antagonistic peptide can be administered to a subject in combination with several of the anti-cancer agents described hereinabove.
• the order in which the CXCR4 antagonistic peptide and the anti-cancer agent are administered to the subject can vary according to the method of treating.
• administering the peptide is effected prior to administering the agent.
• administering the peptide is effected following administering the agent.
• administering the peptide is effected concomitantly with administering the agent.
• administering comprises multiple administrations of the peptide.
• administration of the anti-cancer agent is effected following at least one administration of the CXCR4 antagonistic peptide.
• administering comprises multiple administrations of the anti-cancer agent.
• administering the CXCR4 antagonistic peptide of the present invention is effected following at least one administration of the agent.
• administering the peptide and administering the agent are effected sequentially.
• the CXCR4 antagonistic peptide and the anti-cancer agent of the invention can be administered to a subject in combination with other established or experimental therapeutic regimen to treat cancer including analgetics, chemotherapeutic agents, radiotherapeutic agents, hormonal therapy, immune modulators and other treatment regimens (e.g., surgery, cell transplantation e.g. hematopoietic stem cell transplantation) which are well known in the art.
• other established or experimental therapeutic regimen to treat cancer including analgetics, chemotherapeutic agents, radiotherapeutic agents, hormonal therapy, immune modulators and other treatment regimens (e.g., surgery, cell transplantation e.g. hematopoietic stem cell transplantation) which are well known in the art.
• the CXCR4 antagonistic peptides and/or the anti-cancer agent described hereinabove can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
• a pharmaceutical composition comprising as active ingredients a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof and an anti-cancer agent selected from the group consisting of:
• a vaccine selected from the group consisting of IMCgplOO, Prophage G- 100 & G-200, GV-1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAdl, GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as GI-4000;
• MNBCs anti-cancer reactive mononuclear blood cells
• a cytokine capable of inducing activation and/or proliferation of a T cell
• an immune-check point regulator wherein said immune-check point regulator is not a PD1 antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TEVI-3 antagonist, KIR antagonist, IDO antagonist, OX40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS agonist;
• CSF1R colony stimulating factor- 1 receptor
• a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• active ingredient refers to the CXCR4 antagonistic peptides and/or the anti-cancer agent accountable for the biological effect.
• physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
• An adjuvant is included under these phrases.
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
• excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, intradermal, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
• neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
• molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
• pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
• the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
• each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
• the CXCR4 antagonistic peptide of the invention, the anti-cancer agent or the pharmaceutical composition comprising same can be administered in the same route or in separate routes.
• the CXCR4 antagonistic peptide of the invention or the pharmaceutical composition comprising same is administered subcutaneously.
• the CXCR4 antagonistic peptide of the invention or the pharmaceutical composition comprising same is administered intravenously.
• the anti-cancer agent or the pharmaceutical composition comprising same is administered intravenously.
• the anti-cancer agent or the pharmaceutical composition comprising same is administered via a subcutaneous route.
• compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
• Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
• the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
• Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
• the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
• a suitable vehicle e.g., sterile, pyrogen-free water based solution
• compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• Alternative embodiments include depots providing sustained release or prolonged duration of activity of the active ingredient in the subject, as are well known in the art.
• compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, according to specific embodiments, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.
• a disorder e.g., cancer
• the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
• a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l). Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
• dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
• compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• the CXCR4 antagonistic peptide of the invention or the pharmaceutical composition comprising same is administered in a dose ranging between 0.1 to 10 mg/kg of body weight, between 0.1 to 2 mg/kg of body weight, between 0.1 to 1 mg/kg of body weight, between 0.3 to 10 mg/kg of body weight, between 0.3 to 2 mg/kg of body weight, between 0.3 to 1 mg/kg of body weight or between 0.3 to 0.9 mg/kg of body weight.
• the CXCR4 antagonistic peptide of the invention or the pharmaceutical composition comprising same is administered in a dose ranging between 0.5-2 mg / kg.
• the CXCR4 antagonistic peptide of the invention or the pharmaceutical composition comprising same is administered at a dose of 0.5-1 mg / kg.
• the anti-cancer antibody is administered in a dose ranging between 0.001 to 30 mg/kg body weight, between 0.001 to 20 mg/kg body weight, between 0.001 to 10 mg/kg body weight, between 0.001 to 1 mg/kg body weight, between 0.01 to 30 mg/kg body weight, between 0.01 to 20 mg/kg body weight, between 0.01 to 10 mg/kg body weight, between 0.01 to 1 mg/kg body weight, between 0.1 to 30 mg/kg body weight, between 0.1 to 20 mg/kg body weight, between 0.1 to 10 mg/kg body weight, between 0.1 to 1 mg/kg body weight, between 1 to about 30 mg/kg, between 1 to about 20 mg/kg or between 1 to about 10 mg/kg.
• the desired dose can be administered at one time or divided into sub-doses, e.g., 2-4 sub-doses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
• the CXCR4 antagonistic peptide of the invention, the anti-cancer agent or the pharmaceutical composition comprising same is administered multiple times e.g. 2-10, over a period of time e.g. for several days to several weeks at appropriate intervals e.g. once a day, twice a week, once a week, once every two weeks, once a month, once every 3 to 6 months.
• compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may, for example, comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
• Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
• an article of manufacture identified for use in treating cancer comprising a packaging material packaging a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof and an anti-cancer agent selected from the group consisting of:
• a vaccine selected from the group consisting of IMCgplOO, Prophage G-
• MNBCs anti-cancer reactive mononuclear blood cells
• a cytokine capable of inducing activation and/or proliferation of a T cell
• an immune-check point regulator wherein said immune-check point regulator is not a PDl antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist, KIR antagonist, IDO antagonist, OX40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS agonist;
• CSF1R colony stimulating factor- 1 receptor
• the peptide and the agent may be packaged in the same container or in separate containers; each possibility represents a separate embodiment of the present invention.
• the peptide and the agent are in separate containers.
• the peptide and the agent are in separate formulations.
• the peptide and the agent are in a co- formulation.
• anti-cancer agent it is expected that during the life of a patent maturing from this application many relevant anti-cancer agents will be developed and the scope of the term "anti-cancer agent" is intended to include all such new technologies a priori.
• compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
• a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
• BL-8040 is safe and well tolerated drug that was shown to induce rapid mobilization of hematopoietic stem/progenitor cells and mesenchymal stem cells as well as T cells, B cells, NK cells, NKT cells and ImDC to the peripheral blood. Therefore, BL-8040 can be used to induce the mobilization and dissemination of immature DC and T effector and memory cells into tumors.
• BL-8040 is injected into a cancer patient at a dose of 0.5-1 mg / kg for 3-10 days and then 1-3 times a week in combination with a tumor vaccine such as a dendritic cells vaccine, IMCgplOO, Prophage G-100 & G-200, GV- 1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAdl, GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as GI-4000.
• a tumor vaccine such as a dendritic cells vaccine, IMCgplOO, Prophage G-100 & G-200, GV- 1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak,
• BL-8040 is injected into a cancer patient at a dose of 0.5-1 mg / kg for 3-10 days and then 1-3 times a week in combination with immunomodulatory cytokines such as IL-2, IFN-a, IL-12.
• BL-8040 is injected into a cancer patient at a dose of 0.5-1 mg / kg for 3-10 days and then 1-3 times a week in combination with a tumor vaccine such as a dendritic cells vaccine, IMC gp 100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX- 594, ColoAdl, GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as GI-4000 and immunomodulatory cytokines such as IL-2, IFN-a and IL-12.
• a tumor vaccine such as a dendritic cells vaccine, IMC gp 100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201
• BL-8040 is injected into a cancer patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3 times a week in combination with anticancer adoptive T cell transfer such as T cells transduced with an anti-cancer T cell receptor (TCR) or a chimeric antigen receptor (CAR).
• anticancer adoptive T cell transfer such as T cells transduced with an anti-cancer T cell receptor (TCR) or a chimeric antigen receptor (CAR).
• BL-8040 is injected into a cancer patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3 times a week in combination with an immune-check point regulator targeting e.g. B7-H3, CD19 and CD70.
• an immune-check point regulator targeting e.g. B7-H3, CD19 and CD70.
• BL-8040 is injected into a cancer patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3 times a week in combination with an anti-cancer agent (e.g. antibody or T cell) capable of binding a cancer cell expressing an immune-check point protein.
• an anti-cancer agent e.g. antibody or T cell

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• 2016
  • 2016-07-14 CN CN201680044602.4A patent/CN107921087A/zh not_active Withdrawn
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  • 2016-07-14 EP EP20176901.5A patent/EP3744340A3/en not_active Withdrawn
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