WO2016162867A1 - Thérapie combinée composée d'un facteur d'activation des macrophages et d'inhibiteurs de la voie de signalisation pd-1 - Google Patents

Thérapie combinée composée d'un facteur d'activation des macrophages et d'inhibiteurs de la voie de signalisation pd-1 Download PDF

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WO2016162867A1
WO2016162867A1 PCT/IL2016/050365 IL2016050365W WO2016162867A1 WO 2016162867 A1 WO2016162867 A1 WO 2016162867A1 IL 2016050365 W IL2016050365 W IL 2016050365W WO 2016162867 A1 WO2016162867 A1 WO 2016162867A1
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pharmaceutical compositions
gcmaf
antibody
treating cancer
cancer
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PCT/IL2016/050365
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Michal Shahar
Uri Yogev
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Efranat Ltd.
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    • 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
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • 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
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to methods for treating cancer or infectious diseases comprising administering to a subject an agent that reduces or inhibits the signal transduction mediated by PD-1 in combination with macrophage activating factor derived from Gc protein (GcMAF).
  • GcMAF macrophage activating factor derived from Gc protein
  • the present invention provides methods of treating cancer comprising administering to a subject an anti-PD-1 antibody and GcMAF.
  • PD-1 is an immunoinhibitory receptor that belongs to the immunoglobulin superfamily related to CD28. PD-1 has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2). PD-1 is type I transmembrane glycoprotein containing an Ig Variable- type domain responsible for ligand binding and a cytoplasmic tail that is responsible for the binding of signaling molecules.
  • the cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs, an ITIM (immunoreceptor tyrosine-based inhibition motif) and an ITSM (immunoreceptor tyrosine-based switch motif).
  • PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motif within its cytoplasmic tail, leading to dephosphorylation of effector molecules such as CD3 zeta, PKC theta and ZAP70, thereby downregulating T cell responses.
  • PD-1 was shown to be expressed on activated lymphocytes, i.e., peripheral CD4 + and CD8 + T cells, B cells and monocytes.
  • the ligands for PD-1 are type I transmembrane receptors containing both Ig Variable- and Ig Constant- like domains in the extracellular region. Both ligands contain short cytoplasmic regions with no known signaling motifs.
  • PD- Ll and PD-L2 are constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic tissues as well as various tumor types.
  • PD-L1 is expressed on B, T, myeloid and dendritic cells (DCs), but also on peripheral cells, like microvascular endothelial cells and non-lymphoid organs like heart, lung etc.
  • PD-L2 is found on macrophages and DCs.
  • PD-L1 has been shown to be expressed on a number of mouse and human tumors and to be induced by IFN gamma on the majority of PD-L1 negative tumor cell lines.
  • expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 (on tumor cells) has been found in a number of primary tumor biopsies assessed by immunohistochemistry.
  • Such tissues include cancers of the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast, kidney, esophagus, stomach, pancreas as well as tumors of the head and neck. More strikingly, PD-ligand expression on tumor cells has been correlated to poor prognosis of cancer patients across multiple tumor types.
  • Blockade of the PD-1/PD-L1 interaction has been found to enhance tumor- specific T-cell immunity and to augment the clearance of tumor cells by the immune system.
  • administration of either PD-1 or PD-L1 directed antibody significantly inhibited tumor growth.
  • Antibody blockade effectively promoted tumor reactive CD8 + T cell infiltration into the tumor resulting in the up-regulation of anti-tumor effectors including IFN gamma, granzyme B and perforin. It was also shown that PD-1 blockade in combination with chemotherapy yielded a synergistic effect against the pancreatic tumor growth.
  • Macrophages are widely distributed immune cells that play an essential role in the innate and adaptive immune response to pathogens and in cancer cell destruction. Activation of macrophages which leads to their increased phagocytic and cytotoxic activity is mediated inter alia by Gc protein derived macrophage activation factor.
  • Gc protein also known as vitamin D-binding protein, is a glycoprotein of the oc-2 macroglobulin fraction of human plasma having an apparent molecular weight of 52 kDa, constituting about 0.5% of plasma proteins in healthy human subjects. Two genes of Gc protein have been identified: Gel and Gc2 which differ from each other by four amino acids.
  • Gel protein carries a trisaccharide composed of N-acetylgalactosamine attached to the core protein, sialic acid and galactose (the Gclf and Gels* subtypes), or N-acetylgalactosamine, mannose and galactose (the Gels subtype) while Gc2 protein carries a disaccharide composed of N-acetylgalactosamine and galactose.
  • GcMAF has been shown to activate macrophages and to play a tumoricidal role. It was demonstrated that GcMAF inhibited proliferation and migration of cancer cells (Pacini et.al, 2011 , Cancer Immunol. Immunother. 60(4):479-485). In addition, it was shown that systemic administration of GcMAF to tumor bearing mice inhibited the rate of tumor growth of various solid tumors and caused regression of established tumors (Yamamoto et al., 1997, Cancer Research, 57:2187-2192; Korbelik et al., 1997, Br. J. Cancer 75(2): 202-207; Kisker et al, 2003, Neoplasia 5(1): 32-40).
  • WO 2012/137199 to the applicant of the present invention discloses pharmaceutical compositions comprising GcMAF, particularly GcMAF compositions which are essentially devoid of glycosidase enzymes, and method of producing same.
  • WO 2014/199373 to the applicant of the present invention discloses storage- stable pharmaceutical compositions comprising GcMAF and at least one pharmaceutically acceptable surfactant and/or a synthetic water-soluble polymer having surface activity and uses thereof for treating diseases associated with macrophage activation.
  • the present invention provides methods for treating cancer or infectious diseases comprising administering to a subject in need of such treatment a pharmaceutical composition comprising an agent that reduces or inhibits the signal transduction mediated by PD-1 in combination with a pharmaceutical composition comprising a macrophage activating factor derived from the Gc protein denoted GcMAF.
  • a pharmaceutical composition comprising an antibody which specifically binds to PD-1 and a pharmaceutical composition comprising GcMAF.
  • the present invention discloses for the first time that a combination therapy of an anti PD-1 antibody and GcMAF was efficient in arresting tumor growth. While administration of GcMAF as a sole medicament to a patient having pulmonary metastatic sarcoma was capable of arresting tumor growth for several months, administration of a combination therapy of Keytruda®, a humanized anti- PD-1 antibody, and GcMAF to that patient stabilized the disease and even eradicated pulmonary metastases.
  • GcMAF oral cavity squamous cell carcinoma
  • the present invention therefore discloses the therapeutic efficacy of a combination of anti PD-1 antibody and GcMAF in arresting tumor growth.
  • the combination therapy of anti PD-1 antibody and GcMAF is particularly advantageous in cancer patients whose expression of PD-L1 on the tumors cells and/or adjacent cells might become high during GcMAF treatment, as the combination potentiates the immune response, leading to tumor growth arrest and even to tumor eradication.
  • the combination of two therapies (i) the therapy targeted to inhibition of PD-1 signaling, e.g., anti PD-1 antibody; and (ii) GcMAF, provides improved treatment than each one of the therapies alone.
  • the present invention provides a method for treating cancer comprising administering to a subject in need of such treatment a first pharmaceutical composition comprising a therapeutically effective amount of an agent that reduces or inhibits the signal transduction mediated by PD-1, and a second pharmaceutical composition comprising a therapeutically effective amount of a macrophage activating factor derived from Gc protein, denoted GcMAF, or a biologically active fragment thereof.
  • a first pharmaceutical composition comprising a therapeutically effective amount of an agent that reduces or inhibits the signal transduction mediated by PD-1
  • a second pharmaceutical composition comprising a therapeutically effective amount of a macrophage activating factor derived from Gc protein, denoted GcMAF, or a biologically active fragment thereof.
  • the agent that reduces or inhibits the signal transduction mediated by PD-1 is an antibody selected from the group consisting of: (i) an anti-PD-1 antibody; (ii) an anti-PD-Ll antibody; (iii) an anti-PD-L2 antibody; (iv) an antigen binding fragment of (i), (ii), or (iii); and (v) a combination of two or more of (i), (ii), (iii) and (iv).
  • an antibody selected from the group consisting of: (i) an anti-PD-1 antibody; (ii) an anti-PD-Ll antibody; (iii) an anti-PD-L2 antibody; (iv) an antigen binding fragment of (i), (ii), or (iii); and (v) a combination of two or more of (i), (ii), (iii) and (iv).
  • the antibody is selected from the group consisting of a humanized antibody, a chimeric antibody, a non-human antibody, and a human antibody. Each possibility represents a separate embodiment of the invention. According to a certain embodiment, the antibody is a monoclonal antibody.
  • the anti-PDl antibody is selected from the group consisting of Pembrolizumab, Nivolumab and Pidilizumab. Each possibility represents a separate embodiment of the invention. According to a certain embodiment, the anti-PD-1 antibody is Pembrolizumab.
  • the anti-PD-Ll antibody is Atezolizumab.
  • the agent that reduces or inhibits the signal transduction mediated by PD-1 is a fusion protein comprising a PD-1 polypeptide or a PD-L1 polypeptide or a PD-L2 polypeptide or a biologically active fragment thereof conjugated to a carrier protein including, but not limited to, an immunoglobulin or a fragment thereof.
  • the fusion protein is PD-L2/IgGl fusion protein named AMP-224.
  • the GcMAF comprises an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO: 3 (Gclf, Gels and Gc2, respectively) or any combination thereof. Each possibility represents a separate embodiment of the invention. According to yet further embodiments, the GcMAF consists of the amino acid sequence selected from the group consisting of SEQ ID NOs:l-3 or any combination thereof. Each possibility represents a separate embodiment of the invention.
  • the GcMAF fragment comprises an amino acid sequence corresponding to amino acids 400-435 of the Gc Protein. According to still further embodiments, the GcMAF fragment comprises the amino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:5. According to yet further embodiments, the GcMAF fragment consists of the amino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:5.
  • the cancer is selected from the group consisting of solid tumors and hematologic malignancies.
  • the cancer is selected from the group consisting of solid tumors and hematologic malignancies.
  • the solid tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, colorectal cancer, liver cancer, head/neck cancer, brain cancer, glioblastoma, kidney cancer, bladder cancer, stomach cancer, uterus cancer, cervical cancer, oropharyngeal cancer, skin cancer, and metastases thereof.
  • the cancer is lung cancer and metastases thereof.
  • the cancer is oral cavity squamous cell carcinoma (SCC).
  • the hematologic malignancy is selected from the group consisting of leukemias, lymphomas, and multiple myeloma.
  • leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphoblastic leukemia (CLL).
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • the lymphoma is selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma. Each possibility represents a separate embodiment of the invention.
  • administering the first and second pharmaceutical compositions is performed by parenteral administration route including, but not limited to, intravenous, intramuscular, or subcutaneous administration route.
  • the first pharmaceutical composition comprising an anti-PD-1 antibody is administered by intravenous injection and the second pharmaceutical composition comprising GcMAF is injected intramuscularly or subcutaneously.
  • the therapeutically effective amount of an anti-PD-1 antibody ranges from about 0.1 mg/kg body weight to about 20 mg/kg body weight, alternatively from about 1 mg/kg body weight to about 10 mg/kg body weight.
  • the therapeutically effective amount GcMAF ranges from about 10 ng/injection to about 10 mg/injection, alternatively from about 50 ng/injection to about 1 ⁇ g/injection.
  • the first pharmaceutical composition e.g., the pharmaceutical composition comprising an anti-PD-1 antibody
  • the second pharmaceutical composition comprising GcMAF are administered concurrently.
  • said first and second pharmaceutical compositions are administered at different regimens. Examples of regimens for injections include, but are not limited to, twice a week, once a week, once in two weeks, once in three weeks, once a month, etc.
  • the present invention provides a method for treating cancer comprising administering to a subject in need of such treatment a first pharmaceutical composition comprising a therapeutically effective amount of an anti-PD-1 antibody and a second pharmaceutical composition comprising a therapeutically effective amount of GcMAF, wherein the anti PD-1 antibody is Pembrolizumab administered by intravenous injection and GcMAF is administered by intramuscular or subcutaneous injection.
  • the cancer is a lung cancer or metastases thereof.
  • Pembrolizumab in a dosage of 2 mg/kg body weight is injected once in three weeks and GcMAF in a dosage of 100 ng/injection is injected once a week for a period of at least two months, three months, four months, five months, six months, or until cancer is eradicated.
  • the present invention provides a method for treating an infectious disease comprising administering to a subject in need of such treatment a first pharmaceutical composition comprising a therapeutically effective amount of an agent that reduces or inhibits the signal transduction mediated by PD-1, and a second pharmaceutical composition comprising a therapeutically effective amount of macrophage activating factor derived from the Gc protein (GcMAF) or a biologically active fragment thereof according to the principles of the present invention.
  • a first pharmaceutical composition comprising a therapeutically effective amount of an agent that reduces or inhibits the signal transduction mediated by PD-1
  • a second pharmaceutical composition comprising a therapeutically effective amount of macrophage activating factor derived from the Gc protein (GcMAF) or a biologically active fragment thereof according to the principles of the present invention.
  • GcMAF Gc protein
  • the infectious disease is caused by a virus selected from the group consisting of human immunodeficiency virus (HIV), human papilloma virus (HPV), herpes simplex virus (HSV), hepatitis C virus (HCV), and hepatitis B virus (HPV).
  • HAV human immunodeficiency virus
  • HPV human papilloma virus
  • HSV herpes simplex virus
  • HCV hepatitis C virus
  • HPV hepatitis B virus
  • the present invention provides a first pharmaceutical composition comprising an agent that reduces or inhibits the signal transduction mediated by PD-1 and a second pharmaceutical composition comprising a macrophage activating factor derived from the Gc protein (GcMAF) or a biologically active fragment thereof for use in treating cancer or infectious disease according to the principles of the present invention.
  • a first pharmaceutical composition comprising an agent that reduces or inhibits the signal transduction mediated by PD-1 and a second pharmaceutical composition comprising a macrophage activating factor derived from the Gc protein (GcMAF) or a biologically active fragment thereof for use in treating cancer or infectious disease according to the principles of the present invention.
  • GcMAF macrophage activating factor derived from the Gc protein
  • FIGs. 1A-C show the effect of a combination therapy of anti-PD- 1 antibody and GcMAF on pulmonary metastatic sarcoma.
  • FIG. 1A is a CD scan of the lung of a patient who had pulmonary metastatic sarcoma and treated with Keytruda® only.
  • FIG. IB is a CD scan of the lung of the same patient after three months of treatment with Keytruda® and GcMAF
  • FIG. 1C is a CD scan of the same patient after seven months of treatment with Keytruda® and GcMAF.
  • FIG. 2 shows the number of PD-L1 expressing cells in tumor biopsies collected from cancer patients before and 57 days after GcMAF treatment as evaluated by the expression level of PD-L1.
  • FIGs. 3A-B show micrographs of histochemical staining of PD-L1 in a biopsy collected from a patient having oral cavity squamous cell carcinoma (SCC) who was treated with Keytruda® prior to treatment with GcMAF.
  • FIG. 3A shows PD-L1 staining in the biopsy before GcMAF treatment and
  • FIG. 3B shows PD-L1 staining after 57 days of GcMAF treatment.
  • FIGs. 4A-B show micrographs of histochemical staining of CD8 + cells in a biopsy collected from a patient having oral cavity SCC who was treated with Keytruda® prior to treatment with GcMAF.
  • FIG. 4A shows CD8 + cell staining before GcMAF treatment and FIG. 4B after 57 days of GcMAF treatment.
  • the present invention provides methods for treating cancer or infectious diseases comprising administering to a subject in need of such treatment an agent that reduces or inhibits the signal transduction mediated by PD-1, particularly an anti-PD- 1 antibody or an anti-PD-Ll antibody or an anti-PD-L2 antibody or a combination thereof, and GcMAF.
  • an agent that reduces or inhibits the signal transduction mediated by PD-1 particularly an anti-PD- 1 antibody or an anti-PD-Ll antibody or an anti-PD-L2 antibody or a combination thereof, and GcMAF.
  • Gc protein also designated vitamin D binding protein (DBP)
  • DBP vitamin D binding protein
  • Gc protein can be purified from blood serum or plasma by any method known to a person skilled in the art.
  • Gc protein of high purity can be isolated from serum or plasma by 25-hydroxyvitamin D 3 -Sepharose affinity chromatography.
  • the Gc protein can also be purified by actin-agarose affinity chromatography which takes advantage of the binding specificity of the Gc protein for actin.
  • glycosidases ⁇ -galactosidase in combination with sialidase and/or mannosidase to produce macrophage activating factor (MAF) preparations (see, for example, U.S. Pat. No. 5,177,002 and WO 2012/137199, the content of which is incorporated by reference as if fully set forth herein).
  • MAF macrophage activating factor
  • the Gc protein can be obtained from isolated cDNA encoding the Gc protein or the Gc protein small domain (domain III). Cloning and expression of the Gc protein and the Gc domain III was described in U.S. Patent No. 6,410,269. The method described therein employs a human liver cDNA library in bacteriophage gtl 1 (Clontech, Palo Alto, CA) for isolating a full length cDNA encoding the human Gc protein, and the use of the baculoviral expression system in insect cells for the protein expression. However, mammalian cell systems are preferred for expressing a cDNA encoding the Gc protein or active fragment thereof.
  • expression is performed in eukaryotic cells so that the Gc protein or its active domain is correctly glycosylated.
  • Any such cell system known in the art can be used, for example Chinese hamster ovary (CHO) cells, BHK cells, human embryonic kidney HEK293 cells and Saccharomyces cerevisiae.
  • any eukaryotic expression vector can be used, including, but not limited to, pCI-NEO, pWE3, pcDNA3.1 and pCM182.
  • Insertion of the vector into the selected cell system can be performed, for example, by electroporation, by lipid transfection such as TransFectin or by any chemical method known to a person skilled in art, with or without amplification. The transfection may result in transient or stable expression, both forms being adequate to obtain the desired Gc protein or fragment thereof.
  • the expressed protein being the precursor of active MAF, can then be extracted from the cells or collected from the growth media by any method known in the art.
  • Gc protein is a polymorphic protein which appears in two major phenotypes as demonstrable by gel electrophoresis analysis: Gel and Gc2.
  • the entire nucleotide coding sequences of the Gel and Gc2 genes and the predicted amino acid sequences have been reported (Cook et al, 1985. J. Clin. Invest.76: 2420; Yang et al., 1985. Proc. Natl. Acad. Sci. USA 82 7994).
  • Gel is further divided into Gclf and Gels subtypes, which migrate electrophoretically as two bands ("fast” and "slow"), due to a variation in one amino acid residue.
  • Gel protein is the major subtype of human Gc protein. It carries a trisaccharide composed of N-acetylgalactosamine (GalNAc) attached to the core protein with sialic acid and galactose (in Gclf) or mannose and galactose (in Gel s); galactose is believed to be the outer oligosaccharide moiety.
  • Gc2 has a simple glycosylation pattern with a core GalNAc linked to a terminal galactose moiety.
  • Gclf and Gels* oligosaccharides can be hydrolyzed in vitro by ⁇ -galactosidase, preferably immobilized ⁇ -galactosidase such as to acrylic beads, to yield a macrophage pro-activating factor, which in turn can be hydrolyzed by sialidase (also known as neuraminidase), preferably immobilized sialidase, to yield a macrophage activating factor denoted GcMAF.
  • Animal, e.g., mouse or dog, DBP carries a disaccharide composed of N-acetylgalactosamine with a terminal galactose, similarly to that of Gc2. Hydrolysis of this disaccharide by ⁇ - galactosidase generates a potent MAF denoted GcMAF,.
  • Gc protein or “vitamin D-binding protein” as used herein refer to human or animal Gc protein, to all genotypes and polymorphic forms, including glycosylation forms, e.g., Gel, Gc2, Gclf, Gels and Gels*, and biologically active variants and fragments thereof.
  • biologically active variant or fragment refers to any variant or fragment of Gc protein which upon deglycosylation produces a GcMAF variant or fragment, the GcMAF variant or fragment thus produced is capable of activating macrophages and has an N- acetylgalactosamine group linked to an amino acid residue, most likely to a threonine residue.
  • activating macrophages refers to the ability of the MAFs derived from Gc protein to induce cytotoxicity or phagocytosis of a target cell by macrophages and/or to induce cytokine or chemokine release from macrophages and/or to induce Fc receptor expression or translocation.
  • GcMAF refers to a macrophage activating factor (MAF) derived from Gc protein which has been subjected to deglycosylation with ⁇ -galactosidase alone or in combination with sialidase and/or mannosidase.
  • GcMAF is produced by stepwise in vitro deglycosylation of isolated human Gc protein with ⁇ -galactosidase followed by sialidase. The GcMAF produced is therefore partially deglycosylated as compared to the untreated glycosylated Gc protein, having the N-acetylgalactosamine group linked to the core protein, most likely to a threonine residue.
  • the GcMAF comprises the amino acid sequence as set forth in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3, , having an GalNAc moiety linked to a threonine residue at position 418 or 420 of the Gc protein.
  • GcMAF consists of the amino acid sequence selected from the group consisting of: SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3. Each possibility represents a separate embodiment of the present invention.
  • fragment refers to any portion of the full length amino acid sequence of Gc protein which has less amino acids than the full length amino acid sequence of Gc protein, e.g., less than the 458 amino acids of Gc proteins of SEQ ID NOs:l to 3, which portion still contains N-acetylgalactosamine group linked to an amino acid residue, typically to a threonine, and still retains macrophage activating activity.
  • a portion of a full length protein is a peptide or a polypeptide.
  • peptide it is meant an amino acid sequence consisting of not more than 50 amino acids.
  • polypeptide it is meant an amino acid sequence generally consisting of more than 50 amino acid residues, typically up to 200 amino acid residues.
  • the Gc protein fragment comprises an amino acid sequence corresponding to amino acids 400-435 of Gc Protein.
  • the Gc fragment comprises the Gc protein domain III corresponding to amino acids 375- 458 of the mature protein.
  • the Gc fragment Domain III corresponding to amino acids 375-458 of the mature Gc protein, consists of the amino acid sequence as set forth in either SEQ ID NO:4 or SEQ ID NO:5.
  • the N-acetylgalactosamine in these amino acid sequences is linked to threonine at positions 44 or 46.
  • GcMAF variants or fragments retain the desired biological activities of the native GcMAF such that the variants or fragments have the same therapeutic effect as of the native GcMAF when administered to a subject. That is, according to some embodiments, the variant polypeptide will serve as a therapeutically active component in a pharmaceutical composition in a manner similar to that observed for the native polypeptide.
  • variant relates to either the native Gc protein or a fragment of the native Gc protein comprising one or more amino acid substitutions, insertions, or deletions, and or other modifications such as glycosylation.
  • Gc protein fragments have at least 50%, preferably at least 60%, more preferably at least 70%, 80%, 90%, 95%, and most preferably about 98% amino acid sequence identity to the corresponding full length amino acid sequence of the native Gc protein or fragment thereof, respectively, which serve as the basis for comparison.
  • the biologically active variants have at least 70%, 80%, 90%, 95%, and most preferably about 98% amino acid sequence identity to Domain III of the Gc protein, which serves as the basis for comparison.
  • Each possibility represents a separate embodiment of the present invention.
  • sequence identity relates to having the same amino acid residues within a variant polypeptide and the corresponding polypeptide molecule that serves as a reference when a specified, contiguous segment of the amino acid sequence of the variant is aligned and compared to the amino acid sequence of the reference molecule.
  • the percentage sequence identity between two amino acid sequences is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the segment undergoing comparison to the reference molecule, and multiplying the result by 100 to yield the percentage of sequence identity.
  • substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Antibodies and antagonists to PD-1 signaling
  • the present invention provides pharmaceutical compositions comprising an agent that reduces or inhibits the signal transduction mediated by PD-1 in combination with pharmaceutical compositions comprising GcMAF or a biologically active fragment thereof for treating cancer or infectious diseases.
  • the present invention thus provides pharmaceutical compositions comprising isolated anti-PD-1 antibodies and/or anti-PD-Ll antibodies and/or anti-PD-L2 antibodies in combination with pharmaceutical compositions comprising GcMAF or a biologically active fragment thereof for treating cancer or infectious diseases.
  • the present invention further provides pharmaceutical compositions comprising antibody fragments that bind to PD-1 and/or to PD-L1 and /or to PD-L2 in combination with pharmaceutical compositions comprising GcMAF or a biologically active fragment thereof for treating cancer or infectious diseases.
  • the antibody or antibody fragment useful in the present invention can be a human antibody, humanized antibody, a chimeric antibody, a non-human antibody, or an antigen binding fragment thereof.
  • antibody refers to any form of antibody that exhibits the desired biological activity, such as inhibiting binding of a ligand to its receptor, or inhibiting ligand-induced signaling of a receptor.
  • antibody is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies).
  • antibody fragment and “antibody binding fragment” used interchangeably throughout the application mean antigen-binding fragments, typically including at least a portion of the antigen binding or variable regions (e.g. one or more CDRs) of the parental antibody.
  • An antibody fragment retains at least some of the binding specificity of the parental antibody. Typically, an antibody fragment retains at least 10% of the parental binding activity when that activity is expressed on a molar basis. Preferably, an antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the parental antibody's binding affinity for the target.
  • Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv fragments; single-chain antibody molecules, e.g., sc-Fv.
  • the "Fab fragment” is comprised of one light chain and the CRI and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a "Fab' fragment” contains one light chain and a portion of one heavy chain that contains the V H domain and the CRI domain and also the region between the CRI and CR2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab') 2 molecule.
  • a “F(ab') 2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab') 2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • the "Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • single-chain Fv antibody refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding.
  • An "Fc" region contains two heavy chain fragments comprising the CHI and CR2 domains of an antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • an “isolated” antibody is an antibody that has been separated and/or recovered from a component of its natural environment.
  • the antibody will be purified to greater than 95 % by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight.
  • a “human antibody” is an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human. This definition specifically excludes a humanized antibody that comprises non-human antigen- binding residues.
  • a “chimeric” antibody refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a “complementarity determining region” or "CDR".
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method, or may be made by recombinant DNA methods.
  • the monoclonal antibodies may also be isolated from phage antibody libraries.
  • telomere binding affinity ⁇ 10 "5 M.
  • the antibody via its antigen-binding domain can bind to the antigen with a Kd of ⁇ 10 "6 M or ⁇ 10 " M.
  • Kd refers to the ratio of the dissociation rate to the association rate (ko ff /k on ), and may be determined using any suitable methods known in the art.
  • Anti-PD-1 antibodies useful in practicing the present invention include, but are not limited to, the humanized antibody MK-3945 named Lambrolizumab, now named Pembrolizumab (Keytruda ® ; see U.S. Patent No. 8,952,136, the content of which is incorporated by reference as if fully set forth herein); the human monoclonal antibody (mAB) BMS-936558/MDX-1106/ONO-4538 named Nivolumab; and the humanized antibody CT-011 named Pidilizumab.
  • the humanized antibody MK-3945 named Lambrolizumab
  • Pembrolizumab Keytruda ®
  • Nivolumab the human monoclonal antibody
  • CT-011 named Pidilizumab.
  • anti-PD-1 antibodies include, but are not limited to, the antibodies described in the following publications: WO/2003/099196 to Hardy et al.; WO/2006/121168 and WO/2007/005874 to Korman et al; WO/2009/014708 to Li et al.; WO/2004/004771 and WO/2004/072286 to Honjo et al; WO/2004/056875 to Collins et al; WO/2008/083174 to Ahmed et al; WO/2009/073533 to Terrett et al; Berger et al, Clin. Cancer Res., Vol.
  • Anti-PD-Ll antibodies useful in practicing the present invention include, but are not limited to, the human mAb named BMS-936559; RG7446/MPDL3280A; MEDI4736; and the human antibody MDX-1105 (see, for example, WO/2007/005874, the content of which is incorporated by reference as if fully set forth herein).
  • anti-PD-Ll antibodies include, but are not limited to, the antibodies described in the following publications: WO/2006/133396; WO/2008/083174; and US 2006/0110383 (the content of the publications is incorporated by reference as if fully set forth herein).
  • the present invention further encompasses a bi-specific antibody that comprises an antibody that binds to the PD-1 receptor bridged to an antibody that binds to a ligand of PD-1, such as PD-L1.
  • a bi-specific antibody that comprises an antibody that binds to the PD-1 receptor bridged to an antibody that binds to a ligand of PD-1, such as PD-L1.
  • the antibodies or antibody fragments disclosed herein reduce or inhibit signal transduction through the PD- 1 receptor.
  • the antibody or antibody fragment of the invention may bind to human PD- 1 with a KD of about 100 pM or lower. In another embodiment, the antibody or antibody fragment may bind to human PD-1 with a K D of about 30 pM or lower. Similarly, the antibody or antibody fragment may bind to human PD-L1 or PD-L2 with a K D of about 100 pM or lower.
  • the isolated antibodies and antibody fragments of the invention may lead to an increase in T cell activation and/or to an increase in macrophage activation as measured by typical means known to one skilled in the art (including, without limitation, increased T cell/macrophage proliferation and/or increased cytokine secretion and/or increased number of T cells/macrophages in a tumor site).
  • antibodies to PD-1 or to its ligands are preferable for reducing or inhibiting the signal transduction mediated by the PD-1 receptor
  • antagonists to PD-1 or to its ligands are also encompassed in the present invention. These antagonists are agents that, similarly to the antibodies, either specifically bind to a ligand of PD-1 to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 without inducing inhibitory signal transduction through the PD-1.
  • an agent that reduces or inhibits the signal transduction mediated by PD-1 refers to an agent, e.g., antibody or an antagonist, which reduces or inhibits the inhibitory signal mediated by PD-1 and thereby elicits a more pronounced immune response.
  • the increased immune response can be determined by T cell and/or macrophage proliferation and/or T cell or macrophage infiltration into a tumor site and/or cytokine or chemokine secretion.
  • the PD-1 antagonist like an anti PD-1 antibody, binds directly to the PD-1 without triggering inhibitory signal transduction.
  • An antagonist to PD-Ll like an anti PD-Ll antibody, binds to the ligand of the PD-1 , i.e., PD-Ll, to reduce or inhibit the ligand from triggering signal transduction through the PD-1.
  • An antagonist to PD-L2, like an anti PD-L2 antibody binds to the ligand of the PD-1 , i.e., PD-L2, to reduce or inhibit the ligand from triggering signal transduction through the PD-1.
  • Exemplary PD-1 antagonists include, but are not limited to, PD-Ll or PD-L2 polypeptides, including homologs and variants thereof, as well as active fragments of any of the foregoing, and fusion proteins that incorporate any of these.
  • the fusion protein comprises the soluble portion of PD-L2 coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human PD-L2.
  • Such PD-1 antagonists interfere with PD-1 signaling, thereby not triggering inhibitory signal transduction through the PD-1 receptor.
  • PD-Ll polypeptides PD-L2 polypeptides, PD-1 polypeptides, including variants as well as active fragments of any of the foregoing, are listed, for example, in U.S. Patent No. 8,709,416, the content of which is incorporated by reference as if fully set forth herein.
  • the present invention provides a pharmaceutical composition comprising an agent that interferes or inhibits the signaling through PD-1 in combination with a pharmaceutical composition comprising GcMAF or a biologically active fragment thereof, for use in treating cancer or infectious diseases.
  • an agent that interferes or inhibits the signaling through PD-1 includes an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, and any antigen binding fragment thereof, PD-1 polypeptide, PD-L1 polypeptide, and PD-L2 polypeptide, and a fusion protein comprising a PD-1 polypeptide or a PD-L1 polypeptide or a PD-L2 polypeptide or a fragment thereof conjugated to a carrier protein such as to an immunoglobulin or a fragment thereof.
  • the active agents i.e., the agent that interferes or inhibits the signaling of PD- 1 and GcMAF
  • the active agents i.e., the agent that interferes or inhibits the signaling of PD-1 such as the antibodies disclosed herein above, and GcMAF
  • the compositions can further comprise pharmaceutically acceptable carriers and/or excipients.
  • carrier refers to a diluent or vehicle with which the active agent is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the pharmaceutical compositions can further comprise as an excipient a pharmaceutically acceptable surfactant.
  • the surfactant can be a nonionic, anionic, cationic, zwitterionic, and amphoteric pharmaceutically acceptable surfactant which is useful in medicaments for human, or for animals. TWEEN® and Poloxamer surfactants are preferred because they are FDA approved for human use.
  • the pharmaceutical composition comprising GcMAF comprises a pharmaceutically acceptable surfactant present in an amount of from 0.001 % to 10% by weight of the total weight of the composition.
  • the pharmaceutical compositions can further comprise agents for adjustment of tonicity including, but not limited to, sodium chloride or dextrose.
  • Sodium chloride is used in order to keep the osmotic pressure of the pharmaceutical composition of the present invention suitable for an injectable preparation.
  • the pharmaceutical composition comprising GcMAF can comprise sodium chloride in a concentration ranging from about 25 mM to about 300 mM.
  • compositions can further comprise a buffering agent.
  • buffering agents that can be included in the compositions of the present invention are, for example, phosphate buffer, acetate buffer, Tris buffer and citrate buffer.
  • the pharmaceutical composition comprising GcMAF comprises a nonionic surfactant, a tonicity agent, a buffering agent, and water.
  • the pharmaceutical composition comprising GcMAF further comprises polysorbate 80, sodium chloride, phosphate buffer, and water.
  • the pharmaceutical composition comprises GcMAF, phosphate buffered saline, and 0.005 % (w/w) polysorbate 80.
  • the pharmaceutical compositions of the present invention can be formulated as a liquid, such as a solution, suspension, or emulsion, i.e., oil-in-water emulsion, water- in-oil emulsion, microemulsion or nanoemulsion.
  • the pharmaceutical composition can be prepared in a dried form, such as a lyophilized powder, which can be reconstituted into a liquid solution, suspension, or emulsion before administration, for example, by parenteral administration such as by subcutaneous, intramuscular or intravenous administration.
  • the pharmaceutical compositions are preferably sterilized by membrane filtration and are stored in unit-dose or multi-dose containers such as sealed vials or ampoules.
  • the pharmaceutical compositions can also take the form of tablets, capsules, sustained-release formulations and the like. Each possibility is a separate embodiment of the invention.
  • the pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.
  • the present invention further provides a kit comprising: (i) a pharmaceutical composition comprising an agent that interferes or inhibits the signaling through PD- 1 ; and (ii) a pharmaceutical composition comprising GcMAF or a biologically active fragment thereof.
  • a pharmaceutical composition comprising an agent that interferes or inhibits the signaling through PD- 1 ; and a pharmaceutical composition comprising GcMAF or a biologically active fragment thereof.
  • each one of the pharmaceutical compositions of the kit is present or contained in a separate container.
  • the pharmaceutical compositions of the kit are present or contained in the same container.
  • the present invention provides a method for increasing, augmenting, enhancing or stimulating an immune response comprising administering to a subject in need of such treatment: (i) a pharmaceutical composition comprising a therapeutically effective amount of an agent that interferes, reduces or inhibits the signaling through PD-1 ; and (ii) a pharmaceutical composition comprising a therapeutically effective amount of Gc macrophage activating factor (GcMAF), or a biologically active fragment thereof.
  • GcMAF Gc macrophage activating factor
  • the pharmaceutical composition of the present invention are particularly suitable for treating subjects having a disease that can be treated by increasing T-cell mediated immune response.
  • the pharmaceutical compositions of the present invention are therefore useful for treating subjects having cancer or an infectious disease.
  • compositions of the present invention provide synergistic effect and are advantageous in cases where an anti-PD-l/anti-PD-Ll/anti-PD-L2 antibody or GcMAF alone are not sufficiently potent.
  • the cancer to be treated by the methods of the present invention is selected from the group consisting of solid tumors and hematologic malignancies.
  • Solid tumors include, but are not limited to, lung cancer, breast cancer, ovarian cancer, prostate cancer, uterus cancer, pancreatic cancer, colorectal cancer, liver cancer, stomach cancer, head/neck cancer, brain cancer, glioblastoma, kidney cancer, bladder cancer, skin cancer, and any cancer associated with HPV infection, including cervical cancer, anal cancer, penile cancer, and oropharyngeal cancer.
  • the cancer is lung cancer.
  • Leukemias and lymphomas include, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblasts, promyelocytic, myelomonocytic, and erythroleukemia syndrome, chronic leukemias such as chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia (CLL), and hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, childhood leukemia, and childhood lymphoma.
  • acute leukemia acute lymphocytic leukemia
  • acute myelocytic leukemias such as myeloblasts, promyelocytic, myelomonocytic, and erythroleukemia syndrome
  • chronic leukemias such as chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia (CLL), and hairy cell
  • the cancers to be treated by the methods of the present invention include, but are not limited to, squamous cell carcinoma, sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma bile duct carcinoma, choriocarcinoma, seminoma, embryon
  • the present invention thus provides a method for treating cancer or an infectious disease comprising administering to a subject in need of such treatment: (i) a pharmaceutical composition comprising a therapeutically effective amount of an agent that interferes, reduces or inhibits the signaling through the PD-1 ; and (ii) a pharmaceutical composition comprising a therapeutically effective amount of Gc macrophage activating factor (GcMAF), or a biologically active fragment thereof.
  • a pharmaceutical composition comprising a therapeutically effective amount of an agent that interferes, reduces or inhibits the signaling through the PD-1 ; and (ii) a pharmaceutical composition comprising a therapeutically effective amount of Gc macrophage activating factor (GcMAF), or a biologically active fragment thereof.
  • GcMAF Gc macrophage activating factor
  • the present invention further provides a pharmaceutical composition comprising an agent that interferes, reduces or inhibits the signaling of PD-1, preferably an antibody which specifically binds to PD-1 and/or an antibody which specifically binds to PD-L1 and/or an antibody which specifically binds to PD-L2, and a pharmaceutical composition comprising GcMAF, or a biologically active fragment thereof, for use in treating cancer or an infectious disease in a subject.
  • a pharmaceutical composition comprising an agent that interferes, reduces or inhibits the signaling of PD-1, preferably an antibody which specifically binds to PD-1 and/or an antibody which specifically binds to PD-L1 and/or an antibody which specifically binds to PD-L2, and a pharmaceutical composition comprising GcMAF, or a biologically active fragment thereof, for use in treating cancer or an infectious disease in a subject.
  • compositions of the present invention on cancer cell replication is measured in comparison to the growth or replication of untreated or control treated subjects.
  • a control treated subject is a subject having the diseases and being treated by administering either the antibody or GcMAF.
  • Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with cancer or infectious disease, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay the onset of one or more symptoms or slow the progression of one or more symptoms.
  • the efficacy of the treatment may be measured in any manner.
  • treatment may result in an inhibition or lessening of adverse symptoms of the disease, may ameliorate the disease, or may cure the disease.
  • this may be measured by inhibition of metastases, reduction of tumor size, reducing progression of a cancer to a less aggressive form, reducing proliferation of cancer cells, reducing the rate of tumor growth, reducing the likelihood of recurrence of a cancer in a subject, prolonging overall survival of a subject or prolonging the progression-free time period or disease-free time period.
  • treatment may result in decreased symptoms.
  • a “therapeutically effective amount” as used herein means the amount of an active agent that is sufficient to effect a treatment (as defined above).
  • the therapeutically effective amount will vary depending on the active agent, formulation, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject to be treated.
  • the agents are administered to a subject, e.g., a cancer patient, in an amount effective to enhance treatment of the disease by increasing clearance of the diseased cells, e.g., cancer cells, from the subject or blocking proliferation of the diseased cells, e.g., cancer cells, such that a therapeutic effect is achieved in the subject.
  • compositions of the invention can be administered via a parenteral route of administration, such as by intravenous, intramuscular, subcutaneous, intraarterial and intraperitoneal injection or infusion.
  • parenteral route of administration such as by intravenous, intramuscular, subcutaneous, intraarterial and intraperitoneal injection or infusion.
  • the pharmaceutical compositions can be administered topically, nasally, or by oral, rectal or vaginal administration route.
  • the pharmaceutical compositions comprising anti-PD-1 and GcMAF are administered by injection; the anti PD-1 antibody is administered by intravenous injection and GcMAF is administered by intramuscular or subcutaneous injection.
  • compositions of the invention may be administered locally to the area in need of treatment; this may be achieved, for example, and not by way of limitation, by direct injection, e.g., via a syringe, at the site of a tumor or neoplastic tissue or by topical application, e.g., in conjunction with a wound dressing after surgery, by means of a medical patch or by means of an implant, said implant being of a porous, non-porous, or gelatinous material.
  • the GcMAF can be administered at a dose ranging from 10 ng/injection to 10 mg/injection, alternatively at a dose ranging from 50 ng/injection to 1 ⁇ g/injection, or any integer in between. According to an exemplary embodiment, GcMAF is administered at a dose of 100 ng/injection.
  • the dosage of GcMAF ranges from about 10 pg/kg to about 200 g/kg of the subject's weight or any integer in between. According to some embodiments, the dosage of GcMAF ranges from about 100 pg/kg to about 100 ng/kg of the subject's weight. According to yet further embodiments, the dosage of GcMAF ranges from about 0.5 ng/kg to about 20 ng/kg of the subject's weight. According to further embodiments, the dosage of GcMAF ranges from about
  • the anti-PD-1 antibody can be administered at a dose ranging from about 0.1 mg/kg to 20 mg/kg body weight, alternatively at a dose ranging from 1 mg/kg to 10 mg/kg body weight, or at a dose of
  • composition comprising the antibody and the composition comprising GcMAF may be administered as two separate dosage forms via the same or different routes.
  • the composition comprising the antibody and the composition comprising GcMAF may be administered such that one is administered before the other with a difference in administration time of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours or more or any integer in between.
  • composition comprising the antibody and the composition comprising GcMAF can be administered more than once.
  • administrations may be repeated, for example, every 1 , 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • the maximal dosage of an active agent for a subject is the highest dosage that does not cause undesirable or intolerable side effects.
  • a therapeutically effective amount of an antibody and GcMAF according to the present invention arrests tumor growth.
  • a therapeutically effective amount of an antibody and GcMAF according to the present invention reduces the size of a tumor in a subject by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%.
  • a therapeutically effective amount of an antibody and GcMAF according to the present invention decreases or eradicates tumor metastases.
  • the method of treating cancer according to the present invention can further comprise administering a therapeutic agent, such as, but not limited to, a chemotherapeutic agent.
  • a therapeutic agent such as, but not limited to, a chemotherapeutic agent.
  • the chemotherapeutic agent is selected from the group consisting of an alkylating agent, an anti-metabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic and a combination thereof.
  • a therapeutic agent such as, but not limited to, a chemotherapeutic agent.
  • the chemotherapeutic agent is selected from the group consisting of an alkylating agent, an anti-metabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic and a combination thereof.
  • the combination therapy of the present invention can also be coupled with surgery, radiation therapy, hormonal therapy or any other approach to affect cancer treatment.
  • the subject is a human subject.
  • the subject is a mammal such as, but not limited to, a dog, a cat, a horse, a pig and a cow.
  • compositions of the present invention while intended for use in the treatment of a disease or condition, may also find use as prophylactic compositions to prevent any of the diseases recited herein.
  • the following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. All references cited herein are hereby incorporated by reference in their entireties.
  • a patient having pulmonary metastatic sarcoma received a compassionate treatment with GcMAF.
  • GcMAF was prepared as previously described (Yamamoto et al., 2008, Cancer Immunol. Immunother. 57: 1007-1016). Briefly, Gc protein was purified from human plasma using 25-hydroxyvitamin D3-affnity chromatography. The purified Gc was incubated sequentially with immobilized ⁇ -galactosidase and sialidase, preferably to acrylic beads, to form GcMAF. The GcMAF was filtered through a 0.22 micron filter for sterilization, and then diluted to a final concentration of 200 ng/ml in PBS pH 7.4 containing 0.005 % (w/w) polysorbate 80 (TWEEN ® 80).
  • the patient having pulmonary metastatic sarcoma received weekly injections of GcMAF (100 ng/each injection) intramuscularly (IM) for one year. As a result, disease stabilization was achieved for about 6 months. Thereafter, relapse accompanied with new lesions in the lungs occurred. At that stage, the patient received a combination therapy: GcMAF was administered by weekly IM injections (100 ng/each injection) and anti-PD-1 antibody (Keytruda®, 2 mg/kg body weight) was administered by intravenous (IV) injections every three weeks. The combination therapy continued for additional 6 months. This combination therapy resulted in disease stabilization and complete disappearance of one of the lung metastases (FIGs. IB and 1C). Thus, the combination therapy of GcMAF and anti PD-1 antibody was found to be more efficacious than the GcMAF treatment alone.
  • GcMAF Phase 1 clinical study of GcMAF.
  • the patients received chemotherapy alone or chemotherapy in combination with anti PD-1 antibody (Keytruda®) or with EGF- receptor inhibitor Erlotinib (Table 1).
  • the treatments were terminated, and after one month GcMAF, prepared as described in Example 1 herein above, was injected subcutaneously (SC) or intramuscularly (IM) at a dose of 100 ng/each injection to 1000 ng/each injection (Table 1).
  • the patients were followed up for clinical response by CT scans and by assessment of the Response Evaluation Criteria in Solid Tumors (RECISTl.l) or of the immune related Criteria (irRC).
  • the immune profile of the tumor site was also assessed by immunohistochemistry of tumor biopsies collected before GcMAF treatment and on day 57 of GcMAF treatment.
  • the biopsies were stained with different CD markers and analyzed by specialized algorithms for the quantification of stained cells and cell expression levels.
  • the patient having oral cavity squamous cell carcinoma (SCC) treated with GcMAF showed stabilization of the disease (SD) as detected by the Response Evaluation Criteria in Solid Tumors (RECISTl.l) and by the immune related criteria (irRC).
  • FIG. 3 shows histochemical staining of PD-Ll in a biopsy sample collected from the patient having oral cavity SCC before GcMAF treatment and after 57 days of GcMAF treatment.
  • FIG. 3 clearly shows higher staining of PD-Ll after 57 days of GcMAF treatment.
  • FIG. 4 shows histochemical staining of CD8 + T cells in a biopsy collected from the patient having oral cavity SCC and treated with GcMAF for 57 days. As seen in FIG. 4, a significant increase (115% compared to baseline) was noted in the level of CD8 + cytotoxic T cells in this biopsy after GcMAF treatment.

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Abstract

La présente invention concerne des procédés de traitement du cancer ou de maladies infectieuses consistant à administrer à un sujet un agent qui réduit ou inhibe la transduction du signal à médiation par PD-1 en association avec un facteur d'activation des macrophages dérivé de la protéine Gc (GcMAF). En particulier, la présente invention consistant à administrer à un sujet un anticorps anti-PD-1 et GcMAF.
PCT/IL2016/050365 2015-04-08 2016-04-07 Thérapie combinée composée d'un facteur d'activation des macrophages et d'inhibiteurs de la voie de signalisation pd-1 WO2016162867A1 (fr)

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US11071730B2 (en) 2018-10-31 2021-07-27 Gilead Sciences, Inc. Substituted 6-azabenzimidazole compounds
CN113453685A (zh) * 2019-02-18 2021-09-28 美迪维尔公司 使用口服给药的二氧戊环核苷酸与抗pd1或抗pdl1单克隆抗体的组合治疗肝癌的方法
US11203591B2 (en) 2018-10-31 2021-12-21 Gilead Sciences, Inc. Substituted 6-azabenzimidazole compounds
US11453681B2 (en) 2019-05-23 2022-09-27 Gilead Sciences, Inc. Substituted eneoxindoles and uses thereof

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WO2014113641A1 (fr) * 2013-01-18 2014-07-24 Kline Ellis Régime de glycosidase sélective pour la programmation immunitaire et le traitement du cancer
WO2014199373A1 (fr) * 2013-06-09 2014-12-18 Efranat Ltd. Composition comprenant le facteur d'activation des macrophages gc et utilisations associées

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10441654B2 (en) 2014-01-24 2019-10-15 Children's Hospital Of Eastern Ontario Research Institute Inc. SMC combination therapy for the treatment of cancer
US11071730B2 (en) 2018-10-31 2021-07-27 Gilead Sciences, Inc. Substituted 6-azabenzimidazole compounds
US11203591B2 (en) 2018-10-31 2021-12-21 Gilead Sciences, Inc. Substituted 6-azabenzimidazole compounds
US11897878B2 (en) 2018-10-31 2024-02-13 Gilead Sciences, Inc. Substituted 6-azabenzimidazole compounds
US11925631B2 (en) 2018-10-31 2024-03-12 Gilead Sciences, Inc. Substituted 6-azabenzimidazole compounds
CN113453685A (zh) * 2019-02-18 2021-09-28 美迪维尔公司 使用口服给药的二氧戊环核苷酸与抗pd1或抗pdl1单克隆抗体的组合治疗肝癌的方法
US11453681B2 (en) 2019-05-23 2022-09-27 Gilead Sciences, Inc. Substituted eneoxindoles and uses thereof
US12037342B2 (en) 2019-05-23 2024-07-16 Gilead Sciences, Inc. Substituted eneoxindoles and uses thereof

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