WO2021245095A1 - Method to improve immunotherapies - Google Patents

Method to improve immunotherapies Download PDF

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Publication number
WO2021245095A1
WO2021245095A1 PCT/EP2021/064699 EP2021064699W WO2021245095A1 WO 2021245095 A1 WO2021245095 A1 WO 2021245095A1 EP 2021064699 W EP2021064699 W EP 2021064699W WO 2021245095 A1 WO2021245095 A1 WO 2021245095A1
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WIPO (PCT)
Prior art keywords
sepiolite
clay mineral
use according
cells
cancer
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PCT/EP2021/064699
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French (fr)
Inventor
Bernard Lopez
Sandrine RAGU
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique (Cnrs)
Université de Paris
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Publication of WO2021245095A1 publication Critical patent/WO2021245095A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A61K33/12—Magnesium silicate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates a clay mineral for use to improve an immunotherapy in a subject in need thereof.
  • Sepiolite show different structural and textural characteristics depending on the geological origin. So, according to Suarez and Garcia-Romero (Suarez and Garcia-Romero, 2012), the presence of crystalline defects, the diversity in the chemical composition and the variability in size and aggregation state of sepiolite fibres result from the growth conditions, which in turn depend on the genetic environment (hydrothermal or sedimentary) and on the involved generation processes (neoformation, transformation or mechanical heritage). In this manner, the main physico-chemical properties of sepiolite, as for instance its surface characteristics, show great variability depending on the geological origin of the silicate.
  • Sepiolite has been used to transport small molecules into cells such as tetracycline, nitric oxide, antisense oligonucleotides, and in combination with carbon nanotubes to detect DNA as well as DNA-drug interactions (Ruiz-Hitzky et al., 2011; Erdem et al., 2012; Fernandes et al., 2013; Mitsudome et al., 2014; Giir et al., 2017; Olivato et al., 2017; Pietrement et al., 2018).
  • sepiolite can transfer DNA into mammalian cells, opening alluring avenues for biotechnological and biomedical applications (Castro- Smirnov et al., 2016, 2017; Pietrement et al., 2018).
  • mammalian cells spontaneously internalize sepiolite, facilitating the spontaneous transport and delivery of bound molecules (Castro-Smirnov et al., 2017).
  • non-asbestos minerals belonging to fibrous silicates such as sepiolite raised health concerns about possible asbestos-like health effects (Gonzalez-Tortuero et al., 2018).
  • fibrous-clay minerals and asbestos from the point of view of fibres morphology and topochemistry, crystallinity, composition and surface properties, which can be directly related to their biological activity, possible carcinogenic behavior and, therefore, to potential health hazards (Santaren and Alvarez, 1994).
  • sepiolite can transfer DNA into bacteria through the Yoshida effect, which was first described with asbestos (Yoshida and Sato, 2009; Wilharm et al., 2010; Gonzalez-Tortuero et al., 2018, Castro-Smirnov et al., 2020). Friction forces by the fibres perforate the bacterial membrane, allowing DNA transfer via holes generated. Besides, it has been reported that sepiolite can generate breaks into the bacterial genome (Gonzalez- Tortuero et al., 2018).
  • mammalian cells can also spontaneously expelled sepiolite fibres (Castro-Smirnov et al., 2017).
  • mammals are also pluri- cellular organisms (metazoans), in contrasts with bacteria, that have developed numerous defence systems, both at cell and organism level, including cell cycle checkpoint, apoptosis, senescence and innate immunity.
  • metalazoans pluri- cellular organisms
  • sepiolite is poorly toxic at the doses used for mammalian cells transfection (Castro- Smirnov et al., 2017; Pietrement et al., 2018). Therefore, extrapolation from bacteria to mammals constitutes an over-interpretation.
  • ROS Reactive oxygen species
  • ROS intracellular ROS
  • 2-Cells generate inflammatory cytokines upon exogenous as well as endogenous stress (Ragu et al., 2020), activating the innate immune system that eliminates damaged cells, which potentially could become pathological, thus protecting the whole organism;
  • 3-Genome integrity is routinely jeopardized by exogenous (radiations, chemicals, ...) as well as endogenous (replication stress, ROS) assaults.
  • Faithful genome replication and transmission is essential to maintain genetic stability during cell division. DNA is replicated in the S phase of cell cycle, and then is compacted into chromosomes that are segregated to the daughter cells during mitosis (M).
  • DDR DNA damages response
  • cells activate the DNA damages response (DDR) that coordinates a network of pathways including DNA replication and repair, cell cycle progression, chromosome segregation and apoptosis.
  • DDR DNA damages response
  • cells activate the DDR, which arrest cell cycle progression, giving time to repair DNA before the sensitive S and M phases and avoiding conflict between DNA replication and repair. If the cell is unable to repair DNA (for example when there are too much damages), then they activate the apoptosis program that kill and eliminate the damage cell. Therefore, activation of DDR, cell cycle arrest and apoptosis induction are markers of genome insult.
  • Defect in DDR results in genome instability, associated with tumour initiation and development, and with premature aging (Kastan and Bartek, 2004; Bartkova et ak, 2005, 2006; Gorgoulis et ak, 2005; Bartek et ak, 2007; Halazonetis et ak, 2008; Hoeijmakers, 2009; Jackson and Bartek, 2009; Gorgoulis and Halazonetis, 2010; Negrini et ak, 2010).
  • mammalian cells respond to sepiolite exposure, inducing the production of ROS and the expression of inflammatory cytokines genes, showing that cells detect sepiolite contamination and respond.
  • cancer osteosarcoma cells (U20S), SV40 immortalized human fibroblasts and normal primary human skin fibroblasts)
  • sepiolite exposure did not alter cell cycle distribution and trigger neither DDR nor apoptosis, suggesting that it does not significantly assault the genetic material in mammalian cells.
  • this ability to induce inflammatory cytokines opens promising avenues in strategies aiming at boosting the efficacy of immunotherapy.
  • the present invention relates to a clay mineral for use to improve an immunotherapy in a subject in need thereof.
  • the invention is defined by its claims.
  • a first aspect of the invention relates to a clay mineral for use to improve an immunotherapy in a subject in need thereof. Improvement of an immunotherapy can be very useful to treat diseases through the stimulation of the efficiency of the immune system.
  • the invention relates to a clay mineral for use in the treatment of a disease, involving the immune system in a subject in need thereof.
  • a disease involving the immune system denotes a cancer, an infectious disease and all diseases eligible for immunotherapy and where stimulation of the efficiency of the immune system constitutes a therapeutic gain.
  • the invention also relates to a clay mineral for use in the treatment of a cancer or an infectious disease in a subject in need thereof.
  • the clay mineral of the invention can also be used in combination with a treatment already used to treat a disease involving the immune system and/or to improve an immunotherapy.
  • the invention also relates to a i) clay mineral and ii) a treatment used to treat a disease involving the immune system as a combined preparation for simultaneous, separate or sequential for use in the improvement of an immunotherapy in a subject in need thereof.
  • the invention relates to a clay mineral and ii) a treatment used to treat a disease involving the immune system as a combined preparation for simultaneous, separate or sequential for use in the treatment of a cancer or an infectious disease in a subject in need thereof.
  • the clay mineral is the sepiolite.
  • a treatment used to treat a disease involving the immune system denotes a treatment used to treat a cancer or an infectious disease.
  • treatment used to treat a cancer or an infectious disease refers to anti-cancer agent or anti-infectious agents.
  • the anti-cancer agent is a checkpoint blockade cancer immunotherapy agent.
  • the checkpoint blockade cancer immunotherapy agent is an anti- PD1 antibody.
  • the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer
  • the cancer is resistant to immunotherapy, and in particular resistant to checkpoint blockade cancer immunotherapy agent.
  • the infectious diseases can be induced by a virus selected in the group consisting of viral disease Genital warts, common warts, plantar warts, respiratory polynuclear virus (RSV), hepatitis B, hepatitis C, dengue virus, herpes simplex virus (for example, HSV-I, HSV-II, CMV or VZV), molluscum contagiosum, vaccinia, pressure ulcer, lentivirus, human immunodeficiency virus (HIV), human papilloma virus (HPV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rhino Virus, enterovirus, adenovirus, influenza, para-influenza, mumps virus, measles virus , Papovavirus, hepadnavirus, flavivirus, retrovirus, arenavirus (for example, LCM, Junin virus, Machupovirus, Guanarito virus and Lassa fever) and filovirus (
  • the infectious diseases can be induced by a bacterium selected in the group consisting of Streptococcus pneumoniae; Staphylococcus aureus; Haemophilus influenza, Myoplasma species, Moraxella catarrhalis, Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella enterica serovar, Typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, Campylobacter, Mycobacterium tuberculosis, Helicobacter pylori and Streptomyce.
  • a bacterium selected in the group consisting of Streptococcus pneumoniae; Staphylococcus aureus; Haemophilus influenza,
  • clay mineral denotes hydrous aluminium phyllosilicates, sometimes with variable amounts of iron, magnesium, alkali metals, alkaline earths, and other cations found on or near some planetary surfaces.
  • clay mineral includes but is not limited to layered silicates (phyllosilicates) and micro/nano fibrous clays.
  • layered silicates includes but is not limited to the kaolin group (halloysite) including the kaolinite, dickite and nacrite, and the smectite layered silicate group including montmorillonite, beidellite, saponite and hectorite.
  • micro/nano fibrous clays includes but is not limited to the paligorskite and sepiolite fibre.
  • the sepiolite is a sepiolite fibre.
  • sepiolite or sepiolite fibre
  • Sepiolite shows an alternation of blocks and tunnels that grow up in the fiber direction.
  • the blocks are constituted by two layers of tetrahedral silica sandwiching a central magnesium oxide-hydroxide layer.
  • the discontinuity of the silica sheets gives rise to the presence of silano 1 groups (Si-OH) at the edges of the channels, which are the tunnels open to the external surface of the sepiolite particles.
  • the total specific surface area of the sepiolite fibers ranges from 100 to 500 m2/g, preferably from 200 to 400 m2/g.
  • the mean length of the sepiolite fibers is less than or equal to 2pm, or is less than or equal to 1 p , and better still less than or equal to 800nm. More particularly, the mean length of the sepiolite fibres ranges from 150 to 850 nm.
  • the sepiolite used according to the invention contains advantageously less than 1 % by weight of fibres having a length of at least 5 pm, preferably less than 5 % by weight of fibres having a length of at least 2.5pm.
  • the size distribution (length and diameter) of the fibers can be determined by transmission electron microscopy (TEM), using a Zeiss 912AB transmission electron microscope (dark-field mode with a tilted illumination).
  • TEM transmission electron microscopy
  • the mean diameter of the sepiolite fibres of the present invention is preferably less than or equal to 50nm, more preferably less than or equal to 25nm, and better still ranges from 8 to 1 8nm.
  • sepiolite fibres that can be used in the present invention are the products sold by the company Tolsa S .A., under the name Pangel S9, and by the company Kremer Pigmente GmbH&Co, under the number 58945.
  • the sepiolites fibres can be sonicated prior their use according to the invention.
  • the clay mineral of the invention can also be used with other molecules like, small molecules (natural or not), nucleic acids or antibodies to improve/boost an immune response of the organism or to allow a targeting of the clay mineral.
  • the clay mineral will be linked to a small molecule, at least one nucleic acid or at least one antibody.
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
  • the clay mineral and particularly the sepiolite can be used in combination with one or more nucleic acids that are linked to the clay mineral and particularly the sepiolite.
  • the nucleic acids can be in particular chosen from DNA, RNA, DNA/RNA hybrids and chemically modified nucleic acids. More particularly, the nucleic acids are chosen from DNA chains and RNA chains. According to a particular embodiment, the nucleic acids are chosen from linear or circular, single stranded or double-stranded nucleic acid chains.
  • the nucleic acids can be present in any kind of vector. Particularly, the vector is a plasmid, such as pCMV.
  • the nucleic acids are chosen from single stranded or double stranded RNA chains.
  • the double stranded RNA is a siRNA.
  • the nucleic acid can come from a human or another species.
  • the nucleic acids can be salmon sperm DNA (Castro-Smimov et. al 2016).
  • the nucleic acids when linked to the clay mineral and particularly the sepiolite, can be very useful to improve the answer of the immune system.
  • the nucleic acids will be recognized as a stranger DNA (Ragu et. al., 2020).
  • the clay mineral and particularly the sepiolite can also be used in combination with an antibody that is linked to the clay mineral and particularly the sepiolite.
  • the antibody will be used to allow the targeting of the clay mineral to a specific cell or tissue.
  • the antibody can be directed to a specific antigen expressed by a cancerous cell and thus allow the improvement of the immune system against this specific cancerous cell.
  • the specific antigen can be the CD99 which is specific of the Ewing sarcoma.
  • the clay mineral and particularly the sepiolite is used in combination with a checkpoint blockade cancer immunotherapy antiboy linked to the clay mineral and particularly the sepiolite
  • the clay mineral and particularly the sepiolite is used in combination with an anti -PD 1 antibody linked to the clay mineral and particularly the sepiolite.
  • the clay mineral can be linked to one or more nucleic acids and to a specific antibody.
  • the clay mineral can be intravenously, parenterally, intramuscularly injected or can be injected directly to a tumour and particularly a solid tumour.
  • the clay mineral will be injected as a suspension.
  • the antibody linked to the clay mineral When injected directly in the systemic system, the antibody linked to the clay mineral will be useful to target the tumoral cells / the tumour.
  • the clay mineral can be orally administered.
  • the clay mineral is administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms.
  • the invention also relates to a method for improving an immunotherapy in a subject in need thereof comprising administering to said subject in need thereof a therapeutically effective amount of a clay mineral.
  • the invention in another embodiment, relates to a method for treating a disease involving the immune system in a subject in need thereof comprising administering to said subject in need thereof a therapeutically effective amount of a clay mineral.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • Another object of the invention relates to a therapeutic composition comprising a clay mineral for use in the improvement of an immunotherapy in a subject in need thereof.
  • the therapeutic composition comprises sepiolite.
  • the therapeutic composition comprises sepiolite and a treatment used to treat a disease involving the immune system according to the invention.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles that are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles that are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions of the present invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent.
  • an anti-cancer agent used to treat cancer may be added to the pharmaceutical composition as described below.
  • Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).
  • Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole
  • additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors, Ca2+ ATPase inhibitors and PARP inhibitors.
  • Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
  • the further therapeutic active agent can be an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol s, thiefhylperazine, thioproperazine and tropisetron.
  • the antiemetic agent is granisetron or ondans
  • the further therapeutic active agent can be an hematopoietic colony stimulating factor.
  • Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the other therapeutic active agent can be an opioid or non opioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofmac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • the further therapeutic active agent can be an anxiolytic agent.
  • Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.
  • the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • CTL4 cytotoxic T lymphocyte-associated protein 4
  • PDCD1 programmed cell death 1
  • NK cells like various members of the killer cell immunoglobulin like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • the checkpoint blockade cancer immunotherapy agent is an antibody.
  • the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti -PD 1 antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti -IDO 1 antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.
  • the clay mineral and particularly the sepiolite is combined with an anti-PDl antibody.
  • the clay mineral of the invention can be also combined with radiotherapy.
  • an anti-infectious agent used to treat infectious diseases may be added to the pharmaceutical composition as described below.
  • further anti-infectious agents may be selected in the group consisting bronchodilators like b2 agonists and anticholinergics, corticosteroids, beta2-adrenoceptor agonists like salbutamol, anticholinergic like ipratropium bromide or adrenergic agonists like epinephrine.
  • Further agent may be also an antiviral compound like amantadine, rimantadine or pleconaril.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Expression of inflammatory cytokine genes (real-time qPCR).
  • FIG. 1 Induction of the expression of Inflammatory cytokines genes by different clays or nano-diamonds.
  • the expression of cytolines genes (A. IL-6, B. IL-18, C. TNFa, D. IL_8, E. CCL2) were measured by RT-QPCR 24 (black bars) or 48 hours (white bars) after exposure (the doses are indicated on the figure).
  • the Nano-diamonds+ and Nano-diamonds- panels correspond to the exposure to cationic (+) or anionic (-) Nano-diamonds.
  • the data from at least three independent experiments are presented ( ⁇ SEM).
  • FIG. 1 Production (in protein) and excretion of cytokines monitored by cytokine array 48 after exposure of U20S cells to different clays (indicated on the left part). Three different exposure of the same array are shown. The rectangles focussed on the induced cytokines. The numbers identifies the induced cytokines.
  • U20S Ctrl control of cells without clays.
  • FIG. 4 Impact of association sepiolite/anti-PDl antibodies.
  • C Size of the tumors at the end of the experiment (Day 22). The values represent the mean size of 6 tumors in 6 mice were measured for each point. The values are shown normalize to the size of the tumors at Day 0 (first day of injection).
  • Sepiolite of rheological grade commercialized by TOLSA S. A as Pangel S9 (> 95% of pure sepiolite) was used in this work.
  • This sepiolite originated from the Vallecas-Vicalvaro clay deposits (Madrid, Spain) located in the Tagus Basin area.
  • this sepiolite sonicated in an ultrasonic bath shows from TEM analyses nano/micro size particles with fibre length distribution ⁇ 1200 nm, being 80% of fibres in the 200 to 400 nm range with a maximal length of 800 nm.
  • sepiolite fibres show width distribution in the 10-25 nm range with a maximal width of 15 nm, also deduced from TEM observations (Castro-Smirnov et ah, 2016).
  • CM-H2DCFDA (2’,7’-dichlorofluorescein diacetate) (Life Technologies, USA) assay kit was used according to the manufacturer’s protocol. 105 cells/well plated in 6-well plates were incubated at 37°C (5% C02). After washing with PBS cells were incubated with 10 mM CM-H2DCFDA in DMEM supplemented with 1% FBS for 45 min at 37°C in the dark. After trypsinization, cells were re-suspended in DMEM supplemented with 1% FBS.
  • RNA extraction reverse transcription and quantitative RT-PCR of cytokine genes expression RNA extraction reverse transcription and quantitative RT-PCR of cytokine genes expression.
  • the cDNAs were generated from 2 pg of total RNA using random hexamers and RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific).
  • the following primers were used for SYBR real-time PCR assay (Applied Biosystems): IL-6,
  • IL-8, IL-1B, CCL2, IL-18, TNFa and GAPDH served as the internal control.
  • the sequences of the primers used for SYBR assays are in Table 1. Quantitative RT-PCR was performed using the Applied Biosystems 7300 Real-Time PCR System. All experiments were performed in triplicate.
  • Proteins were extracted from cell pellets with lysis buffer (50mM Tris pH 7.5, 20mM NaCl, lmMMgC12, 0.1% SDS, Benzonase 25KU/ml Santa Cruz sc-202391, protease inhibitors Roche 5892970001, phosphatase inhibitors cocktails 2 and 3 Sigma Aldrich P5726 and P0044) for 15 min at room temperature. 30 pg of protein were loaded on the separation gel, for each sample. Electrophoresis, transfer to a nitrocellulose membrane and antibody probing were performed using standard procedures.
  • Sepiolite stimulates the production of intracellular ROS in U2QS cells.
  • Sepiolite triggers inflammatory cytokines genes expression.
  • One cell response to the presence of foreign molecules/compounds is the production of inflammatory cytokines, which acts through the induction of inflammatory cytokines genes expression.
  • sepiolite induces the expression of inflammatory cytokines genes.
  • GC92 immortalized SV40- transformed fibroblasts
  • DDR is activated by the phosphorylation of effectors by the signalling kinases ATM/ATR.
  • ATR phosphorylates Chkl .
  • ATM phosphorylates Chk2. Therefore, the phosphorylation of Chkl (pChkl) or Chk2 (pChk2) are markers of the activation of ATR and ATM pathway, respectively, and indirectly of the production of single-strand and double-strand breaks into the DNA.
  • Sepiolite does not trigger apoptosis.
  • apoptosis induction is also a marker of genotoxic stress.
  • Many cancer cells (but not all) are resistant to apoptosis induction, escaping to this protection system and thus participating to their increased proliferation capacities. Therefore, here it is essential to verify potential apoptosis induction by sepiolite in cancer cells (U20S), immortalized (CG92) and primary skin fibroblasts. It is important to verify as whether U20S and GC92 cells are actually able to induce apoptosis, as we did here upon HU exposure (data not shown).
  • Apoptosis induction program leads to the activation of proteases such as caspase 3 that inactivate DNA repair proteins by cleaving them at specific sites, making cell death more efficient.
  • PARP1 is a essential DNA damge signalling and DNA repair protein.
  • PARP1 is a target of caspase 3.
  • PARP1 is cleave at a specific site, resulting in shorter polypeptides. Therefore, using anti -P ARP 1 antibodies, we monitored the cleavage of PARP1 upon exposure to sepiolite (data not shown). The exposure to a genotoxic stress (HU) indeed, generates the appearance of the diagnosis shorter PARP1 polypeptides (89 kDa).
  • Cell lines, U20S (human osteosarcoma) were maintained at 37°C with 5% CO2 in modified Eagle’s medium (MEM).
  • MEM modified Eagle’s medium
  • concentrations (10 or 50ug/ml) of sepiolite or Cloisite (Wyoming) or Halloysite (IMERYS), or Laponite (Laporte) or cationic and anionic nanodiamonts (5 or 25ug/ml) were added to the culture medium. Cells were collected one or two days after exposure.
  • RNA extraction reverse transcription and quantitative RT-PCR of cytokine genes Total RNA was isolated using the Macher el -Nagel NucleoSpin RNA kit, according to the manufacturer’s instructions. The cDNAs were generated from 2 pg of total RNA using random hexamers and RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific). The following primers were used for SYBR real-time PCR assay (Applied Biosystems): IL-6, IL-8, IL-IB, CCL2, IL-18, TNFa and GAPDH served as the internal control. Quantitative RT-PCR was performed using the Applied Biosystems 7300 Real-Time PCR System. All experiments were performed in triplicate.
  • the supernatants from cultured U20S in the sepiolit or cloisite or laponite treatment groups were analyzed with a human Cytokine Array Kit (Cat: ARY005B, R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
  • MET-1 cells were subcutaneously injected in FVB/N mice (syngeneic) backs. After tumors growth (one month), sepiolite (300 pg/ml) was injected into the tumors associated or not with intraperitoneal injections of an anti-PD-1 antibody (200 pg, RMP1-14 clone; BioXcell). Injections were performed every 4 days.
  • MET-1 cells were derived from tumors of the the PyMT spontaneous breast cancer mouse model.
  • the PyMT derived from the FVB/N mouse strain.
  • injection of MED-1 cells in FVB/N generate tumors (see Fig. 4A-C).
  • MET-1 tumors are "old" for immunotherapy, meaning that injection of anti-immune checkpoint antibody (anti-PDl Ac) does not affect tumors progression.
  • anti-PDl Ac anti-immune checkpoint antibody
  • anti- PDl Ac alone did not block tumor progression (Fig. 4B and C), at the concentration used, sepiolite alone has no significant effect (Fig. 4C).
  • the combination of sepiolite and anti-PDl Ac impaired tumor progression (Fig. 4B and C).
  • Clay nanoparticles for regenerative medicine and biomaterial design A review of clay bioactivity. Biomaterials 159, 204-214. https://doi.Org/10.1016/j.biomaterials.2017.12.024

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Abstract

The present invention relates to the improvement of immunotherapies. In this study, the inventors showed that mammalian cells respond to sepiolite exposure, inducing the production of ROS and the expression of inflammatory cytokines genes, showing that cells detect sepiolite contamination and respond. Remarkably, in three different and complementary types of cells (cancer osteosarcoma cells, (U2OS), SV40 immortalized human fibroblasts and normal primary human skin fibroblasts), sepiolite exposure did not alter cell cycle distribution and trigger neither DDR nor apoptosis, suggesting that it does not significantly assault the genetic material in mammalian cells. Thus, this ability to induce inflammatory cytokines opens promising avenues in strategies aiming at boosting the efficacy of immunotherapy. Thus, the present invention relates to a clay mineral for use to improve an immunotherapy in a subject in need thereof.

Description

METHOD TO IMPROVE IMMUNOTHERAPIES
FIELD OF THE INVENTION:
The present invention relates a clay mineral for use to improve an immunotherapy in a subject in need thereof.
BACKGROUND OF THE INVENTION:
Increasing number of biomedical applications are based on the use of clay minerals, taking advantage of their capacity to interact with many biomolecules, drugs, up to microorganisms (Ferris, 2006; Alimova et al., 2009; Ruiz-Hitzky et ah, 2012, 2013; Wicklein et al., 2012; Dong, 2012; Erdem et al., 2012; Calabrese et al., 2013; Fernandes et al., 2013; Mitsudome et al., 2014; Mueller, 2015; Castro-Smirnov et al., 2016, 2017; Rautureau et al., 2017; Sun et al., 2017; Giir et al., 2017; Massaro et al., 2018; Mousa et al., 2018; Mousavi et al., 2018; Pietrement et al., 2018; Ren et al., 2018; Gonzalez-Tortuero et al., 2018). Among clay minerals, sepiolite, a fibrous natural hydrated magnesium silicate (Ruiz-Hitzky, 2001), is used in domestic and industrial, cosmetic and pharmaceutical formulations among other applications (Carretero and Pozo, 2009, 2010).
Sepiolite show different structural and textural characteristics depending on the geological origin. So, according to Suarez and Garcia-Romero (Suarez and Garcia-Romero, 2012), the presence of crystalline defects, the diversity in the chemical composition and the variability in size and aggregation state of sepiolite fibres result from the growth conditions, which in turn depend on the genetic environment (hydrothermal or sedimentary) and on the involved generation processes (neoformation, transformation or mechanical heritage). In this manner, the main physico-chemical properties of sepiolite, as for instance its surface characteristics, show great variability depending on the geological origin of the silicate.
In the present application, the inventors refer to largely commercialized sepiolite of sedimentary origin from Tagus Basin (Spain) of well-known surface characteristics (Ruiz-
Hitzky, 2001) showing fibres inferior to around 5 mih length (Suarez and Garcia-Romero, 2012).
After grinding of very pure samples of mineral, a partial defibrillation of sepiolite takes place and the fibres decreases in length. The resulting solids are commercialized (e.g. PangelTM) being mainly addressed to applications based on their sorbent and rheological properties. We have shown that the mean length of the fibres was 200 nM [Castro-Smimov et. al. 2016).
Sepiolite has been used to transport small molecules into cells such as tetracycline, nitric oxide, antisense oligonucleotides, and in combination with carbon nanotubes to detect DNA as well as DNA-drug interactions (Ruiz-Hitzky et al., 2011; Erdem et al., 2012; Fernandes et al., 2013; Mitsudome et al., 2014; Giir et al., 2017; Olivato et al., 2017; Pietrement et al., 2018). More specifically, we have shown that sepiolite can transfer DNA into mammalian cells, opening alluring avenues for biotechnological and biomedical applications (Castro- Smirnov et al., 2016, 2017; Pietrement et al., 2018). Noteworthy, mammalian cells spontaneously internalize sepiolite, facilitating the spontaneous transport and delivery of bound molecules (Castro-Smirnov et al., 2017).
As above signalled, non-asbestos minerals belonging to fibrous silicates such as sepiolite raised health concerns about possible asbestos-like health effects (Gonzalez-Tortuero et al., 2018). However, it can be invoked important differences between fibrous-clay minerals and asbestos from the point of view of fibres morphology and topochemistry, crystallinity, composition and surface properties, which can be directly related to their biological activity, possible carcinogenic behavior and, therefore, to potential health hazards (Santaren and Alvarez, 1994).
Indeed, based on the fibrous structure, sepiolite can transfer DNA into bacteria through the Yoshida effect, which was first described with asbestos (Yoshida and Sato, 2009; Wilharm et al., 2010; Gonzalez-Tortuero et al., 2018, Castro-Smirnov et al., 2020). Friction forces by the fibres perforate the bacterial membrane, allowing DNA transfer via holes generated. Besides, it has been reported that sepiolite can generate breaks into the bacterial genome (Gonzalez- Tortuero et al., 2018). Because DNA damage can lead to genetic instability, which is a hallmark of cancer cells (Negrini et al., 2010), potential carcinogenicity of sepiolite has been extrapolated from bacteria data to mammals (Gonzalez-Tortuero et al., 2018). However, as discussed in (Castro-Smirnov et. al., 2020), one can object that bacteria and mammalian cells exhibit strong differences including their respective sizes, their subcellular organization (genome in embedded into nucleus in eukaryotes, in contrast with bacteria) and their membrane composition. These parameters are very important for sepiolite to reach genetic material. Moreover, mammallian cells do not survive to friction forces. In addition, mammalian cells can also spontaneously expelled sepiolite fibres (Castro-Smirnov et al., 2017). Moreover, mammals are also pluri- cellular organisms (metazoans), in contrasts with bacteria, that have developed numerous defence systems, both at cell and organism level, including cell cycle checkpoint, apoptosis, senescence and innate immunity. Noteworthy, sepiolite is poorly toxic at the doses used for mammalian cells transfection (Castro- Smirnov et al., 2017; Pietrement et al., 2018). Therefore, extrapolation from bacteria to mammals constitutes an over-interpretation. Accordingly, several studies, toxicological as well as epidemiological analyses, consistently conclude to the absence of asbestos-like effects of Tagus Basin’s sepiolite (Denizeau et al., 1985; McConnochie et al., 1993; Santaren and Alvarez, 1994; Maisanaba et al., 2015). Hence, the International Agency of Research on Cancer (IARC, a World Health Organization agency) does not classified sepiolite as hazardous or carcinogenic (Wilbourn et al., 1997).
Nevertheless, because of the concerns, the inventors address here the question whether human cells respond to interactions with sepiolite, in order to document further potential risks. They tested three classical cell responses to stress: 1-Reactive oxygen species (ROS) are produced as by-products of cell metabolism. Therefore, detection and cell response to the presence of an exogenous compound such as sepiolite, might generate ROS. On the one hand, ROS can oxidize all kinds of biological molecules, threaten cell viability and genome integrity, which favours ageing and carcinogenesis. On another hand, ROS can also be used as secondary messengers in cell physiological transactions (Ameziane-El-Hassani et al., 2016). The production of intracellular ROS is thus a marker of a cell response to a treatment; 2-Cells generate inflammatory cytokines upon exogenous as well as endogenous stress (Ragu et al., 2020), activating the innate immune system that eliminates damaged cells, which potentially could become pathological, thus protecting the whole organism; 3-Genome integrity is routinely jeopardized by exogenous (radiations, chemicals, ...) as well as endogenous (replication stress, ROS) assaults. Faithful genome replication and transmission is essential to maintain genetic stability during cell division. DNA is replicated in the S phase of cell cycle, and then is compacted into chromosomes that are segregated to the daughter cells during mitosis (M). Two preparation phases (G1 and G2) precede the sensitive S and M phase, in the following succession: G1-S-G2-M. To protect genome integrity, cells activate the DNA damages response (DDR) that coordinates a network of pathways including DNA replication and repair, cell cycle progression, chromosome segregation and apoptosis. When the DNA is damaged, cells activate the DDR, which arrest cell cycle progression, giving time to repair DNA before the sensitive S and M phases and avoiding conflict between DNA replication and repair. If the cell is unable to repair DNA (for example when there are too much damages), then they activate the apoptosis program that kill and eliminate the damage cell. Therefore, activation of DDR, cell cycle arrest and apoptosis induction are markers of genome insult. Defect in DDR results in genome instability, associated with tumour initiation and development, and with premature aging (Kastan and Bartek, 2004; Bartkova et ak, 2005, 2006; Gorgoulis et ak, 2005; Bartek et ak, 2007; Halazonetis et ak, 2008; Hoeijmakers, 2009; Jackson and Bartek, 2009; Gorgoulis and Halazonetis, 2010; Negrini et ak, 2010).
SUMMARY OF THE INVENTION:
In this study, the inventors surprisingly showed that mammalian cells respond to sepiolite exposure, inducing the production of ROS and the expression of inflammatory cytokines genes, showing that cells detect sepiolite contamination and respond. Remarkably, in three different and complementary types of cells (cancer osteosarcoma cells, (U20S), SV40 immortalized human fibroblasts and normal primary human skin fibroblasts), sepiolite exposure did not alter cell cycle distribution and trigger neither DDR nor apoptosis, suggesting that it does not significantly assault the genetic material in mammalian cells. Thus, this ability to induce inflammatory cytokines opens promising avenues in strategies aiming at boosting the efficacy of immunotherapy.
Thus, the present invention relates to a clay mineral for use to improve an immunotherapy in a subject in need thereof. Particularly, the invention is defined by its claims.
DETAILED DESCRIPTION OF THE INVENTION:
A first aspect of the invention relates to a clay mineral for use to improve an immunotherapy in a subject in need thereof. Improvement of an immunotherapy can be very useful to treat diseases through the stimulation of the efficiency of the immune system.
Thus, in one embodiment the invention relates to a clay mineral for use in the treatment of a disease, involving the immune system in a subject in need thereof.
As used herein, the term “a disease involving the immune system” denotes a cancer, an infectious disease and all diseases eligible for immunotherapy and where stimulation of the efficiency of the immune system constitutes a therapeutic gain.
Thus, the invention also relates to a clay mineral for use in the treatment of a cancer or an infectious disease in a subject in need thereof.
The clay mineral of the invention can also be used in combination with a treatment already used to treat a disease involving the immune system and/or to improve an immunotherapy. Thus, the invention also relates to a i) clay mineral and ii) a treatment used to treat a disease involving the immune system as a combined preparation for simultaneous, separate or sequential for use in the improvement of an immunotherapy in a subject in need thereof.
In a particular embodiment, the invention relates to a clay mineral and ii) a treatment used to treat a disease involving the immune system as a combined preparation for simultaneous, separate or sequential for use in the treatment of a cancer or an infectious disease in a subject in need thereof.
In a particular embodiment, the clay mineral is the sepiolite.
According to the invention, “a treatment used to treat a disease involving the immune system” denotes a treatment used to treat a cancer or an infectious disease.
According to the invention, “treatment used to treat a cancer or an infectious disease” refers to anti-cancer agent or anti-infectious agents.
In some embodiment, the anti-cancer agent is a checkpoint blockade cancer immunotherapy agent.
In some embodiment, the checkpoint blockade cancer immunotherapy agent is an anti- PD1 antibody.
According to the invention, the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.
In some embodiment, the cancer is resistant to immunotherapy, and in particular resistant to checkpoint blockade cancer immunotherapy agent.
According to the invention, the infectious diseases can be induced by a virus selected in the group consisting of viral disease Genital warts, common warts, plantar warts, respiratory polynuclear virus (RSV), hepatitis B, hepatitis C, dengue virus, herpes simplex virus (for example, HSV-I, HSV-II, CMV or VZV), molluscum contagiosum, vaccinia, pressure ulcer, lentivirus, human immunodeficiency virus (HIV), human papilloma virus (HPV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rhino Virus, enterovirus, adenovirus, influenza, para-influenza, mumps virus, measles virus , Papovavirus, hepadnavirus, flavivirus, retrovirus, arenavirus (for example, LCM, Junin virus, Machupovirus, Guanarito virus and Lassa fever) and filovirus (for example, Ebola virus or Marburg virus), coronavirus such as SARS-coronavirus (SARS-Covl or SARS-Cov2) .
According to the invention, the infectious diseases can be induced by a bacterium selected in the group consisting of Streptococcus pneumoniae; Staphylococcus aureus; Haemophilus influenza, Myoplasma species, Moraxella catarrhalis, Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella enterica serovar, Typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, Campylobacter, Mycobacterium tuberculosis, Helicobacter pylori and Streptomyce.
As used herein, the term “clay mineral” denotes hydrous aluminium phyllosilicates, sometimes with variable amounts of iron, magnesium, alkali metals, alkaline earths, and other cations found on or near some planetary surfaces.
According to the invention, the term “clay mineral” includes but is not limited to layered silicates (phyllosilicates) and micro/nano fibrous clays.
As used herein, the term “layered silicates” includes but is not limited to the kaolin group (halloysite) including the kaolinite, dickite and nacrite, and the smectite layered silicate group including montmorillonite, beidellite, saponite and hectorite. As used herein, the term “micro/nano fibrous clays” includes but is not limited to the paligorskite and sepiolite fibre.
In a particular, the sepiolite is a sepiolite fibre.
As used herein, the term “sepiolite” or sepiolite fibre” has its general meaning in the art and denotes a natural hydrated magnesium silicate of theoretical unit cell formula Sii 203oMg8(OH,F) 4(H20) 4 , 8H20. Sepiolite shows an alternation of blocks and tunnels that grow up in the fiber direction. The blocks are constituted by two layers of tetrahedral silica sandwiching a central magnesium oxide-hydroxide layer. The discontinuity of the silica sheets gives rise to the presence of silano 1 groups (Si-OH) at the edges of the channels, which are the tunnels open to the external surface of the sepiolite particles.
In a particular embodiment, the total specific surface area of the sepiolite fibers, determined by BET measurements, ranges from 100 to 500 m2/g, preferably from 200 to 400 m2/g. In another particular embodiment, the mean length of the sepiolite fibers is less than or equal to 2pm, or is less than or equal to 1 p , and better still less than or equal to 800nm. More particularly, the mean length of the sepiolite fibres ranges from 150 to 850 nm.
The sepiolite used according to the invention contains advantageously less than 1 % by weight of fibres having a length of at least 5 pm, preferably less than 5 % by weight of fibres having a length of at least 2.5pm.
The size distribution (length and diameter) of the fibers can be determined by transmission electron microscopy (TEM), using a Zeiss 912AB transmission electron microscope (dark-field mode with a tilted illumination).
In addition, the mean diameter of the sepiolite fibres of the present invention is preferably less than or equal to 50nm, more preferably less than or equal to 25nm, and better still ranges from 8 to 1 8nm.
Examples of sepiolite fibres that can be used in the present invention are the products sold by the company Tolsa S .A., under the name Pangel S9, and by the company Kremer Pigmente GmbH&Co, under the number 58945.
According to a particular embodiment, the sepiolites fibres can be sonicated prior their use according to the invention.
The clay mineral of the invention can also be used with other molecules like, small molecules (natural or not), nucleic acids or antibodies to improve/boost an immune response of the organism or to allow a targeting of the clay mineral.
According to the invention, the clay mineral will be linked to a small molecule, at least one nucleic acid or at least one antibody.
The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
Thus, and according to the invention, the clay mineral and particularly the sepiolite can be used in combination with one or more nucleic acids that are linked to the clay mineral and particularly the sepiolite.
In the present invention, the nucleic acids can be in particular chosen from DNA, RNA, DNA/RNA hybrids and chemically modified nucleic acids. More particularly, the nucleic acids are chosen from DNA chains and RNA chains. According to a particular embodiment, the nucleic acids are chosen from linear or circular, single stranded or double-stranded nucleic acid chains. The nucleic acids can be present in any kind of vector. Particularly, the vector is a plasmid, such as pCMV.
According to another particular embodiment, the nucleic acids are chosen from single stranded or double stranded RNA chains. Particularly, the double stranded RNA is a siRNA.
Particularly, the nucleic acid can come from a human or another species. Particularly, the nucleic acids can be salmon sperm DNA (Castro-Smimov et. al 2016).
According to the invention, when linked to the clay mineral and particularly the sepiolite, the nucleic acids can be very useful to improve the answer of the immune system. In this case, the nucleic acids will be recognized as a stranger DNA (Ragu et. al., 2020).
According to the invention, the clay mineral and particularly the sepiolite can also be used in combination with an antibody that is linked to the clay mineral and particularly the sepiolite.
In this case, the antibody will be used to allow the targeting of the clay mineral to a specific cell or tissue.
For example, the antibody can be directed to a specific antigen expressed by a cancerous cell and thus allow the improvement of the immune system against this specific cancerous cell.
Particularly, the specific antigen can be the CD99 which is specific of the Ewing sarcoma.
In some embodiment, the clay mineral and particularly the sepiolite is used in combination with a checkpoint blockade cancer immunotherapy antiboy linked to the clay mineral and particularly the sepiolite
In some embodiment, the clay mineral and particularly the sepiolite is used in combination with an anti -PD 1 antibody linked to the clay mineral and particularly the sepiolite.
In a particular embodiment, the clay mineral can be linked to one or more nucleic acids and to a specific antibody.
According to the invention, the clay mineral can be intravenously, parenterally, intramuscularly injected or can be injected directly to a tumour and particularly a solid tumour.
Particularly, the clay mineral will be injected as a suspension.
When injected directly in the systemic system, the antibody linked to the clay mineral will be useful to target the tumoral cells / the tumour.
According to the invention, the clay mineral can be orally administered. When orally administered, the clay mineral is administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms.
The invention also relates to a method for improving an immunotherapy in a subject in need thereof comprising administering to said subject in need thereof a therapeutically effective amount of a clay mineral.
In another embodiment, the invention relates to a method for treating a disease involving the immune system in a subject in need thereof comprising administering to said subject in need thereof a therapeutically effective amount of a clay mineral.
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
Therapeutic composition
Another object of the invention relates to a therapeutic composition comprising a clay mineral for use in the improvement of an immunotherapy in a subject in need thereof.
Particularly, the therapeutic composition comprises sepiolite.
More particularly, the therapeutic composition comprises sepiolite and a treatment used to treat a disease involving the immune system according to the invention.
Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
Preferably, the pharmaceutical compositions contain vehicles that are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used. Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent.
For example, an anti-cancer agent used to treat cancer may be added to the pharmaceutical composition as described below.
Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).
Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors, Ca2+ ATPase inhibitors and PARP inhibitors.
Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof. In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol s, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.
In another embodiment, the further therapeutic active agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
In still another embodiment, the other therapeutic active agent can be an opioid or non opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofmac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.
Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1). Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.
In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti -PD 1 antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti -IDO 1 antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.
In some embodiment, the clay mineral and particularly the sepiolite is combined with an anti-PDl antibody. In another embodiment, the clay mineral of the invention can be also combined with radiotherapy.
For example, an anti-infectious agent used to treat infectious diseases may be added to the pharmaceutical composition as described below. For example, further anti-infectious agents may be selected in the group consisting bronchodilators like b2 agonists and anticholinergics, corticosteroids, beta2-adrenoceptor agonists like salbutamol, anticholinergic like ipratropium bromide or adrenergic agonists like epinephrine. Further agent may be also an antiviral compound like amantadine, rimantadine or pleconaril.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1. Expression of inflammatory cytokine genes (real-time qPCR). A. Scheme of the experiment. B. Quantification of the expression of six inflammatory cytokines. The data from at least three independent experiments are presented (± SEM).
Figure 2. Induction of the expression of Inflammatory cytokines genes by different clays or nano-diamonds. The expression of cytolines genes (A. IL-6, B. IL-18, C. TNFa, D. IL_8, E. CCL2) were measured by RT-QPCR 24 (black bars) or 48 hours (white bars) after exposure (the doses are indicated on the figure). The Nano-diamonds+ and Nano-diamonds- panels correspond to the exposure to cationic (+) or anionic (-) Nano-diamonds. The data from at least three independent experiments are presented (± SEM).
Figure 3. Production (in protein) and excretion of cytokines monitored by cytokine array 48 after exposure of U20S cells to different clays (indicated on the left part). Three different exposure of the same array are shown. The rectangles focussed on the induced cytokines. The numbers identifies the induced cytokines. U20S Ctrl: control of cells without clays.
Figure 4. Impact of association sepiolite/anti-PDl antibodies. A. Scheme of the experiment: 106 MET-1 cells were injected into FVB/N mice. After tumor growth (1 month) sepiolite (300 pg/ml) and/or anti-PDl antibodies (intraperitoneal) were injected every 4 days. Tumors sizes was measured during the 22 days of the experiment. One example of tumor is shown on the upper panel. B. Evolution of tumors sizes in the course of the experiment. C. Size of the tumors at the end of the experiment (Day 22). The values represent the mean size of 6 tumors in 6 mice were measured for each point. The values are shown normalize to the size of the tumors at Day 0 (first day of injection).
EXAMPLE 1:
Materials and Methods:
Sepiolite sample.
Sepiolite of rheological grade commercialized by TOLSA S. A, as Pangel S9 (> 95% of pure sepiolite) was used in this work. This sepiolite originated from the Vallecas-Vicalvaro clay deposits (Madrid, Spain) located in the Tagus Basin area. We previously reported that this sepiolite sonicated in an ultrasonic bath shows from TEM analyses nano/micro size particles with fibre length distribution < 1200 nm, being 80% of fibres in the 200 to 400 nm range with a maximal length of 800 nm. These sepiolite fibres show width distribution in the 10-25 nm range with a maximal width of 15 nm, also deduced from TEM observations (Castro-Smirnov et ah, 2016). A sepiolite suspension of 2 mg/ml was prepared in 10 mM Tris-HCl buffer, pH = 7.5. Sepiolite suspension was sonicated, 3 times at 30% amplitude for 10 sec each time using QSonica Q125 sonicator. The solution was then sterilized by autoclaving. Importantly, we also previously show that sepiolite added to the culture medium, was spontaneously internalized by the cell (Castro-Smirnov et ah, 2017).
Cell culture treatments.
Cell lines, U20S (human osteosarcoma) or GC92 (SV40-transformed human fibroblasts) were maintained at 37°C with 5% C02 in modified Eagle’s medium (MEM). Primary human skin fibroblasts (GM03348) were grown in MEM (Gibco, Life Technologies) supplemented with 20% foetal calf serum (FCS; Lonza Group, Ltd.). One day after seeding of the cells, different concentrations of sepiolite were added to the culture medium. Cells were collected one or two days after exposure to sepiolite. Intracellular ROS measurement.
The CM-H2DCFDA (2’,7’-dichlorofluorescein diacetate) (Life Technologies, USA) assay kit was used according to the manufacturer’s protocol. 105 cells/well plated in 6-well plates were incubated at 37°C (5% C02). After washing with PBS cells were incubated with 10 mM CM-H2DCFDA in DMEM supplemented with 1% FBS for 45 min at 37°C in the dark. After trypsinization, cells were re-suspended in DMEM supplemented with 1% FBS. After washing, living pelleted cells were re-suspended in PBS and analysed with a BD Accuri C6 flow cytometer (BD Biosciences, San Diego, CA) equipped with an FL1 laser (515-545 nm). The data are presented as the mean percentages of four independent experiments.
RNA extraction reverse transcription and quantitative RT-PCR of cytokine genes expression.
Total RNA was isolated using the Macherel -Nagel NucleoSpin RNA kit, according to the manufacturer’s instructions. The cDNAs were generated from 2 pg of total RNA using random hexamers and RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific). The following primers were used for SYBR real-time PCR assay (Applied Biosystems): IL-6,
IL-8, IL-1B, CCL2, IL-18, TNFa and GAPDH served as the internal control. The sequences of the primers used for SYBR assays are in Table 1. Quantitative RT-PCR was performed using the Applied Biosystems 7300 Real-Time PCR System. All experiments were performed in triplicate.
Cell cycle analysis.
Cell pellets were re-suspended in propidium iodide solution (PI 5pg/ml Sigma-Aldrich P4864, ribonuclease A 50pg/ml Sigma-Aldrich R4642, Triton X-100 0.1%, EDTA 10 mM, PBS). Flow cytometry analyses were performed using an Accuri C6 flow cytometer (BD Biosciences).
Western blot.
Proteins were extracted from cell pellets with lysis buffer (50mM Tris pH 7.5, 20mM NaCl, lmMMgC12, 0.1% SDS, Benzonase 25KU/ml Santa Cruz sc-202391, protease inhibitors Roche 5892970001, phosphatase inhibitors cocktails 2 and 3 Sigma Aldrich P5726 and P0044) for 15 min at room temperature. 30 pg of protein were loaded on the separation gel, for each sample. Electrophoresis, transfer to a nitrocellulose membrane and antibody probing were performed using standard procedures. Primary Antibodies (incubation in PBS-0.005% Tween20): PARP1 (Cell Signalling #9542, 1/1000), pChkl (S317) (Novus Biologicals AF2054, 1/500), Chkl (Cell Signalling #2360, 1/500), pChk2 (T68) (Cell Signalling #2661, 1/500). Secondary antibodies (incubation in PBS-0.005% Tween20): Goat anti-Mouse Antibody HRP Conjugated (Bethyl Laboratories A90-116P), Goat anti -Rabbit Antibody HRP Conjugated (Bethyl Laboratories A120-101P), The proteins were visualized by chemiluminescence detection kit (ECL, Pierce).
Immunofluorescence of sepiolite into mammalian cells.
Detection of intracellular sepiolite by immunofluorescence microscopy was performed as previously describes (Castro-Smirnov et al., 2017). For confocal microscopy. Images were acquired on a confocal Leica SpE with the objective 63x
(ACS AP063.0xl.30 oil). The pictures were 1024 by 1024 pixels, taille pixel: 174.6 m m.
Statistical analyses.
Student’s t-test was used to compare differences between two groups. P < 0.05 was considered statistically significant.
Results
Sepiolite stimulates the production of intracellular ROS in U2QS cells.
We analysed whether the contact with sepiolite affects the intracellular level of ROS. Indeed, increase in intracellular ROS level diagnoses metabolic changes of the cell. Intracellular ROS were monitored by flow cytometry (FACS: Fluorescence Activated Cell Sorting) using the CM-H2DCFDA (2’,7’-dichlorofluorescein diacetate) fluorescent probe (data not shown).
Remarkably, as soon as 5 pg/ml, sepiolite significantly induced the intracellular level of ROS, both after 24 and 48 hours of contact, then the level of ROS increased in a dose dependent manner, in the U20S (human osteosarcoma) cell line (data not shown). In SV40- tranformed fibroblasts, sepiolite did not significantly affect the intracellular level of ROS (data not shown). However, one can object that even in the absence of exogenous challenge SV40- immortilized cells exhibit a spontaneous high level of ROS, due to the inactivation of detoxifying enzymes such as catalase and superoxide dismutase by the SV40 large T antigen (Hoffschir et al., 1998; Yen et al., 2005). Therefore, any potential induction of the ROS level is difficult to monitor due to this high basal level that masks potential variations. These data show that cell lines might differently react but the results with U20S cells (data not shown) reveal metabolic change suggesting that cells can detect and thus might respond to the presence of sepiolite.
Sepiolite triggers inflammatory cytokines genes expression. One cell response to the presence of foreign molecules/compounds is the production of inflammatory cytokines, which acts through the induction of inflammatory cytokines genes expression. Thus, we tested whether sepiolite induces the expression of inflammatory cytokines genes. We choose to test the gene expression of six classical inflammatory cytokines (IL-8,
CC12, IL1B, IL-6, IL-18 and TNFa) in U20S cells exposed to sepiolite (Figure 1 A). Cells were exposed to 2 different doses of sepiolite (10 or 50 pg/ml) for 24 or 48 hours; then cells were harvested and RNA was extracted. After reverse transcription, the level of mRNA of each gene was measured by quantitative real-time PCR (qPCR), using specific primers for each of the six genes (Figure 2).
While after only 24 hours of exposure, only IL-8 gene expression was induced, the expression all six cytokines genes were induced after 48 hours of exposure, with both doses (Figure IB). Collectively, these data show that cells detect the presence of sepiolite and respond, leading the expression of inflammatory cytokines genes, according different dose response and kinetics for each cytokine.
Cell cycle analysis.
DNA damages elicit the DDR, which results in cell cycle distribution modification, and apoptosis induction. Thus, we tested here the impact of sepiolite exposure on cell cycle distribution. Cell cycle distribution was monitored by FACS using propidium iodine that measures, in each cell, the DNA content, which differs in each cell cycle phase. At all the doses tested, the cell cycle progression was not significantly altered in both U20S (data not shown).
Tumour cells possess high proliferation rate, especially compared to normal (primary cells); this might affect the cell cycle distribution and their response to genotoxic agents. Therefore, we verified the impact of sepiolite in another cell line, i.e. immortalized SV40- transformed fibroblasts (GC92). In this second cell line, GC92 cells, exposure to sepiolite did not alter cell cycle distribution, confirming the data obtained with U20S cells (data not shown). Same data were obtained after 24 hours of contact with sepiolite (data not shown).
However, DDR and cell cycle regulation are frequently corrupted in tumour and immortalized cells such as U20S and GC92. This leaky DDR is part of the high proliferation rate of tumour and immortalized cells compared to primary cells that are fully competent for DDR. Thus, we checked the impact of sepiolite exposure in human primary skin fibroblasts (GM3348), which are fully competent for DDR. First, we verified the toxicity of sepiolite in these cells (data not shown). Both immortalized/transformed cell lines (U20S and GC92) exhibited similar sensitivity to sepiolite (data not shown), in a comparable range to previously described sensitivity for U20S, using an-other method (Castro-Smirnov et al., 2017). Primary skin fibroblasts exhibited higher sensitivity to sepiolite exposure than cell lines (data not shown). These differences might reflect, in part, the difference in proliferation rates between tumour/immortalized and primary cells. Then, we analysed cell cycle distribution of primary skin fibroblasts (data not shown). In spite of higher toxicity, sepiolite did not cause significant cell cycle modification in primary skin fibroblast (data not shown). A slight decrease in G1 cells (while activation of DDR should lead to an increase) is accompanied by a slight increase in S phase cells, and no effect on G2 cells was observed (data not shown). However, these differences are not statistically significant. These data confirm, in primary cells, that sepiolite does not significantly lead to modifications of cell cycle distribution.
Sepiolite does not trigger DDR
Then we verified the above conclusions at molecular level. DDR is activated by the phosphorylation of effectors by the signalling kinases ATM/ATR. In response to replication stress and accumulation of single-strand DNA, ATR phosphorylates Chkl . In response to DNA double-strand breaks, ATM phosphorylates Chk2. Therefore, the phosphorylation of Chkl (pChkl) or Chk2 (pChk2) are markers of the activation of ATR and ATM pathway, respectively, and indirectly of the production of single-strand and double-strand breaks into the DNA.
Using specific antibodies against the phosphorylated forms of pChkl (residue serine 317; S317) and pChk2 (residue Threonine 68, T68)) we monitor whether sepiolite induced the phosphorylation of Chkl and Chk2, i.e. trigger DDR (data not shown).
As positive control of induction, we used cells exposed to hydroxyurea (HU), which generates a genotoxic stress and thus that activates the ATR/CHK1 pathway. In addition, prolonged exposure to HU generates DNA double-strand breaks (Saintigny et al., 2001) that activate the ATM/CHK2 pathway, Therefore HU exposure is used as control of activation of both pathways. Indeed, 1 mM HU induced pChkl phosphorylation in U20S in human primary skin fibroblasts (GM03348, passage 12). In contrast sepiolite did not induced the phosphorylation of pChkl, suggesting that sepiolite does not generate a replication stress and single-strand Breaks (data not shown). Moreover, in contrast with 1 mM HU, sepiolite did not induced the phosphorylation of pChk2, (data not shown), suggesting that sepiolite does not generate a lot of double-strand breaks.
Sepiolite does not trigger apoptosis.
When DNA damages are too numerous and DNA repair systems are overwhelmed, cells engage then program cell death, also called apoptosis. This allows avoiding that cells bearing DNA damages, which could generate genetic instability, to proliferate. Therefore, apoptosis induction is also a marker of genotoxic stress. Many cancer cells (but not all) are resistant to apoptosis induction, escaping to this protection system and thus participating to their increased proliferation capacities. Therefore, here it is essential to verify potential apoptosis induction by sepiolite in cancer cells (U20S), immortalized (CG92) and primary skin fibroblasts. It is important to verify as whether U20S and GC92 cells are actually able to induce apoptosis, as we did here upon HU exposure (data not shown).
Apoptosis induction program leads to the activation of proteases such as caspase 3 that inactivate DNA repair proteins by cleaving them at specific sites, making cell death more efficient. PARP1 is a essential DNA damge signalling and DNA repair protein. PARP1 is a target of caspase 3. Thus, upon apoptosis activation PARP1 is cleave at a specific site, resulting in shorter polypeptides. Therefore, using anti -P ARP 1 antibodies, we monitored the cleavage of PARP1 upon exposure to sepiolite (data not shown). The exposure to a genotoxic stress (HU) indeed, generates the appearance of the diagnosis shorter PARP1 polypeptides (89 kDa). Noteworthy, HU exposure induced apoptosis also in cancer and immortalized cells, showing that apoptosis can indeed be induced also in such kind of cells. Consistently with the above results, sepiolite did not induced the cleavage of PARP1 in all kind of cells analysed, i.e. in cancer and immortalized cells (in which apoptosis can be induced) as well as in primary skin fibroblats (data not shown), suggesting that sepiolite does not induce apoptosis.
Collectively, these data suggest that, sepiolite does not cause significant injuries to the genome, either directly or indirectly. Accounting for these results, taking advantage of spontaneous fluorescence of sepiolite (Castro-Smirnov et ah, 2017), we show that intracellular sepiolite does not migrate into the nucleus, as monitored by confocal fluorescence microscopy (data not shown). Therefore, sepiolite is not directly in contact with DNA in mammalian cells, in contrast with bacteria.
Conclusion.
Here the inventors showed that cells detect the presence of sepiolite and respond, leading the expression of inflammatory cytokines genes, according different dose response and kinetics for each cytokine. Inflammatory cytokines are excreted from the cell, modifying its microenvironment, communicating with neighbour cells and activating the innate immune response that recruit cells of the immune system (macrophages, lymphocytes). Thus stressed cell are eliminated by the immune system, avoiding its maintenance and proliferation, which might become deleterious to the whole organism. Transient induction of inflammation can thus be beneficial to boost an immune response. Therefore, the ability to induce inflammatory cytokines opens promising avenues in strategies aiming at boosting the efficacy of immunotherapy.
EXAMPLE 2:
Materials and Methods:
Cell culture treatments.
Cell lines, U20S (human osteosarcoma) were maintained at 37°C with 5% CO2 in modified Eagle’s medium (MEM). One day after seeding of the cells different concentrations (10 or 50ug/ml) of sepiolite or Cloisite (Wyoming) or Halloysite (IMERYS), or Laponite (Laporte) or cationic and anionic nanodiamonts (5 or 25ug/ml) (Nanomaterials 2020, 10, 553; doi: 10.3390/nanol0030553) were added to the culture medium. Cells were collected one or two days after exposure.
RNA extraction reverse transcription and quantitative RT-PCR of cytokine genes. Total RNA was isolated using the Macher el -Nagel NucleoSpin RNA kit, according to the manufacturer’s instructions. The cDNAs were generated from 2 pg of total RNA using random hexamers and RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific). The following primers were used for SYBR real-time PCR assay (Applied Biosystems): IL-6, IL-8, IL-IB, CCL2, IL-18, TNFa and GAPDH served as the internal control. Quantitative RT-PCR was performed using the Applied Biosystems 7300 Real-Time PCR System. All experiments were performed in triplicate.
Proteome profiler array for human cytokines
The supernatants from cultured U20S in the sepiolit or cloisite or laponite treatment groups were analyzed with a human Cytokine Array Kit (Cat: ARY005B, R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Statistical analyses.
Student’s t-test was used to compare differences between two groups. P < 0.05 was considered statistically significant.
Results:
We analysed the induction of the expression of Inflammatory cytokines genes by different clays or nano-diamonds. The results are depicted in figure 2A-E. Here we see that clays induce the expression of cytokine genes as well as nano-diamonds. However, the nano diamonds seem less efficient than the clays. Then we studied the production (in protein) and excretion of cytokines monitored by cytokine array 48 after exposure of U20S cells to different clays. The results are depicted in figure 3. We show that cytokines are induced (at the protein level) and excreted after exposure to clays.
These data show that cells detect the presence of clays and respond, leading the expression of inflammatory cytokines (genes and at protein level) and that the induction efficiency varies depending on cytokine and contact time.
EXAMPLE 3: Impact of association sepiolite/anti-PDl antibodies.
In order to study the combination sepiolite and anti-PDl antibodies, 106 MET-1 cells were subcutaneously injected in FVB/N mice (syngeneic) backs. After tumors growth (one month), sepiolite (300 pg/ml) was injected into the tumors associated or not with intraperitoneal injections of an anti-PD-1 antibody (200 pg, RMP1-14 clone; BioXcell). Injections were performed every 4 days.
MET-1 cells were derived from tumors of the the PyMT spontaneous breast cancer mouse model. The PyMT derived from the FVB/N mouse strain. Thus injection of MED-1 cells in FVB/N generate tumors (see Fig. 4A-C). MET-1 tumors are "old" for immunotherapy, meaning that injection of anti-immune checkpoint antibody (anti-PDl Ac) does not affect tumors progression. We have injected MET-1 cells in FVB/N mice, and after tumor growth (one month) we injected sepiolite in association or not with anti-PDl Ac. As expected, anti- PDl Ac alone did not block tumor progression (Fig. 4B and C), at the concentration used, sepiolite alone has no significant effect (Fig. 4C). However, the combination of sepiolite and anti-PDl Ac impaired tumor progression (Fig. 4B and C).
These data demonstrate that synergy effect of clay mineral, such as sepiolite, and anti- PDl antibody. They show that sepiolite can shift a tumor "cold" (resistant to anti-PDl antibody) to "hot" (at least partially sensitive to anti-PDl antibody) to immunotherapy,
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Claims

CLAIMS:
1. A clay mineral for use to improve an immunotherapy in a subject in need thereof.
2. A clay mineral for use in the treatment of a disease involving the immune system in a subject in need thereof.
3. A i) clay mineral and ii) a treatment used to treat a disease involving the immune system as a combined preparation for simultaneous, separate or sequential for use in the improvement of an immunotherapy in a subject in need thereof.
4. A clay mineral for use according to claims 1 or 2 or a combined preparation for use according to claim 3 wherein the disease involving the immune system is a cancer or an infectious disease.
5. A combined preparation for use according to claim 4, wherein the treatment used to treat cancer is an checkpoint blockade cancer immunotherapy agent.
6. A combined preparation for use according to claim 5, wherein the checkpoint blockade cancer immunotherapy is an anti -PD 1 antibody.
7. A clay mineral for use according to claims 1, 2 or 4 or a combined preparation for use according to claims 3 to 6 wherein the clay mineral is layered silicates (phyllosilicates) or micro/nano fibrous clays.
8. A clay mineral for use according to claim 7 or a combined preparation for use according to claim 7 wherein the layered silicates is a kaolinite, a dickite or a nacrite.
9. A clay mineral for use according to claim 7 or a combined preparation for use according to claim 7 wherein the layered silicates is a montmorillonite, a beidellite, a saponite, or a hectorite.
10. A clay mineral for use according to claim 7 or a combined preparation for use according to claim 7 wherein the micro-nano fibrous silicates is a paligorskite or a sepiolite.
11. A clay mineral for use according to claim 8 or a combined preparation for use according to claim 9 wherein the micro-nano fibrous is a sepiolite.
12. A clay mineral for use according to claims 1, 2 and 4 to 10 or a combined preparation for use according to claims 3 to 10 wherein the clay mineral is linked to a small molecule, at least one nucleic acid or at least one antibody.
13. A clay mineral for use according to claim 12 or a combined preparation for use according to claim 12 wherein the clay mineral is linked to at least one nucleic acid and at least one antibody.
14. A therapeutic composition comprising a clay mineral for use in the improvement of an immunotherapy in a subject in need thereof.
15. A method for improving an immunotherapy in a subject in need thereof comprising administering to said subj ect in need thereof a therapeutically effective amount of a clay mineral.
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