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  • A61K40/00—Cellular immunotherapy
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  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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  • A61K40/00—Cellular immunotherapy
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  • A61K40/00—Cellular immunotherapy
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2510/00—Genetically modified cells

Definitions

• CD8 cluster of differentiation 8
• TCR T cell receptor
• CD8 forms a dimer, consisting of a pair of CD8 chains.
• the most common form of CD 8 in T cells is composed of a CD8-a and CD8-P chain and CD8 + CD45RC low/" Treg cells express both chains.
• the naturally occurring human CD8-a protein has an amino acid sequence provided in the UniProt database under accession number P01732.
• the naturally occurring human CD8-P protein has an amino acid sequence provided in the UniProt database under accession number P10966.
• the population of CD8 + CD45RC low/" Treg cells is genetically modified using a vector particle such as a viral vector (or a recombinant virus) or a virus-like particle (VLP).
• a vector particle such as a viral vector (or a recombinant virus) or a virus-like particle (VLP).
• the heterologous nucleic acid is, for example, introduced into a recombinant virus which is then used to infect population of CD8 + CD45RC low/" Treg cells.
• Different types of recombinant viruses can be used, in particular recombinant retroviruses. Retroviruses are preferred vectors since retroviral infection results in stable integration into the genome of the cells.
• the nucleic acid used to genetically modify the population of CD8+CD45RC low/" Treg cells may encode various biologically active products, including polypeptides (e.g., proteins, peptides, etc.), RNAs, etc.
• the nucleic acid encodes a polypeptide having an immuno-suppressive activity.
• the nucleic acid encodes a polypeptide which is toxic or conditionally toxic to the cells.
• Preferred examples include a thymidine kinase (which confers toxicity in the presence of nucleoside analogs), such as HSV- 1 TK, a cytosine desaminase, etc.
• the intracellular domain of "third generation” CARs comprise two co- stimulatory domains in tandem with an activation moiety, such as the combination of CD28, a tumor necrosis factor receptor (TNFr), such as OX40 or 4- IBB, and CD3 ⁇ .
• CARs are generally obtained by fusing the extracellular antigen-binding domain with the intracellular signaling domains derived from the CDS- ⁇ chain of the T-cell receptor, in tandem with costimulatory endo-domains to support survival and proliferative signals. Because CAR- modified T cells function independently of a patient's MHC and can readily be generated for clinical use, the targeting of pathogenic antigens as described below with a CAR based-approach is useful.
• CAARs comprise an extracellular autoantigen, such as an autoantigen involved in an autoimmune disease, fused to intracellular signaling domains.
• intracellular signaling domains of a CAAR include, without being limited to, T or NK receptor signaling domains such as CD137CD3 ⁇ signaling domain.
• the CD8 + CD45RC " Treg cells of the invention are genetically modified and lack expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA), e.g., HLA class I and/or HLA class II.
• TCR T cell receptor
• HLA human leukocyte antigen
• the genetically modified CD8 + CD45RC low/" Treg cells of the invention lacking a functional TCR and/or HLA are allogeneic Tregs.
• the population of CD8 + CD45RC low/" Treg cells is cultured in an appropriate culture medium.
• the term “medium” refers to a medium for maintaining a cell population, or culturing a cell population (e.g. "culture medium") containing nutrients that maintain cell viability and support proliferation.
• the medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as growth factors, cytokines etc.
• Media ordinarily used for particular cell types are known to those skilled in the art.
• the medium of the invention may be based on a commercially available medium such as RPMI 1640 from Invitrogen.
• the culture medium comprises an amount of rapamycin of about
• the culture medium comprises an amount of rapamycin of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47; 48; 49, 50, 51, 52, 53, 54, or 55ng/ml.
• the method of the present invention is particular useful for adoptive T cell transfer for preventing or reducing transplant rejection or GVHD.
• Acute rejection is the rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplant tissue by immune cells of the recipient, which carry out their effector function and destroy the transplant tissue. The onset of acute rejection is rapid and generally occurs in humans within a few weeks after transplant surgery. Generally, acute rejection can be inhibited or suppressed with immunosuppressive drugs such as rapamycin, cyclosporin and the like. "Chronic rejection” generally occurs in humans within several months to years after engraftment, even in the presence of successful immunosuppression of acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ transplants.
• the cell composition of the present invention is particularly suitable of the treatment of genetic diseases in which activation of the immune system is involved and wherein inhibition of immune responses would be beneficial.
• diseases include but are not limited to monogenic genetic diseases affecting the immune system associated to autoimmunity, such as IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked syndrome) and APECED (autoimmune polyendocrinopathy-candidiasis- ectodermal dystrophy), B cell primary immunodeficiencies, Muckle-Wells syndrome, mixed autoinflammatory and autoimmune syndrome, NLRP12-associated hereditary periodic fever syndrome, tumor necrosis factor receptor 1 associated periodic syndrome) and monogenic hereditary diseases, such as Duchenne muscular dystrophy (DMD), cystic fibrosis, lysosomal diseases and alphal-anti-trypsin deficiency.
• DMD Duchenne muscular dystrophy
• cystic fibrosis cystic fibrosis
• lysosomal diseases alphal-
• the term "therapeutically effective combination” as used herein refers to an amount the population of Tregs together with the amount of the rapamycin compound that is sufficient to prevent or reduce transplant rejection or GVHD.
• the “therapeutically effective amount” is determined using procedures routinely employed by those of skill in the art such that an "improved therapeutic outcome” results. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts.
• the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
• the population of CD8+CD45RC low/" Tregs and the rapamycin compound are administered to the subject in the form of a pharmaceutical composition.
• the Population of Tregs and the rapamycin compound may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
• pharmaceutically acceptable excipients such as biodegradable polymers
• sustained-release matrices such as biodegradable polymers
• saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
• 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 pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
• Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the population of Tregs and the rapamycin compound can be formulated into a composition in a neutral or salt form.
• Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
• Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• sterile powders for the preparation of sterile injectable solutions the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
• solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
• aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
• the inventors also showed that a combination of cyclosporine and methylprednisolone increases the immunosuppressive capacity of the population of CD8 + CD45RC low/" Tregs.
• a further object of the present invention also relates to a method of preventing or reducing transplant rejection or GVHD comprising administering a therapeutically effective combination of a population of CD8+CD45RC low/" Tregs, cyclosporine and methylprednisolone. All the embodiments for the method of preventing or reducing transplant rejection or GVHD described with rapamycin apply mutatis mutandis to the combination of cyclosporine and methylprednisolone.
• methylprednisolone has its general meaning in the art and refers to 6a, l ip)-l l,17,21-trihydroxy-6-methyl-pregna-l,4-diene-3,20-dione.
• Tregs were harvested, counted, washed, and plated at 5xl0 5 Tregs/well/3ml in p6 plate previously coated with anti-CD3 (OKT3 clone, ⁇ g/ml in PBS, lml/well, lh at 37°C then washed with PBS 3 times), in RPMI1640 medium supplemented with 10% AB serum, Penicillin (lOOU/ml), Streptomycin (O.lmg/ml), Sodium pyruvate (ImM), Glutamine (2mM), Hepes Buffer (ImM), non-essential amino acids (IX), IL-2 (lOOOU/ml) and IL-15 (lOng/ml), anti-CD28 mAbs (clone CD28.2, soluble, ⁇ g/ml), and supplemented or not with an immunosuppressive drug (cyclosporine A (45ng/ml), rapamycin (45ng/ml),

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• 2018
  • 2018-07-12 EP EP18737628.0A patent/EP3652306A1/en not_active Withdrawn
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  • 2018-07-12 WO PCT/EP2018/068882 patent/WO2019012024A1/en not_active Ceased
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