Patent Application
20200407421
This application contains a sequence listing submitted electronically via EFS-web, which serves as both the paper copy and the computer readable form (CRF) and consists of a file entitled “ST-CT1-PCT_sequence.txt”, which was created on Sep. 5, 2018, which is 9,831 bytes in size, and which is herein incorporated by reference in its entirety.
The present disclosure relates to a method of targeting stems cells, in particular non-apoptotic stem cells, employing a GLA domain, for example to facilitate entry into the cell.
GLA domains are contained in a number of GLA proteins, such as Thrombin, Factor VII, Factor IX, Factor X, Protein C, Protein S (PrS), Protein Z, Osteocalcin, Matrix GLA protein, GAS6, Transthretin, Periostin, Proline rich GLA 1, Proline rich GLA 2, Proline rich GLA 3 and Proline rich GLA 4.
The GLA domains of so-called GLA proteins are able to bind phosphatidylserine (PtdS also referred to as PS) on the surface of apoptotic cells, such as cancer cells and pathogen infected cells. Molecules excluding the catalytic domain, which specifically bind phosphatidyl serine are disclosed in WO2014/151535 and WO2014/151683, incorporated herein by reference.
GLA domains (Vitamin K-dependent carboxylation/gamma-carboxyglutamic) are protein domains which have been modified by vitamin K dependent post-translational carboxylation of glutamate residues in the amino sequence to provide gamma-carboxyglutamate (Gla).
The GLA domain binds calcium ions by chelating them between two carboxylic acid residues. These residues are part of a region that starts at the N-terminal extremity of the mature form of GLA proteins, and that ends with a conserved aromatic residue. This results in a conserved Gla-x(3)-Gla-x-Cys motif that is found in the middle of the domain, which seems to be important for substrate recognition by the carboxylase.
Phosphatidyl serine was thought to be a conserved marker for apoptotic cells and part of the mechanism by which diseased cells reduce immune clearance or induced immune tolerance. Thus, it was hypothesized that the GLA domains only bound to apoptotic cells. However, surprisingly the present inventors have established that the GLA domains of the present disclosure can be employed to target stem cells, such as non-apoptotic stem cells and/or cancer stem cells. This is even more surprising because the inventors have evidence to suggest that normally healthy differentiated cells are not bound by the GLA domains employed in the present disclosure.
This has important implications, for example for stem therapy, which is used to treat conditions such as haematological cancers, such as leukemia. The stem cell therapy can only be given once the patient's own bone marrow/stems cells/immune system has been wiped clean.
This wiping clean process requires “obliteration therapies”, for example high doses of chemo, radiation therapy, and/or B cell depletion therapy.
This “obliteration therapy” has many side effects, for example mouth and throat pain (which may make it difficult for the patient to eat), nausea and vomiting, susceptibility to infection, such as pneumonia and CMV infection, anemia, bleeding, infertility, cognitive dysfunction, etc. These side effects are very severe, and are difficult for patients, especially children to cope with. It would greatly improve patient quality of life if these side effects could be minimized or eliminated.
Wiping out the immune system and rebooting it has also been found to put aggressive forms of MS into the remission. However, the treatment is reserved for only the severest of cases because the risk associated with the treatment are significant. However, the present disclosure allows the chemotherapy to be specifically targette to the stems by employing the GLA-component.
The present invention provides a mechanism for “specifically” targeting stem cells, in particular non-apoptotic stem cells. Stem cells targeted by the method can, for example be isolated, treated (including genetic correction, augmentation, addition), labelled, transformed and/or eliminated. Thus, the method of the present disclosure can be employed to deliver therapeutics interventions to stem cells, for example genetic material and/or proteineous material and/or chemical therapies.
By linking the GLA domain of the present disclosure to a detectable label, such as fluorescent label, his-tag or a magnetic bead, then stems cells can isolated and sorted etc. This may be useful in a diagnostic or isolating stems cells for further manipulation to render them useful in therapeutic application.
The present disclosure will now be summarised in the “numbered” paragraphs below:
In one embodiment the GLA-component binds surface exposed phosphatidylserine on the cells, before internalisation.
Whilst not wishing to be bound by theory the present inventors believe that not all phosphatidylserine is equivalent from a biological perspective. The inventors believe that the phosphatidylserine exposes by the enzyme TMEM16F is involved in immune suppression and is the one “seen” by the molecules of the present disclosure.
In one embodiment the stem cell is an adult stem cells or vesicle derived therefrom, for example a somatic stem cells, such as a hematopoetic stem cell, a mesenchymal stem cell, or a stromal stem cell.
In one embodiment the stem cell is an embryonic stem cell or a vesicle derived therefrom. In one embodiment the cell is not an embryonic stem cell.
In one embodiment the method relates to mammalian stem cells, for example human stem cells. The stem cell discussed herein are primarily human stem cells. However, the skilled person is able to identify the relevant or corresponding stem cell population for other mammals, as required. For example SSEA-1 is a marker for murine embryonic stem cells, human germline cells and embryonal carcinoma cells; SSEA-3 is a marker for primate embryonic stem cells, human embryonic germline cells, human embryonic stem cells and embryonal carcinoma cells; SSEA-4 is a marker for primate embryonic stem cells, human embryonic germ cells, human stem cells, embryonal carcinoma cells; CD324 is a marker for human & murine embryonic stem cells, embryonal cancer cells; CD90 is a marker for human & murine embryonic stems cells, hematopoietic stem cells, embryonal carcinoma cells; CD117 is a marker for human & murine embryonic stem cells, hematopoietic stem progenitor cells, neural crest-derived melanocytes, primordial germ cells, embryonal carcinoma cells; CD326 is a marker for human & murine embryonic stem cells, embryonal carcinoma cells; CD9 is a marker for human & murine embryonic stems; CD24 is a marker for human & murine embryonic stems; CD29 is a marker for human & murine embryonic stems; CD59 is a marker for human & murine embryonic stems; CD133 is a marker for human & murine embryonic stems, embryonal carcinoma cells, hematopoietic stem cells; CD31 is a marker for human & murine embryonic stems; TRA-1-60 is a marker for human embryonic stem cells, teracarcinoma, embryonic germ cells, embryonal carcinoma cells; TRA-1-81 is a marker for human embryonic stem cells, teracarcinoma, embryonic germ cells, embryonal carcinoma cells; Frizzled5 is a marker for human & murine embryonic stem cells; Stem cell factor (SCF) is a marker for human & embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, embryonal carcinoma cells; and Cripto is a marker for human & murine embryonic stem cells, cardiomyocytes and embryonal carcinoma cells.
In one embodiment the payload comprises a therapeutic agent.
In one embodiment the payload comprises a detectable label.
In one embodiment the payload comprises a DNA or RNA sequence, for example cDNA comprising a transgene or an RNAi sequence (such as miRNA, siRNA including shRNA). The DNA encoding a transgene may be delivered a transcriptionally active DNA or a plasmid for transient or stable expression.
In one embodiment the payload is suitable for inducing differentiation of the stem cell, for example to activate and/or mature the cell into a specific lineage.
In one embodiment the method of the present disclosure comprises a pre-treatment of a patient, for example to induce or augment expression of PS on stem cells, for example the pre-treatment step may be treatment with radiation therapy, in particular irradiation of bone marrow cells.
The disclosure also extends to pharmaceutical compositions comprising a molecule for use according to the present disclosure, in particular for use as described herein. Thus, in one embodiment the molecules according to the present disclosure are employed in the treatment of an intra-cellular target.
The present disclosure also extends to the use of a GLA-component comprises a GLA domain or an active fragment thereof, wherein said GLA-component does not comprise an active catalytic domain from a GLA protein, for intracellular targeting and delivery (including intracellular delivery of the payload).
The present disclosure also extends to the use of a GLA-component comprises a GLA domain or an active fragment thereof, wherein said GLA-component does not comprise an active catalytic domain from a GLA protein, for the manufacture of a medicament for intracellular targeting and delivery (including intracellular delivery of the payload, in particular where the payload comprises a therapeutic entity/molecule).
The present technology may be used to wipe out the immune cells of patient before stem cells transplantation, for example the payload will generally be a chemotherapy, for example comprising carmustine.
A current regime for immune cell ablation is (BCNU) 300 mg/m2 on day −6, etoposide 200 mg/m2 and cytarabine 200 mg/m2 daily from day −5 to −2, and melphalan 140 mg/m2 on day −1 (BEAM). Rabbit antithymocyte globulin (2.5 mg/kg/d) was administered on days −2 and −1. This regime can be adapted by conjugating each of the agents to a GLA molecule of the present disclosure.
In one embodiment the immune obliteration is for cancer, for a hematological cancer (for example is selected from myeloma, lymphoma, leukaemia, such as acute myeloid leukaemia (AML), chronic myeloproliferative disease, monoclonal gammopathy of uncertain significance, myelodysplastic syndrome and amyloidosis, such as AML, CML, CLL or ALL)
In one embodiment the myeloma is selected from multiple myeloma, amyloidosis and plasmacytoma.
In one embodiment the myeloma is selected from monoclonal gammopathy of undetermined significance, asymoptomatic myeloman, symptomatic myeloma and Kahler's disease.
In one embodiment the lymphoma is selected from anaplastic large cell lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma, lymphoblastic lymphoma, MALT lymphoma, mantle cell lymphoma, mediastinal large B-cell lymphoma, nodal marginal zone B-cell lymphoma, small lymphocytic lymphoma, thyroid lymphoma, and Waldenstrom's macroglobulinaemia.
In one embodiment the chronic myeloproliferative disease is selected from essential thrombocythaemia, chronic idiopathic myelofibrosis, and polycythaemia rubra vera.
In one embodiment the leukaemia is selected from acute myeloid leukaemia (AML), hairy cell leukaemia, acute lymphoblastic leukaemia, and chronic lymphoblastic leukaemia, such as AML.
It has recently become apparent that for severe autoimmune diseases, such as severe multiple sclerosis and severe arthritis obliteration of immune cells followed by stem transplant can put the disease into remission. Thus, the ablation therapy of the present disclosure may be employed in an autoimmune disease, such as multiple sclerosis and arthritis.
In one embodiment the GLA molecule of the present disclosure is linked, for example conjugated, to a payload which comprises a detectable label. Examples of detectable labels are given below. The detectable label can be employed to sort or isolate the stem cells, for example employing FACs sorting, magnetic sorting or similar. Thus, in one embodiment there is provided a method isolating or enriching stem cells employing a GLA-molecule of the present disclosure. This is advantages because historically the isolation of the certain stem cell populations, such as cancer stem cells has been very difficult.
The labelled GLA molecule may also be employed in vivo as an imaging agent, in particular as a diagnostic tool, for example to identify cancer stem cells in primary tumors or metastasise. This may be important for monitoring patients after surgery and/or chemotherapy to ensure the cancer is in remission.
The DNA transgene payloads and/or RNA payloads linker to the GLA molecule can be employed as an alternative intracellular delivery to a viral vector delivery (transduction) or traditional transfection. This can be employed to in vitro to express exogenous or endogenous proteins in the cell (for example where the modified stem cells are for reinfusion into a patient) or can be effected in vivo. The genes can be expressed transiently or can be designed to be stably integrated into the stem cell.
Surprisingly the present inventors have shown that the molecules according to the present disclosure not only bind stem cells they are rapidly internalised therein along with the payload attached thereto.
Intra-cellular delivery as employed herein refers conveying, for example the payload to inside the cell.
In one embodiment the payload is not internalized.
In one embodiment the GLA-component and the payload is not internalised.
Payload as employed herein refers to a molecule which is linked to the GLA domain, in particular for the purpose of intracellular delivery. The link may be a link through chemical conjugation using, for example maleimide chemistry or click chemistry to anchor to moiety to a solvent exposed lysine. Alternatively, the link may be a fusion, for example a peptide bond where the linked entity is expressed as a fusion protein with the GLA component, for example this may be suitable for certain detectable labels, such as fluorescent proteins or antibodies. Linkers may be employed between the GLA-component and the payload. Payloads may comprising a drug, a toxin, a polymer, a biologically active protein, therapeutic virus, oncolytic virus, viral vector, radionuclides, a metal chelating agent and/or a reporter group (such as a label).
In one embodiment 1, 2, 3, 4 or 5 payloads are linked per GLA-component.
GLA-component (also referred to herein as a gamma-carboxyglutamic acid component) refers to a polypeptide comprising a GLA-domain in the absence of catalytic domain from a GLA protein, such as protein S. The polypeptide may further comprise an EGF domain and/kringle domain, for example from protein S. In one embodiment the GLA-component comprises 30 to 300 amino acid residues, for example 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 residues. In one embodiment the GLA component is in the range of 4.5 to 30 kDa. In one embodiment the GLA-component comprises the sequence shown in SEQ ID NO: 1. In one embodiment the GLA-component comprises a sequence shown in SEQ ID NO: 6 or a derivative thereof excluding the his-tag.
GLA domains (Vitamin K-dependent carboxylation/gamma-carboxyglutamic) as employed herein are protein domains which have been modified by vitamin K dependent post-translational carboxylation of glutamate residues in the amino sequence to provide gamma-carboxyglutamate (Gla). In one embodiment the GLA domain employed in the molecules of the present disclosure comprises 30 to 45 consecutive residues from a native (wild-type) GLA domain. In one embodiment the GLA domain comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 GLA residues.
In one embodiment 30% or less of the GLA-component is GLA residues.
In one embodiment the GLA-component comprises 1 to 5 disulfide bonds, for example 1, 2, 3, 4 or 5 disulfide bonds.
The GLA domain binds calcium ions by chelating them between two carboxylic acid residues. These residues are part of a region that starts at the N-terminal extremity of the mature form of Gla proteins, and that ends with a conserved aromatic residue. This results in a conserved Gla-x(3)-Gla-x-Cys motif that is found in the middle of the domain, and which seems to be important for substrate recognition by the carboxylase.
GLA domains are contained in a number of proteins, such as Thrombin, Factor VII, Factor IX, Factor X, Protein C, Protein S (PrS), Protein Z, Osteocalcin, Matrix GLA protein, GAS6, Transthretin, Periostin, Proline rich GLA 1, Proline rich GLA 2, Proline rich GLA 3, and Proline rich GLA 4.
GLA domain as employed herein also extends to proteins where 1 to 10 percent (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%) of the amino acids in the native GLA domain may be replaced and deleted, provided that modified domain retains at least 70% (such as 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) of the native activity of the native (unmodified GLA domain) in a suitable in vitro assay.
EGF domain as employed herein refers is a conserved protein domain. It comprises about 30 to 40 amino-acid residues and has been found in a large number of mostly animal proteins. Most occurrences of the EGF-like domain are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted. The EGF-like domain includes 6 cysteine residues. The main structure of EGF-like domains is a two-stranded β-sheet followed by a loop to a short C-terminal, two-stranded β-sheet. These two β-sheets are usually denoted as the major (N-terminal) and minor (C-terminal) sheets. EGF-like domains frequently occur in numerous tandem copies in proteins: these repeats typically fold together to form a single, linear solenoid domain block as a functional unit. In one embodiment the domain employed is the full-length native domain.
EGF domain as employed herein also extends to proteins where 1 to 10 percent (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%) of the amino acids in the native EGF domain may be replaced and deleted, provided that modified domain retains at least 70% (such as 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) of the native activity of the native (unmodified EGF domain) in a suitable in vitro assay. In one embodiment the protein is the full-length native domain.
Kringle domain as employed herein refers to autonomous protein domains that fold into large loops stabilized by 3 disulfide bonds. They are characterized by a triple loop, 3-disulfide bridge structure, whose conformation is defined by a number of hydrogen bonds and small pieces of anti-parallel beta-sheet. They are found throughout the blood clotting and fibrinolytic proteins, in a varying number of copies, in some plasma proteins including prothrombin and urokinase-type plasminogen activator, which are serine proteases belonging to MEROPS peptidase family S1A.
Kringle domain as employed herein also extends to proteins where 1 to 10 percent (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%) of the amino acids in the native kringle domain may be replaced and deleted, provided that modified domain retains at least 70% (such as 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) of the native activity of the native (unmodified Kringle domain) in a suitable in vitro assay. In one embodiment the domain employed is the full-length native domain.
An active fragment of a protein as employed herein is a less than the whole native protein (or relevant domain), which retains at least 50% (such as 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) of the active of the native full-length domain or protein in a relevant in vitro assay.
Catalytic domain as employed herein is a domain (or fragment) downstream of the EGF domain in the C-terminal direction, for example as illustrated in
In vitro as employed herein refers to laboratory work not performed in a human or animal body.
In vivo as employed herein refer to work/testing/treatment in a living organism, in particular a human or animal.
Stem cell as employed herein refers to undifferentiated cells that are capable of differentiation and includes embryonic stem cells and adult stem cells, in particular adult stem cells.
Hematopoietic stem cells (HSCs) or hemocytoblasts are the stem cells that give rise to all the other blood cells through the process of haematopoiesis. They are derived from mesoderm and located in the red bone marrow, which is contained in the core of most bones.
Cancer stem cell as employed herein refers to tumorigenic cells (i.e. cancer cells found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. See, for example Identification and Targeting of Cancer Stem Cells, BioessayS 2009 October; 31 (10) 1038-1049. Cancer stem cells are defined by three distinct properties: i) a selective capacity to initiate tumour and drive neoplastic proliferation: ii) an ability to create endless copies of themselves through self-renewal, and iii) the potential to give rise to more mature non-stem cell cancer progeny though differentiation. Cancer stem cells are not necessarily derived from a healthy stem cell but may originate from a differentiated cell.
CD34 is also known as hematopoietic progenitor cells antigen CD34, has a function as cell-cell adhesion factor. It can be employed as a marker to enrich stem populations.
Molecule as employed herein is used in the broadest sense and includes a synthetic chemical molecule but also macromolecules such as proteins, polymers (natural or otherwise), ribonucleic acid molecules, labels etc.
Payloads may comprising a drug, a toxin, a polymer, a biologically active protein, radionuclides, a metal chelating agent and/or a reporter group.
A drug as employed herein, unless the context indicates otherwise, is intended to refer to a small chemical entity, for example which has been synthesised by organic chemistry methods, in particular a molecule approved or licensed or in the process of being licensed for therapeutic use, especially in humans. Drug as employed herein also includes an anti-viral compound, an antibiotic, and an anti-cancer therapy.
An antiviral compound (antiviral agent) as employed herein refers to the class of medicaments used specifically for treating viral infections, including broad spectrum anti-viral agents and also “narrow” spectrum specific to a particular virus or particular family of viruses.
Antibiotic as employed herein refers to medicine or agent that inhibits the growth of bacteria or destroys bacteria. Anti-bacterial and antibiotic are used interchangeable here unless the context indicates otherwise.
Anti-parasitic as employed herein in refers to a medicine or agent that inhibits the growth of parasite, destroys parasite or removes parasites from the host.
Anti-cancer therapy is a broad term which includes anti-cancer drugs, chemotherapy, radiotherapy, immune-oncology therapies, etc.
Anti-cancer drug as employed herein generally refers to a small molecule cancer therapy.
Chemotherapy as herein generally refers to a cytotoxic agent and includes antineoplastics.
A biological therapeutic (also referred to as a biopharmaceutical, biological or biologic) is a therapeutic product “derived” from a biological source, for example a recombinant proteins and fragments, including antibodies molecules, including antibodies, antibody binding fragments and multispecific antibody molecules and complex combinations of such materials. A biologically active protein is a subgroup of a biological therapeutics and includes recombinant proteins and active fragments thereof (including antibody molecules).
Antibody molecules as employed herein include a complete antibody having full length heavy and light chains or a fragment thereof and a molecule comprising any one of the same for example a Fab, modified Fab, Fab′, modified Fab′, F(ab′)2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews—Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab′ fragments described in International patent applications WO2005/003169, WO2005/003170 and WO2005/003171. Multi-valent antibodies may comprise multiple specificities e.g bispecific or may be monospecific (see for example WO 92/22853 and WO05/113605). Bispecific and multispecific antibody variants are especially considered in this example since the aim is to neutralise two independent target proteins. Variable regions from antibodies disclosed herein may be configured in such a way as to produce a single antibody variant which is capable of binding to and neutralising two target antigens.
Antibody and binding fragments thereof, in particular small antibody fragments such as domain antibodies, VHHs, single chain Fvs (scFvs), ds-scFvs and dsFv, may be delivered intracellularly using the present technology.
In one embodiment the antibody or binding fragment thereof is a checkpoint inhibitor, for example an anti-PD-1 or anti-PD-L1 inhibitor.
In one embodiment the antibody molecule is human or humanised.
A toxin is a poisonous substance, especially derived from a natural source, in particular a protein. Many toxins, such as calicheamicin are used in cancer therapy. In addition chemotherapeutic agents can be considered toxic (or toxins). Thus the definition of toxin overlaps with other definitions herein. However, neurotoxins like snake venom are toxin but not a chemotherapeutic. However, those skilled in the art are familiar with these technical definitions and are capable of understanding the meaning the context of the present disclosure.
Diagnostic as employed herein is agent used in analysis or imaging to diagnose, label or monitor or understand a disease status. A diagnostic will generally comprise a reporter molecule, such as a label or similar that can visualised, measured or monitored in some way.
Radionuclides suitable for use the present disclosure include thallium-201, technetium-99m, Iodine-123, Iodine 131, Iodine-125, Fluorine-18 and Oxygen-15.
Also, of particular interest, is using GLA-component to deliver intrabodies, for example via GLA-fusions, for example where the intrabody is fused to the N or C terminus of the GLA-component. Intrabodies are able to target intracellular antigens.
In one embodiment antibodies that interact and inhibit RAS or proteins in the RAS signaling pathway are employed in the payload. RAS genes constitute a multigene family that includes HRAS, NRAS, and KRAS. RAS proteins are small guanosine nucleotide-bound GTPases that function as a critical signaling hub within the cell. The RAS/MAPK pathway has been studied extensively in the context of oncogenesis because its somatic dysregulation is one of the primary causes of cancer. RAS is somatically mutated in approximately 20% of malignancies (Bos J L, Cancer Res. 49: 4682-4689, 1989). In this particular case, it is envisioned that, for example the GLA-component is fuses to a RAS intrabody (described in Cetin M et al., J Mol Biol. 429:562-573, 2017).
Apoptosis as employed herein is cell death pathway which occurs as normal and controlled part an organism growth. Cell death by apoptosis is less damaging to surrounding tissue than cell death mechanisms, such as necrosis.
Necrosis as employed herein is cell death from disease or injury. It releases cytokines and factors into the surrounding tissue that may damage surrounding cells. Gangrene is an example of necrotic cell death.
Chemotherapeutic agent and chemotherapy or cytotoxic agent are employed interchangeably herein unless the context indicates otherwise.
Chemotherapy as employed herein is intended to refer to specific antineoplastic chemical agents or drugs that are “selectively” destructive to malignant cells and tissues, for example alkylating agents, antimetabolites including thymidylate synthase inhibitors, anthracyclines, anti-microtubule agents including plant alkaloids, topoisomerase inhibitors, parp inhibitors and other antitumour agents. Selectively in this context is used loosely because of course many of these agents have serious side effects.
The preferred dose may be chosen by the practitioner, based on the nature of the cancer being treated.
Examples of alkylating agents, which may be employed in the method of the present disclosure include an alkylating agent nitrogen mustards, nitrosoureas, tetrazines, aziridines, platins and derivatives, and non-classical alkylating agents.
Example a platinum containing chemotherapeutic agent (also referred to as platins), such as cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin and lipoplatin (a liposomal version of cisplatin), in particular cisplatin, carboplatin and oxaliplatin.
The dose for cisplatin ranges from about 20 to about 270 mg/m2 depending on the exact cancer. Often the dose is in the range about 70 to about 100 mg/m2.
Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan.
Nitrosoureas include N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin. Tetrazines include dacarbazine, mitozolomide and temozolomide.
Aziridines include thiotepa, mytomycin and diaziquone (AZQ). Examples of antimetabolites, which may be employed in the method of the present disclosure, include anti-folates (for example methotrexate and pemetrexed), purine analogues (for example thiopurines, such as azathiopurine, mercaptopurine, thiopurine, fludarabine (including the phosphate form), pentostatin and cladribine), pyrimidine analogues (for example fluoropyrimidines, such as 5-fluorouracil and prodrugs thereof such as capecitabine [Xeloda®]), floxuridine, gemcitabine, cytarabine, decitabine, raltitrexed(tomudex) hydrochloride, cladribine and 6-azauracil.
Examples of anthracyclines, which may be employed in the method of the present disclosure, include daunorubicin (Daunomycin), daunorubicin (liposomal), doxorubicin (Adriamycin), doxorubicin (liposomal), epirubicin, idarubicin, valrubicin currenity used only to treat bladder cancer and mitoxantrone an anthracycline analog, in particular doxorubicin.
Examples of anti-microtubule agents, which may be employed in the method of the present disclosure, include include vinca alkaloids and taxanes.
Vinca alkaloids include completely natural chemicals for example vincristine and vinblastine and also semi-synthetic vinca alkaloids, for example vinorelbine, vindesine, and vinflunine
Taxanes include paclitaxel, docetaxel, abraxane, carbazitaxel and derivatives of thereof. Derivatives of taxanes as employed herein includes reformulations of taxanes like taxol, for example in a micelluar formulations, derivatives also include chemical derivatives wherein synthetic chemistry is employed to modify a starting material which is a taxane.
Topoisomerase inhibitors, which may be employed in a method of the present disclosure include type I topoisomerase inhibitors, type II topoisomerase inhibitors and type II topoisomerase poisons. Type I inhibitors include topotecan, irinotecan, indotecan and indimitecan. Type II inhibitors include genistein and ICRF 193 which has the following structure:
Type II poisons include amsacrine, etoposide, etoposide phosphate, teniposide and doxorubicin and fluoroquinolones.
In one embodiment the chemotherapeutic is a PARP inhibitor.
In one embodiment the payload comprises a fluorescent label, a chemi-lluminescent label, a radio label, an enzyme, a dye or a ligand.
A label in accordance with the present disclosure is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.
Label conjugates are generally preferred for use as diagnostic agents. Diagnostic agents generally fall within two classes, those for use in in vitro diagnostics, and those for use in vivo diagnostic protocols, generally known as “directed imaging.” Many appropriate imaging agents are known in the art, as are methods for their attachment to peptides and polypeptides (see, for e.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.
In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt, copper67, 152Eu, gallium67, 3hydrogen, iodine123, iodine125, iodine131, indium111, 59iron, 32phosphorus, rhenium186, rhenium188, 75selenium, 35sulphur, technicium99m and/or yttrium90. 125I is suitable for use in certain embodiments, and technicium99m and/or indium111 are particularly suitable due to their low energy and suitability for long range detection. Radioactively labeled peptides and polypeptides may be produced according to well-known methods in the art. For instance, peptides and polypeptides can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Peptides may be labeled with technetium99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the peptide to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the peptide. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to peptide are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).
Fluorescent labels suitable for use as payloads include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAM RA, TET, Tetramethylrhodamine, and/or Texas Red.
Another type of payload is that suitable for use in vitro, is where a peptide is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and is described, for example, in U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.
Other methods are known in the art for the attachment for linking a peptide to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948). Peptides or polypeptides may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
In one embodiment the label is able to stain or label the nucleus of a stem cell.
In one embodiment the virus employed in the present disclosure is an envelope virus, for example selected from a herpesvirus (such as Herpes simplex 1), a poxvirus (such as vaccina virus), a hepadnavirus, a flavivirus, a togavirus, a coronavirus, hepatitis D, orthomyxovirus, paramyxovirus (such as measles or Newcastle disease virus), rhabdovirus, bunyavirus, filovirus, and Rhabdoviridae (such as vesicular stomatitis Indiana virus (VSV).
In one embodiment the virus employed in the present disclosure is a non-envelope virus, for example selected from adenoviridae (such as an adenovirus), papilomaviridae, picornaviridae (such as coxsackie virus or Seneca Valley virus (eg Senecavirus)), reovirus.
In one embodiment the virus is an adenovirus, for example a human adenovirus, such as selected from a group B virus (in particular Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad51 or a chimeria thereof, such as Enadenotucirev), a group C virus (in particular Ad1, 2, 5, 6 or a chimeria thereof), a group D virus (in particular Ad8, Ad10, Ad13, Ad15, Ad17, Ad19, Ad20, Ad22, Ad30, Ad32, Ad33, Ad36, Ad37, Ad38, Ad39, Ad42, Ad43, Ad44, Ad45, A46, Ad47, Ad48, Ad49, Ad50 or a chimeria thereof), a group E virus (in particular Ad4), a group F virus (in particular Ad40, Ad41 or a chimeria thereof) and a chimeria of two or more of group B, C, D, E or F viruses.
The vast majority of viruses have well described proteins associated with target cell recognition and uptake. Modification of their tropism to re-direct or enable more selective tumor targeting into oncolytic viruses may be introduced using methods described in rev. in Verheije and Rottier, Adv. Virology 2012: 798526, 2012.
Additional viral cell surface proteins not involved in native viral targeting can have targeting motifs engineered onto them (e.g. Ad virion minor coat protein IX Salisch et al., PLoS One 12: e0174728, 2017).
Envelope viruses have an outer membrane (envelope) covering the virus capsid. The envelope is typically derived from the portions of the host cell membranes (phospholipids and proteins) but also include some viral proteins. Glycoproteins on the surface of the envelope serve to identify and bind to receptor sites on the host's membrane. The viral envelope then fuses with the host's membrane, allowing the capsid and viral genome to enter and infect the host.
Various oncolytic viruses are disclosed in WO2014/13834, incorporated herein by reference.
Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins—gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. Retargeting of HSV has been achieved by the insertion of ligands and scFvs into the gC and/or gD protein or gH (Campadelli-Fiume, G et al., Rev in Med Virol 21: 213-226, 2011, Gatta, V PLoS Pathog 11: e1004907, 2015). Oncolytic herpes simplex virus type 1 vectors have been developed for clinical use. These viruses are replication competent and have mutations in the genes that affect viral replication, neuropathogenicity, and immune evasiveness, and for example include first generation viruses such as NV1020 (R7020), dlsptk, d18.36tk, hrR3, R3616, 1716, second generation viruses such as G207 (MGH-1), 3616UB, SUP, NV1023, third generation viruses such as G47Δ, transcriptional expressing vectors such as G92A, d12.CALP, Myb34.5, transgene expressing vectors such as rRP450, and other viruses such as Talimogene laherparepvec (T-Vec). The HSV-1 vectors are the thought to be useful in the treatment of a wide of solid tumors, for example including glioma, melanoma, breast, prostate, colon, ovarian, and pancreatic cancers. The HSV-1 virus infects a broad range of cells types and species, it is cytolytic by nature, the replicative life cycle of the virus results in host cell destruction, it has a well characterised and large genome (152K) but contains many non-essential genes providing up to 30K of space for the insertion of therapeutic genes. Generally, HSV viruses are not mutated in the thymidine kinase gene for safety reasons. Talimogene laherparepvec is an oncolytic herpes virus, which is approved for use in the treatment of melanoma. Other herpes bases viruses include G207, SEPREHVIR (HSV-1716), by Virttu Biologics, HSV-1 R3616 mutant, HSV-1 1716 mutant, NV1020 (R7020), R3616 mutant (deleted RL1), KM100 mutant has insertions in UL48 (encodes the transactivator tegument protein pUL48 [VP16]) and RL2 genes, G92A, mutants, Myb34.5 and rQNestin34.5.
Poxvirus—Vaccina virus, such as Modified Vaccinia Ankara (MVA) may be employed (Galmiche M C et al., J Gen Virol 78: 3019-3027, 1997), MVA may be replaced with a p14 fusion molecule carrying an inserted scFv directed against the tumor associate antigen MUC-1 (Paul, S et al., Viral Immunol 20: 664-671, 2007) See also rev. in Liang L et al., Viruses 6: 3787-3808, 2014, Hsiao J C et al., J Virol 73: 8750-8761, 1999, rev. in Chen T L and Roffler S, Med Res. Rev. 28: 885-928, 2008 and Kinoshita T et al., J Biochem 144: 287-294, 2008. JX-594, by Jennerex, is a thymidine kinase-deleted Vaccinia virus plus GM-CSF. GL-ONC1 is an attenuated vaccinia virus (Lister strain) that causes regression and elimination of a wide range of solid tumors in preclincal mouse models
Paramyxovirus (such as measles or Newcastle disease virus),
Measles virus (MeV) is a single-stranded, negative-sense, enveloped (non-segmented) RNA virus of the genus Morbillivirus within the family Paramyxoviridae. Measles virus has two envelope glycoproteins: the hemagglutinin (H) attachment protein and the fusion (F) protein. Attachment, entry and subsequent cell-cell fusion is mediated via 2 measles receptors, CD46 and the signaling lymphocyte activation molecule (SLAM). See for example rev. in Msaouel P et al., Methods Mol Biol 797: 141-162, 2012, Robinson S. and Galanis, E. Expert Opin Biol Ther. 17: 353-363, 2017, Aref S et al., Viruses 8. Pii:E294, 2016); (rev. in Chen T L and Roffler S, Med Res. Rev. 28: 885-928, 2008 and Kinoshita T et al., J Biochem 144: 287-294, 2008), and (Russell S J and Peng K W, Curr Topic Microbiol. Immunol 330: 213-241, 2009, Robinson S and Galanis, E Expert Opin Biol. Ther 17: 353-363, 2017, Aref S et al., Viruses 8. Pii: E294, 2016). Measles virus encoding the human thyroidal sodium iodide symporter or MV-NIS is an attenuated oncolytic Edmonston (Ed) strain of measles virus. Radioactive Iodine imaging provides a novel technique for NIS gene expression monitoring.
Newcastle disease virus may also be employed.
Adenoviridae Adenoviruses are among the most extensively studied viruses being used as oncolytic agents. An array of peptides and proteins have been engineered into virion associated viral proteins to alter the native tropism of the virus (rev. in Verheije M H and Rottier P J M Adv Virol 2012: 798526, 2012). However, all of these are dependent upon viral assembly in the nucleus which presents significant challenges.
Other non-enveloped viruses include Coxsackievirus, Poliovirus and Reovirus. See for example rev. in Altan-Bonnet, N, Curr Opin Microbiol 32: 77-81, 2016 and Chen Y H et al., Cell 160: 619-630, 2015, rev. in Chen T L and Roffler S, Med Res. Rev. 28: 885-928, 2008 and Kinoshita T et al., J Biochem 144: 287-294, 2008 and rev. in Verheije M H and Rottier P J M Adv Virol 2012: 798526, 2012).
There are a numerous adenoviruses for example Ad5-yCD/mutTKSR39rep-hIL12, such as for the treatment of prostate cancer was initiated, CGTG-102 (Ad5/3-D24-GMCSF), by Oncos Therapeutics, for example for the treatment soft tissue sarcomas, Oncorine (H101), CG0070, Enadenotucirev (EnAd) WO2005/118825, OvAd1 and OvAd2 disclosed in WO2008/080003, ONCOS-102, for example for Unresectable Malignant Pleural Mesothelioma, and DNX-2401 for example for glioma.
Cavatak is the trade name for a preparation of wild-type Coxsackievirus A21, useful in the treatment of malignant melanoma. Seneca Valley virus (NTX-010) and (SVV-001), for example for small cell lung cancer and neuroblastoma
Reovirus-Reolysin® (pelareorep; Wild-Type Reovirus; Serotype 3 Dearing; Oncolytics Biotech), for example for the treatment of various cancers and cell proliferative disorders.
Vesicular Stomatitis Virus (VSV) VSV is another enveloped virus being explored as on oncolytic agent. See for example Betancourt D et al., J Virol 89: 11786-11800, 2015) and rev. in Hastie E and Grdzelishvili V Z J Gen Virol 93: 2529-2545, 2012).
In one embodiment a virus or vector employed in the method of the present disclosure comprises a transgene, for example where the transgene is to replace defective genetic material in the cell, to provide a new or augmented function in the cell, to sensitize the cell to treatment, to block a function in the cell, or to express a therapeutic protein or peptide.
In one embodiment the virus employed as the payload according to the present disclosures, comprises a transgene or transgenes, for example encoding an agent independently selected from an RNAi sequence, a protein, polypeptide or peptide (for example an antibody molecule or binding fragment thereof, a chemokine, a cytokine, an immunomodulator, a fluorescent tag or an enzyme).
This includes but is not limited to unique formats that have shown preclinical promise but have lacked an effective and economical means for delivery e.g. peptides, intrabodies and alternative scaffolds (rev. in Boldicke T, Protein Sci 26: 925-945, 2017, Marschall and Dubel, Comput Struct Biotechnol J 14: 304-308, 2016, Miersch and Sidhu F1000Res 5.pii.F1000 Faculty Rev. 1947, 2016, Peptides, Tsomaia Eur J Med Chem 94:459-470, 2015, Marschall A L J et al, Mabs 7: 1010-1035, 2015, AlDeghaither D et al., J Clin Pharmacol. 55: S4-S20, 2015))) and includes agents with therapeutic effects on the tumor cells tumor stem cells, tumor associated endothelium and tumor associated stroma. Of special interest are molecules that could serve multiple functions, for example as therapeutics, biomarkers and/or diagnostics. The herpes simplex virus thymidine kinase (HSV-TK) gene is a well-established pro-drug converting enzyme with a clinically approved pro-drug (ganciclovir—GCV) see for example Holder et al., Cancer Res. 53: 3475-3485, 1993, Touraine R L et al., Gene Therapy 5: 1705-1711, 1998),
In addition, the thymidine kinase protein expression can also be exploited to image and track the activity of the virotherapy during the course of treatment. Positron emission tomography and single photon emission computed tomography are both methods that are routinely used for the detection and monitoring of cancer and cancer therapies and are both viable means to detect the expression of the thymidine kinase protein when an appropriate thymidine kinase substrate is administered (Wang J Q et al., Bioorg Med Chem 13: 549-556, 2005, Tjuvajev J G et al, J Nucl Med 43: 1072-1083, 2002). Alternatively, the NIS gene may be used and has been explored as an agent for diagnostic and therapeutic purposes in oncolytic viruses, much like TK (Miller A and Russell S Expert Opin Biol Ther 16: 15-32, 2016, Ravera S et al., Annu Rev Physiol 79: 261-289, 2017, Portulano et al., Endocr Rev. 35: 106-149, 2014).
In one embodiment antibodies that interact and inhibit RAS or proteins in the RAS signaling pathway are encoded in the virus of the present disclosure, for example as fusion protein with the GLA-component. RAS genes constitute a multigene family that includes HRAS, NRAS, and KRAS. See for example Bos J L, Cancer Res. 49: 4682-4689, 1989; and Cetin M et al., J Mol Biol. 429:562-573, 2017.
In one embodiment the GLA-component is employed in combination with a second therapy, for example an anti-cancer therapy. This is therapy that is administered separately to the GLA-component (i.e. is not linked to the GLA-component).
In one embodiment a combination of chemotherapeutic agents employed is a chemotherapeutic described herein, for example a platin and 5-FU or a prodrug thereof, for example cisplatin or oxaplatin and capecitabine or gemcitabine, such as FOLFOX.
In one embodiment the chemotherapy comprises a combination of chemotherapy agents, in particular cytotoxic chemotherapeutic agents.
In one embodiment the chemotherapy combination comprises a platin, such as cisplatin and fluorouracil or capecitabine.
In one embodiment the chemotherapy combination in capecitabine and oxaliplatin (Xelox).
In one embodiment the chemotherapy is a combination of folinic acid and 5-FU, optionally in combination with oxaliplatin.
In one embodiment the chemotherapy is a combination of folinic acid, 5-FU and irinotecan (FOLFIRI), optionally in combination with oxaliplatin (FOLFIRINOX). The regimen consists of: irinotecan (180 mg/m2 IV over 90 minutes) concurrently with folinic acid (400 mg/m2 [or 2×250 mg/m2] IV over 120 minutes); followed by fluorouracil (400-500 mg/m2 IV bolus) then fluorouracil (2400-3000 mg/m2 intravenous infusion over 46 hours). This cycle is typically repeated every two weeks. The dosages shown above may vary from cycle to cycle.
In one embodiment the chemotherapy combination employs a microtubule inhibitor, for example vincristine sulphate, epothilone A, N-[2-[(4-Hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide (ABT-751), a taxol derived chemotherapeutic agent, for example paclitaxel, abraxane, or docetaxel or a combination thereof.
In one embodiment the combination therapy employs an mTor inhibitor. Examples of mTor inhibitors include: everolimus (RAD001), WYE-354, KU-0063794, papamycin (Sirolimus), Temsirolimus, Deforolimus(MK-8669), AZD8055 and BEZ235(NVP-BEZ235).
In one embodiment the combination therapy employs a MEK inhibitor. Examples of MEK inhibitors include: AS703026, CI-1040 (PD184352), AZD6244 (Selumetinib), PD318088, PD0325901, AZD8330, PD98059, U0126-EtOH, BIX 02189 or BIX 02188.
In one embodiment the combination therapy employs an AKT inhibitor. Examples of AKT inhibitors include: MK-2206 and AT7867.
In one embodiment the combination employs an aurora kinase inhibitor. Examples of aurora kinase inhibitors include: Aurora A Inhibitor I, VX-680, AZD1152-HQPA (Barasertib), SNS-314 Mesylate, PHA-680632, ZM-447439, CCT129202 and Hesperadin.
In one embodiment the combination therapy employs a p38 inhibitor, for example as disclosed in WO2010/038086, such as N-[4-({4-[3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl) ureido]naphthalen-1-yloxy}methyl) pyridin-2-yl]-2-methoxyacetamide.
In one embodiment the combination employs a Bcl-2 inhibitor. Examples of Bcl-2 inhibitors include: obatoclax mesylate, ABT-737, ABT-263(navitoclax) and TW-37.
In one embodiment the combination therapy comprises a checkpoint inhibitor, for an anti-PD-1 inhibitor or an anti-PD-L1 inhibitor.
In one embodiment the chemotherapy combination comprises an antimetabolite such as capecitabine (xeloda), fludarabine phosphate, fludarabine (fludara), decitabine, raltitrexed (tomudex), gemcitabine hydrochloride and cladribine.
In one embodiment the chemotherapy combination comprises ganciclovir, which may assist in controlling immune responses and/or tumour vasculation.
In one embodiment the chemotherapy includes a PARP inhibitor.
In one embodiment the combination therapy includes an inhibitor of cancer metabolism with specific inhibition of the activity of the DHODH enzyme.
In one embodiment one or more therapies employed in the method herein are metronomic, that is a continuous or frequent treatment with low doses of anticancer drugs, often given concomitant with other methods of therapy.
In one embodiment, there is provided the use of multiple cycles of treatment (such as chemotherapy) for example 2, 3, 4, 5, 6, 7, 8.
In one embodiment the chemotherapy is employed in a 28 day cycle.
In one embodiment the molecules of the present disclosure are provided in a pharmaceutical composition comprising a excipient, diluent and/or carrier. In one embodiment the composition is as a parenteral formulation.
Parenteral formulation means a formulation designed not to be delivered through the GI tract. Typical parenteral delivery routes include injection, implantation or infusion.
In one embodiment the parenteral formulation is in the form of an injection. Injection includes intravenous, subcutaneous, intra-cranial, intrathecal, intra-tumoural or intramuscular injection. Injection as employed herein means the insertion of liquid into the body via a syringe.
In one embodiment the parenteral formulation is in the form of an infusion.
Infusion as employed herein means the administration of fluids at a slower rate by drip, infusion pump, syringe driver or equivalent device. In one embodiment, the infusion is administered over a period in the range of 1.5 minutes to 120 minutes, such as about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 65, 80, 85, 90, 95, 100, 105, 110 or 115 minutes.
In one embodiment, the formulation is for intravenous (i.v.) administration. This route is particularly effective because it allows rapid access to the majority of the organs and tissue and is particular useful for the treatment of metastases, for example established metastases especially those located in highly vascularised regions such as the liver and lungs.
Therapeutic formulations typically will be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other parenteral formulation suitable for administration to a human and may be formulated as a pre-filled device such as a syringe or vial, particular as a single dose.
As discussed above the formulation will generally comprise a pharmaceutically acceptable diluent or carrier, for example a non-toxic, isotonic carrier that is compatible with the virus, and in which the virus is stable for the requisite period of time.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a dispersant or surfactant such as lecithin or a non-ionic surfactant such as polysorbate 80 or 40. In dispersions the maintenance of the required particle size may be assisted by the presence of a surfactant. Examples of isotonic agents include sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
In one embodiment there is provided a kit of parts comprising a GLA-component according to the present disclosure and a payload, wherein the payload is linked or unlinked to said GLA-component.
“Comprising” in the context of the present specification is intended to mean “including”.
Where technically appropriate, embodiments of the invention may be combined.
Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.
Technical references such as patents and applications are incorporated herein by reference.
The technical backgrounds is part of the technical disclosure of the present specification and may be used as basis for amendments because the discussion therein is not limited to discussing the prior art as it also includes a discussion of the technical problems encountered in the field and the application of the present technology.
Any embodiments specifically and explicitly recited herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments.
The present application claims priority from U.S. Ser. Nos. 62/554,530, 62/569,403, 62/554,533, 62/569,411, 62/584,565 and 62/593,014. Each of these applications are incorporated by reference. These applications may be employed as the basis for a correction to the present specification.
The invention will now be described with reference to the following examples, which are merely illustrative and should not in any way be construed as limiting the scope of the present invention.
This specification also includes sequences 1 to 6, in the associated sequence listing.
This project initiated the testing of labeled recombinant PrS as an in vivo imaging agent for SPECT (Single Photon Computed Tomography). Surprisingly it was found that the molecule rapidly internalized into apoptotic cells. This unexpected finding led us to explore the phenomenon further, whereupon we found that PrS was also internalized into a subset of non-apoptotic stem cells of several types.
PrS is protein S GLA domain and protein S EGF domain as shown in SEQ ID NO: 6.
For fluorescence, conjugation of Cy5 and FITC was achieved using Amersham (GE Heathcare) and Molecular Probes (Invitrogen) labeling kits, respectively, according to the instructions of the manufacturers. Both kits provide columns for the removal of unconjugated fluorophore. Initially, 0.77 mg of PrS (Fraction 2) in 1 ml and 0.77 mg of annexin in 1 ml were labeled with FITC to test for specificity of binding to apoptotic cells. For co-localization and competition studies 0.68 mg of PrS (Fraction 3) in 1 ml and 0.68 mg of annexin were labeled with Cy5. For confocal microscopy, 0.76 mg of PrS from the second shipment was labeled with FITC and the previously labeled Cy5-conjugated annexin was used. It should be noted that the precise efficiency of labeling was not determined and the recovery from the columns was assumed to be 85%, according to the instructions of the manufacturers of the labeling kits. Thus, the relative staining intensity of the two proteins in any case may reflect these contingencies. The cells were stained for 30 min initially, but it was subsequently determined that less than 5 min was sufficient. To test PrS for apoptotic cell-specificity, four breast cancer cell lines were initially employed; human MDA-231 and MCF7 and murine 4T1 and MET-1. Subsequently, COS-1 monkey kidney cells were also used. Apoptosis was induced with hydrogen peroxide or tertiary-Butyl hydroperoxide (t-BHP). The cells were plated in 24-well plates at 6×104 cells per well or Eppendorf chamber slides at 1×104 cells per well, and apoptosis was induced the next day, using 2 mM H2O2, or t-BHP for time points from 30 min to 2 hrs. After induction, the wells were washed with Annexin Binding Buffer (AB; Santa Cruz Biotech), and stained with labeled protein. From past experience and the literature, 5.5 μg/ml of annexin protein was used for staining. This amount was adjusted for equimolar addition of PrS by assuming the molecular weights of annexin to be 36 kD and the recombinant PrS to be 30 kD, based on the gel images provided. The cells were stained for 15 min. Hoechst 33342 dye was used for visualizing nucleic acid. The wells were then washed with AB and observed using the EVOS fluorescence microscope while still viable. For confocal microscopy, the Leica SP8 microscope in the Stanford Cell Sciences Imaging Facility was employed. The wells were then washed with AB and observed using the Leica sp8 microscope. Hoechst 33342 dye was used for visualizing nuclei. For toxicity studies, PrS was added to trophoblast stem cells (TSCs) and the viability tested with trypan blue using a Nexcelom Cellometer.
To test the labeled proteins for the ability to detect tumors, 5×104 4 T1-luc cells were implanted into groups of 5 male BALB/c mice, in the left axillary fat pad. The mice were imaged with in vivo bioluminescence imaging (BLI) each day to monitor tumor growth, starting at 1 week post implantation. The mice were then treated on day 11 post implantation with 13 mg/kg body weight of intraperitoneal (IP) doxorubicin, and BLI was performed the next day. Control mice bearing tumors were left untreated with doxorubicin. 48 hrs post treatment the mice were imaged 1 hr after intravenous tracer injection (anesthesia 1.3 g/kg of urethane IP), with single head A-SPECT gamma camera (Gamma Medica); 1 mm pin hole collimator, 128 steps into a 128×128 imaging matrix, 15 seconds per step, 2.7 cm ROR; FOV=upper chest/neck. The injected dose of each protiein was 160 μl (800 μCO. The animals were then sacrificed and biodistribution was performed. For the cyclohexamide treatment experiment, groups of 5 young (7 week old) male Swiss Webster mice were anesthetized (1.3 g/kg of urethane IP) and injected intravenously with 50 mg/kg cycloheximide. 1 hr 45 min after cycloheximide injection, tracer was injected (PrS=180 ul/1.2 mCi per dose; annexin V =170 μl/1.05 mCi per dose). 45 min after tracer injection, the mice were imaged with 10 min static whole body images using a single head parallel hole collimator (128×128 matrix) on the A-SPECT gamma camera.
To test for the specific localization of fluorescent PrS to apoptotic sites due to infection in live animals, CD1 mice were injected intravenously with bioluminescent Listeria monocytogenes. This bacterial pathogen infects many organs including the spleen, in which extensive apoptosis of monocytes and granulocytes occurs. At certain times post infection, spleen is the primary site of bacterial replication and so splenic BLI signals from the bacteria can be correlated with the localization of probes for apoptosis. Mice were infected and imaged each day. When splenic signals were evident (day 2 post infection for 2×105 colony forming units of bacteria in 8 week old CD1 female mice), 300 mg/kg body mass of Cy5 PrS was injected into mice, the animals were sacrificed 30 min later, and the spleens removed, frozen in OCT, and sectioned for fluorescence microscopy. Uninfected control mice were employed.
Flow cytometry was performed. Freshly labeled FITC PrS, prepared as described above, was employed. Murine hematopoietic stem cells (HSCs) are routinely purified in this laboratory. The cells were isolated from normal mouse bone marrow by staining for c-Kit+, lineage-negative cells. To further characterize the cells, SLAM marker staining was also performed. These markers stain cells that self-renew and differentiate, whereas non-staining HSCs can only differentiate. Subsequent staining with FITC PrS revealed the percent positive in SLAM-staining cells, as shown in the Results. The cells were then sorted for FITC and examined with confocal microscopy, using Hoechst 33342 for nuclear visualization.
To assess PrS binding specificity in the context of apoptosis in cell culture, we employed several human and murine breast cancer cell lines. Apoptosis was induced with peroxide as described above, and FITC PrS binding was assessed. Examples of these experiments are shown in
EVs, specifically exosomes, microvesicles (MVs) and apoptotic bodies (ABs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these types of EVs differs, and they originate from either the endosomal (exosomes) or plasma membranes (MV) or are products of programmed cell death (ABs). All mammalian cells are thought to secrete EVs. Each type of EV can transfer molecular cargo to both neighboring and distant cells, affecting cellular behaviors such as those involved in tumor development and progression. In fact, EVs may play a role in nearly all the hallmarks of cancer, including sustaining proliferative signaling, evading growth suppression, resisting cell death, reprogramming energy metabolism, acquiring genomic instability, and developing the tumor microenvironment. They have also been implicated in the induction of angiogenesis, control of invasion, initiation of premetastatic niches, sustaining inflammation, and evading immune surveillance. Immune cells appear to also communicate through EVs and my recognize EVs as signals from tumor cells, infected tissues and wounds. A deeper understanding of the biology of EVs and their contribution to the hallmarks of cancer is leading to new possibilities for diagnosis and treatment of cancer. Development of additional EV surface markers is essential to advancing this field and PrS may be such a determinant.
Following these studies with fluorescence microscopy, the subcellular localization of the staining by PrS and annexin was then evaluated via confocal microscopy. Murine 4T1 cells (lacking the Luc-GFP reporters) were plated on 8-part chamber slides at 1×104 cells per chamber and apoptosis was induced with 2 mM H2O2 or t-BHP (2 hr exposure) the next day. The cells were then washed and stained for 15 min with PrS and annexin. Hoechst 33342 dye was used to stain nucleic acid. In all cases, the most brightly staining cells were stained with both probes. However, in many cells labeled PrS was observed in the cytoplasm, whereas the labeled annexin was not (
SPECT imaging of mice treated with cyclohexamide, which induces apoptosis in the liver, was also performed (
The localization of fluorescent PrS to 4T1 tumors treated with doxorubicin was then tested. Mice implanted with tumors were treated with doxorubicin as described above and Cy5 PrS was injected intravenously 30 min prior to sacrifice and removal of the tumors for sectioning and fluorescence microscopy. The results are shown in
Stem cells are distinct in phenotype from differentiated cells and may express PS non-apoptotically to avoid the induction of immune responses. Trophoblast stem cells (TSCs) differentiate into several types of trophoblasts in culture. TSCs are prepared from mouse uterine scrapings grown in the presence of fibroblast growth factor, activin, and heparin. TSCs spontaneously differentiate into giant cells when these factors are removed from the medium (
To assess subcellular staining pattern, undifferentiated MSC were stained with PrS and annexin, as well as Hoechst nuclear staining reagent, and observed with confocal microscopy. Results of the observations are shown in
We have succeeded in staining hematopoietic stem cells (HSC) with PrS. Using flow cytometry we determined that HSC stain with PrS, and have observed internalization of PrS in these cells with confocal microscopy. HSC were identified and isolated using fluorescence activated cell sorting (FACS). The cells were identified in bone marrow as lineage-negative, SCA/c-kit positive cells (
We then proceeded to test for internalization of PrS into HSC. This experiment was complicated by many factors. Perhaps the most difficult was the survival in culture of HSC, which die in large numbers in medium overnight. We therefore had to time the experiment such that flow cytometry analysis and confocal microscopy occurred on the same day. Furthermore, the cells are not adherent, making microscopy less than optimal. To make microscopy more efficient, the cells were resuspended in a small drop of medium. Finally, we needed to make sure that the PrS-stained cells analyzed by microscopy were still alive. Many HSC died during the processes of analysis and isolation. Therefore, PI was added and scanned in addition to the Hoescht nuclear stain, and another channel was employed. The presence of PI-bright nuclei indicated dead cells. Despite these difficulties and the complexities of timing, we were able to perform the experiment, and confirmed internalization of PrS into live HSC (
Finally, in
The above results have shown that PrS is rapidly internalized into an array of cells expressing PrS, including stem cells of many types, which suggests that PrS possesses unique characteristics amenable to manipulation toward the goal of developing a therapeutic agent. In addition, the difference in specificity between PrS and annexin such as seen in
Stem cells are distinct in phenotype from differentiated cells and may express PS non-apoptotically to avoid the induction of immune responses. Stem cells were stained with a GLA domain molecule of the present disclosure comprising a payload of a fluorescent label, without the induction of apoptosis.
Trophoblast stem cells, (
This data the molecules of the present disclosure may be employed to target cells in vivo or in ex vivo samples.
This application claims the benefit of U.S. Provisional Application No. 62/554,530 filed Sep. 5, 2017, U.S. Provisional Application No. 62/554,533 filed Sep. 5, 2017, U.S. Provisional Application No. 62/569,403 filed Oct. 6, 2017, U.S. Provisional Application No. 62/569,411 filed Oct. 6, 2017, U.S. Provisional Application No. 62/584,565 filed Nov. 10, 2017, and U.S. Provisional Application No. 62/593,014 filed Nov. 30, 2017, each of which applications is herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/049618 | 9/5/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62593014 | Nov 2017 | US | |
| 62584565 | Nov 2017 | US | |
| 62569411 | Oct 2017 | US | |
| 62569403 | Oct 2017 | US | |
| 62554533 | Sep 2017 | US | |
| 62554530 | Sep 2017 | US |
