WO2024161015A1 - Méthode de traitement de maladies liées à l'âge - Google Patents

Méthode de traitement de maladies liées à l'âge Download PDF

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WO2024161015A1
WO2024161015A1 PCT/EP2024/052643 EP2024052643W WO2024161015A1 WO 2024161015 A1 WO2024161015 A1 WO 2024161015A1 EP 2024052643 W EP2024052643 W EP 2024052643W WO 2024161015 A1 WO2024161015 A1 WO 2024161015A1
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csf
inhibitor
cancer
cells
treatment
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PCT/EP2024/052643
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English (en)
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Frédéric PENDINO
Isabelle MUNOZ
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Institut National de la Santé et de la Recherche Médicale
Centre National De La Recherche Scientifique
Université Paris Cité
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/243—Colony Stimulating Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a G-CSF inhibitor for use in the treatment of an age- related disease in a subject in need thereof.
  • hematopoietic cells undergo dramatic changes during normal aging, including an increase in myeloid cell production while lymphoid cell production wanes. This increased myeloid-to-lymphoid ratio contributes to age-related diseases and impairs immunity against tumor cells or viral infections.
  • Several inflammatory cytokines CCL5, TNFa, ILip have been identified during that process, but the epigenetic mechanisms driving their secretion in the bone marrow (BM) niche are not fully understood.
  • Cxxc5 a tumor suppressor gene candidate encoding an epigenetic factor that they have previously identified during human ex vivo granulopoiesis [1] and erythropoiesis [2]
  • Cxxc5 a tumor suppressor gene candidate encoding an epigenetic factor that they have previously identified during human ex vivo granulopoiesis [1] and erythropoiesis [2]
  • murine leukemia development as also suggested by a mutagenesis screen performed in a model of myelodysplasia [3]
  • hematopoietic aging hematopoietic aging.
  • CXXC5 Human CXXC5 gene is located at 5q31.2, a region commonly deleted in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the consequences of Cxxc5 Knockout on mouse hematopoiesis are not described yet in aging animals.
  • CXXC5 encodes an epigenetic factor that we named RINF (Retinoid-Inducible Nuclear Factor) and that harbors a CXXC zinc finger domain (conserved in TET1/3 but lacking in TET2) that is essential for chromatin binding.
  • RINF Retinoid-Inducible Nuclear Factor
  • RINF protein has been proposed by several groups as a platform for TET2 enzymatic activity [2, 4-6], Controversially, RINF would interact with DVL in the cytoplasm and inhibit the WNT/p-catenin signaling pathway, [7-9] indicating that its mechanism of action (MO A) remains to be clarified.
  • the inventors have compared the hematopoietic phenotype of two mouse models functionally invalidated for Cxxc5, one constitutive knockout (Rinf /_ ), and one conditional knockout (Vav-Cre / 'Rinf rio /rio ) specifically invalidated in the hematopoietic tissue.
  • Cytokines By analyzing blood cells counts and blood parameters, Cytokines, BM cells (immune cells and mesenchymal stem cells (MSCs)), they noticed an accelerated aging of hematopoietic cells in Rinf /_ mouses (as early as 6 months of age) but, unexpectedly, this phenotype was not observed in Vav-Cre /_ Rinf lo /no mouses, revealing a key role of Rinf in the BM niche and the fact that the Rinf /_ mouse can be used as a model for age-related diseases.
  • MSCs mesenchymal stem cells
  • the present invention relates to a G-CSF inhibitor for use in the treatment of an age-related disease in a subject in need thereof.
  • the invention is defined by its claims.
  • a first aspect, the invention relates to a G-CSF inhibitor for use in the treatment of an age-related disease in a subject in need thereof.
  • age-related disease denotes a disease whose probability to occur increases with age and health problem links to the age.
  • age-related disease are for example cancer like leukemia, Alzheimer disease, low immune response, arthritis, type 2 diabetes, atherosclerosis, cardiovascular disease, myelodysplasia, anemia.
  • the inhibitor of G-CSF can be used in the treatment of anemia in a subject in need thereof.
  • anemia has its general meaning in the art and refers to a blood disorder occurring when there aren’t enough healthy red blood cells to carry oxygen. Anemia can be caused by blood loss, decreased red blood cell production, and increased red blood cell breakdown. Anemia is a common condition of cancer patients. This is because cancers cause inflammation that decrease red blood cell production. In addition, many chemotherapies are myelosuppressive, meaning they slow down the production of new blood cells by the bone marrow.
  • the inventors demonstrate that the inhibitor of G-CSF decrease/attenuate the anemia in a triple-negative breast cancer mouse model.
  • the anemia is associated to a cancer.
  • the invention refers to an inhibitor of G-CSF for use in the treatment of anemia in a subject suffering from a cancer.
  • the inhibitor of G-CSF can be used in the treatment of cancer.
  • an inhibitor of G-CSF resolved both lymphopenia and neutrophilia, which means reverse the increasing of the myeloid-to-lymphoid ratio observed during ageing.
  • this kind of inhibitor can be used to improve antiviral and/or anti turn oral immunity.
  • the inhibitor of G-CSF can be used to improve antiviral or anti turn oral immunity in the case of cancer, viral disease or during a vaccinal protocol.
  • the inhibitor of G-CSF can be used to improve the humoral response in the case of a vaccinal protocol.
  • the cancer may be a liquid or a solid cancer.
  • the cancer may be a cancer selected from the group consisting in adrenal cortical cancer, anal cancer, bile duct cancer (e.g. perihilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma), brain and central nervous system cancer (e.g.
  • adrenal cortical cancer e.g. perihilar cancer, distal bile duct cancer, intrahepatic bile duct cancer
  • bladder cancer e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma,
  • meningioma astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g.
  • adenocarcinoma endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell
  • esophagus cancer gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g.
  • lung cancer e.g. small cell lung cancer, non-small cell lung cancer
  • mesothelioma plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g.
  • rhabdomyosarcoma embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma,), vaginal cancer, vulvar cancer, uterine cancer (e.g.
  • leukaemia like acute myeloid leukaemia, acute lymphoid leukaemia, chronic myelomonocytic leukemia (CMML)
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic syndrome
  • the cancer is a cancer that expresses/secretes G-CSF.
  • the cancer is a selected from the group consisting of glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, stomach cancer, liver cancer, pancreatic cancer, renal cancer, urothelial cancer, prostate cancer, testis cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma and acute myeloblastic leukemia.
  • the cancer is a resistant cancer.
  • resistant refers to cancer, which does not respond to a treatment.
  • the cancer may be resistant at the beginning of treatment or it may become resistant during treatment.
  • the resistance to drug leads to rapid progression of metastasis.
  • the cancer is resistant to anti-immune checkpoint inhibitor.
  • the cancer is resistant to anti-PD-Ll antibody.
  • the cancer is associated with an anemia.
  • the inhibitor of G-CSF decreases or attenuate anemia associated with the cancer.
  • the viral diseases is due to a pathogen like a virus.
  • the viral disease is induced by a respiratory virus.
  • the respiratory virus can be Influenza virus, such as the Influenza A virus (IAV) or the Influenza B virus (IAB), adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), the human rhinovirus (HRV), the Human respiratory syncytial virus (HRSV) or a coronavirus.
  • Influenza virus such as the Influenza A virus (IAV) or the Influenza B virus (IAB)
  • adenovirus such as the Influenza A virus (IAV) or the Influenza B virus (IAB)
  • adenovirus such as the Influenza A virus (IAV) or the Influenza B virus (IAB)
  • metapneumovirus e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4
  • HRV human rhinovirus
  • HRSV Human respiratory syncytial virus
  • coronavirus has its general meaning in the art and refers to any member of the Coronaviridae family.
  • Coronavirus is a virus whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular virus.
  • the virion RNA has a cap at the 5’ end and a poly A tail at the 3’ end.
  • the length of the RNA makes coronaviruses the largest of the RNA virus genomes.
  • coronavirus RNAs encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; plus (4) three non-structural proteins.
  • the coronavirus particle comprises at least the four canonical structural proteins E (envelope protein), M (membrane protein), N (nucleocapsid protein), and S (spike protein).
  • E envelope protein
  • M membrane protein
  • N membrane protein
  • S spike protein
  • the S protein is cleaved into 3 chains: Spike protein SI, Spike protein S2 and Spike protein S2'. Production of the replicase proteins is initiated by the translation of ORF la and ORF lab via a -1 ribosomal frame-shifting mechanism.
  • ppi a and pplab that are further processed by two virally encoded cysteine proteases, the papain-like protease (PLpro) and a 3C-like protease (3CLpro), which is sometimes referred to as main protease (Mpro).
  • PLpro papain-like protease
  • 3CLpro 3C-like protease
  • Coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions.
  • Coronaviruses are exemplified by, but not limited to, human enteric coV (ATCC accession # VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCC accession # VR-920), Middle East respiratory syndrome-related coronavirus (MERS-Cov) and SARS-coronavirus (Center for Disease Control), in particular SARS-Covl and SARS-Cov2.
  • human enteric coV ATCC accession # VR-1475
  • human coV 229E ATCC accession # VR-740
  • human coV OC43 ATCC accession # VR-920
  • Middle East respiratory syndrome-related coronavirus MERS-Cov
  • SARS-coronavirus Center for Disease Control
  • the coronavirus can be a MERS-CoV, SARS-CoV, SARS- CoV-2 or any new future family members.
  • the inhibitor of the invention can be administrated to a subject in need thereof during a vaccinal protocol to boost its antiviral immunity and/humoral immunity.
  • the inhibitor of the invention can be administered at the same time than a vaccine against a viral pathogen and particularly against a respiratory virus like a coronavirus.
  • a subject denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a subject according to the invention is a human. More particularly, the subject is suffering from a cancer or a viral disease or will receive a vaccine against a viral pathogen. More particularly, the subject is suffering from a cancer, and more particularly is suffering from a cancer and an anemia.
  • the patient is an elderly patient, i.e. an adult patient aged more than 50, 55 or 60 years and which suffer from a cancer or viral disease, or which will receive a vaccine against a viral pathogen. More particularly, the subject is an old person, aged more than 65 years and which suffer from a cancer or viral disease, or which will receive a vaccine against a viral pathogen.
  • G-CSF G-CSF
  • GSCF GSCF for “Granulocyte colony-stimulating factor”
  • G-CSF has its general meaning in the art and denotes a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream. Functionally, it is a cytokine and an hormone, a type of colony-stimulating factor (also known as CSF3), and is produced by a number of different tissues.
  • CSF3 colony-stimulating factor 3
  • the pharmaceutical analogs of naturally occurring G-CSF are called filgrastim and lenograstim.
  • G-CSF also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. Its entrez reference number is 1440 and its Uniprot reference number is P09919.
  • G-CSF inhibitor denotes a molecule or compound which can inhibit its function like the initiation of the proliferation and differentiation of mature granulocytes, or a molecule or compound which destabilizes G-CSF.
  • G-CSF inhibitor also denotes an inhibitor of the expression of the gene coding for the protein.
  • 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.]).
  • the inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).
  • 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 inhibitor according to the invention is an antibody.
  • Antibodies or directed against G-CSF can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against G-CSF can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti- G-CSF single chain antibodies.
  • Coumpounds useful in practicing the present invention also include anti- G-CSF antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to G-CSF.
  • Humanized anti- G-CSF antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • an anti-G-CSF antibody can be the MAB414-500 Mouse G-CSF Mab from R&D Systems, MAB005 Rat IgGl isotype from R&D systems or Anti-hG-CSF mAb (clone BVD11-37G10) from SouthernBiotech. the MAB214 human G-CSF antibody from R&D Systems, the MAS- 23758 from ThermoFisher Scientific.
  • the compound according to the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • the compound according to the invention is a polypeptide.
  • the polypeptide is an antagonist of G-CSF and is capable to prevent the function of G-CSF.
  • the polypeptide can be a mutated G-CSF protein or a similar protein without the function of G-CSF.
  • the polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell.
  • Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others.
  • Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown.
  • a common, preferred bacterial host is E coli.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
  • modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability is the utilization of water-soluble polymers.
  • Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.).
  • the PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages.
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate.
  • the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • the compound according to the invention is an isolated G-CSF receptor polypeptide.
  • G-CSF receptor also known as CD 114, has its general meaning in the art and refers to a cell-surface receptor for the granulocyte colony-stimulating factor. Its Entrez reference is 1441 and its Uniprot reference is Q99062.
  • G-CSF receptor polypeptide refers to a polypeptide that specifically bind to G-CSF can be used as G-CSF inhibitor that bind to and sequester the G- CSF (G-CSF Trap) , thereby preventing it from signaling.
  • the G-CSF receptor polypeptide is soluble.
  • a soluble G- CSF receptor polypeptide exerts an inhibitory effect on the biological activity of the G-CSF by binding to the protein, thereby preventing it from binding to G-CSF receptor present on the surface of target cells. It is undesirable for a G-CSF receptor polypeptide not to become associated with the cell membrane.
  • the soluble G-CSF receptor polypeptide lacks any amino acid sequences corresponding to the transmembrane and intracellular domains from the G-CSF receptor.
  • said polypeptide is a soluble G-CSF receptor (sG-CSF receptor) or a functional equivalent thereof.
  • soluble G-CSF receptor or "sG-CSF receptor”, as used herein, refer to a polypeptide comprising or consisting of the extracellular region of the G-CSF receptor or a fragment thereof.
  • the soluble G-CSF receptor polypeptide comprises the extracellular domain of the G-CSF receptor.
  • a “functional equivalent of sG-CSF receptor” is a molecule which is capable of binding to G-CSF.
  • the term “functional equivalent” includes fragments and variants of sG-CSF receptor as above described.
  • binding specifically means that the biologically active fragment has high affinity for G-CSF but not for control proteins. Specific binding may be measured by a number of techniques such as ELISA, flow cytometry, western blotting, or immunoprecipitation.
  • the G-CSF receptor domain or a fragment thereof are linked to an Fc portion of an antibody.
  • the antibody portion confers increased stability on the conjugate and/or reduces the patient's immune response against the G-CSF inhibitor.
  • the G-CSF receptor domain is linked to an Fc portion of an antibody via a linker, e.g., a peptide linker.
  • the G-CSF inhibitor according to the invention is an inhibitor of G-CSF gene expression.
  • Small inhibitory RNAs can also function as inhibitors of G-CSF expression for use in the present invention.
  • G-CSF gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that G-CSF gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • Ribozymes can also function as inhibitors of G-CSF gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of G-CSF mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of G-CSF gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-m ethyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing G-CSF.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40- type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno-associated virus
  • SV40- type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno- associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • one object of the present invention relates to a method of treating an age- related disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of G-CSF.
  • G-CSF inhibitors In order to test the functionality of a putative G-CSF inhibitor a test is necessary. For that purpose, to identify G-CSF inhibitors, measurement of the G-CSF concentration in a liquid (blood, serum, plasma) before and after depletion/inhibition of this liquid. To detect G-CSF, we could use standard protocols for cytokine/chemokine detection (ELISA, luminex).
  • ELISA cytokine/chemokine detection
  • G-CSFR G-CSF receptor
  • the invention in another aspect, relates to a therapeutic composition comprising a G- CSF inhibitor for use in the treatment of an age-related disease in a subject in need thereof.
  • the invention in another aspect, relates to a therapeutic composition comprising a G- CSF inhibitor and an immune checkpoint inhibitor for use in the treatment of an age-related disease in a subject in need thereof.
  • the immune checkpoint inhibitor is an anti-PD-Ll antibody.
  • the G-CSF inhibitor or the therapeutic composition of the invention are administrated in a therapeutically effective amount.
  • 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.
  • the term "therapeutically effective amount” or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of the inhibitor or the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the inhibitor or the composition are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the inhibitor or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art.
  • a physician having ordinary skill in the art may readily determine and prescribe the effective amount of the inhibitor or the composition of the invention required.
  • the physician could start doses of the inhibitor or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable dose of the inhibitor or the composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1- 20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period.
  • the dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1- 100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination
  • Administration may be intravenous, intramuscular, intraperitoneal, intratumoral or subcutaneous, and for instance administered proximal to the site of the target.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of the G-CSF inhibitor of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of the G-CSF inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
  • the quantity of the immune cell or the population of immune cells administered to a subject in need thereof is between 10 4 to 10 9 cells per kg.
  • the quantity of cells injected is 10 6 or 10 7 cells per kg.
  • the immune cell or the population of immune cells of the invention can be administrated is 1, 2, 3, 4 or 5 times to the subject in need thereof.
  • “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 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 subject, 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 which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in 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.
  • these may be in organic solvent such as DMSO, castor oil, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • the G-CSF inhibitor of the invention are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought.
  • an effective amount of the G-CSF inhibitor of the invention administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)).
  • MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
  • the inhibitor of the invention or the pharmaceutical composition of the invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising an inhibitor according to the invention and a further therapeutic active agent.
  • anti-cancer agents may be added to the pharmaceutical composition or used in combination with the inhibitor of the invention in the case of the treatment of a cancer.
  • Anti-cancer agents or conventional cancer therapies can be surgery, radiotherapy, chemotherapy, or combinations thereof.
  • Anti-cancer agents include other anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti -angiogenic agents, anti-cancer immunogens, cell cycle control/apoptosis regulating agents, hormonal regulating agents, and other agents described below.
  • the inhibitor or composition of the present invention is used in combination with a chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its ad
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33:183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the inhibitor or composition of the present invention is used in combination with a targeted cancer therapy.
  • Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer.
  • Targeted cancer therapies are sometimes called “molecularly targeted drugs,” “molecularly targeted therapies,” “precision medicines,” or similar names.
  • the targeted therapy consists of administering the subj ect with a tyrosine kinase inhibitor.
  • tyrosine kinase inhibitor refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007/0254295, which is incorporated by reference herein in its entirety.
  • a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase.
  • tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to, dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl-l,2,4-triazolo[3,4-
  • the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication.
  • inhibitors include, but are not limited to, Gefitinib, Erlotinib, Lapatinib, Canertinib, BMS- 599626 (AC-480), Neratinib, KRN-633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM- 475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP-547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KRN-951, Dovitinib, Seliciclib, SNS-032, PD-0332991, MKC-I (Ro- 317453; R-440), Sorafenib, ABT
  • the inhibitor or composition of the present invention is used in combination with an immunotherapeutic agent.
  • immunotherapeutic agent refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies.
  • Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents.
  • Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy.
  • immunotherapeutic agents examples include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and noncytokine adjuvants.
  • the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells).
  • Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents.
  • Nonspecific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system.
  • Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines).
  • Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents.
  • Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines.
  • Non-specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.
  • cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-P) and IFN-gamma (IFN-y). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy.
  • IFNs Interferons
  • IFN-a IFN-alpha
  • IFN-P IFN-beta
  • IFN-y IFN-gamma
  • IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages.
  • Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).
  • Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc.
  • Combination compositions and combination administration methods of the present invention may also involve "whole cell” and "adoptive” immunotherapy methods.
  • such methods may comprise infusion or re-infusion of immune system cells (for instance tumor-infiltrating lymphocytes (TILs), such as CD2+ and/or CD8+ T cells (for instance T cells expanded with tumor-specific antigens and/or genetic enhancements), antibody-expressing B cells or other antibody-producing or -presenting cells, dendritic cells (e.g., dendritic cells cultured with a DC-expanding agent such as GM-CSF and/or Flt3-L, and/or tumor-associated antigen-loaded dendritic cells), anti-tumor NK cells, so-called hybrid cells, or combinations thereof.
  • TILs tumor-infiltrating lymphocytes
  • CD2+ and/or CD8+ T cells for instance T cells expanded with tumor-specific antigens and/or genetic enhancements
  • antibody-expressing B cells or other antibody-producing or -presenting cells for instance dendritic cells cultured with a DC-expanding agent such as GM-CSF and/or Flt
  • Cellular “vaccines” in clinical trials that may be useful in such aspects include CanvaxinTM, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens shed from cancer cells, and mixtures thereof (see for instance Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally admixed with adjuvants such as alum, may also be components in such methods and combination compositions.
  • Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient.
  • the source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)).
  • Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium- 137, iridium- 192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium- 111.
  • the G-CSF inhibitor or composition of the present invention is used in combination with an immune checkpoint inhibitor.
  • immune checkpoint protein has its general meaning in the art and refers to a molecule that is expressed by T lymphocytes in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules).
  • Immune checkpoints are the regulators of the immune system. They are crucial for selftolerance, which prevents the immune system from attacking cells indiscriminately. Immune checkpoints are targets for cancer immunotherapy due to their potential for use in multiple types of cancers. Typically, by using immune checkpoint inhibitors, the anti -turn oral response is reactivated by reactivation of cytotoxic T- lymphocytes.
  • Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489).
  • stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, 0X40, GITR, and ICOS.
  • inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA.
  • Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine.
  • B7-H4 also called VTCN1
  • B7-H4 also called VTCN1
  • B and T Lymphocyte Attenuator (BTLA) and also called CD272 has HVEM (Herpesvirus Entry Mediator) as its ligand.
  • CTLA-4 Cytotoxic T-Lymphocyte- Associated protein 4 and also called CD 152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation.
  • IDO Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis.
  • KIR Killer-cell Immunoglobulin-like Receptor
  • LAG3, Lymphocyte Activation Gene-3 works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells.
  • PD-1 Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014.
  • An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment.
  • TIM-3 short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines.
  • TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9.
  • VISTA Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response.
  • an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade.
  • the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.
  • the G-CSF inhibitor or composition of the invention as described above can be combined with an immune checkpoint inhibitor to enhance the potency of the immune checkpoint inhibitor.
  • the G-CSF inhibitor sensitizes cancer cells to immune checkpoint inhibitor.
  • the G-CSF inhibitor composition of the present invention is used in combination with an immune checkpoint inhibitor, wherein the G-CSF inhibitor decreases toxicities caused by immune checkpoint inhibitor. In other words, the G-CSF inhibitor decreases the immune checkpoint inhibitor-related side effects.
  • immune checkpoint inhibitor-related adverse events or “immune-related adverse events (irAEs) “ has its general meaning in the art and refers to diarrhea, colitis, rash, pruritus, hepatitis and endocrinopathies, such as hypophysitis and thyroiditis, piscleritis and/or uveitis, pancreatitis, nephritis, myasthenia gravis, autoimmune autonomic ganglionopathy, Guillain-Barre syndrome (GBS) and other neuropathies, sarcoidosis-like reactions, autoimmune thrombocytopenia, toxic epidermal necrolysis and Stevens-Johnson-like syndromes, headaches, encephalopathy, endocrinopathies and meningitis, fatal hypersensitivity reactions specifically to immune checkpoint inhibitor.
  • irAEs immune-related adverse events
  • the immune checkpoint inhibitor is an antibody.
  • the inhibitor or composition of the present invention is used in combination with an antibody that is specific for a costimulatory molecule.
  • antibodies that are specific for a costimulatory molecule include but are not limited to anti- CTLA4 antibodies (e.g. Ipilimumab), anti-PDl antibodies, anti-PD-Ll antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies.
  • the antibody that is specific for a costimulatory molecule is an anti-PD-1 antibody such as Pembrolizumab (Keytruda), Nivolumab (Opdivo) and Cemiplimab (Libtayo).
  • the antibody that is specific for a costimulatory molecule is an anti-PD-Ll antibody such as Atezolizumab (Tecentriq), Durvalumab (Imfinzi), Avelumab and BMS-936559 (BMS).
  • the antibody that is specific for a costimulatory molecule is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.
  • the antibody that is specific for a costimulatory molecule is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and W02013006490.
  • the antibody that is specific for a costimulatory molecule is an anti-CTLA-4 antibody such as Ipilimumab (Yervoy) and tremelimumab (Imjuno).
  • the antibody that is specific for a costimulatory molecule is an anti -LAG-3 antibody such as Relatlimab.
  • the G-CSF inhibitor or composition of the present invention is used in combination with an anti-PD-Ll antibody.
  • the second agent is an agent that induces, via ADCC, the death of a cell expressing an antigen to which the second agent binds.
  • the agent is an antibody (e.g. of IgGl or IgG3 isotype) whose mode of action involves induction of ADCC toward a cell to which the antibody binds.
  • NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second agent.
  • the second agent is an antibody specific for a cell surface antigens, e.g., membrane antigens.
  • the second antibody is specific for a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (e.g., CD20).
  • a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (e.g., CD20).
  • a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (
  • Antibodies of interest for the methods of the invention act through ADCC, and are typically selective for tumor cells, although one of skill in the art will recognize that some clinically useful antibodies do act on non-tumor cells, e.g. CD20.
  • CD20 There are a number of antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies.
  • One popular target antigen is CD20, which is found on B cell malignancies.
  • Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen.
  • CD20 has an important functional role in B cell activation, proliferation, and differentiation.
  • the CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia.
  • CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapyresistant hairy cell leukemia.
  • Monoclonal antibodies targeting CD20 also include tositumomab and ibritumomab.
  • Monoclonal antibodies useful in the methods of the invention, which have been used in solid tumors include without limitation edrecolomab and trastuzumab (herceptin).
  • Edrecolomab targets the 17-1 A antigen seen in colon and rectal cancer, and has been approved for use in Europe for these indications.
  • Trastuzumab targets the HER- 2/neu antigen. This antigen is seen on 25% to 35% of breast cancers. Trastuzumab is thought to work in a variety of ways: downregulation of HER-2 receptor expression, inhibition of proliferation of human tumor cells that overexpress HER-2 protein, enhancing immune recruitment and ADCC against tumor cells that overexpress HER-2 protein, and downregulation of angiogenesis factors.
  • Alemtuzumab (Campath) is used in the treatment of chronic lymphocytic leukemia; colon cancer and lung cancer; Gemtuzumab (Mylotarg) finds use in the treatment of acute myelogenous leukemia; Ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma; Panitumumab (Vectibix) finds use in the treatment of colon cancer. Cetuximab (Erbitux) is also of interest for use in the methods of the invention.
  • the antibody binds to the EGF receptor (EGFR), and has been used in the treatment of solid tumors including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).
  • EGFR EGF receptor
  • anti-viral agents may be added to the pharmaceutical composition or used in combination with the inhibitor of the invention in the case of the treatment of a viral disease.
  • the further agent may be selected in the group consisting of bronchodilators like P2 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.
  • Another aspect of the present invention relates to i) a G-CSF inhibitor, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of an age-related disease in a subject in need thereof.
  • the term “simultaneous use” denotes the use of a G-CSF inhibitor and at least one therapeutic active agent occurring at the same time.
  • the term “separate use” denotes the use of a G-CSF inhibitor and at least one therapeutic active agent not occurring at the same time.
  • sequential use denotes the use of a G-CSF inhibitor and at least one therapeutic active agent occurring by following an order.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Hematopoietic premature ageing of Cxxc5 knockout animals is characterized by a mild anaemia, a drop in the lymphocytes (especially B cells) percentage and an increase in monocytes/granulocytes (especially neutrophiles) percentage.
  • FIG. 2 G-CSF concentration was higher in blood serum of animals that age prematurely (Cxxc5 ) compared to wild-type littermate control animals.
  • Figure 3 Non hematopoietic cells (CD45 negative) and Mesenchymal Stem Cells (MSC) isolated from bone marrow Cxxc5 animals (that age prematurely) secrete and produce more G-CSF (Csf3) in a proinflammatory context (here triggered by LPS or poly- IC treatment), a) Multiplex cytokines/chemokines screening analysis of the supernatant issue from wild type control and Cxxc5-/- MSC cells (S) or not stimulated (NS) with LPS. The mean cytokines/chemokines concentration is presented in the table.
  • n 3 mice per group performed in duplicate, (*, P ⁇ 0.1; **, P ⁇ 0.01; ***, P ⁇ 0.001; **** P ⁇ 0.0001, analysed by unpaired t test), b) Total mRNA was prepared from bone marrow CD45- cells of control and Cxxc5-/- mice and quantified Thpo, 117, Tnf-a and Sppl gene expression. Values are presented relative to those of Gapdh mRNA.
  • Figure 4 In vivo administration of anti-G-CSF antibodies reestablishes bone marrow production of B cells of ageing animals (>6 months) and rescued the accelerated ageing phenotype of hematopoietic cells in Cxxc5 animals, a) Schematic representation of the treatment experiment with neutralizing anti-G-CSF antibodies.
  • Figure 5 A brief in vivo administration of anti-GCSF antibodies attenuates anemia and subsequent stress erythropoiesis that is responsible for splenomegaly in tumor bearing mouse
  • Histogram represents the spleen weight of 8 BALB/C animals at sacrifice time (day 35) and treated with PBS or anti- GCSF antibodies (4 intraperitoneal injections from day 10 to 16).
  • Histrogram represents the spleen weight of 8 BALB/C animals at sacrifice time (day 35) and treated with PBS of anti-GCSF antibodies (4 intraperitoneal injections from day 10 to 16).
  • PBS or anti-GCSF the spleen was weight for 4 animals at day 35.
  • mice were generated at the Mouse Clinical Institute in France (http://www-mci.u-strasbg.fr) by deleting exon 3 from the Cxxc5 gene, which contains the starting ATG site and that is encoding more than 90% of the full-length protein coding sequence. All mice were bred and maintained in a pathogen-free animal facility at Cochin Institute of Paris. Animal care was performed in compliance with all relevant ethical regulations for animal testing and research of the Federation of European Laboratory Animal Science Association. All the samples and animal tissues (bone marrow, spleen, blood) were taken on freshly sacrificed animals.
  • Real-time quantitative PCR was performed on the LightCycler 480 Instrument II (LC480 Roche) and the amplifications were done using the LightCycler 480 SYBR Green I Master Mix (Roche 04887352001). Relative expression levels of target genes were quantitatively normalized against the expression of GAPDH. All samples were assayed in duplicate.
  • a rat monoclonal antibody targeting murine G-CSF (Clone 67604, MAB414-500, R&D Systems, Biotechne) or a control antibody (Clone 43414, MAB005, Rat IgGl Isotype Control, R&D Systems, Biotechne) was administered intraperitoneally (25pg/mouse) every couple of days for two weeks (according to experimental design indicated on Figure 4A). After 17 days of treatment, the animals were sacrificed, and their bone marrow was isolated for flow cytometry analysis to quantify the production B cells and neutrophiles.
  • Thymus, spleen and bone marrow cells suspension were harvested from mice (3 months old or 6 months old) after the organ’s dissociation and the bone marrow flush. Cells were next counted, incubated with conjugated antibodies for Ih at 4°C in MACS buffer, washed and finally fixed in PFA 4%. Stained cells were quantified using an LSR Fortessa flow cytometer (Becton Dickinson) and analyzed with Flow Jo software (Treestar).
  • mice cytokine/chemokine Magnetic 25-Plex Bead Panel (MCYTMAG70PMX25BK, Milipore) was used to quantify the levels of mice serum cytokines/chemokines on a BioPlex200 machine from Biorad (Hercules, Califomie, USA) according to the manufacturer’s instructions.
  • MYTMAG70PMX25BK Magnetic 25-Plex Bead Panel
  • Mouse were treated with neutralizing anti-GCSF antibodies (4 intraperitoneal injections from day 10 to 16) and/or anti-PD-Ll post-tumor orthotopic injection. Mouse were treated for 6 days (by 4 intraperitoneal injections performed every couple of days, from Day 10 to 16) with 20pg/mouse of anti-mGCSF or anti-Iggl control antibodies (4 intraperitoneal injections at day 14, 16, 18 and 26 days). Tumor size was followed by caliper measurement every couple of days from Day 10 to Day 31 post-transplantation. Each group were initially constituted of 6 mice except for one group , the anti-GCSF+anti-PD-Ll group, that was constituted of only 5 mice (because one tumor did not grew for one )
  • results are expressed as means ⁇ SEM. All experiments were repeated at least twice, yielding similar results. Data were analyzed with GraphPad Prism8 software, p values were calculated by the unpaired Student's t test. *p ⁇ 0.1; **p ⁇ 0.01; ***p ⁇ 0.001. Results
  • cytokines/chemokines G-CSF, TNFa, CXCL10 and CCL4 were deregulated out of the twenty-two cytokines/chemokines detected in our screen (GM-CSF, CCL2, CCL5, CXCL1, CXCL2, IFNg, IL-la, IL-lb, IL-2, IL-5, IL-6, IL-7, IL-9, IL-10, IL- 12p40, IL-12p70, IL-13, and IL-17).
  • G-CSF also known as CSF3
  • concentration of this cytokine was significantly increased in serum of the Cxxc5 /_ animals (a ⁇ 2.2-fold increase was noticed, in average).
  • Non-hematopoietic cells (CD45 -negative) of the bone marrow microenvironment were studied. We did not find any statistical changes in the total number of MSC subtypes characterized by flow cytometry. However, a quantitative RT-PCR analysis (Figure 3B) confirmed that CD45-negative cells freshly isolated from bone marrow of Cxxc5' /_ animals expressed more G-csf (Csf3) mRNA than the controls.
  • 3 mRNA were also more upregulated in Cxxc5- deficient MSC, while it was not the case for other investigated factors (Csf2 (Gm-csf), Tnf-a, 116, 117, Tgf-P, Thpo, Cxcll2, Sppl, Mmp9, and Angptl (angiopoietin).
  • Csf3 expression was not increased in CD45+ bone marrow cells, suggesting that this increase was restricted to CD45-compartment.
  • MSC cells were expanded ex vivo and analyzed for their ability to secrete cytokines, following (or not) a stimulation with LPS, a molecule that is well-known to increase secretion of proinflammatory cytokines.
  • the release of cytokines in the supernatant was first quantified by a multiplex screening approach (Figure 3 A) and then confirmed by qRT-PCR ( Figure 3C).
  • Figure 3 A A multiplex screening approach
  • qRT-PCR Figure 3C
  • a strong increase in the secretion of CCL5 and G-CSF was noticed in the supernatant of Cxxc5' /_ MSC compared to the control.
  • this result was confirmed by qRT-PCR and by using another type of stimulation (Poly (I:C)).
  • ILip cytokine which was raised in CD45-compartment, was not more secreted by Cxxc5-deficient MSC than controls, even after LPS stimulation. Moreover, although its basal secretion appeared increased, this finding was not confirmed by qRT-PCR experiments.
  • the 4T1 model a triple-negative breast cancer model, is also characterized by an inefficient bone marrow erythropoiesis, and thus, a stress erythropoiesis occurs in the spleen to compensate the lack of myeloid red cell production. It was already suggested that a high G-CSF production by 4T1 cells could be responsible or contribute to this phenotype. However, it is the first time that we demonstrated that a brief anti-GCSF treatment could attenuate this symptom.

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Abstract

La présente invention concerne le traitement de maladies liées à l'âge. Les inventeurs ont comparé le phénotype hématopoïétique d'un knockout constitutif (Rinf/_) et un modèle de souris knockout conditionnel (Vav-Cre+/-Rintfox/flox) invalidé spécifiquement dans le tissu hématopoïétique pour Cxxc5. Le vieillissement accéléré de cellules hématopoïétiques chez des souris Rinf/_ mais pas de manière inattendue pas chez des souris Vav-Cre+/'Rinfflox/flox a révélé un rôle clé de Rinf dans la niche BM et que la souris Rinf/- peut être utilisée en tant que modèle pour des maladies liées à l'âge. Ils ont identifié la cytokine G-CSF comme particulièrement dérégulée et fortement exprimée dans le microenvironnement BM des animaux Rinf/-. Ils ont montré que le ciblage in vivo du G-CSF avec une lymphopénie et une neutrophilie résolues par un anticorps de déplétion. L'invention concerne un inhibiteur de G-CSF destiné à être utilisé dans le traitement de maladies liées à l'âge chez un sujet en ayant besoin.
PCT/EP2024/052643 2023-02-03 2024-02-02 Méthode de traitement de maladies liées à l'âge WO2024161015A1 (fr)

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