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  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
  • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
  • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
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  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K39/0005—Vertebrate antigens
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• • A—HUMAN NECESSITIES
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  • 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 [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • A—HUMAN NECESSITIES
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  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
  • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
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  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55588—Adjuvants of undefined constitution
  • A61K2039/55594—Adjuvants of undefined constitution from bacteria
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6006—Cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

• iNKT Invariant natural killer T
• iNKT cells express an invariant a chain T cell receptor (Va24-Jal8 in humans and Val4-Jal8 in mice) that is specifically activated by certain glyco lipids presented in the context of the non-polymorphic MHC class T-like protein, CD Id.
• CD Id binds to a variety of dialkyl lipids and glycolipids, such as the glycosphingolipid a-galactosylceramide (a-GalCer).
• iNKT cell TCR recognition of the CD Id- lipid complex results in the release of pro -inflammatory and regulatory cytokines, including the Thl cytokine interferon gamma (IENg). The release of cytokines in turn activates adaptive cells, such as T and B cells, and innate cells, such as dendritic cells and NK cells.
• IENg Thl cytokine interferon gamma
• a-GalCer also known as KRN7000, chemical formula C50H99NO9, is a synthetic glyco lipid derived from structure- activity relationship studies of galactosylceramides isolated from the marine sponge Agelas mauritianus.
• a-GalCer is a strong immunostimulant and shows potent anti-tumor activity in many in vivo models
• a major challenge to using a-GalCer for immunotherapy is that it induces anergy in iNKT cells because it can be presented by other CD Id expressing cells, such as B cells, in the peripheral blood. Delivery of a-GalCer also has been shown to induce liver toxicity.
• compositions and methods are needed for delivery of a-GalCer to phagocytic cells and inducing an effective immune response against tumor cells.
• the present invention satisfies these needs.
• adjuvant compositions comprising an immunogenically effective amount of intact, bacterially derived minicells or killed bacterial cells that encapsulate a CD ld-restricted invariant Natural Killer T (iNKT) cell antigen.
• iNKT Natural Killer T
• the encapsulated CD ld-restricted iNKT cell antigen is capable of uptake by a phagocytic cell, such as a dendritic cell or a macrophage.
• the CD ld-restricted iNKT cell antigen form complexes with CD Id within the lysosomes of the phagocytic cells and is subsequently transported to the surface of the phagocytic cells where the CD ld-restricted iNKT cell antigen bound to CD Id is presented for recognition by an iNKT cell.
• the CD ld-restricted iNKT cell antigen induces a Thl cytokine response by an iNKT cell that recognizes the CD ld-restricted iNKT cell antigen bound to CD Id on the surface of the phagocytic cell.
• the CD ld-restricted iNKT cell antigen is a glycosphingolipid.
• the glycosphingolipid is selected from among a-galactosylceramide (a- GalCer), C-glycosidific form of a-galactosylceramide (a-C-GalCer), 12 carbon acyl form of galactosylceramide (b-GalCer), b-D-glucopyranosylceramide (b-GlcCer), l,2-Diacyl-3-0- galactosyl-sn-glycerol (BbGL-II), diacylglycerol containing glycolipids (Glc-DAG-s2), ganglioside (GD3), gangliotriaosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), a- glucuro
• the glycosphingolipid is a-GalCer. In some embodiments, the glycosphingolipid is a synthetic a-GalCer analog.
• the synthetic a-GalCer analog is selected from among 6'-deoxy-6'-acetamide a- GalCer (PBS57), napthylurea a-GalCer (NU-a-GC), NC-a-GalCer, 4ClPhC-a-GalCer, PyrC-a- GalCer, a-carba-GalCer, carba-a-D-galactose a-GalCer analog (RCAI-56), 1-deoxy-neo-inositol a-GalCer analog (RCAI-59), 1-O-methylated a-GalCer analog (RCAI-92), and HS44 aminocyclitol ceramide.
• the CD ld-restricted iNKT cell antigen is derived from a bacterial antigen, a fungal antigen, or a protozoan antigen.
• the adjuvant composition comprises (a) an immunogenically effective amount of an encapsulated CD ld-restricted invariant Natural Killer T (iNKT) cell antigen and (b) a therapeutically effective dose of an antineoplastic agent.
• iNKT Natural Killer T
• the CD ld-restricted iNKT cell antigen and the antineoplastic agent are packaged within two or more intact bacterially derived minicells or killed bacterial cells.
• the adjuvant composition comprises the CD ld-restricted iNKT cell antigen and the antineoplastic agent, wherein: (a) the CD ld-restricted iNKT cell antigen and the antineoplastic agent are comprised within the same intact bacterially-derived minicell or killed bacterial cell; or (b) the CD ld-restricted iNKT cell antigen is comprised within a first intact bacterially-derived minicell or killed bacterial cell, and the antineoplastic agent is comprised within a second intact bacterially-derived minicell or killed bacterial cell.
• the intact bacterially-derived minicell comprising the antineoplastic agent comprises at least one targeting agent.
• the intact bacterially-derived minicell comprising the CD ld-restricted iNKT cell antigen does not comprise a targeting agent, and the intact bacterially-derived minicell comprising the antineoplastic agent comprises a targeting agent.
• the targeting agent is a bispecific ligand.
• the bispecific ligand comprises a first arm that carries specificity for a minicell surface structure and a second arm that carries specificity for a non-phagocytotic mammalian cell surface receptor.
• the mammalian cell surface receptor is the Epidermal Growth Factor receptor (EGFR).
• the minicell surface structure is an O-polysaccharide component of a lipopolysaccharide on the minicell surface.
• the non-phagocytotic mammalian cell surface receptor is capable of activating macropinocytosis or receptor-mediated endocytosis of the minicell.
• the bispecific ligand comprises a bispecific antibody or antibody fragment.
• the antibody or antibody fragment comprises a first multivalent arm that carries specificity for a bacterially derived minicell surface structure and a second multivalent arm that carries specificity for a cancer cell surface receptor, wherein the cancer cell surface receptor is capable of activating macropinocytosis or receptor-mediated endocytosis of the minicell.
• the second multivalent arm carries specificity for EGFR.
• the therapy that induces the death of neoplastic cells comprises
• the encapsulated CD ld-restricted iNKT cell antigen is capable of uptake by a phagocytic cell, such as a dendritic cell or a macrophage.
• the CD ld-restricted iNKT cell antigen induces a Thl cytokine response by an iNKT cell that recognizes the antigen presented by CD Id.
• the CD ld-restricted iNKT cell antigen is a glycosphingo lipid.
• the glycosphingolipid is selected from among a-galactosylceramide (a- GalCer), C-glycosidific form of a-galactosylceramide (a-C-GalCer), 12 carbon acyl form of galactosylceramide (b-GalCer), b-D-glucopyranosylceramide (b-GlcCer), l,2-Diacyl-3-0- galactosyl-sn-glycerol (BbGL-II), diacylglycerol containing glycolipids (Glc-DAG-s2), ganglioside (GD3), gangliotriaosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), a- glucuronosylceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide (iGb3),
• the glycosphingolipid is a-GalCer. In some embodiments, the glycosphingolipid is a synthetic a-GalCer analog.
• the synthetic a-GalCer analog is selected from among 6'-deoxy-6'-acetamide a- GalCer (PBS57), naphtylurea a-GalCer (NU-a-GC), NC-a-GalCer, 4ClPhC-a-GalCer, PyrC-a- GalCer, a-carba-GalCer, carba-a-D-galactose a-GalCer analog (RCAI-56), 1-deoxy-neo-inositol a-GalCer analog (RCAI-59), 1-O-methylated a-GalCer analog (RCAI-92), and HS44 aminocyclitol ceramide.
• the CD ld-restricted iNKT cell antigen is derived from a bacterial antigen, a fungal antigen, or a protozoan antigen.
• the antineoplastic agent is selected from the group consisting of a radionuclide, a chemotherapy drug, a functional nucleic acid, and a polynucleotide from which a functional nucleic acid can be transcribed.
• the chemotherapeutic drug is a cytotoxin.
• the chemotherapeutic drug is selected from the group consisting of morpholinyl anthracycline, a maytansinoid, duocarmycin, auristatins, calicheamicins (DNA damaging agents), a-amanitin (RNA polymerase II inhibitor), centanamycin, pyrrolobenzodiazepine, streptonigtin, nitrogen mustards, nitrosorueas, alkane sulfonates, pyrimidine analogs, purine analogs, antimetabolites, folate analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors, hormonal agents, and a combination thereof.
• the morpholinyl anthracycline is selected from the group consisting of nemorubicin, PNU- 159682, idarubicin, daunorubicin, caminomycin, and doxorubicin.
• the functional nucleic acid is selected from the group consisting of a siRNA, a miRNA, a shRNA, a lincRNA, an antisense RNA, and a ribozyme.
• the functional nucleic acid inhibits a gene that promotes tumor cell proliferation, angiogenesis or resistance to chemotherapy and/or that inhibits apoptosis or cell cycle arrest.
• the therapy that induces the death of neoplastic cells comprises radiation therapy or surgery.
• the methods comprise administering an adjuvant composition that comprises (a) an immunogenically effective amount of intact, bacterially derived minicells or killed bacterial cells that encapsulate CD ld-restricted iNKT cell antigen and (b) a
• the CD ld-restricted iNKT cell antigen and the antineoplastic agent are packaged within two or more purified, intact bacterially derived minicells or killed bacterial cells.
• the adjuvant composition comprises the CD ld-restricted iNKT cell antigen and the antineoplastic agent, wherein: (a) the CD ld-restricted iNKT cell antigen and the antineoplastic agent are comprised within the same minicell or killed bacterial cell or (b) the CD ld-restricted iNKT cell antigen is comprised within a first minicell or killed bacterial cell, and the antineoplastic agent is comprised within a second minicell or killed bacterial cell.
• the intact bacterially-derived minicell comprising the antineoplastic agent comprises a targeting agent.
• the intact bacterially-derived minicell comprising the CD ld-restricted iNKT cell antigen does not comprise a targeting agent
• the intact bacterially-derived minicell comprising the antineoplastic agent comprises a targeting agent.
• the targeting agent is a bispecific ligand.
• the bispecific ligand comprises a first arm that carries specificity for a minicell surface structure and a second arm that carries specificity for a non-phagocytotic mammalian cell surface receptor.
• the mammalian cell surface receptor is the Epidermal Growth Factor receptor (EGFR).
• the minicell surface structure is an O-polysaccharide component of a
• the non-phagocytotic mammalian cell surface receptor is capable of activating macropinocytosis or receptor-mediated endocytosis of the minicell.
• the bispecific ligand comprises a bispecific antibody or antibody fragment.
• the antibody or antibody fragment comprises a first multivalent arm that carries specificity for a bacterially derived minicell surface structure and a second multivalent arm that carries specificity for a cancer cell surface receptor, wherein the cancer cell surface receptor is capable of activating macropinocytosis or receptor- mediated endocytosis of the minicell.
• the cell surface receptor is EGFR.
• the encapsulated CD ld-restricted iNKT cell antigen e.g., a- GalCer
• the therapy that induces the death of neoplastic cells e.g., antineoplastic agent
• the encapsulated CD ld-restricted iNKT cell antigen and the therapy that induces the death of neoplastic cells are administered sequentially.
• the encapsulated CD ld-restricted iNKT cell antigen and/or the therapy that induces the death of neoplastic cells is/are administered multiple times.
• the encapsulated CD ld-restricted iNKT cell antigen and/or the therapy that induces the death of neoplastic cells is/are administered at least once a week over the course of several weeks. In some embodiments, the encapsulated CD ld-restricted iNKT cell antigen and/or the therapy that induces the death of neoplastic cells is/are administered at least once a week over several weeks to several months.
• the encapsulated CD Id- restricted iNKT cell antigen and/or the therapy that induces the death of neoplastic cells is/are administered at least once a week for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 weeks or more. In some embodiments, the encapsulated CD Id- restricted iNKT cell antigen and/or the therapy that induces the death of neoplastic cells is/are administered about twice every week.
• the encapsulated CD ld-restricted iNKT cell antigen and/or the therapy that induces the death of neoplastic cells is/are administered twice a week for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 weeks or more.
• the subject that is treated with an adjuvant composition provided herein is a mammal, a human, a non-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.
• the neoplastic disease is cancer.
• the cancer is selected from the group consisting of lung cancer, breast cancer, brain cancer, liver cancer, colon cancer, pancreatic cancer, and bladder cancer.
• the cancer is selected from the group consisting of an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor; breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myecytic leukemia; chronic myecytic
• craniopharyngioma cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors;
• esthesioneuroblastoma Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer;
• gastrointestinal carcinoid tumor gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma;
• medulloepithelioma melanoma
• Merkel cell carcinoma Merkel cell skin carcinoma
• mesothelioma metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer;
• osteosarcoma other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer;
• papillomatosis paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer;
• pharyngeal cancer pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm/multiple myeloma; pleuropulmonaryblastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer;
• transitional cell cancer transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom’s macroglobulinemia; and Wilms’ tumor.
• the cancer is malignant.
• the cancer is recurrent or relapsed cancer.
• compositions comprising an adjuvant composition provided herein and at least one pharmaceutically acceptable carrier.
• an adjuvant composition comprising an immunogenically effective amount of (a) an encapsulated CD ld-restricted iNKT cell antigen that is capable of uptake by a phagocytic cell and (b) a pharmaceutically acceptable carrier for the treatment of a neoplastic disease.
• an adjuvant composition comprising an immunogenically effective amount of (a) an encapsulated CD ld-restricted iNKT cell antigen that is capable of uptake by a phagocytic cell and (b) a pharmaceutically acceptable carrier in the preparation of a medicament for the treatment of a neoplastic disease.
• FIG. 1 is a graphical depiction of an EnGeneIC Dream Vehicle (EDV) (e.g., a bacterial minicell) loaded with the CD ld-restricted iNKT cell antigen a-galactosylceramide (a- GalCer).
• EDV EnGeneIC Dream Vehicle
• FIG. 2 is a graphical depiction of an EDV (e.g., a bacterial minicell) comprising a bispecific antibody for O-polysaccharide and human epidermal growth factor receptor antigens and loaded with the anti-cancer drug PNU- 159682 (an anthracycline analogue).
• EDV e.g., a bacterial minicell
• PNU- 159682 an anthracycline analogue
• FIG. 3 shows combination treatment using Ep minicell Dox and minicell a-GC in a syngeneic mouse model ( Ep CT26 colon tumors in Balb/c mice).
• FIG. 4 shows combination treatment of Ep minicell Dox and minicell a-GC is effective in reducing large tumors in Balb/c mice bearing CT26 isograft.
• FIG. 5 shows effect of Ep minicell Dox and minicell a-GC on tumor regression in Balb/c mice with CT26 isograft.
• FIGs. 6A-F show different sized CT26 isografts treated with (FIGS. 6A and 6B) Ep minicellDox and minicell a-GC ,
• FIG. 6A EpminicellDox ( lxlO 9 ) + EDV a -Gc ( lxl0 7 ))
• FIG. 6B Epminicel ox ( lxl0 9 )+ EDV a -Gc (lxl0 6 )
• FIG. 6C saline
• FIG. 6E minicell a-G c (lxl0 6 )
• FIGs. 7A and 7B show different sized CT26 isografts treated with Ep minicell Dox and minicell a-GC.
• FIG. 7A EpminicellDox (lxl0 9 )+ minicell a-GC (lxlO 7 ); 16hr;
• EpminicellDox (lxl0 9 )+ minicella-GC (lxlO 7 ); 24hr.
• FIGs. 8A-E show aGC-CDld presentation of JAWS II cells following minicell aGC treatment at various time points (FIGS. 8A-D).
• FIG. 8A 8 hrs;
• FIG. 8B 16 hrs;
• FIG. 8C 24 hrs;
• FIG. 8D 48 hrs;
• FIG. 8E shows aGC-CDID positive JAWSII cells during the course of treatment.
• FIG. 9 shows IL-12 production by JAWSII cells treated with ED V a oc at 48h post treatment.
• FIG. 10 shows concentrations of IFNy secreted by iNKT cells co-cultured with JAWSII cells treated with EDV a oc-
• FIG. 11 shows concentrations of IL4 secreted by iNKT cells co-cultured with JAWSII cells treated with EDV a oc-
• FIG. 12 shows concentrations of TNFa secreted by iNKT cells co-cultured with JAWSII cells treated with EDV a oc-
• FIGs. 14A-D show the percentage of CD45+ cells within the tumours during the course of the in vivo treatment for five different tested compositions: saline, minicell,
• FIG. 18 shows CD Id mRNA expression in PBMCs 8h post- injection (fold increase as compared to saline) for four different mouse treatment groups: saline, Minicell a oc,
• FIG. 19 shows CD Id mRNA expression in DCs isolated from thymus 8h post injection (fold increase as compared to saline) for four different mouse treatment groups: saline, Minicellaoc, Epcam MinieellDox + Minicellaoc, and Epcam Minieell682 + MinicellaGC.
• compositions comprising an immunogenically effective amount of intact, bacterially derived minicells or killed bacterial cells that encapsulate a CD ld-restricted invariant Natural Killer T (iNKT) cell antigen (e.g., a sphingolipid, such as a-galactosylceramide), when administered with an antineoplastic agent, synergistically improve cancer treatment strategies.
• iNKT Natural Killer T
• an encapsulated a-galactosylceramide (a-GalCer), which is contained within a bacterially-derived minicell, is taken up by phagocytic cells and expressed on the surface of the cell in a complex with CD Id.
• the encapsulated a-GalCer was able to significantly and synergistically augment the antitumor response when administered in combination with an antineoplastic agent, doxorubicin.
• the components of this combined therapy include induction of an immune response elicited by an antineoplastic agent (e.g., a cytotoxic agent comprised in a bacterially derived minicell) to trigger tumor cell killing and activate a potent CD4+ and CD8+ anti-tumor responses and use of bacterially derived minicells carrying a-GalCer, which biases the antigen processing and presentation towards MHC class 1 (CD ld-restricted) to mount a potent iNKT cell-based anti-tumor response.
• the minicells themselves elicit an innate immune response via recognition of damage-associated molecular pattern (DAMP) molecules, which are released by dying tumor cells, by the antigen presenting cells (APC).
• DAMP damage-associated molecular pattern
• Induction of the innate immune response involving pro-inflammatory cytokines are critical to this process since it elicits the activation and differentiation of bone marrow derived macrophages and dendritic cells. This process dramatically enhances anti-tumor immunity.
• a carrier such as a liposome or polymer is incapable of eliciting an innate immune response. Therefore, anti-tumor efficacy will be lower compared to the bacterially derived minicells of the present disclosure since the anti-tumor activity of the liposome or polymer carried a-GalCer is solely reliant on iNKT cell activation and not on the formation of Ml macrophages and activation of dendritic cells.
• tumor cell antigens which may be derived in situ via tumor cell death, or delivered exogenously must be taken up by dendritic cells (DC)
• DCs need to receive a proper maturation signal prompting differentiation and enhanced processing and presentation of antigens such that antitumor function as opposed to tolerance is promoted (Anguille et al., 2015; Emens et al., 2017; Jung et al., 2018; Mellman et al., 2011 ; Simmons et al., 2012).
• Checkpoint inhibitors such as cytotoxic T lymphocyte antigen 4 (CTLA-4), and programmed cell death 1/programmed cell death 1 ligand (PD-l/PDL-1) function by blocking the transmission of immune-suppressive signals and direct stimulation to activate cytotoxic T lymphocytes within the tumor microenvironment (Dine et al., 2017; Jenkins et al., 2018; Sharpe, 2017). Inhibitors of these pathways have shown dramatic clinical results in specific cancers, but overall response rates across different cancers remains low (-15-25%) and immune related toxicities associated with these therapies can be high (Dine et al., 2017; Emens et al., 2017; Jenkins et al., 2018; Sharpe, 2017; Ventola, 2017).
• CTLA-4 cytotoxic T lymphocyte antigen 4
• PD-l/PDL-1 programmed cell death 1/programmed cell death 1 ligand
• CAR-T cell therapy which entails the genetic engineering of a patient’s T-cells to express membrane fusion receptors with defined tumor antigen specificities and capable of eliciting robust T-cell activation to initiate killing of the target tumor cells (D'Aloia et al., 2018’; Farkona et al., 2016; Mellman et al., 2011; Sharma et al., 2017).
• the EnGeneIC Dream Vector is a bacterially-derived delivery system consisting of nonviable nanocells that are about 400 ⁇ 20 nm in diameter, generated by reactivating polar sites of cell division in bacteria (MacDiarmid et al., 2007b). It has been demonstrated that these nanocells can be packaged with a cytotoxic drug, siRNA, or miRNA and specifically targeted to tumor cell- surface receptors via attachment of bispecific antibodies to the surface polysaccharide of the nanocells (MacDiarmid et al., 2009; MacDiarmid et al., 2007b; Reid et al., 2013).
• bacterially-derived minicells to deliver chemotherapeutic agents to cancer cells has previously been described.
• This delivery method to treat cancer packages a toxic chemotherapy agent or drug, or functional nucleic acid, into a bacterially-derived minicell, which are typically about 400 nm in diameter.
• the minicell carrying a chemotherapeutic agent an antibody targeting specific cancer cells.
• the antibodies attach to the surface of cancer cells and the minicell is internalized by the cancer cell.
• the toxic chemotherapy agents are not widely distributed throughout the body, and therefore reduce the chance of side effects and intolerability as the toxic drug or compound is delivered inside the cancer cell.
• Using antibody-targeted minicells as a delivery vehicle for toxic chemotherapy agents results in much less drug needed to kill the cancer cell, thus improving the therapeutic index.
• minicells or EnGeneIC Dream
• EDVs can deliver chemotherapy drugs, such as paclitaxel or doxorubicin, to xenograft tumors in mice, dogs, and monkeys.
• chemotherapy drugs such as paclitaxel or doxorubicin
• the targeted delivery ensures that the cancer cells receive most of the chemotherapeutic agent, resulting in a low level of toxicity. See MacDiarmid et al., 2007b; MacDiarmid et al., 2007a; MacDiarmid et al., 2009; and MacDiarmid et al., 2016.
• minicells do not induce a significant immune response in the xenograft models, and the minicells are well tolerated.
• intact bacterially derived minicells are a well- tolerated vehicle for delivering anti-cancer drugs to patients, with examples including doxorubicin targeted to advanced solid tumors, doxorubicin targeted to glioblastoma, and MicroRNA-16a targeted to mesothelioma.
• these treatment strategies did not result in complete remission or cure of all cancers in all patients. Accordingly, there is a need for improved cancer treatment therapies.
• the present inventors discovered that using a combination of bacterially-derived minicells comprising a CD ld-restricted invariant Natural Killer T (iNKT) cell antigen and an antineoplastic agent produced surprisingly dramatic and effective clinical efficacy.
• the bacterially-derived minicells comprising a CD ld-restricted invariant Natural Killer T (iNKT) cell antigen do not comprises a targeting agent and the bacterially-derived minicells comprising an antineoplastic agent comprises a targeting agent.
• minicells comprising a CD ld- restricted invariant Natural Killer T (iNKT) cell antigen, such as a-GalCer, combined with minicells comprising an antineoplastic agent (e.g., doxorubicin), resulted in synergistic anti tumor effects.
• iNKT Natural Killer T
• antineoplastic agent e.g., doxorubicin
• the data reveal a unique pathway for anti-tumor immunity based on innate and adaptive immune responses.
• the minicells comprising a-GalCer and the minicells comprising an antineoplastic agent extravasate into the tumor microenvironment via the leaky vasculature associated with a solid tumor.
• Targeted minicells comprising the antineoplastic agent and an anti-EGFR targeting agent are internalized by the tumor cells via macropinocytosis. Once inside the tumor cells the minicells are broken down, resulting in the release of the antineoplastic drug and inducing tumor cell apoptosis.
• apoptotic tumor cells rapidly expose calreticulin on their surface followed by phosphatidylserine (a marker of apoptosis) and release damage-associated molecular pattern (DAMP) molecules such as ATP (during apoptosis) and HMGB1 upon secondary necrosis.
• DAMP damage-associated molecular pattern
• the minicells that do not make it to the tumor microenvironment are engulfed by cells of the immune system (e.g., macrophages and dendritic cells) found in the vasculature associated with the liver, spleen and lymph nodes.
• the minicells are broken down in lysosomes and the released LPS escapes from lysosomal membranes and binds to caspase 4 and caspase 5 via the CARD (caspase recruit domain) domains. This LPS binding facilitates rapid
• oligomerization of caspase 4/5 resulting in pyroptosis of the macrophage or dendritic cell and the secretion of pro-inflammatory cytokines IL-Ib and IL-18.
• the process of pyroptosis also triggers the release of a plethora of pro-inflammatory cytokines such as TNF-a, IL-6, IL-8, and IL-10.
• proinflammatory signals are picked up by monocytes in the bone marrow and these cells differentiate into activated macrophages and dendritic cells, which extravasate from the bone marrow and enter into the general circulation.
• the DAMPs (ATP and HMGB1) released from dying tumor cells generates a strong “find-me” signal for dendritic cells and macrophages, upon its binding to P2Y2 receptors expressed on the surface of the target cells.
• Extracellular ATP not only attracts immune cells into the tumor microenvironment, but also modulates their activity.
• ATP can induce the maturation of myeloid-derived DCs, which is accompanied by increased expression of CD40, CD80, CD83, and CD86, and also promote macrophage expansion through formation of lamellipodial membrane protrusions. These newly differentiated active macrophages and dendritic cells follow the“find-me” signals and enter into the tumor microenvironment.
• Calreticulin on the surface of apoptotic tumor cells is functionally considered as an “eat-me” signal to the immune system.
• Cells with calreticulin expression on their surface are recognized and engulfed by CD91+ cells (e.g., dendritic cells and macrophages).
• Calreticulin acts on target dendritic cells via CD91 expressed on their surface to promote the release of pro- inflammatory cytokines e.g., TNF-a and IF-6 and modulate the activity of type 17 helper T (Thl7) cells in an immunosuppressive tumor bed.
• the binding of calreticulin to CD91 also facilitates the recruitment of antigen presenting cells e.g., dendritic cells into the tumor microenvironment, engulfment of tumor cells by dendritic cells, and optimal antigen presentation to T cells, eventually leading to activation of the immune system.
• antigen presenting cells e.g., dendritic cells into the tumor microenvironment, engulfment of tumor cells by dendritic cells, and optimal antigen presentation to T cells, eventually leading to activation of the immune system.
• HMGB 1 triggers a strong inflammatory response. HMGB 1 activates dendritic cells and stimulates an optimal presentation of tumor-associated antigens to T cells, upon its binding to TFR4.
• RAGE receptor for advanced glycation end products
• MAPKs p38 and ERK1/2
• NF-kB NF-kB
• CD8+ cytotoxic T cells Cross-priming of CD8+ cytotoxic T cells is triggered by mature dendritic cells and gdT cells in an IL-Ib- and IL- 17-dependent manner. Primed CD8+ cytotoxic T cells then elicit a direct cytotoxic response to kill remaining tumor cells through the generation of IFN-g, perforin- 1 and granzyme B.
• a-GalCer Many of the circulating minicells comprising a-GalCer are engulfed by the macrophages and dendritic cells present within the vasculature associated with the liver, spleen and lymph nodes.
• the minicells are broken down within the lysosomes and the a- galactosylceramide is released.
• a-galactosylceramide forms complexes with CD Id. These complexes are transported to the cell surface and localize predominantly to cholesterol rich microdomains, the lipid rafts, in the plasma membrane.
• Thymus derived invariant Natural Killer T cells recognize lipid antigens presented by CD Id via their unique T Cell Receptor (TCR) repertoire Va24Jal8.
• iNKT cells carry pre-existing rriRNA for IFNy and hence rapidly secrete IFNy post- TCR/Cdld/a-GalCer binding.
• iNKT cells also rapidly secrete multiple cytokines upon TCR triggering which is accompanied by an increased CD ld-restricted cytotoxic capacity of various cells of the immune system.
• Cytokines released by iNKT include both regulatory cytokines (e.g., IL-4, IL-10, IL-13) as well as proinflammatory cytokines such as IL-2, IL-17, and IFNy.
• iNKT cells can directly kill tumor cells mediated by classical granule-mediated mechanisms.
• CD I d lipid complexes and the costimulatory molecules CD80/86 on the surface of dendritic cells
• iNKT cells up-regulate the IL-12R and CD40L molecule. Subsequently, and mediated by CD40L, iNKT induce dendritic cell maturation and release of IL-12.
• iNKT cytotoxic CD8+ T lymphocytes
• the minicells comprising the neoplastic agent starts the tumor killing process
• the minicells comprising a-GalCer and minicells comprising the neoplastic agent trigger the innate immune response
• minicells comprising a-GalCer activate the adaptive immune response resulting in the immune system taking over the tumor-specific killing process.
• the inventors of the present disclosure have found that a CD ld-restricted iNKT cell antigen a-GalCer encapsulated within a bacterially derived minicell combined an antineoplastic agent surprisingly produced synergistic anti-tumor effects.
• the synergistic effect appears to depend in part on cell death- inducing activities of the antineoplastic agent
• any antineoplastic agent that is able to induce cell death of cancer cells is suitable for use in combination with an encapsulated CD ld-restricted iNKT cell antigen.
• the antineoplastic agent is also contained within a bacterially-derived minicell.
• the antineoplastic agent is not contained within a bacterially-derived minicell.
• CD ld- restricted iNKT cell antigens in addition to a-GalCer are known and have been shown to effect iNKT cell activation and can be used in place of or in combination with a-GalCer in the compositions and methods provided herein.
• this invention relates to the surprising discovery that
• compositions comprising a combination of a minicell-packaged antineoplastic agent and a minicell packaged type II interferon agonist, such as for example alpha-galactosylceramide (a- GC), and in the absence of a type I interferon agonist, demonstrates surprising anticancer efficacy.
• a minicell-packaged antineoplastic agent such as for example alpha-galactosylceramide (a- GC)
• a minicell packaged type II interferon agonist such as for example alpha-galactosylceramide (a- GC)
• Example 1 describes data illustrating the efficacy of a dual combination of minicell contained antineoplastic therapeutic and minicell contained CD ld-restricted iNKT cell antigen (e.g., a-GalCer) against tumors.
• a-GalCer CD ld-restricted iNKT cell antigen
• FIGS. 3-6 The experimental results showed a marked halt in tumor progression for combination treatment groups receiving Ep minicell Dox + minicell a-GC (a-GalCer) as compared to saline and Ep minicell Dox treatments. This result supports the theory of an immune adjuvant effect by the addition of minicell a-GC treatment to Ep minicell Dox.
• saline treated control tumors demonstrated dramatic tumor regression following a treatment change to drug and a-GalCer EDV mediated dual combination therapy (FIG. 6); e.g., a combination of minicell packaged antineoplastic agent and minicell packaged type II interferon agonist (e.g., a CD ld-restricted iNKT cell antigen, such as a- GalCer).
• minicell packaged antineoplastic agent e.g., a CD ld-restricted iNKT cell antigen, such as a- GalCer
• tumors that had reached 800mm 3 dropped to below 600mm 3 in 3 days before the experiment was terminated - a markedly dramatic tumor size reduction ( ⁇ 25%) in a short period of time.
• the ability for the dual combination composition to dramatically decrease large tumors in a short period of time was not known prior to the present invention.
• the dual combination composition e.g., a minicell packaged antineoplastic agent in combination with a minicell packaged CD ld-restricted iNKT cell antigen, such as a-GalCer
• a tumor’s size including the size of a large tumor, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about
• the reduction in tumor size can be measured over any suitable time period, such as about
• Bacterial minicell treatment represents a unique cancer therapeutic strategy capable of delivering conventional and novel drug therapies directly to the tumor site and subsequently eliciting an antitumor immune response.
• a dual assault on the tumor occurs, first through cell death in response to the delivered therapeutic and followed by innate immune cell activation leading to an adaptive immune response.
• This type of therapy has certain advantages over current immunotherapy strategies in that immune cell activation occurs both in vivo and primarily at the tumor site, which is a rapidly changing, dynamic environment. Further, it creates an immunogenic tumor environment and elicits effects on multiple immune cell subsets avoiding problems associated with patients who show little to no immune response to their tumors or adaptations to therapies which only target single immune cell subsets.
• the study described below highlights the potential of bacterial minicells as a novel cancer
• compositions of the invention comprise an encapsulated CD Id- restricted iNKT cell antigen (e.g., a-GalCer) that is administered in combination with an antineoplastic agent that induces the death of neoplastic cells in the subject.
• an encapsulated CD Id- restricted iNKT cell antigen e.g., a-GalCer
• an antineoplastic agent that induces the death of neoplastic cells in the subject.
• the CD ld-restricted iNKT cell antigen is encapsulated in a bacterially-derived minicell or killed bacterial cell as described herein.
• the CD ld-restricted iNKT cell antigen is encapsulated with an antineoplastic agent in a bacterially-derived minicell or killed bacterial cell.
• the CD ld-restricted iNKT cell antigen and the antineoplastic agent are encapsulated in separate bacterially-derived minicells or killed bacterial cells.
• the CD ld-restricted iNKT cell antigen and the antineoplastic agent are contained in the same composition.
• the CD ld-restricted iNKT cell antigen and the antineoplastic agent are contained in the separate compositions.
• the CD ld-restricted iNKT cell antigen is encapsulated in a bacterially-derived minicell or killed bacterial cell and the antineoplastic agent is encapsulated in a bacterially-derived minicell or killed bacterial cell.
• the CD ld-restricted iNKT cell antigen is encapsulated in a bacterially-derived minicell or killed bacterial cell and the antineoplastic agent is not encapsulated in a bacterially-derived minicell or killed bacterial cell.
• Type II IFNs play an important role in anti-tumor immunity by activating cytotoxic T cells. See, e.g., Chikuma et al., 2017. IFN gamma cytokines are released by innate Natural Killer cells upon binding of natural antigen, but glycosphingolipid compounds function as potent activators of both innate and acquired immune responses.
• the present inventors discovered that ecapsulated CD ld-restricted iNKT cell antigens, such as the glycosphingolipid a-GalCer, are engulfed by phagocytic cells, such as macrophages and dendritic cells, and then expressed on the surface of the cells bound to the surface glycoprotein CD Id.
• iNKT innate natural killer T
• Examples of CD ld-restricted iNKT cell antigens useful for the compositions described herein include, but are not limited to, glycosphingolipids, such as a- galactosylceramide (a-GalCer), C-glycosidific form of a-galactosylceramide (a-C-GalCer), 12 carbon acyl form of galactosylceramide (b-GalCer), b-D-glucopyranosylceramide (b-GlcCer), l,2-Diacyl-3-0-galactosyl-sn-glycerol (BbGL-II), diacylglycerol containing glycolipids (Glc- DAG-s2), ganglioside (GD3), gangliotriaosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), a-glucuronosy
• the glycosphingolipid is a synthetic a-GalCer analog.
• the synthetic a-GalCer analog is selected from among 6'-deoxy-6'-acetamide a- GalCer (PBS57), naphtylurea a-GalCer (NU-a-GC), NC-a-GalCer, 4ClPhC-a-GalCer, PyrC-a- GalCer, a-carba-GalCer, carba-a-D-galactose a-GalCer analog (RCAI-56), 1-deoxy-neo-inositol a-GalCer analog (RCAI-59), 1-O-methylated a-GalCer analog (RCAI-92), and HS44 aminocyclitol ceramide.
• the CD ld-restricted iNKT cell antigen is derived from a bacterial antigen, a fungal antigen, or a protozoan antigen.
• the CD ld- restricted iNKT cell antigen is a glycosphingolipid from the bacterial species Sphinomonadacae spp..
• the glycosphingolipid is Sphinomonadacae spp. glycosphingolipid- 1 (GSL-1), GSL-1', GSL-2, GSL-3, or GSL-4.
• the CD ld-restricted iNKT cell antigen is a glyco lipid from the bacterial species Streptococcus spp.. In some embodiments, the glyco lipid is S. pneumoniae Glc-diacyl glycerol (DAG) or Gal-Glc-DAG. In some embodiments, the CD ld-restricted iNKT cell antigen is a glyco lipid from the bacterial species Borrelia spp.. In some embodiments, the glycolipid is B. burgdorferi BbGL-IIc. In some embodiments, the CD ld-restricted iNKT cell antigen is a glycolipid from the bacterial species Heliobacter pylori.
• the glycolipid is H. pylori PI57.
• CD ld- restricted iNKT cell bacterial antigens useful in the compositions and methods provided herein include, but are not limited to, monoglycosylceramides derived from Spongemonas,
• lipopphosphoglycans derived from Leishmania donovi.
• the CD ld-restricted iNKT cell antigen is a fungal glycolipid from Aspergillus spp., such as A. fumigatus or A. niger. In some embodiments, the glycolipid is A. fumigatus aperamide B. In some embodiments, the CD ld-restricted iNKT cell antigen is a glycolipid from the protozoan Entamoeba histolytica. In some embodiments, the glycolipid is E. histolytica EhPlb.
• Additional exemplary CD ld-restricted iNKT cell antigens including additional a- GalCer derivatives, useful for the compositions provided herein include those described in US2017/0368002, Birkholz and Kronenberg, 2015, and Anderson, 2013, which are each incorporated by reference in their entirety.
• minicell a-G c tumor containing mice that were administered intact bacterially-derived minicells containing the chemotherapeutic doxorubicin ( Ep minicell Dox ) and minicells containing the CD ld-restricted iNKT cell antigen a-GalCer (minicell a-G c) displayed a marked halt in tumor progression over mice administered only Ep minicell Dox .
• minicell compositions incorporating a CD ld- restricted iNKT cell antigen are effective at treating tumors in mice.
• the minicell can deliver type II IFN agonists, such as CD ld-restricted iNKT cell antigens, directly to cells of the immune system, for enhancing iNKT cell activation and type II interferon IFN-g production in vivo.
• type II IFN agonists such as CD ld-restricted iNKT cell antigens
• non-targeted encapsulated CD ld-restricted iNKT cell antigens are taken up by phagocytic cells of the immune system, where they are broken down in endosomes, and aGC is presented to iNKT cells for immune activation.
• compositions described herein provide targeted delivery of type II interferon agonists, such as the CD ld-restricted iNKT cell antigens.
• compositions disclosed herein comprise a non-targeted delivery of the CD ld-restricted iNKT cell antigens.
• the inventive methodology results in increase of serum IFN-g concentration that is not higher than about 30,000 pg/mL.
• the serum IFN-g concentration is increased to not higher than about 5000 pg/mL, 1000 pg/mL, 900 pg/mL, 800 pg/mL, 700 pg/mL, 600 pg/mL, 500 pg/mL, 400 pg/mL, 300 pg/mL, 200 pg/mL, or 100 pg/mL.
• the resulting serum IFN- gamma concentration is at least about 10 pg/mL, or at least about 20 pg/mL, about 30 pg/mL, about 40 pg/mL, about 50 pg/mL, about 60 pg/mL, about 70 pg/mL, about 80 pg/mL, about 90 pg/mL, about 100 pg/mL, about 150 pg/mL, about 200 pg/mL, about 300 pg/mL, about 400 pg/mL or about 500 pg/mL.
• anticancer agent denotes a drug, whether chemical or biological, that prevents or inhibits the growth, development, maturation, or spread of neoplastic cells.
• anticancer agent is used interchangeably with“anticancer agent” and“chemotherapy agent.”
• selecting an antineoplastic agent for treating a given tumor depends on several factors. These factors include but are not limited to the patient’s age, the stage of the tumor, and whatever previous therapy the patient may have received.
• the composition can comprise at most about 1 mg of the antineoplastic or chemotherapeutic drug.
• the amount of the chemotherapeutic drug can be at most about 750 pg, about 500 pg, about 250 pg, about 100 pg, about 50 pg, about 10 pg, about 5 pg, about 1 pg, about 0.5 pg, or about 0.1 pg.
• the composition comprises a chemotherapeutic drug having an amount of less than about 1/1,000, or alternatively less than about 1/2,000, 1/5,000, 1/10,000, 1/20,000, 1/50,000, 1/100,000, 1/200,000 or 1/500,000 of the therapeutically effective amount of the drug when used without being packaged into minicells.
• the composition can comprise at least about 1 nmol of the chemotherapeutic drug. Accordingly, the disclosure also encompasses embodiments where the amount of the chemotherapeutic drug is at least about 2 nmol, about 3 nmol, about 4 nmol, about 5 nmol, about 10 nmol, about 20 nmol, about 50 nmol, about 100 nmol, or about 800 nmol.
• a chemotherapeutic drug can be selected from one of the classes detailed below for administration with the encapsulated CD ld-restricted iNKT cell antigens provided herein.
• a chemotherapeutic drug is packed into intact, bacterially derived minicells as described herein. These drugs can also be synthetic analogs designed from drug design and discovery efforts.
• Any known chemotherapy agent can be utilized in the compositions of the invention. Examples of known chemotherapy agents include, but are not limited to:
• alkylating agents such as mustard gas derivatives (Mechlorethamine, Cyclophosphamide (Cytoxan), Chlorambucil (Leukeran), Melphalan, and Ifosfamide), ethylenimines (Thiotepa (Thioplex) and Hexamethylmelamine), alkylsulfonates (Busulfan (Myleran)), hydrazines and triazines (Altretamine (Hexalen), Procarbazine (Matulane),
• DTIC dacarbazine
• Temozolomide Temozolomide
• nitrosureas Carmustine, Lomustine and
• Plant alkaloids, terpenoids and topoisomerase inhibitors such as vinca alkaloids (Vincristine (Oncovin), Vinblastine (Velban), Vindesine, and Vinorelbine), taxanes (Paclitaxel (Taxol) and Docetaxel (Taxotere)), podophyllotoxins (Etoposide and Tenisopide), and camptothecan analogs (Irinotecan and Topotecan);
• antitumor antibiotics such as anthracyclines (Doxorubicin (Adriamycin, Rubex, Doxil), Daunorubicin, Epirubicin, Mitoxantrone, Idarubicin, Duocarmycin, and Dactinomycin (Cosmegen)), chromomycins (Dactinomycin and Plicamycin (Mithramycin)), and miscellaneous (Mitomycin and Bleomycin (Blenoxane));
• anthracyclines Doxorubicin (Adriamycin, Rubex, Doxil) Daunorubicin, Epirubicin, Mitoxantrone, Idarubicin, Duocarmycin, and Dactinomycin (Cosmegen)
• chromomycins Dactinomycin and Plicamycin (Mithramycin)
• miscellaneous Mitsubishi (Mitomycin and Bleomycin (Blenoxane)
• antimetabolites such as folic acid antagonists (Methotrexate), pyrimidine antagonists (5-Fluorouracil, Foxuridine, Cytarabine, Flurouracil (5-FU), Capecitabine, and Gemcitabine), purine antagonists (6-Mercaptopurine (Purinethol) and 6-Thioguanine), 6- Thiopurines, and adenosine deaminase inhibitor (Cladribine (Leustatin), Fludarabine, Nelarabine and Pentostatin), Azacitidine, Thioguanine, and Cytarabine (ara-C);
• folic acid antagonists Metalhotrexate
• pyrimidine antagonists (5-Fluorouracil, Foxuridine, Cytarabine, Flurouracil (5-FU), Capecitabine, and Gemcitabine
• purine antagonists (6-Mercaptopurine (Purinethol) and 6-Thioguanine), 6- Thiopurines, and adenosine deamin
• topoisomerase Inhibitors such as topoisomerase I inhibitors (Ironotecan, topotecan), and topoisomerase II inhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide);
• estrogen and Androgen Inhibitors Tamoxifen and Flutamide
• Gonadotropin-Releasing Hormone Agonists Leuprolide and Goserelin
• PARP Poly( adenosine diphosphate [ADP]-ribose) polymerase
• PBK/Akt/mTOR pathway inhibitors e.g., Everolimus
• Histone deacetylase (HD AC) inhibitors e.g., Vorinostat, Entinostat (SNDX- 275), Mocetinostat (MGCD0103), Panobinostat (LBH589), Romidepsin, Valproic acid.
• Histone deacetylase (HD AC) inhibitors e.g., Vorinostat, Entinostat (SNDX- 275), Mocetinostat (MGCD0103), Panobinostat (LBH589), Romidepsin, Valproic acid.
• Cyclin-dependent kinase (CDK) inhibitors e.g., Flavopiridol, Olomoucine, Roscovitine, Kenpaullone, AG-024322 (Pfizer), Fascaplysin, Ryuvidine, Purvalanol A, NU2058, BML-259, SU 9516, PD-0332991, P276-00.
• CDK Cyclin-dependent kinase
• HSP90 Heat shock protein (HSP90) inhibitors, e.g., Geldanamycin, Tanespimycin, Alvespimycin, Radicicol, Deguelin, and BIIB021 ;
• Murine double minute 2 (MDM2) inhibitors e.g., Cis-imidazoline, Benzodiazepinedione, Spiro-oxindoles, Isoquinolinone, Thiophene, 5-Deazaflavin, Tryptamine;
• Anaplastic lymphoma kinase (ALK) inhibitors e.g., Aminopyridine, Diaminopyrimidine, Pyridoisoquinoline, Pyrrolopyrazole, Indolocarbazole, Pyrrolopyrimidine, Dianilinopyrimidine ;
• PARP Poly [ADPribose] polymerase
• miscellaneous anticancer drugs exemplified by Amsacrine, Asparaginase (El-spar), Hydroxyurea, Mitoxantrone (Novantrone), Mitotane (Lysodren), Maytansinoid, Retinoic acid Derivatives, Bone Marrow Growth Factors (sargramostim and filgrastim), Amifostine, agents disrupting folate metabolism, e.g., Pemetrexed, ribonucleotide reductase inhibitors (Hydroxyurea), adrenocortical steroid inhibitors (Mitotane), enzymes (Asparaginase and Pegaspargase), anti microtubule agents (Estramustine), and retinoids (Bexarotene,
• Chemotherapy drugs that are illustrative of the small molecule drug subcategory are Actinomycin-D, Alkeran, Ara-C, Anastrozole, BiCNU, Bicalutamide, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carboplatinum, Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine, CPT-11, Cyclophosphamide, Cytarabine, Cytosine arabinoside, Cytoxan, dacarbazine, Dactinomycin, Daunorubicin, Dexrazoxane, Docetaxel, Doxorubicin, DTIC, Epirubicin, Ethyleneimine, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide, Fotemustine, Gemcitabine, Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide, Irinote
• Vincristine, Vindesine, Vinorelbine, VP- 16, and Xeloda Vincristine, Vindesine, Vinorelbine, VP- 16, and Xeloda.
• Maytansinoids (molecular weight: -738 Daltons) are a group of chemical derivatives of maytansine, having potent cytotoxicity. Although considered unsafe for human patient use, due to toxicity concerns, maytansinoids are suitable for delivery to brain tumor patients via minicells, pursuant to the present invention.
• Duocarmycins (molecular weight: -588 Daltons) are a series of related natural products, first isolated from Streptomyces bacteria. They also have potent cytotoxicity but are considered as unsafe for human use. Like maytansinoids, duocarmycins are suitable
• the subcategory of biologic chemotherapy drugs includes, without limitation, Asparaginase, AIN-457, Bapineuzumab, Belimumab, Brentuximab, Briakinumab, Canakinumab, Cetuximab, Dalotuzumab, Denosumab, Epratuzumab, Estafenatox, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab, Girentuximab (WX-G250), Herceptin, Ibritumomab, Inotuzumab, Ipilimumab, Mepolizumab, Muromonab-CD3, Naptumomab, Necitumumab, Nimotuzumab, Ocrelizumab, Ofatumumab, Otelixizumab, Ozogamicin, Pagibaximab, Panitumumab,
• the antineoplastic agent comprises a compound selected from the group consisting of actinomycin-D, aikeran, ara-C, anastrozole, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine, carboplatin, carboplatinum, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, cytosine arabinoside, cytoxan, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxuridine, fludarabine, fluorouracil, flutamide, fotemustine, gemcitabine, hexamethylamine, hydroxyurea, idarubicin, ifo
• pagibaximab panitumumab, pertuzumab, ramucirumab, reslizumab, rituximab, REGN88, solanezumab, tanezumab, teplizumab, tiuxetan, tositumomab, trastuzumab, tremelimumab, vedolizumab, zalutumumab, zanolimumab, 5FC, accutane hoffmann-la roche, AEE788 novartis, AMG-102, anti neoplaston, AQ4N (Banoxantrone), AVANDIA (Rosiglitazone Maleate), avastin (Bevacizumab) genetech, BCNU, biCNU carmustine, CCI-779, CCNU, CCNU lomustine, celecoxib (Systemic), chloroquine, cilen
• mercaptopurine Purinethol
• fludarabine phosphate (Leustatin), flurouracil (5-FU), cytarabine (ara-C), azacitidine, vinblastine (Velban), vincristine (Oncovin), podophyllotoxins (etoposide ⁇ VP- 16 ⁇ and teniposide ⁇ VM-26 ⁇ ), camptothecins (topotecan and irinotecan ), taxanes such as paclitaxel (Taxol) and docetaxel (Taxotere), (Adriamycin, Rubex, Doxil), dactinomycin
• benzimidazole indazole, pyrrolocarbazole, isoindolinone, morpholinyl anthracycline, a maytansinoid, ducarmycin, auristatins, caliche amicins (DNA damaging agents), a-amanitin (RNA polymerase II inhibitor), centanamycin, pyrrolobenzodiazepine, streptonigtin, nitrogen mustards, nitrosorueas, alkane sulfonates, pyrimidine analogs, purine analogs, antimetabolites, folate analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors, hormonal agents, and any combination thereof.
• Active agents useable in accordance with the present disclosure are not limited to those drug classes or particular agents enumerated above.
• Different discovery platforms continue to yield new agents that are directed at unique molecular signatures of cancer cells; indeed, thousands of such chemical and biological drugs have been discovered, only some of which are listed here.
• the surprising capability of intact, bacterially derived minicells and killed bacterial cells to accommodate packaging of a diverse variety of active agents, hydrophilic or hydrophobic means that essentially any such drug, when packaged in minicells, has the potential to treat a cancer, pursuant to the findings in the present disclosure.
• antineoplastic agents are radionuclides, chemotherapy drugs, and functional nucleic acids, including but not limited to regulatory RNAs. Members of the class are discussed further below.
• the encapsulated CD ld-restricted iNKT cell antigens are administered in combination with an antineoplastic agent that is a radionuclide.
• A“radionuclide” is an atom with an unstable nucleus, i.e., one characterized by excess energy available to be imparted either to a newly created radiation particle within the nucleus or to an atomic electron.
• Radionuclides herein may also be referred to as“radioisotopes,”“radioimaging agents,” or “radiolabels.” Radionuclides can be used imaging and/or therapeutic purposes.
• the radionuclide is administered using an intact, bacterially derived minicell.
• chelators can be contained within the minicell or attached to a ligand, peptide, or glyco lipid on the outer surface of a minicell described herein. Attachments may be directly or via a linker, a linker containing a chelating moiety comprising chelators such as mercaptoacetyltriglycine (MAG3), DOTA, EDTA, HYNIC, DTPA or crown ethers may be used.
• MAG3 mercaptoacetyltriglycine
• DOTA EDTA
• HYNIC EDTA
• DTPA mercaptoacetyltriglycine
• crown ethers such as mercaptoacetyltriglycine (MAG3), DOTA, EDTA, HYNIC, DTPA or crown ethers may be used.
• the chelators may be attached directly the minicell surface component or attached to the minicell via a linker.
• radionuclides are known in the art, and a number of them are known to be suitable for medical use, such as yttrium-90, technetium-99m, iodine- 123, iodine- 124, iodine- 125, iodine-131, rubidium-82, thallium- 201 , gallium-67, fluorine- 18, xenon- 133, and indium-111.
• the radioisotope comprises a radioisotope selected from the group consisting of yttrium-90, yttrium-86, terbium-152, terbium- 155, terbium-149, terbium-161, technetium-99m, iodine-123, iodine- 131, rubidium-82, thallium-201, gallium-67, fluorine-18, copper-64, gallium-68, xenon-133, indium-111, lutetium-177, and any combination thereof.
• Radioisotopes useful for attaching to minicells for both imaging and therapeutic purposes include, for example, Iodine-131 and lutetium-177, which are gamma and beta emitters. Thus, these agents can be used for both imaging and therapy.
• iodine- 123 gamma emitter
• iodine- 131 gamma and beta emitters
• Newer examples are yttrium-86/yttrium-90 or terbium isotopes (Tb): 152 Tb (beta plus emitter), 155 Tb (gamma emitter), 149 Tb (alpha emitter), and 161 Tb (beta minus particle) (Miiller et al., 2012; Walrand et al., 2015).
• Nuclear imaging utilizes gamma and positron emitters (b+).
• Gamma emitters such as technetium-99m ( 99m Tc) or iodine- 123 ( 123 I)
• gamma cameras plane imaging
• SPECT single photon emission computed tomography
• Beta particles have a potential cytocidal effect, but they also spare the surrounding healthy tissue due to having a tissue penetration of only a few millimeters.
• beta emitters in routine nuclear oncology practices include lutetium-177 ( 177 Lu, tissue penetration: 0.5-0.6 mm, maximum: 2 mm, 497 keV, half-life: 6.7 days) and yttrium-90 ( 90 Y, tissue penetration: mean 2.5 mm, maximum: 11 mm, 935 keV, half- life: 64 hours) (Teunissen et al., 2005; Kwekkeboom et al., 2008; Ahmadzadehfar et al., 2010; Pillai et al., 2013; Ahmadzadehfar et al., 2016).
• Radionuclides have found extensive use in nuclear medicine, particularly as beta-ray emitters for damaging tumor cells.
• radionuclides are suitably employed as the antineoplastic agents.
• Radionuclides can be associated with intact, bacterially derived minicells by any known technique.
• a protein or other minicell-surface moiety can be labeled with a radionuclide, using a commercially available labeling means, such as use of Pierce Iodination reagent, a product of Pierce Biotechnology Inc. (Rockford, Ill.), detailed in Rice et al., Semin. Nucl. Med., 41, 265-282 (2011).
• radionuclides can be incorporated into proteins that are inside minicells.
• minicell-producing bacterial strain is transformed with plasmid DNA encoding foreign protein.
• minicells are formed during asymmetric cell division, several copies of the plasmid DNA segregate into the minicell cytoplasm.
• the resultant recombinant minicells are incubated in the presence of radiolabeled amino acids under conditions such that foreign protein expressed inside the minicell, from the plasmid DNA, incorporates into the radionuclide-carrying amino acids.
• recombinant minicells are incubated in minimal growth medium that contains 35s methionine, whereby newly expressed, plasmid-encoded proteins incorporate the 35s methionine.
• minimal growth medium that contains 35s methionine
• plasmid-encoded proteins incorporate the 35s methionine.
• a similar approach can be used so that recombinant minicells become packaged with other radiolabels, as desired.
• Oligosaccharides on the minicell surface also can be radiolabeled using, for example, well-established protocols described by Fukuda, Curr. Protocols Molec. Biol. (Suppl. 26), 17.5.1-17.5.8 (1994).
• Illustrative of such oligosaccharides that are endemic to minicells is the O- polysaccharide component of the lipopolysaccharide (LPS) found on the surface of minicells derived from Gram-negative bacteria (see below).
• LPS lipopolysaccharide
• a preferred methodology in this regard is to radio label a bispecific antibody used as a tumor targeting ligand that is used to target minicells to specific tumors. See US Patent
• the bispecific antibody“coated” on a minicell exposes a significant amount of additional surface protein for radiolabeling. Accordingly, it is possible to achieve a higher specific activity of the radiolabel associated with the antibody-coated minicell.
• the radiolabeling of non- coated minicells i.e., when the radionuclide labels only endemic moieties, can result in weaker labeling (lower specific activity). In one embodiment, this weaker labeling is thought to occur because the outer membrane-associated proteins of minicells derived from Gram-negative bacteria are masked by LPS, which, as further discussed below, comprises long chains of O- polysaccharide covering the minicell surface.
• a composition of the disclosure would be delivered in a dose or in multiple doses that affords a level of in-tumor irradiation that is sufficient at least to reduce tumor mass, if not eliminate the tumor altogether.
• the progress of treatment can be monitored along this line, on a case-by-case basis.
• the amount of radioactivity packaged in the composition typically will be on the order of about 30 to about 50 Gy, although the invention also contemplates a higher amount of radioactivity, such as for example about 50 to about 200 Gy, which gives an overall range between about 30 Gy and about 200 Gy.
• the amount of radioactivity packaged in the composition can be even lower than mentioned above, given the highly efficient and specific delivery of the minicell-borne radionuclides to a tumor. Accordingly, in one aspect the composition comprises from about 20 to about 40 Gy, or about 10 to about 30 Gy, or about 1 to about 20 Gy, or less than about 10 Gy.
• Some tumor targeting ligands may include a radioisotope that functions to deliver radiation to the tumor while the ligand binds the tumor cell.
• the ligand comprises Arg-Gly-Asp (RGD) peptide, bombesin (BBN)/gastrin-releasing peptide (GRP), cholecystokinin (CCK)/gastrin peptide, a-melanocyte-stimulating hormone (a-MSH), neuropeptide Y (NPY), neutrotensin (NT), [ 68 Ga]Ga-PSMA-HBED-CC ([ 68 Ga]Ga-PSMA-l l [PET]), [ 177 LU]LU/[ 90 Y]Y-J591, [ 123 I]I-MIP-1072, [ 131 I]I-MIP-1095, 68 Ga or 177 Lu labeled PSMA-I&T, 68 Ga or 177 Lu labeled DKFZ-PSMA-6
• the radioisotope is conjugated to the tumor targeting ligand.
• the conjugation is via a linker.
• the tumor targeting ligand comprises a peptide comprising functional group(s) for conjugation of a radioisotope or chelator moiety that chelates a radioisotope.
• the functional groups of peptides available for conjugation include but are not limited to the e-amino group on lysine side chains, the guanidinium group on arginine side chains, the carboxyl groups on aspartic acid or glutamic acid, the cysteine thiol, and the phenol on tyrosine.
• the radioisotope functions as a radioimaging agent.
• radioisotopes have been used for peptide labeling including 99m Tc, 123 I, and 11 'in for SPECT imaging and 1S F, 64 Cu and 68 Ga for PET imaging (Chatalic et al., 2015).
• these radioisotopes are attached to the peptides via chelators.
• Some widely-used chelators are described in (Sun et al., 2017).
• Most therapeutic radiopharmaceuticals are labeled with beta- emitting isotopes (b-).
• the minicells of the present invention targeted to the tumor cells will also deliver targeted radiation from the radioisotope to the tumor cell to which the minicell is bound.
• the radioisotope functions as a therapeutic radiation emitting agent, and wherein the amount of radiation provided by the radioisotope is sufficient to provide a therapeutic effect on the tumor.
• the therapeutic effect is a reduction in tumor size.
• the tumor may be reduced in size by about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%.
• Radiolabeled phosphonates have a high bone affinity and can be used for imaging and palliation of painful bone metastases. Depending on the degree of osseous metabolism, the tracer accumulates via adhesion to bones and, preferably, to osteoblastic bone metastases. Therapy planning requires a bone scintigraphy with technetium-99m-hydroxyethylidene diphosphonate (HEDP) to estimate the metabolism and the extent of the metastasis involvement.
• HEDP technetium-99m-hydroxyethylidene diphosphonate
• Bisphosphonate HEDP can be labeled for therapy either with rhenium- 186 (beta-emitter, half- life: 89 hours, 1.1 MeV maximal energy, maximal range: 4.6 mm) or rhenium-188 (beta-emitter [to 85%, 2.1 MeV] and gamma-emitter [to 15%, 155 keV], half-life: 16.8 hours, maximal range in soft tissue: 10 mm) (Palmedo, 2007).
• New promising radiopharmaceuticals for bone palliation therapy include radiolabeled complexes of zoledronic acid. Zoledronic acid belongs to a new, most potent generation of bisphospho nates with cyclic side chains.
• the bone affinity of zoledronic acid labeled with scandium-46 or lutetium-177 has shown excellent absorption (98% for [177Lu]Lu-zoledronate and 82% for [46Sc]Sc-zoledronate), which is much higher than of bisphosphonates labeled with samarium- 153 (maximum: 67%) (Majkowska et al., 2009). These bisphosphonates can be conjugated to intact minicells for use as diagnostics or treatment for bone metastasis.
• the encapsulated CD ld-restricted iNKT cell antigens are administered in combination with an antineoplastic agent that is a chemotherapy drug.
• antineoplastic agent that is a chemotherapy drug.
• “chemotherapeutic drug,”“chemotherapeutic agent,” and“chemotherapy” are employed interchangeably to connote a drug that has the ability to kill or disrupt a neoplastic cell.
• a chemotherapeutic agent can be a small molecule drug or a biologic drug, as further detailed below.
• the chemotherapy drug is administered using an intact, bacterially derived minicell.
• The“small molecule drug” subcategory encompasses compounds characterized by having (i) an effect on a biological process and (ii) a low molecular weight as compared to a protein or polymeric macromolecule.
• Small molecule drugs typically are about 800 Daltons or less, with a lower limit of about 150 Daltons, as illustrated by Temodar® (temozolomide), at about 194 Daltons, which is used to treat glioblastoma and other types of brain cancer.
• Temodar® temozolomide
• about indicates that the qualified molecular- weight value is subject to variances in measurement precision and to experimental error on the order of several Daltons or tens of Daltons.
• a small molecule drug can have a molecular weight of about 900 Daltons or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, or about 400 Daltons or less, e.g., in the range of about 150 to about 400 Daltons. More specifically, a small molecule drug can have a molecular weight of about 400 Daltons or more, about 450 Daltons or more, about 500 Daltons or more, about 550 Daltons or more, about 600 Daltons or more, about 650 Daltons or more, about 700 Daltons or more, or about 750 Daltons or more.
• the small molecule drug packaged into the minicells has a molecular weight between about 400 and about 900 Daltons, between about 450 and about 900 Daltons, between about 450 and about 850 Daltons, between about 450 and about 800 Daltons, between about 500 and about 800 Daltons, or between about 550 and about 750 Daltons.
• suitable small molecule drugs include but are not limited to those listed above, such as nitrogen mustards, nitrosorueas, ethyleneimine, alkane sulfonates, tetrazine, platinum compounds, pyrimidine analogs, purine analogs, anti-metabolites, folate analogs, anthracyclines, taxanes, vinca alkaloids, and topoisomerase inhibitors, inter alia.
• a small molecule drug for use in the present invention can be selected from among any of the following, inter alia: enediynes, such as dynemicin A, unicalamycin, calicheamicin g ⁇ and calicheamicin-theta- 1 ; meayamicin, a synthetic analog of FR901464; benzosuberene derivatives as described, for example, by Tanpure et a , Bioorg. Med. Chem., 21 : 8019-32 (2013);
• auristatins such as auristatin E, mono-methyl auristatin E (MMAE), and auristatin F, which are synthetic analogs of dolastatin; duocarmysins such as duocarmycin SA and CC-1065;
• maytansine and its derivatives maytansinoids
• maytansinoids such as DM1 and DM4
• irinotecan maytansinoids
• topoisomerase inhibitors such as topotecan, etoposide, mitoxantrone and teniposide; and yatakemycin, the synthesis of which is detailed by Okano et al., 2006.
• any one or more or all of the specific small molecule drugs detailed herein are illustrative of those suitable for use in this invention: actinomycin-D, alkeran, ara-C, anastrozole, BiCNU, bicalutamide, bisantrene, bleomycin, busulfan, capecitabine (Xeloda®), carboplatin, carboplatinum, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, cytosine arabinoside, cytoxan, dacarbazine,
• mitoxantrone oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, streptozocin, STI-571, tamoxifen, temozolomide, teniposide, tetrazine, thioguanine, thiotepa, tomudex, topotecan, treosulphan, trimetrexate, vinblastine, vincristine, vindesine, vinorelbine, and VP- 16.
• a“biologic drug” is defined, by contrast, to denote any biologically active macromolecule that can be created by a biological process, exclusive of “functional nucleic acids,” discussed below, and polypeptides that by size qualify as small molecule drugs, as defined above.
• The“biologic drug” subcategory thus is exclusive of and does not overlap with the small molecule drug and functional nucleic acid subcategories.
• Illustrative of biologic drugs are therapeutic proteins and antibodies, whether natural or recombinant or synthetically made, e.g., using the tools of medicinal chemistry and drug design.
• the encapsulated CD ld-restricted iNKT cell antigens are administered in combination with an antineoplastic agent that is a supertoxic chemotherapy drug.
• the supertoxic chemotherapy drug is administered using an intact, bacterially derived minicell described herein.
• “Highly toxic chemotherapy drug” or“supertoxic chemotherapy drugs” in this description refer to chemotherapy drugs that can overcome the resistance to conventional drugs due to their relatively low lethal dose to normal cells as compared to their effective dose for cancer cells.
• a highly toxic chemotherapy drug has a median lethal dose (LD50) that is lower than its median effective dose (ED50) for a targeted cancer.
• LD50 median lethal dose
• ED50 median effective dose
• a highly toxic or supertoxic chemotherapy drug can have an LD50 that is lower than about 500%, about 400%, about 300%, about 250%, about 200%, about 150%, about 120%, or about 100% of the ED50 of the drug for a targeted cancer.
• a highly toxic or supertoxic chemotherapy drug has a maximum sub- lethal dose (i.e., the highest dose that does not cause serious or irreversible toxicity) that is lower than its minimum effective dose, e.g., about 500%, about 400%, about 300%, about 250%, about 200%, about 150%, about 120%, about 100%, about 90%, about 80%, about 70%, about 60% or about 50% of the minimum effective dose.
• the targeted cancer can be, for example, (1) a cancer type for which the drug is designed, (2) the first cancer type in which a pre-clinical or clinical trial is run for that drug, or (3) a cancer type in which the drug shows the highest efficacy among all tested cancers.
• Illustrative, non-limiting examples of supertoxic chemotherapy drugs include but are not limited to maytansinoids, duocarmycins, morpholinyl anthracycline, and their derivatives.
• Maytansinoids molecular weight: about 738 Daltons
• maytansinoids are suitable for delivery to tumor patients via minicells, pursuant to the present invention.
• Duocarmycins molecular weight: about 588 Daltons
• maytansinoids are suitable chemotherapy drugs for use in the invention.
• nemorubicin (3’-deamino-3’-[2(S)-methoxy-4-morpholinyl]doxorubicin) (MMDX)
• MMDX mitochondrial-derived neuropeptide
• PNU-159682 (3’-deamino-3”-4’-anhydro-[2”(S)-methoxy- 3”(R)-hydroxy-4”-morpholinyl- ] doxorubicin
• the minicell comprises the supertoxic chemotherapy drug 3’-deamino-3”,4’-anhydro-[2”(S)-methoxy-3”(R)-oxy-4”- morpholinyl] doxorubicin (PNU-159682).
• PNU-159682 is a potent drug that appears to overcome drug resistance in a number of different tumor cell lines and is much more potent than a range of conventional chemotherapeutics in cytotoxicity assays against many different tumor cell lines. See Examples 8 and 9. Further, it was shown in in vivo mouse xenograft experiments that human tumor xenografts resistant to doxorubicin can be treated effectively with IV administration of EGFR-targeted and PNU-159682-loaded EDVs.
• composition comprises an EGFR-targeted minicell comprising PNU- 159682 as an active anticancer drug.
• cancer chemotherapy drugs that may exhibit supertoxic chemotherapy properties include auristatins, calicheamicins (DNA damaging agents), a-amanitin (RNA polymerase II inhibitor), centanamycin, geldanamycin, pyrrolobenzodiazepine, streptonigtin, nitrogen mustards, nitrosorueas, ethyleneimine, alkane sulfonates, tetrazine, platinum compounds, pyrimidine analogs, purine analogs, antimetabolites, folate analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors, and hormonal agents, inter alia.
• auristatins calicheamicins (DNA damaging agents), a-amanitin (RNA polymerase II inhibitor), centanamycin, geldanamycin, pyrrolobenzodiazepine, streptonigtin, nitrogen mustards, nitrosorueas, ethyleneimine, alkane sulfonates,
• the encapsulated CD ld-restricted iNKT cell antigens are administered in combination with an antineoplastic agent that is a biologic chemotherapy drug.
• antineoplastic agent that is a biologic chemotherapy drug.
• drugs include but are not limited to asparaginase, AIN-457, bapineuzumab, belimumab, brentuximab, briakinumab, canakinumab, cetuximab, dalotuzumab, denosumab, epratuzumab, estafenatox, farletuzumab, figitumumab, galiximab, gemtuzumab, girentuximab (WX-G250), ibritumomab, inotuzumab, ipilimumab, mepolizumab, muromonab-CD3, naptumomab, necitumumab, nimotuzumab, o
• the biologic chemotherapy drug is administered using an intact, bacterially derived minicell.
• the encapsulated CD ld-restricted iNKT cell antigens are administered in combination with a functional nucleic acid.
• “Functional nucleic acid” refers to a nucleic acid molecule that, upon introduction into a host cell, specifically interferes with expression of a protein.
• the functional nucleic acid is administered using an intact, bacterially derived minicell. With respect to treating cancer, it is preferable that a functional nucleic acid payload delivered to cancer cells via intact, bacterially derived minicells inhibits a gene that promotes tumor cell proliferation, angiogenesis or resistance to
• RNAs such as siRNA, shRNA, short RNAs (typically less than 400 bases in length), micro-RNAs (miRNAs), ribozymes and decoy RNA, antisense nucleic acids, and LincRNA, inter alia.
• “ribozyme” refers to an RNA molecule having an enzymatic activity that can repeatedly cleave other RNA molecules in a nucleotide base sequence-specific manner.
• “Antisense oligonucleotide” denotes a nucleic acid molecule that is complementary to a portion of a particular gene transcript, such that the molecule can hybridize to the transcript and block its translation.
• An antisense oligonucleotide can comprise RNA or DNA.
• The“LincRNA” or“long intergenic non-coding RNA” rubric encompasses non-protein coding transcripts longer than 200 nucleotides. LincRNAs can regulate the transcription, splicing, and/or translation of genes, as discussed by Khalil et al., 2009.
• Each of the types of regulatory RNA can be the source of functional nucleic acid molecule that inhibits a tumor-promoting gene as described above and, hence, that is suitable for use according to the present disclosure.
• the intact minicells carry siRNA molecules mediating a post-transcriptional, gene-silencing RNA interference (RNAi) mechanism, which can be exploited to target tumor-promoting genes.
• RNAi gene-silencing RNA interference
• siRNA generally refers to double- stranded RNA molecules from about 10 to about 30 nucleotides long that are named for their ability specifically to interfere with protein expression.
• siRNA molecules are about 12 to about 28 nucleotides long, more preferably about 15 to about 25 nucleotides long, still more preferably about 19 to about 23 nucleotides long and most preferably about 21 to about 23 nucleotides long. Therefore, siRNA molecules can be, for example, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, or about 29 nucleotides in length.
• the length of one strand designates the length of an siRNA molecule.
• an siRNA that is described as 21 ribonucleotides long could comprise two opposing strands of RNA that anneal for 19 contiguous base pairings. The two remaining ribonucleotides on each strand would form an“overhang.”
• the longer of the strands designates the length of the siRNA. For instance, a dsRNA containing one strand that is 21 nucleotides long and a second strand that is 20 nucleotides long, constitutes a 21-mer.
• the intact minicells of the present disclosure carry miRNAs, which, like siRNA, are capable of mediating a post -transcriptional, gene-silencing RNA interference (RNAi) mechanism.
• RNAi gene-silencing RNA interference
• the gene-silencing effect mediated by miRNA can be exploited to target tumor-promoting genes. For example, see Kota et al., 2009 (delivery of a miRNA via transfection resulted in inhibition of cancer cell proliferation, tumor- specific apoptosis and dramatic protection from disease progression without toxicity in murine liver cancer model), and Takeshita et al., 2010 (delivery of synthetic miRNA via transient transfection inhibited growth of metastatic prostate tumor cells on bone tissues).
• miRNA generally refers to a class of about 17 to about 27- nucleotide single- stranded RNA molecules (instead of double- stranded as in the case of siRNA). Therefore, miRNA molecules can be, for example, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, or about 27 nucleotides in length.
• miRNA molecules are about 21 to about 25 nucleotide long.
• siRNAs are very similar to miRNAs.
• the former generally do not fully complement the mRNA target.
• siRNA must be completely complementary to the mRNA target. Consequently, siRNA generally results in silencing of a single, specific target, while miRNA is promiscuous.
• mouse“mir-353” a new miRNA discovered after mouse mir-352 will be named mouse“mir-353.”
• tools to assist the design of regulatory RNA including miRNA are readily available.
• a computer-based miRNA design tool is available on the internet at wmd2.weigelworld.org/cgi-bin/mirnatools.pl.
• miRNA 16a can be administered by targeted minicell-mediated delivery to mesothelioma and Adreno-Cortical cancer cells. See Example 7. Once internalized by the cancer cells, the miRNA16a was found to potently inhibit cancer cell proliferation. Accordingly, in some embodiments the minicells of the present disclosure comprise miRNA16a. Other microRNAs useful in inhibiting the proliferation of neoplastic cells include mir-34 family and let-7 family.
• a functional nucleic acid employed in the compositions of the invention can inhibit a gene that promotes tumor cell proliferation, angiogenesis or resistance to chemotherapy. The inhibited gene also can itself inhibit apoptosis or cell cycle arrest. Examples of genes that can be targeted by a functional nucleic acid are provided below.
• Functional nucleic acids of the disclosure preferably target the gene or transcript of a protein that promotes drug resistance, inhibits apoptosis or promotes a neoplastic phenotype.
• Successful application of functional nucleic acid strategies in these contexts have been achieved in the art, but without the benefits of minicell vectors. See, e.g., Sioud, Trends Pharmacol. Sci., 2004;, Caplen, Expert Opin. Biol. Ther., 2003; Nieth et al., 2003; Caplen and Mousses, 2003; Duxbury et al., 2004; Yague et aL, 2004; and Duan et al., 2004.
• Proteins that contribute to drug resistance constitute preferred targets of functional nucleic acids.
• the proteins may contribute to acquired drug resistance or intrinsic drug resistance.
• diseased cells such as tumor cells
• PGY1, MDR1, ABCB1, MDR-associated protein ATP binding cassette transporters
• MDR-2 and MDR-3 ATP binding cassette transporters
• MRP2 multi-drug resistance associated protein
• BCR-ABL breakpoint cluster region— Abe Ison protooncogene
• STI-571 resistance-associated protein
• lung resistance -related protein cyclooxygenase-2
• nuclear factor kappa nuclear factor kappa
• XRCC1 X-ray cross-complementing group 1
• ERCC1 excision cross-complementing gene
• mutant b-tubulin are additional targets involved in acquired drug resistance.
• Particularly useful targets that contribute to drug resistance include ATP binding cassette transporters such as P-glycoprotein, MDR-2, MDR-3, BCRP, APTl la, and LRP.
• Useful targets also include proteins that promote apoptosis resistance. These include Bcl-2 (B cell leukemia/lymphoma), BC1-XL, Al/Bfl 1, focal adhesion kinase, dihydrodiol dehydrogenase, and p53 mutant protein.
• Useful targets further include oncogenic and mutant tumor suppressor proteins.
• Illustrative of these are beta.-Catenin, PKC-a (protein kinase C), C-RAF, K-Ras (V12), DP97 Dead box RNA helicase, DNMT1 (DNA methyltransferase 1), FLIP (Flice-like inhibitory protein), C-Sfc, 53BPI, Polycomb group protein EZH2 (Enhancer of zeste homologue), ErbBl, HPV-16 E5 and E7 (human papillomavirus early 5 and early 7), Fortilin & MCI IP (Myeloid cell leukemia 1 protein), DIP13a (DDC interacting protein 13a), MBD2 (Methyl CpG binding domain), p21, KLF4 (Kruppel-like factor 4), tpt/TCTP (Translational controlled tumor protein), SPK1 and SPK2 (Sphingosine kinase), P300, PLK
• cyclooxygenase-2 nuclear factor kappa, XRCC1, ERCC1, GSTP1, mutant - b-tubulin, and growth factors.
• CEPB4 cytoplasmic polyadenylation element binding proteins
• glioblastoma and pancreatic cancers where the protein activates hundreds of genes associated with tumor growth, and it is not detected in healthy cells.
• treatment of a glioblastoma could be effected via administration of a composition containing intact, bacterially derived minicells that encompass an agent that counters overexpression of CEPB4, such as an siRNA or other functional nucleic acid molecule that disrupts CEPB4 expression by the tumor cells.
• a further example of useful targets for functional nucleic acids include replication protein A (RPA), a trimeric complex composed of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits, which is essential for DNA replication in all organisms. Iftode et al., 1999.
• RPA replication protein A
• RPA1 a trimeric complex composed of 70-kDa
• RPA2 32-kDa
• RPA3 subunits which is essential for DNA replication in all organisms.
• the minicells of the present disclosure comprise Plkl.
• RR ribonucleotide reductase
• RRM1 ribonucleotide reductase Ml
• Antineoplastic therapies useful for administration with the intact, bacterially derived minicells or killed bacterial cells comprising a CD ld-restricted iNKT antigen of the present disclosure also include non-drug therapies that induce cancer cell death, such as radiation therapies, surgical methods, adoptive cell therapies, enzyme-prodrug therapies and
• microorganism-based anti-tumor therapies are microorganism-based anti-tumor therapies.
• the encapsulated CD ld-restricted iNKT antigens of the present disclosure are administered in combination with an antineoplastic therapy including, but not limited to, targeted radiation therapy, stereotactic radiation, photodynamic therapy, microwave thermal ablation, cryoablation, high intensity ultrasound, radiofrequency ablation, laser beam irradiation, cyberknife, and hyperthermia tumor treatment.
• antineoplastic therapy including, but not limited to, targeted radiation therapy, stereotactic radiation, photodynamic therapy, microwave thermal ablation, cryoablation, high intensity ultrasound, radiofrequency ablation, laser beam irradiation, cyberknife, and hyperthermia tumor treatment.
• the encapsulated CD ld-restricted iNKT antigens of the present disclosure are administered in combination with an antineoplastic prodrug therapy, including, but not limited to, directed enzyme prodrug therapy (DEPT), antibody-directed enzyme prodrug therapy (ADEPT), Gene-directed enzyme prodrug therapy (GDEPT), Virus- directed enzyme prodrug therapy (VDEPT), Polymer-directed enzyme prodrug therapy
• DEPT directed enzyme prodrug therapy
• ADPT antibody-directed enzyme prodrug therapy
• GDEPT Gene-directed enzyme prodrug therapy
• VDEPT Virus- directed enzyme prodrug therapy
• Polymer-directed enzyme prodrug therapy including, but not limited to, directed enzyme prodrug therapy (DEPT), antibody-directed enzyme prodrug therapy (ADEPT), Gene-directed enzyme prodrug therapy (GDEPT), Virus- directed enzyme prodrug therapy (VDEPT), Polymer-directed enzyme prodrug therapy
• PDEPT clostridial-directed enzyme prodrug therapy
• CDEPT clostridial-directed enzyme prodrug therapy
• encapsulated CD ld-restricted iNKT antigens of the present disclosure are administered in combination with an adoptive cell therapy that induces cancer cell death such as a chimeric antigen receptor (CAR) T cell therapy.
• CAR chimeric antigen receptor
• the CAR T cells comprises a chimeric antigen receptor directed against a tumor antigen.
• the CAR T cells comprises a chimeric antigen receptor directed against a a-folate receptor, B-Cell Maturation Antigen (BCMA), carboxyanhydrase-IX (CAIX), carcinoembryonic antigen (CEA), CD22, CD19, CD30, CD133, CLL-1, disialoganglioside (GD2), EPH receptor A2, (EphA2), epithelial cell adhesion molecule, (EpCAM), glypican-3 (GPC3), epidermal growth factor receptor (EGFR), EGFRvIII, fibroblast activation protein a (FAP), hepatocyte growth factor receptor (c-Met), human epidermal growth factor receptor-2 (HER2), IL13Ra2, LI cell adhesion molecule (Ll-CAM), mesothelin, mucin (MUC-1), PSCA, prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (
• BCMA
• the encapsulated CD Id- restricted iNKT antigens of the present disclosure are administered in combination with an immune checkpoint therapy such as an anti-PD-l/PD-Ll or anti-CTLA-4 antibody therapy.
• an immune checkpoint therapy such as an anti-PD-l/PD-Ll or anti-CTLA-4 antibody therapy.
• the CAR T cells comprise an anti-PD-l/PD-Ll or anti-CTLA-4 antibody.
• a non-drug antineoplastic therapy is administered in addition to one or more antineoplastic agents described above.
• Any combination of antineoplastic agents and therapies is suitable for administration with the disclosed encapsulated CD ld-restricted iNKT antigens provided that the antineoplastic agents and/or therapies effect the death of cancer cells.
• the CDld-restricted iNKT antigens of the present disclosure can be effectively delivered to phagocytic cells by encapsulating the antigen using intact, bacterially-derived minicells or killed bacterial cells that can be uptaken by macrophages and/or by dendritic cells.
• the encapsulated CDld-restricted iNKT antigens are administered in combination with an antineoplastic agent that is also encapsulated, for example, using intact, bacterially derived minicells or killed bacterial cells.
• the CDld-restricted iNKT antigens are administered in combination with an antineoplastic agent, where both the CDld-restricted antigen and the antineoplastic agent are encapsulated in intact, bacterially derived minicells or killed bacterial cells.
• the CDld-restricted antigen and the antineoplastic agent are encapsulated in the same minicell or killed bacterial cell.
• the CD ld-restricted antigen and the antineoplastic agent are encapsulated in separate minicells or killed bacterial cells. In some embodiments, the encapsulated CD ld- restricted antigen is administered with an antineoplastic agent that is not encapsulated.
• minicells are distinct from other small vesicles, such as so-called“membrane blebs” (about 0.2 pm or less in size), which are generated and released spontaneously in certain situations but which are not due to specific genetic rearrangements or episomal gene expression.
• intact minicells are distinct from bacterial ghosts, which are not generated due to specific genetic rearrangements or episomal gene expression.
• Bacterially derived minicells employed in this disclosure are fully intact and thus are distinguished from other chromosome-free forms of bacterial cellular derivatives characterized by an outer or defining membrane that is disrupted or degraded, even removed. See U.S. Pat. No. 7, 1 83, 105 at col. I l l , lines 54 et seq.
• the intact membrane that characterizes the minicells of the present disclosure allows retention of the therapeutic payload within the minicell until the payload is released, post-uptake, within a phagocytic cell or tumor cell.
• Minicell or EDVs are anucleate, non-living nanoparticles produced as a result of inactivating the genes that control normal bacterial cell division, thereby de -repressing polar sites of cell. Ma et al., 2004.
• the de -repression means that the bacteria divide in the center as well as at the poles; the polar division resulting in minicells which the inventors of the present disclosure have shown can function as leak-resistant, micro-reservoir carriers that allow efficient packaging of a range of different chemotherapeutic drugs.
• EDVs can readily accommodate payloads of up to 1 million drug molecules. Further, EDVs can be targeted to over-expressed receptors on the surface of cancer cells using bispecific antibodies, see section D infra, which allows highly significant tumor growth-inhibition and/or regression, both in vitro and in vivo.
• DOXIL liposomal doxorubicin
• the minicells employed in the present invention can be prepared from bacterial cells, such as E. coli and S. typhymurium. Prokaryotic chromosomal replication is linked to normal binary fission, which involves mid-cell septum formation. In E. coli, for example, mutation of min genes, such as minCD, can remove the inhibition of septum formation at the cell poles during cell division, resulting in production of a normal daughter cell and a chromosome-less minicell. See de Boer et al., 1992; Raskin & de Boer, 1999; Hu & Lutkenhaus, 1999; Harry, 2001.
• chromosome-less minicells are generated following a range of other genetic rearrangements or mutations that affect septum formation, for example, in the divIVBl in B. subtilis. See Reeve and Cornett, 1975. Minicells also can be formed following a perturbation in the levels of gene expression of proteins involved in cell division/chromosome segregation. For instance, over-expression of minH leads to polar division and production of minicells. Similarly, chromosome-less minicells can result from defects in chromosome segregation, e.g., the smc mutation in Bacillus subtilis (Britton et al., 1998), the spoOJ deletion in B.
• CafA can enhance the rate of cell division and/or inhibit chromosome partitioning after replication (Okada et al., 1994), resulting in formation of chained cells and chromosome-less minicells.
• minicells can be prepared for the present disclosure from any bacterial cell, be it of Gram-positive or Gram- negative origin due to the conserved nature of bacterial cell division in these bacteria.
• the minicells used in the disclosure should possess intact cell walls (i.e., are“intact minicells”), as noted above, and should be distinguished over and separated from other small vesicles, such as membrane blebs, which are not attributable to specific genetic rearrangements or episomal gene expression.
• the parental (source) bacteria for the minicells can be Gram positive, or they can be Gram negative.
• the parental bacteria are one or more selected from Terra-/Glidobacteria (BV1), Proteobacteria (BV2), BV4 including Spirochaetes, Sphingobacteria, and Planctobacteria.
• the bacteria are one or more selected from Firmicutes (BV3) such as Bacilli, Clostridia or Tenericutes/Mollicutes, or
• Actinobacteria such as Actinomycetales or Bifidobacteriales.
• killed bacterial cells are non-living prokaryotic cells of bacteria, cyanobateria, eubacteria and archaebacteria, as defined in the 2nd edition of Bergey’s Manual Of Systematic Biology. Such cells are deemed to be“intact” if they possess an intact cell wall and/or cell membrane and contain genetic material (nucleic acid) that is endogenous to the bacterial species. Methods of preparing killed bacterial cells are described, for instance, in U.S. 2008/0038296.
• the bacteria are one or more selected from Eobacteria
• Chlamydiae/Verrucomicrobia Planctomycetes, Acidobacteria, Chrysiogenetes, Deferribacteres, Fusobacteria, Gemmatimonadetes, Nitrospirae, Synergistetes, Dictyoglomi, Lentisphaerae Bacillales, Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillales, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Streptococcaceae, Clostridiales, Halanaerobiales,
• composition of the disclosure should comprise minicells or killed bacterial cells that are isolated as thoroughly as possible from immunogenic components and other toxic contaminants.
• Methodology for purifying bacterially derived minicells to remove free endotoxin and parent bacterial cells are described, for example, in WO
• the purification process achieves removal of (a) smaller vesicles, such as membrane blebs, which are generally smaller than 0.2 pm in size, (b) free endotoxins released from cell membranes, and (c) parental bacteria, whether live or dead, and their debris, which also are sources of free endotoxins.
• removal can be implemented with, inter alia, a 0.2 pm filter to remove smaller vesicles and cell debris, a 0.45 pm filter to remove parental cells following induction of the parental cells to form filaments, antibiotics to kill live bacterial cells, and antibodies against free endotoxins.
• minicells Underlying the purification procedure is a discovery by the present inventors that, despite the difference of their bacterial sources, all intact minicells are approximately 400 nm in size, i.e., larger than membrane blebs and other smaller vesicles and yet smaller than parental bacteria. Size determination for minicells can be accomplished by using solid-state, such as electron microscopy, or by liquid- based techniques, e.g., dynamic light scattering. The size value yielded by each such technique can have an error range, and the values can differ somewhat between techniques. Thus, the size of minicells in a dried state can be measured via electron microscopy as approximately 400 nm ⁇ 50 nm.
• Dynamic light scattering can measure the same minicells to be approximately 500 nm ⁇ 50 nm in size. Also, drug-packaged, ligand- targeted minicells can be measured, again using dynamic light scattering, to be approximately 400 nm to 600 nm ⁇ 50 nm.
• This scatter of size values is readily accommodated in practice, e.g., for purposes of isolating minicells from immunogenic components and other toxic contaminants, as described above. That is, an intact, bacterially derived minicell is characterized by cytoplasm surrounded by a rigid membrane, which gives the minicell a rigid, spherical structure. This structure is evident in transmission-electron micrographs, in which minicell diameter is measured, across the minicell, between the outer limits of the rigid membrane. This measurement provides the above- mentioned size value of 400 nm ⁇ 50 nm.
• LPS lipopolysaccharide
• the component is a chain of repeat carbohydrate-residue units, with as many as 70 to 100 repeat units of four to five sugars per repeat unit of the chain. Because these chains are not rigid, in a liquid environment, as in vivo, they can adopt a waving, flexible structure that gives the general appearance of seaweed in a coral sea environment; i.e., the chains move with the liquid while remaining anchored to the minicell membrane.
• a composition of the disclosure preferably comprises less than about 350 EU free endotoxin.
• levels of free endotoxin of about 250 EU or less, about 200 EU or less, about 150 EU or less, about 100 EU or less, about 90 EU or less, about 80 EU or less, about 70 EU or less, about 60 EU or less, about 50 EU or less, about 40 EU or less, about 30 EU or less, about 20 EU or less, about 15 EU or less, about 10 EU or less, about 9 EU or less, about 8 EU or less, about 7 EU or less, about 6 EU or less, about 5 EU or less, about 4 EU or less, about 3 EU or less, about 2 EU or less, about 1 EU or less, about 0.9 EU or less, about 0.8 EU or less, about 0.7 EU or less, about 0.6 EU or less, about 0.5 EU or less, about 0.4 EU or less, about 0.3 EU or less, about 0.2 EU or less, about 0.1 EU or less, about 0.05 EU or less
• a composition of the disclosure also can comprise at least about 10 9 minicells or killed bacterial cells, e.g., at least about 1 xlO 9 , at least about 2 x 10 9 , at least about 5 x 10 9 , or at least 8 x 10 9
• the composition comprises no more than about 10 11 minicells or killed bacterial cells, e.g., no more than about 1 x 10 11 or no more than about 9 x 10 10 , or no more than about 8 x 10 10 .
• Active agents such as a CD ld-restricted iNKT cell antigen, or antineoplastic agents, such as small molecular drugs, proteins and functional nucleic acids can be packaged into minicells directly by co-incubating a plurality of intact minicells with the active agent in a buffer.
• the buffer composition can be varied, as a function of conditions well known in this field, to optimize the loading of the active agent in the intact minicells.
• An exemplary buffer suitable for loading includes, but is not limited to, phosphate buffered saline (PBS).
• Active agents such as functional nucleic acids or proteins that can be encoded for by a nucleic acid, can be introduced into minicells by transforming into the parental bacterial cell a vector, such as a plasmid, that encodes the active agents.
• a vector such as a plasmid
• the minicell retains certain copies of the plasmid and/or the expression product, the antineoplastic agent. More details of packaging and expression product into a minicell is provided in WO 2003/033519, the contents of which are incorporated into the present disclosure in its entirety by reference.
• WO 2003/033519 Data presented in WO 2003/033519 demonstrated, for example, that recombinant minicells carrying mammalian gene expression plasmids can be delivered to phagocytic cells and to non-phagocytic cells.
• WO 2003/033519 also described the genetic transformation of minicell- producing parent bacterial strains with heterologous nucleic acids carried on episomally- replicating plasmid DNAs. Upon separation of parent bacteria and minicells, some of the episomal DNA segregated into the minicells. The resulting recombinant minicells were readily engulfed by mammalian phagocytic cells and became degraded within intracellular
• multiple nucleic acids directed to different mRNA targets can be packaged in the same minicell.
• Such an approach can be used to combat drug resistance and apoptosis resistance. For instance, cancer patients routinely exhibit resistance to
• chemotherapeutic drugs Such resistance can be mediated by over-expression of genes such as multi-drug resistance (MDR) pumps and anti-apoptotic genes, among others.
• MDR multi-drug resistance
• minicells can be packaged with therapeutically significant concentrations of functional nucleic acid to MDR-associated genes and administered to a patient before chemotherapy.
• packaging into the same minicell multiple functional nucleic acid directed to different mRNA targets can enhance therapeutic success since most molecular targets are subject to mutations and have multiple alleles. More details of directly packaging a nucleic acid into a minicell is provided in WO 2009/027830, the contents of which are incorporated into the present disclosure in its entirety by reference.
• Small molecule drugs can be packaged in minicells by creating a concentration gradient of the drug between an extracellular medium comprising minicells and the minicell cytoplasm.
• the extracellular medium comprises a higher drug concentration than the minicell cytoplasm
• the drug naturally moves down this concentration gradient, into the minicell cytoplasm.
• the concentration gradient is reversed, however, the drug does not move out of the minicells. More details of the drug loading process and its surprising nature are found, for instance, in U.S. Patent Application Publication No. 2008/0051469, the contents of which are specifically incorporated by reference.
• the drugs initially can be dissolved in an appropriate solvent.
• paclitaxel can be dissolved in a 1: 1 blend of ethanol and cremophore EL (polyethoxylated castor oil), followed by a dilution in PBS to achieve a solution of paclitaxel that is partly diluted in aqueous media and carries minimal amounts of the organic solvent to ensure that the drug remains in solution.
• Minicells can be incubated in this final medium for drug loading.
• the inventors discovered that even hydrophobic drugs can diffuse into the cytoplasm or the membrane of minicells to achieve a high and therapeutically significant cytoplasmic drug load.
• minicell membrane is composed of a hydrophobic phospholipid bilayer, which would be expected to prevent diffusion of hydrophobic molecules into the cytoplasm.
• the loading into minicells of a diversity of representative small molecule drugs has been shown, illustrating different sizes and chemical properties: doxorubicin, paclitaxel, fluoro-paclitaxel, cisplatin, vinblastine, monsatrol, thymidylate synthase (TS) inhibitor OSI-7904, irinotecan, 5-fluorouracil, gemcitabine, and carboplatin.
• doxorubicin doxorubicin
• paclitaxel fluoro-paclitaxel
• cisplatin vinblastine
• monsatrol thymidylate synthase (TS) inhibitor OSI-7904
• irinotecan 5-fluorouracil
• gemcitabine and carboplatin.
• the inventors discovered that blood vessels around tumor cells display a loss of integrity; that is, the vessels have large fenestrations and are“leaky,” even in the blood brain barrier (BBB) environment.
• BBB blood brain barrier
• cancer cells When cancer cells establish, they secrete substances that promote the formation of new blood vessels - a process called angiogenesis.
• These blood vessels grow quickly and, unlike normal blood vessels, they are leaky with“holes” (fenestrations) ranging from 50 nm to 1.2 pm (hyperpermeable vasculature).
• Drug delivery particles such as liposomes are currently believed to effect tumor-targeting by a passive process involving extravasation from the leaky vasculature that supports the tumor microenvironment. Hobbs et al., 1998.
• minicells Upon entering the tumor microenvironment, minicells are able to trigger
• a minicell that is packaged with an antineoplastic agent will release the agent into the cytoplasm of the tumor cell, killing it.
• minicells or killed bacterial cells that contain an antineoplastic agent and/or a CD ld-restricted iNKT cell antigen can be directed to a target mammalian tumor cell via a ligand.
• the ligand is“bispecific.” That is, the ligand displays a specificity for both minicell and mammalian (tumor) cell components, such that it causes a given vesicle to bind to the target cell, whereby the latter engulfs the former.
• Use of bispecific ligands to target a minicell to a tumor cell is further described in WO
• somatostatin SST
• VIP vasoactive intestinal peptide
• RGD Arg-Gly-Asp
• BBN/GRP bombesin/gastrin-releasing peptide
• Tumor-targeting peptide sequences can be selected mainly in three different ways: (1) derivatization from natural proteins (Nagpal et al., 2011); (2) chemical synthesis and structure- based rational engineering (Andersson et al., 2000; Merrifield, 2006); and (3) screening of peptide libraries (Gray and Brown 2013).
• phage display technology is a conventional but most widely used method with many advantages such as ease of handling and large numbers of different peptides can be screened effectively (Deutscher, 2010).
• Arg-Gly-Asp (RGD) peptide RGD specifically binds to integrin receptors
• Integrins constitute two subunits (a and b subunits).
• the integrin family especially 0(vp3) is associated with tumor angiogenesis and metastasis. They are overexpressed on endothelial cells during angiogenesis, but barely detectable in most normal organs. Therefore, they are widely used for diagnostic imaging.
• the bombesin-like peptide receptors have 4-subtypes: the neuromedin B receptor, the bombesin 3 receptor, the GRP receptor, and the bombesin 4 receptor. These receptors are overexpressed in many tumors such as breast cancer, ovarian cancer and gastrointestinal stromal tumors.
• CCK Cholecystokinin
• GPCR central nervous system
• a-Melanocyte-stimulating hormone a-MSHs are linear tridecapeptides, mainly responsible for skin pigmentation regulation (Singh and Mukhopadhyay, 2014).
• a-MSHs and their analogs exhibit binding affinities to melanocortin- 1 receptors (MC-lr) which are expressed in over 80% of human melanoma metastases, and thus, are widely used as vehicles for melanoma-targeted imaging and radiotherapy.
• MC-lr melanocortin- 1 receptors
• NPY Neuropeptide Y
• NPY is a 36 amino acid peptide and belongs to the pancreatic polypeptide family (Tatemoto, 2004). NPY receptors are overexpressed in various tumors including neuroblastomas, sarcomas, and breast cancers.
• NT Neutrotensin
• NT is a 13 amino acid peptide, targeting NT receptor which has been identified in various tumors such as ductal pancreatic adenocarcinomas, small cell lung cancer, and medullary thyroid cancer (Tyler-McMahon et al., 2000). Therefore, it is an attractive candidate for cancer imaging.
• PSMA Prostate Specific Membrane Antigen
• PSMA-617 there are several available radiopharmaceuticals that target PSMA including [ 68 Ga]Ga-PSMA-HBED-CC (also known as [ Ga]Ga-PSMA-l l [PET]), a monoclonal antibody (mAh) [ 177 Lu]Lu/[ 90 Y]Y-J591 (therapy), [ 123 I]I-MIP-1072 (planar/SPECT), [ 131 I]I-MIP-1095 (therapy), and theranostic agents PSMA-I&T and DKFZ-PSMA-617 (PSMA-617), which are labeled with 68 Ga for PET or with 177 Lu for therapy.
• SST Somatostatin
• SSTs are naturally occurring cyclopeptide hormones with either 14 or 28 amino acids (Weckbecker et al., 2003). They can inhibit the secretion of insulin, glucagon and some other hormones.
• Somatostatin receptors SSTRs; five subtypes SSTR1-SSTR5
• SSTRs Somatostatin receptors
• NEN Neuroendocrine neoplasia
• the common characteristic of all GEP-NEN is the compound features of endocrine and nerve cells.
• Well- differentiated NEN overexpresses somatostatin receptors (SSTRs), especially the SSTR-2 subtype.
• Substance P is an undecapeptide belonging to a family of
• Substance P is a specific endogenous ligand known for neurokinin 1 receptor (NKiR) which is found to be expressed on various cancer cells.
• T140 is a 14 amino acid peptide with one disulfide bridge and is an inverse agonist of chemokine receptor type 4 (CXCR4) (Burger et al., 2005). Its derivatives are widely used as CXCR4 imaging agents.
• CXCR4 chemokine receptor type 4
• TMTP1 Tumor molecular targeted peptide 1
• TMTP1 is a 5-amino acid peptide that has been found to specifically bind to highly metastatic cancer cells, especially those from a typical liver micrometastasis (Yang et al., 2008).
• VIP Vasoactive intestinal peptide
• the ligand can be attached to the cell membrane of the vesicles by virtue of the interaction between the ligand and a component on the cell membrane, such as a polysaccharide, a glycoprotein, or a polypeptide.
• the expressed ligand is anchored on the surface of a vesicle such that the tumor surface component-binding portion of the ligand is exposed so that the portion can bind the target mammalian cell surface receptor when the vesicle and the mammalian tumor cell come into contact.
• the ligand can be expressed and displayed by a living counterpart of a bacterially derived vesicle, e.g., by the parent cell of a minicell or by a bacterial cell before it becomes a killed cell.
• the ligand does not require a specificity to the vesicle and only displays a specificity to a component that is characteristic of mammalian cells. That is, such component need not be unique to tumor cells, per se, or even to the particular kind of tumor cells under treatment, so long as the tumor cells present the component on their surface.
• vesicles Upon intravenous administration, vesicles accumulate rapidly in the tumor microenvironment. This accumulation, occurring as a function of the above-described leaky tumor vasculature, effects delivery of vesicle -packaged therapeutic payload to cells of the tumor, which then internalize packaged vesicles.
• ligands that comprise an antibody directed at an anti- HER2 receptor or anti-EGF receptor can bind minicells to the respective receptors on a range of targeted non-phagocytic cells, such as lung, ovarian, brain, breast, prostate, and skin cancer cells.
• a suitable target cell presents a cell surface receptor the binding of which, by a ligand on a vesicle, elicits endocytosis of that vesicle.
• the present inventors discovered that the interaction between (a) the ligand on a minicell or a killed bacterial cell and (b) a mammalian cell surface receptor can activate an uptake pathway, called here a“receptor- mediated endocytosis” (rME) pathway, into the late-endosomal/lysosomal compartment of the target host cell, such as a tumor cell.
• rME receptor- mediated endocytosis pathway
• the inventors found, bacterially derived vesicles are processed through the early endosome, the late endosome and the lysosome, resulting in release of their payload into the cytoplasm of the mammalian host cell.
• a payload that is a nucleic acid not only escapes complete degradation in the late-endosomal/lysosomal compartment but also is expressed by the host cell.
• a tumor targeting ligand for this delivery approach can be“bispecific,” as described above, because it binds to surface components on a payload-carrying vesicle and on a target cell, respectively, and its interaction with the latter component leads to uptake of the vesicle into the rME pathway.
• a given target cell surface receptor can be a candidate for binding by the ligand, pursuant to the invention, if interaction with the component in effect accesses an endocytic pathway that entails a cytosolic internalization from the target cell surface.
• Such candidates are readily assessed for suitability in the invention via an assay in which a cell type that presents on its surface a candidate component is co-incubated in vitro with minicells carrying a ligand that binds the candidate and that also is joined to a fluorescent dye or other marker amenable to detection, e.g., visually via confocal microscopy.
• an in vitro assay of this sort is described by MacDiarmid et al., 2007b, in the legend to Figure 3 at page 436.
• an observed internalization of the marker constitutes a positive indication by such an assay that the tested target cell surface receptor is suitable for the present invention.
• the ligand can be any polypeptide or polysaccharide that exhibits the desired specificity or specificities.
• Preferred ligands are antibodies.
• the term“antibody” encompasses an immunoglobulin molecule obtained by in vitro or in vivo generation of an immunogenic response. Accordingly, the“antibody” category includes monoclonal antibodies and humanized antibodies, such as single-chain antibody fragments (scFv), bispecific antibodies, etc. A large number of different bispecific protein and antibody-based ligands are known, as evidenced by the review article of Caravella and
• Antibodies useful in accordance with the present disclosure can be obtained by known recombinant DNA techniques.
• an antibody that carries specificity for a surface component can be used to target minicells to cells in a tumor to be treated.
• Illustrative cell surface receptors in this regard include any of the RTKs epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet- derived growth factor receptor (PDGFR) and insulin-like growth factor receptor (IGFR), each of which is highly expressed in several solid tumors, including brain tumors, and folate receptor, which is overexpressed in some pituitary adenomas.
• Such a bispecific ligand can be targeted as well to mutant or variant receptors, e.g., the IF-13Ra2 receptor, which is expressed in 50% to 80% of human glioblastoma multiforme tumors, see Wykosky et al., 2008; Jarboe et al., 2007; Debinski et al., 2000; and Okada et al., 1994), but which differs from its physiological counterpart IL4R/IL13R, expressed in normal tissues. See Hershey, 2003. Thus, IL13Ra2 is virtually absent from normal brain cells. See Debinski and Gibo, 2000. Additionally, tumors that metastasize to the brain may overexpress certain receptors, which also can be suitable targets.
• mutant or variant receptors e.g., the IF-13Ra2 receptor, which is expressed in 50% to 80% of human glioblastoma multiforme tumors, see Wykosky et al., 2008; Jarboe et al.,
• HER2 was amplified and overexpressed in 20% of brain metastases
• EGFR was overexpressed in 21% of brain metastases
• HER3 was overexpressed in 60% of brain metastases
• HER4 was overexpressed in 22% of brain metastases.
• HER3 expression was increased in breast cancer cells residing in the brain.
• RKTs receptor tyrosine kinases
• IGF1R IGF1R
• InsRR PDGF PDGFRa PDGFRP
• CSFIR/Fms Kit/SCFR
• VEGFRl/Fitl VEGFR2/KDR, VEGFR3/Fit4 FGF FGFR1, FGFR2, FGFR3, FGFR4 PTK7 PTK7/CCK4 Trk TrkA, TrkB, TrkC Ror Rorl, Ror2 MuSK Met, Ron Axl, Mer, Tyro3 Tie Tiel, Tie2 Eph EphAl-8, EphAlO, EphBl-4, EphB6 Ret Ryk DDR DDR1, DDR2 Ros LMR LMR1, LMR2, LMR3 ALK, LTK STYK1 SuRTK106/STYKl.
• Another candidate for suitable target cell surface receptors are the family of membrane-associated, high-affinity folate binding proteins (folate receptor), which bind folate and reduced folic acid derivatives and which mediate delivery of tetrahydrofolate to the interior of cells; the family of membrane-bound cytokine receptors that play a role in the internalization of a cognate cytokine, such as IL13; the surface antigens such as CD20, CD33, mesothelin and HM1.24, that are expressed on certain cancer cells and that mediate the internalization of cognate monoclonal antibodies, e.g., rituximab in the instance of CD20; and the family of adhesion receptors (integrins), which are transmembrane glycoproteins that are trafficked through the endosomal pathway and are major mediators of cancer cell adhesion.
• folate receptor membrane-associated, high-affinity folate binding proteins
• IL13 the family of membrane-bound cytokine receptors that play
• the tumor cell surface receptor comprises an integrin, neuromedin B receptor, bombesin 3 receptor, GRP receptor, bombesin 4 receptor, CCK2/gastrin, melanocortin- 1 receptor (MC-lr), neuropeptide Y (NPY) receptor, neutrotensin (NT) receptor, prostate specific membrane antigen (PSMA), somatostatin (SST) receptor, neurokinin 1 receptor (NK1R), chemokine receptor type 4 (CXCR4), vasoactive intestinal peptide (VIP), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR), or any combination thereof.
• integrin neuromedin B receptor
• bombesin 3 receptor GRP receptor
• bombesin 4 receptor CCK2/gastrin
• MC-lr melanocortin- 1 receptor
• NPY neuropeptide Y
• NT neutrotens
• the cell surface receptor is an antigen which is uniquely expressed on a target cell in a disease condition, but which remains either non-expressed, expressed at a low level or non-accessible in a healthy condition.
• target antigens which might be specifically bound by a targeting ligand of the invention may advantageously be selected from EpCAM, CCR5, CD19, HER-2 neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5, MUC5, MUC7, BhcG, Lewis-Y.
• Minicells of the invention are substantially free from contaminating parent bacterial cells.
• minicell-comprising formulations preferably comprise fewer than about 1 contaminating parent bacterial cell per 10 7 minicells, fewer than about 1 contaminating parent bacterial cell per 10 s minicells, fewer than about 1 contaminating parent bacterial cell per 10 9 minicells, fewer than about 1 contaminating parent bacterial cell per 10 10 minicells, or fewer than about 1 contaminating parent bacterial cell per 10 11 minicells.
• Methods of purifying minicells are known in the art and described in PCT/IB02/04632.
• One such method combines cross-flow filtration (feed flow is parallel to a membrane surface; Forbes, 1987) and dead-end filtration (feed flow is perpendicular to the membrane surface).
• the filtration combination can be preceded by a differential centrifugation, at low centrifugal force, to remove some portion of the bacterial cells and thereby enrich the supernatant for minicells.
• Another purification method employs density gradient centrifugation in a biologically compatible medium. After centrifugation, a minicell band is collected from the gradient, and, optionally, the minicells are subjected to further rounds of density gradient centrifugation to maximize purity.
• the method may further include a preliminary step of performing differential centrifugation on the minicell-containing sample. When performed at low centrifugal force, differential centrifugation will remove some portion of parent bacterial cells, thereby enriching the supernatant for minicells.
• a minicell purification method can include the steps of (a) subjecting a sample containing minicells to a condition that induces parent bacterial cells to adopt a filamentous form, followed by (b) filtering the sample to obtain a purified minicell preparation.
• Step A Differential centrifugation of a minicell producing bacterial cell culture. This step, which may be performed at 2,000 g for about 20 minutes, removes most parent bacterial cells, while leaving minicells in the supernatant;
• Step B Density gradient centrifugation using an isotonic and non-toxic density gradient medium. This step separates minicells from many contaminants, including parent bacterial cells, with minimal loss of minicells. Preferably, this step is repeated within a purification method;
• Step C Cross-flow filtration through a 0.45 pm filter to further reduce parent bacterial cell contamination.
• Step D Stress-induced filamentation of residual parent bacterial cells. This may be accomplished by subjecting the minicell suspension to any of several stress-inducing environmental conditions;
• Step E Antibiotic treatment to kill parent bacterial cells
• Step F Cross-flow filtration to remove small contaminants, such as membrane blebs, membrane fragments, bacterial debris, nucleic acids, media components and so forth, and to concentrate the minicells.
• a 0.2 pm filter may be employed to separate minicells from small contaminants, and a 0.1 pm filter may be employed to concentrate minicells;
• Step G Dead-end filtration to eliminate filamentous dead bacterial cells.
• a 0.45 um filter may be employed for this step.
• Step H Removal of endotoxin from the minicell preparation.
• Anti-Lipid A coated magnetic beads may be employed for this step.
• the invention includes within its scope compositions, or formulations, comprising intact, bacterially derived minicells or killed bacterial cells that encapsulate CD ld-restricted iNKT cell antigens (e.g., a-GalCer).
• the formulations comprise bacterially-derived minicells or killed bacterial cells comprising a CD ld-restricted iNKT cell antigen alone or in combination with an antineoplastic agent.
• the formulations comprise intact bacterially-derived minicells or killed bacterial cells comprising a CD ld-restricted invariant Natural Killer T (iNKT) cell antigen and bacterially-derived minicells or killed bacterial cells comprising an antineoplastic agent.
• iNKT Natural Killer T
• the CD ld-restricted iNKT cell antigen and the antineoplastic agent can be comprised within the same minicell or killed bacterial cell; or
• the CD ld-restricted iNKT cell antigen can be comprised within a first minicell or killed bacterial cell
• the antineoplastic can be comprised within a second minicell or killed bacterial cell.
• compositions disclosed herein comprise the CD ld- restricted iNKT cell antigen a-galactosylceramide (a-GalCer) and an antineoplastic agent, wherein the a-GalCer and the antineoplastic agent are comprised within one or more intact bacterially-derived minicells.
• compositions disclosed herein comprise the CD ld-restricted iNKT cell antigen a-GalCer and the antineoplastic agent doxurubicin, wherein the a-GalCer and the doxurubicin are comprised within one or more intact bacterially-derived minicells.
• the formulations also optionally comprise at least one bispecific ligand for targeting the minicell to a target cell.
• the minicell and ligand may be any of those described herein.
• the bispecific ligand of the present invention is capable of binding to a surface component of the intact bacterially-derived minicell and to a surface component of a target mammalian cell.
• a formulation comprising minicells, or killed bacterial cells, drugs (e.g., at least one antineoplastic agent) and optionally bispecific ligands of the present invention can be formulated in conventional manner, using one or more pharmaceutically acceptable carriers or excipients.
• Formulations or compositions of the disclosure can be presented in unit dosage form, e.g., in ampules or vials, or in multi-dose containers, with or without an added preservative.
• the formulation can be a solution, a suspension, or an emulsion in oily or aqueous vehicles, and can contain formulatory agents, such as suspending, stabilizing and/or dispersing agents.
• a suitable solution is isotonic with the blood of the recipient and is illustrated by saline, Ringer's solution, and dextrose solution.
• formulations can be in lyophilized powder form, for reconstitution with a suitable vehicle, e.g., sterile, pyrogen- free water or physiological saline.
• the formulations also can be in the form of a depot preparation.
• Such long-acting formulations can be administered by implantation (for instance, subcutaneously or intramuscularly) or by intramuscular injection.
• administering comprises enteral or parenteral administration.
• administering comprises administration selected from oral, buccal, sublingual, intranasal, rectal, vaginal, intravenous, intramuscular, and subcutaneous injection.
• composition comprising an immunogenically effective amount” of an encapsulated CD ld-restricted iNKT cell antigen.
• An“immunogenically effective amount” as used herein refers to the amount of antigen sufficient to elicit an immune response.
• an immunogenically effective amount is the amount of antigen sufficient activate an iNKT cell response.
• the effectiveness of CD ld- restricted iNKT cell antigen as an immunogen can be assessed, for example, by measuring increases in cytokine (e.g., IFNy) production following administration.
• compositions that includes a therapeutically effective amount of an antineoplastic agent are provided.
• A“therapeutically effective” amount of an antineoplastic agent is a dosage of the agent in question, e.g., a siRNA or a super-cytotoxic drug that invokes a pharmacological response when administered to a subject, in accordance with the present disclosure.
• a therapeutically effective amount can be gauged by reference to the prevention or amelioration of the tumor or a symptom of tumor, either in an animal model or in a human subject, when bacterially derived minicells or killed bacterial cells carrying a therapeutic payload are administered, as further described below.
• An amount that proves“therapeutically effective amount” in a given instance, for a particular subject, may not be effective for 100% of subjects similarly treated for the tumor, even though such dosage is deemed a“therapeutically effective amount” by skilled practitioners.
• the appropriate dosage in this regard also will vary as a function, for example, of the type, stage, and severity of the tumor.
• “therapeutically effective” is used to refer to the number of minicells or killed bacterial cells in a pharmaceutical composition
• the number can be ascertained based on what antineoplastic agent is packaged into the minicells or killed bacterial cells and the efficacy of that agent in treating a tumor.
• the therapeutic effect in this regard, can be measured with a clinical or pathological parameter such as tumor mass. A reduction or reduced increase of tumor mass, accordingly, can be used to measure therapeutic effects.
• Formulations of the invention can be administered via various routes and to various sites in a mammalian body, to achieve the therapeutic effect(s) desired, either locally or systemically. Delivery may be accomplished, for example, by oral administration, by application of the formulation to a body cavity, by inhalation or insufflation, or by parenteral, intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, intratumoral, or intradermal administration.
• the encapsulated CD ld-restricted iNKT cell antigens and the antineoplastic agents can be administered by the same route or by different routes of administration.
• the encapsulated CD ld-restricted iNKT cell antigen can be administered systemically and the antineoplastic agent can be administered locally.
• both the encapsulated CD ld-restricted iNKT cell antigen and the antineoplastic agent are administered systemically.
• the mode and site of administration is dependent on the location of the target cells.
• the target phagocytic cells that uptake the encapsulated CD ld-restricted iNKT cell antigen can be found both in the tumor microenvironment and the in the vasculature associated with liver spleen and lymph nodes.
• the encapsulated CD ld-restricted iNKT cell antigen may be delivered via targeted and/or non-targeted bacterially derived minicells or killed bacterial cells.
• the antineoplastic agents can also be administered via targeted and/or non-targeted methods.
• a tumor metastasis may be more efficiently treated via intravenous or intraperitoneal delivery of targeted compositions, such as, for example, intravenous or intraperitoneal delivery of targeted bacterially derived minicells.
• a combination of routes may also be employed.
• cytotoxic drug-loaded and receptor-targeted minicells may be administered locally as well as intravenously, and the encapsulated CD ld-restricted iNKT cell antigen (receptor-targeted or non-targeted) minicells may be administered intravenously.
• the administration of targeted, drug-packaged minicells may target surface-exposed tumors, while the full combination of minicells administered intravenously may target tissue-localized tumors and also elicit the anti-tumor immune response.
• the formulations disclosed herein may be used at appropriate dosages defined by routine testing, to obtain optimal physiological effect, while minimizing any potential toxicity.
• the dosage regimen may be selected in accordance with a variety of factors including age, weight, sex, medical condition of the patient; the severity of the condition to be treated, the route of administration, and the renal and hepatic function of the patient.
• Optimal precision in achieving concentrations of encapsulated CD ld-restricted iNKT cell antigen and drug within the range that yields maximum efficacy with minimal side effects may require a regimen based on the kinetics of the encapsulated CD ld-restricted iNKT cell antigen and antineoplastic drug availability to target sites and target cells. Distribution, equilibrium, and elimination of the encapsulated CD ld-restricted iNKT cell antigen and antineoplastic drug may be considered when determining the optimal concentration for a treatment regimen.
• the dosages of the encapsulated CD ld-restricted iNKT cell antigen and antineoplastic drugs may be adjusted when used in combination, to achieve desired effects.
• the dosage administration of the formulations may be optimized using a pharmacokinetic/pharmacodynamic modeling system.
• one or more dosage regimens may be chosen and a pharmacokinetic/pharmacodynamic model may be used to determine the pharmacokinetic/pharmacodynamic profile of one or more dosage regimens.
• one of the dosage regimens for administration may be selected which achieves the desired pharmacokinetic/pharmacodynamic response based on the particular
• the formulations of encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug may be administered at least once a week over the course of several weeks.
• the formulations encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug are administered at least once a week over several weeks to several months.
• the encapsulated CD ld-restricted iNKT cell antigen and one or more antineoplastic drugs can be administered simultaneously, sequentially, or intermittently in defined intervals.
• the formulations of encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug may be administered at least once a day for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or about 31 days.
• the formulations of encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug may be administered about once every day, about once every about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or about 31 days or more.
• the formulations of encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug may alternatively be administered about once every week, about once every about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 weeks or more.
• the formulations of encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug may be administered at least once a week for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 weeks or more.
• the formulations of encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug may alternatively be administered about twice every week, about twice every about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 weeks or more.
• the formulations of encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug may be administered at least once a week for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 weeks or more.
• the formulations of encapsulated CD ld-restricted iNKT cell antigen and/or antineoplastic drug may be administered about once every month, about once every about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 months or more.
• the formulations may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.
• administration of the antineoplastic agent may occur anytime from several minutes to several hours after administration of the encapsulated CD ld-restricted iNKT cell antigens.
• the antineoplastic agent may alternatively be administered anytime from several hours to several days, possibly several weeks up to several months after the encapsulated CD ld-restricted iNKT cell antigens.
• the encapsulated CD ld-restricted iNKT cell antigens may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23 or about 24 hours after the antineoplastic agent.
• the encapsulated CD ld-restricted iNKT cell antigens may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or about 31 days after the administration of the antineoplastic agent.
• the encapsulated CD ld-restricted iNKT cell antigens may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 weeks or more after the antineoplastic agent.
• the encapsulated CD ld-restricted iNKT cell antigens may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 months after the antineoplastic agent.
• antineoplastic agent may occur anytime from several minutes to several hours before
• the antineoplastic agent may alternatively be administered anytime from several hours to several days, possibly several weeks up to several months before the encapsulated CD ld-restricted iNKT cell antigens.
• the encapsulated CD ld-restricted iNKT cell antigens may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23 or about 24 hours before the antineoplastic agent.
• the encapsulated CD ld-restricted iNKT cell antigens may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or about 31 days before the administration of the antineoplastic agent.
• the encapsulated CD ld-restricted iNKT cell antigens may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 weeks or more before the antineoplastic agent.
• the encapsulated CD ld-restricted iNKT cell antigens may be administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 months before the
• compositions described herein may be used to treat a subject suffering from a cancer.
• the method disclosed herein comprises administering to the subject an immunogenically effective amount of a composition comprising intact, bacterially derived minicells or killed bacterial cells that encapsulate CD ld-restricted iNKT cell antigen and an antineoplastic agent or therapy.
• the CD ld-restricted iNKT cell antigen is comprised in intact bacterially-derived minicells.
• the CD ld-restricted iNKT cell antigen and the antineoplastic agents are comprised in one or more intact bacterially-derived minicells.
• the CD ld-restricted iNKT cell antigen and the antineoplastic agents are comprised in separate intact bacterially-derived minicells. In some embodiments, the CD ld- restricted iNKT cell antigen and the antineoplastic agents are comprised in the same intact bacterially-derived minicell. In some embodiments, tact, bacterially derived minicells or killed bacterial cells that encapsulate CD ld-restricted iNKT cell antigen are administered separately from the antineoplastic agent or therapy. In some embodiments, the CD ld-restricted iNKT cell antigen and the antineoplastic agents are comprised in the same intact bacterially-derived minicell.
• tact, bacterially derived minicells or killed bacterial cells that encapsulate CD ld-restricted iNKT cell antigen are administered simultaneously with the antineoplastic agent or therapy.
• tact, bacterially derived minicells or killed bacterial cells that encapsulate CD ld-restricted iNKT cell antigen are administered in the same composition with the antineoplastic agent.
• the intact, bacterially derived minicells or killed bacterial cells that encapsulate CD ld-restricted iNKT cell antigen are administered as separate compositions with the antineoplastic agent.
• the compositions comprising the encapsulated CD ld-restricted iNKT cell antigen or antineoplastic agent used to treat a subject suffering from cancer further comprises a pharmaceutically acceptable carrier.
• the methods disclosed herein are useful for treating a subject suffering from a cancer, wherein the subject is a human, a non-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.
• the methods disclosed herein are useful for treating a cancer disease.
• the cancer comprises a lung cancer, a breast cancer, a brain cancer, a liver cancer, a colon cancer, a pancreatic cancer, or a bladder cancer.
• the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor (including brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma); breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site
• esthesioneuroblastoma Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer;
• gastrointestinal carcinoid tumor gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma;
• medulloepithelioma melanoma
• Merkel cell carcinoma Merkel cell skin carcinoma
• mesothelioma metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer;
• papillomatosis paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer;
• pharyngeal cancer pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer;
• transitional cell cancer transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom’s macroglobulinemia; or Wilms’ tumor.
• the brain cancer or tumor is selected from the group consisting of brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate
• “Individual,”“subject,”“host,” and“patient,” used interchangeably herein, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired.
• the individual, subject, host, or patient is a human.
• Other subjects may include, but are not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits, rats, primates, and mice.
• Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs.
• Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.
• Leukemia is a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood.
• Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
• Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord. The methods and compositions of this invention particularly apply to precancerous, malignant, pre-metastatic, metastatic, and non-metastatic cells.
• the terms“treatment,”“treating,”“treat,” and the like refer to obtaining a desired pharmacological and/or physiologic effect in a tumor patient.
• the effect can be prophylactic in terms of completely or partially preventing tumor or symptom thereof and/or can be therapeutic in terms of a partial or complete stabilization or cure for tumor and/or adverse effect attributable to the tumor.
• Treatment covers any treatment of a tumor in a mammal, particularly a human.
• a desired effect in particular, is tumor response, which can be measured as reduction of tumor mass or inhibition of tumor mass increase.
• an increase of overall survival, progress-free survival, or time to tumor recurrence or a reduction of adverse effect also can be used clinically as a desired treatment effect.
• administering includes directly administering to another, self-administering, and prescribing or directing the administration of an agent as disclosed herein.
• the phrases“effective amount” and“therapeutically effective amount” mean that active agent dosage or plasma concentration in a subject, respectively, that provides the specific pharmacological effect for which the active agent is administered in a subject in need of such treatment. It is emphasized that an effective amount of an active agent will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be an effective amount by those of skill in the art.
• the term“active agent” is any small molecular drug, protein, functional nucleic acid, or polynucleic acid encoding a functional nucleic acid that is useful for treating a subject.
• the active agent can be any of the antineoplastic drugs, functional acids, interferon type I agonists or type II agonists described herein.
• phrases“pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in vivo without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• endocytosis encompasses (1) phagocytosis and (2) pinocytosis, itself a category inclusive of (2a) macropinocytosis, which does not require receptor binding, as well as of (2b) clathrin-mediated endocytosis, (2c) caveolae-mediated endocytosis and (2d) clathrin- / caveolae-independent endocytosis, all of which tend to access the late-endosome/lysosome pathway.
• the interaction between the ligand on a minicell and a mammalian cell surface receptor the present inventors discovered, activates a particular endocytosis pathway, involving receptor mediated endocytosis (rME) to the late-endosomal/lysosomal compartment.
• rME receptor mediated endocytosis
• the present inventors further discovered that the minicells were able to release their payload into the cytoplasm of the target mammalian cell.
• the payload is an encoding nucleic acid
• the nucleic acid not only is not completely degraded in the late-endosomai/lysosomal compartment, but also is expressed in the target mammalian cell.
• Example 1 EDV a cc treatment of JAWSII cells and the subsequent surface presentation of aGC through CD Id ligand
• This example contrasts encapsulated delivery of aGC via intact bacterial minicells and free aGC against cancer cells.
• Cells used Mouse immature monocytes JAWSII (ATCC ® CRL-11904TM).
• Preparation Perfecta3D 96-Well Hanging Drop Plate The upper and lower side tray reservoirs of the 3D hanging drop plates were filled with melted 1% agarose using a PI 000 pipette (lg agarose dissolve in 100ml of water, dissolved in microwave and allowed to cool to ⁇ 50°C). The plates were allowed to dry and settle at room temperature for at least 30min. The outside wells of the hanging drop plate were then filled with 50m1 of sterile cell culture media (without cells)/well.
• JAWSII cells were treated with lOOOng/ml aGC (positive control); empty minicells and minicells aGC compared to untreated cells and collected at 8h, 16h, 24h and 48h post-treatment (FIGS. 8A-8D).
• JAWSII cells were grown as semi-suspension cultures in T25 or T75 flasks. The culture media was carefully collected into a sterile 50ml tube by pipetting using a pipette-aid and the culture surface of the flask was washed 2x with 5ml of sterile PBS, and collected in the same sterile 50ml tube after each wash. The adherent cells were collected by the addition of 5ml of 0.25% trypsin/EDTA and incubated at 37°C for 3min or until all the cells were lifted from the surface of the flask.
• the lifted cells were carefully broken up into single cells by gentle pipetting using a pipette-aid and transferred into the sample sterile 50ml tube used in previous steps.
• the cell suspension was then centrifuged at 300g for 7 min and the supernatant was carefully decanted.
• the cell pellet was dissociated by flicking the bottom of the tube with a finger and resuspended in 5ml of pre warmed JAWSII culture media.
• the cell suspension was further dissociated into single cells by careful pipetting using a pipette-aid. To determine the cell number, 10m 1 of the cell suspension was mixed with 10m1 of trypan blue solution and analyzed using an EVE automated cell counter.
• each sample was then made up by the addition of sterile culture media.
• 5xl0 4 JAWSII cells were used for each sample and cultured in JAWSII cell culture media in a total volume of 50m1. Appropriate amount of live JAWSII cells were transferred into Eppendorf tubes.
• the final volume of each sample was then made up by the addition of sterile culture media.
• lOOOng/mL of aGC was added directly into the cell suspension for the JAWSII cells treated with lOOOng/ml aGC (positive control) treatment group.
• the samples were then carefully seeded into each well of the hanging drop plates at 50m1 of treatment suspension/well and incubated at 37°C at 5% CO2 until collection.
• Presentation of a-GC is a crucial step which leads to receptor recognition by invariant NKT cells triggering off a type II IFN cascade essential in anti-tumor activity.
• Example 2 In vivo studies using combination treatment of Ep minicelb ox and minicell a-GC in a syngeneic mouse model ( Ep CT26 murine colon cancer in Balb/c mice)
• This example illustrates the efficacy of minicell contained therapeutic and minicell contained CD ld-restricted iNKT cell antigen (e.g., a-GalCer) against tumors.
• CD ld-restricted iNKT cell antigen e.g., a-GalCer
• This result demonstrates that combination of and encapsulated CD ld-restricted iNKT cell antigen with an antineoplastic agent can be used to effectively treat tumors.
• mice and treatments (Experiments 1-3): Balb/c mice, female, 6-7 weeks old were obtained from the Animal Resources Company in Western Australia. The mice were
• CT26 cells mouse colon cancer
• EpCAM antigen EpCAM antigen
• Epclone 12.1 EpCAM antigen
• CT26 (Epclone 12. 1) isografts were established by injecting 2x105 cells per IOOmI PBS subcutaneously on the left flank of each mouse. The tumors grew to the ⁇ 125mm3 starting volume within 8 days post implantation. The mice were randomly distributed into groups with 8 mice for each treatment group. Tumors were treated with EpminicellDox, minicella-GC and EpminicellDox+ minicella-GC (combination) compared to saline treatment alone.
• CT26 (Ep clone 12.1) isograft was established by injecting subcutaneously 2xl0 5 cells/I 00m 1 PBS into the left flank of female, 6-7 weeks old Balb/c mice. The tumors were grown to ⁇ 200- 250mm 3 or 600- 800mm 3 before treatments commenced. The mice were randomized into 6 groups, 3 mice per group. Mice received one dose only. Treatment groups included; Saline (FIG. 6C), Ep minicell Dox (lxl0 9 ) (FIG. 6F), minicella-GC (lxlO 6 ) (FIG. 6E), minicella-GC (lxlO 7 ) (FIG.
• mice were sacrificed at 24 hrs post treatment for 200-250 mm 3 (FIG. 6) tumors and at 16hrs and 24 hrs for 600-800 mm 3 tumors (FIG. 7).
• This example describes secretion of cytokines IL-12 from dendritic cells/monocytes treated with minicell a-GC and cytokines IFNy, TNFa and IL-4 from iNKT cells exposed to these treated dendritic cells.
• APCs antigen presenting cells
• IL-12 antigen presenting cells
• iNKT cells secrete a plethora of cytokines in particular, IFNy, TNFa and IL-4.
• the aim of this experiment was to determine if JAWSII cells (mouse dendritic cell line) when co-incubated with minicell aGC followed by co-incubation with iNKT cells would result in the secretion of these cytokines.
• iNKT cells were isolated from spleens and thymus of the C57 mice using the NK1.1+ iNKT cell isolation kit, mouse (Miltenyi Biotec) following the manufacturer’s instructions. The purity of the isolated iNKT cells were determined by further staining the cells for the expression of CD3 and NK1.1 and analysed using FACS.
• JAWSII cells were treated with minicell aGC in a 96-well Perfecta3D hanging drop plate (Sigma). The cultures were then incubated for 48h at 37°C with 5% CO2 and supernatant was collected by centrifugation. The supernatant was then used for ELISA analysis for IL-12 secretion.
• JAWSII cells presenting aGC as delivered by the initial treatment with minicell aGC were seeded in a 96-well round bottom plate and co-cultured with iNKT cells isolated from C57 mice at 1 :2 iNKT to JAWSII ratio in AIM V serum free medium (Thermo Fisher Scientific). The supernatant was then collected for ELISA analysis for IENg, TNFa and IL-4.
• IL-12p40, IFN-g, TNFa, and IL-4 in the culture supernatants were measured by standard sandwich enzyme-linked immunosorbent assay (ELISA) from R&D Systems according to manufacturer’s instructions.
• This example describes an increase in activated dendritic cells in the spleen of mice treated with EpCAM minicellDox + minicella GC.
• the dissected spleen and thymus were transferred to the Dounce Homogeniser in freshly prepared media (10% FBS into sterile RPMI-1640 medium) by directly emptying the contents into the tube of the glass homogeniser.
• the organs were then gently broken down by using the glass plunger with 3-4 passes.
• the homogenised organs were then transferred into a 50mL centrifuge tube through a 70uM mesh strainer in media.
• the glass homogeniser was then washed with 4mL of RPMI-1640 medium (serum free) and the content was then again passed through the same 70uM mesh strainer into the centrifuge tube.
• the tube was then centrifuged at 330g for lOmins before resuspending the cell pellet in 4mL of RPMI- 1640 medium (serum free). Red Blood Cells were lysed using Red Blood Cell Lysis Buffer Hybri-Max (Merk R7757-100mL) following manufacturer’s instructions. The cells were then re suspended in 5mL of cold sterile autoMACS running buffer (Miltenyi) and passed through a 70uM mesh strainer into a 50mL centrifuge tube before proceeding to cell counting.
• mice Female Balb/c mice, 6-7 weeks old were obtained from the Animal Resources Company in Western Australia. The mice were acclimatized for one week before the experiments commenced. CT26 cells (mouse colon cancer) were stably transformed with a plasmid expressing EpCAM antigen and a stable clone (Ep clone 12.1) was established. This clone expressed EpCAM on the surface of the cells. All animal experiments were performed in compliance with National Health and Medical Research Council, Australia guidelines for the care and use of laboratory animals, and with EnGeneIC Animal Ethics Committee approval.
• CT26 (Epclone 12. 1) isografts were established by injecting 2 x 10 5 cells per 100 m 1 PBS subcutaneously on the left flank of each mouse. The tumors grew to the ⁇ 125mm 3 starting volume within 8 days post implantation. The mice were randomly distributed into groups with 8 mice for each treatment group. Mice were treated intravenously (tail vein injection) with EpCAM minice ll DoX minicell a-GC and EpCAM minicell Dox + minicell a-GC (combination) as compared to saline treatment alone.
• EpCAM minicell Dox was dosed at lxlO 9 minicells per dose in single and in combination treatments.
• mice were sacrificed and total splenocytes were isolated at 4h, 8h, 16h and 24h after the initial dose. The cells were stained for the presence of activated dendritic cells (CD86+ CD40+) and the population was analysed using FACS.
• activated dendritic cells CD86+ CD40+
• the purpose of this example was to evaluate infiltration of immune cells into the tumor microenvironment following treatment of mouse xenografts with EpCAM minicell Dox + minicella-GC.
• This example illustrates the efficacy of minicell contained therapeutic and minicell contained CD ld-restricted iNKT cell antigen (e.g., a-GalCer) against tumors.
• CD ld-restricted iNKT cell antigen e.g., a-GalCer
• This result demonstrates that combination of minicell-encapsulated CD ld-restricted iNKT cell antigen with tumor cells surface targeted, antineoplastic agent-packaged minicells can be used to effectively provoke a significant infiltration of activated cells of the immune system into the tumor.
• CT26 xenografted mice from Example 4 were sacrificed and the tumor mass was removed and all the cells were extracted following tumor dissociation as described below.
• tumour tissues were excised and placed in serum-free media (RPMI 1640 or DMEM).
• Tumour Dissociation Kit for mouse (Miltenyi Biotec) was used to dissociate tumour tissues into single-cell suspensions. Initially, tumour tissues were finely chopped using sterile blades. Tissues were placed in a gentleMACS C Tube (Miltenyi Biotec) containing the enzyme mix, which was prepared according to the manufacturer’s protocol.
• gentleMACS Program for soft/medium tumor was selected on Octo
• Dissociator with Heaters (Miltenyi Biotec). Following incubation on the dissociator, cell suspensions were applied through a MACS SmartStrainer (Miltenyi Biotec) and dissociated cells were collected.
• the cells were stained for CD45+ cells which identifies all cells of the immune system e.g. macrophages, dendritic cells and T cells.
• the samples were analysed by FACS.
• the purpose of this example was to evaluate infiltration of cytotoxic T cells into the tumor microenvironment following treatment of mouse xenografts with EpCAM minicell Dox + minicella-GC-
• the tumors were excised from the mouse xenograft described in Examples 4 and 5 and the cells were isolated as described in Example 4. The cells were stained for CD45+ CD3+ CD8+ cells which identify cytotoxic T cells.
• the purpose of this example was to evaluate the infiltration of iNKT cells into the tumor microenvironment following treatment of mouse xenografts with EpCAM minicell Dox + minicella-GC.
• the tumors were excised from the mouse xenograft described in Examples 4 and 5 and the cells were isolated as described in Example 5. The cells were stained for CD45+ CD3+ CD49B+ cells which identify iNKT cells.
• mice were added and this group received EpCAM minicell682 + minicell a-GC ⁇
• the drug PNU 159682 (designated 682 in this study) is a nemorubicin derivative that is over 2,000 times more toxic than doxorubicin.
• PBMCs peripheral blood mononuclear cells
• RNAs were isolated from the collected specimens using RNeasy mini kit (Qiagen) following manufacturer’s instructions. The quality and quantity of the RNA was determined by measuring the absorbance at 260nm and 280nm. A 260/280 ratio of 1.8 or above was considered acceptable.
• cDNA synthesis was conducted using Superscript Vilo (Thermo Fisher Scientific) per manufacturer’s protocol. lOng of cDNA was used for each qPCR reaction.
• Tissue pan dendritic cells were isolated from single-cell suspensions of the tumor xenografts and internal organs (spleen, thymus) using the Pan Dendritic Cell Isolation Kit, mouse (Miltenyi Biotec) following manufacturer’s instructions. The cells were then counted using a haemocytometer and used for downstream processes. Isolates activated and non-activated DCs.

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