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WO2013014535A1 - Procédés et compositions pour l'augmentation de la diversité des lymphocytes t - Google Patents

Procédés et compositions pour l'augmentation de la diversité des lymphocytes t Download PDF

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Publication number
WO2013014535A1
WO2013014535A1 PCT/IB2012/001784 IB2012001784W WO2013014535A1 WO 2013014535 A1 WO2013014535 A1 WO 2013014535A1 IB 2012001784 W IB2012001784 W IB 2012001784W WO 2013014535 A1 WO2013014535 A1 WO 2013014535A1
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Prior art keywords
cells
leukocytes
cell
individual
naive
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PCT/IB2012/001784
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English (en)
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Sergei A. LUKYANOV
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Evrogen Joint Stock Company
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Publication of WO2013014535A1 publication Critical patent/WO2013014535A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • Aging-related diseases are diseases that are seen with increasing frequency with increasing aging.
  • aging-associated diseases are diseases that are seen more often in the elderly, and more often still in the older elderly. They are different from age-specific diseases such as childhood diseases, or accelerated-aging diseases such as some specific genetic disorders.
  • Examples of aging-associated diseases include cardiovascular disease, arthritis, glaucoma, cataracts, osteoporosis, type 2 diabetes, hypertension, Alzheimer's disease, acute and chronic infectious diseases, and cancer. The incidence of all of these diseases increases rapidly with aging.
  • telomere shortening are the regions of repetitive DNA sequences at the ends of chromosomes which protect the ends of the chromosomes from deterioration or from fusion with neighboring chromosomes. Telomeres shorten with every round of division, until they become so short that they trigger replicative senescence, which blocks cell division. Cellular senescence due to telomere shortening normally occurs after about 50 cell divisions in vitro, but may occur sooner.
  • Some cells become senescent as a result of DNA double strand breaks, toxins, etc.
  • cells may respond to DNA damage by apoptosis (programmed cell death).
  • apoptosis programmed cell death
  • the cells lose the ability to exit mitosis or apoptose, and the cells instead begin to divide unchecked, i.e. they become cancerous.
  • Aging- associated diseases are also due in some part to the senescence of the organism as a whole as the body ages.
  • Organismal senescence is characterized by the declining ability to respond to stress, increased homeostatic imbalance, changes in hormone profiles, progressive deterioration of physiological function, and increased risk of aging-associated diseases.
  • Certain conditions or medical treatments may promote the early onset of cellular or organismal senescence, and hence of aging- associated diseases. For example, chronic inflammation may force the excessive proliferation of immune cells, which may result in premature senescence of immune cell populations and immunosuppression. Overexposure to the ultraviolet light of the sun may cause DNA damage to epidermal cells, which may result in premature senescence or cancer.
  • aromatase inhibitors e.g. letrozole, anastrozole, exemestane
  • to treat breast cancer which blocks the synthesis of estrogen, may lower estrogen levels which may in turn accelerate bone turnover and osteoporosis.
  • the present invention addresses issues pertaining to aging- associated diseases.
  • aspects of the invention include methods, compositions and kits for enhancing T cell diversity in an individual. These methods, compositions and kits find use in a number of applications, such as enhancing immune protection, suppressing lymphopenia, treating T cell exhaustion, suppressing immunoscenescence and/or compensating for T cell loss due to immunoscenescence, and preventing aging-related disorders.
  • a method for enhancing T cell diversity in an individual comprising: harvesting a population of leukocytes comprising naive T cells from the individual; preserving the leukocytes; storing the leukocytes; and transferring the leukocytes back into the individual to enhance T cell diversity in the individual.
  • the harvesting step does not comprise administering a stem cell mobilizing agent to the individual.
  • the harvesting comprises leukapheresis.
  • 0.01-10% of the total number of T cells in the individual is harvested.
  • the preserving comprises cryopreservation.
  • the storing is for 10 years or more.
  • the method further comprises the step of enriching the harvested population of leukocytes for T cells.
  • the enriching comprises flow cytometry, magnetic bead sorting, or immunopanning.
  • the enriching comprises contacting the population of leukocytes with an antibody that is specific for CD3, CD5, CD7, CD4 and/or CD8.
  • the enriching comprises enriching for naive T cells.
  • the enriching comprises contacting the population of leukocytes with one or more antibodies that is specific for CD62L, CD27, CD28, CCR7 or CD45RA.
  • the enriching comprises the depletion of cells other than T cells.
  • the negative depletion comprises contacting the population of leukocytes with one or more antibodies that are specific for markers of cells other than naive T cells, e.g. CD45RO, CDl la, CD44, CD95, CXCR3, CCR4, CD56, CD57, CD244, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD123, HLA-DR, or CD235a.
  • the enriched population consists essentially of naive T cells.
  • the method further comprises the step of evaluating T cell diversity.
  • T cell diversity is evaluated by measuring naive T cell numbers in the blood, lymph nodes, and/or spleen in the individual.
  • the method further comprises the step of evaluating whether the number of naive T cells in the blood, lymph nodes, and/or spleen subsequent to the recovery of cryopreserved population of autologous leukocytes is elevated relative to prior to the recovery.
  • T cell diversity is evaluated by measuring TCR repertoire diversity.
  • the method further comprises the step of evaluating whether the diversity of the TCR repertoire of T cells in the blood, lymph nodes, and/or spleen subsequent to the transferring back is increased relative to prior to the recovery of leukocytes.
  • T cell diversity is measured 2 weeks or longer after the transplanting.
  • the method is a method of enhancing immune protection. In some embodiments, the method is a method of suppressing lymphopenia in an individual. In some embodiments, the method is a method of treating T cell exhaustion. In some embodiments, the method is a method of suppressing immunoscenescence and/or compensation of T cell loss due to suppressing immunoscenescence. In some embodiments, the method is a method for at least inhibiting the progression of aging-related disorders. In some embodiments, the method is a method for preventing aging-related disorders.
  • a method for preventing aging-related disorders in an individual comprising: harvesting a population of leukocytes comprising naive T cells from an individual; preserving the leukocytes; storing the leukocytes; and transferring the leukocytes back into the individual; wherein aging-related disorders are prevented.
  • kits for use in a method of preventing aging disorders, the kits comprising: a collection receptacle; a cryopreservation tube; and a cryopreservative for the preservation of leukocytes.
  • the collection receptacle has a volume ranging from 100 cc to 500 cc.
  • the cryopreservation tube has a volume ranging from 5 cc to 100 cc.
  • the preservative is DMSO (dimethyl sulfoxidum).
  • aspects of the invention include methods, compositions and kits for enhancing T cell diversity in an individual. These methods, compositions and kits find use in a number of applications, such as enhancing immune protection, suppressing lymphopenia, treating T cell exhaustion, suppressing immunoscenescence and/or compensating for T cell loss due to immunoscenescence, and preventing aging-related disorders.
  • aspects of the invention include methods for enhancing T cell diversity in an individual.
  • T cell diversity it is meant the clonal representation of T cells (number of T-cells with different T-cell receptor) in a blood sample, e.g. a sample of whole blood ranging from 1 to 500 ml. T cell diversity is strongly depend on the diversity of naive T- cells.
  • naive T cell diversity it is meant the clonal representation of naive T cells.
  • Naive T cells are mature T cells, in that they have differentiated and successfully undergone positive and negative selection in the thymus. Naive T cells have not yet encountered their cognate antigens in the periphery. Accordingly, naive T cells are mature T cells that have yet to contact the antigen to which they can respond and become activated against.
  • enhanced T cell diversity it is meant that the diversity of T cells in an individual is increased relative to the diversity of T cells observed in the individual prior to use of the methods, e.g. by 2-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 30-fold or more, or 40-fold or more, including 50-fold or more, 100-fold or more, such as 500-fold or more, 1000-fold or more, etc. relative to the diversity of T cells observed in the individual prior to use of the methods.
  • T cell diversity may be measured by determining the number of naive T cells in an individual, for example by immunohistochemistry or flow cytometry of a blood sample or immune tissue, e.g.
  • T cell diversity may be measured by measuring the diversity of the T cell receptor (TCR) repertoire, for example by TCR spectratyping, which utilizes ⁇ - ⁇ or Vot-Cot (variable domains of alpha and beta chains of TCR) specific PCR and denaturing gradient gel electrophoresis; immunoscoping, a PCR-based method that detects large expansions within ⁇ families (see, e.g. Even, JA et al. Res Immunol 1995, 146:65-80); massive sequencing of T cell receptor repertoires (see, e.g., Zhouet al.
  • the diversity of naive T cells is enhanced by performing a time- shifted autologous transplantation of naive T cells.
  • autologous transplantation it is meant that a population of leukocytes, e.g. in a blood sample, e.g. a fraction of blood sample harvested from an individual, is transferred back (restored) into the same individual from which it is initially harvested.
  • time- shifted it is meant that there is a time delay between when the leukocytes are harvested and when the leukocytes are transferred back into the individual. In some instances, the delay is 1 year or longer, e.g., 2.5 years or longer.
  • the time delay may be as little as 5 years, or as many as 60 years or more, e.g. 7, 10, 15, 20, 30, 40, 50, 60 years or more.
  • a population of leukocytes comprising T cells is harvested from an individual, preserved, and transferred back into that individual at an older age.
  • leukocytes are harvested from an individual and preserved for transplantation at a later date.
  • Leukocytes may be harvested at any age when it is convenient. In some instances, harvesting is from an individual in which the T cell repertoire has completely developed and the naive T cells are still young, and before the decrease in T cell diversity.
  • young it is meant that the naive T cells have a high proliferative potential, e.g. they have telomeres that are about 10-12 kb long or at least 7 kb long and they are capable of 5 or more divisions, e.g. 10 or more, 30 or more, 40 or more divisions, such as 50 or 60 divisions or more, i.e. 5-60 divisions.
  • T cells are harvested from an individual that is age 15 to age 50, e.g. between ages 20 and 40, e.g. between 20 and 35 years of age, e.g. between 20 and 25 years of age, that is, from a person that is 15 years old or older, 20 years old or older, 25 years old or older, in some instances 30 or older, 35 or older, or 40 or older, such as 45 or older, or 50.
  • the individual has no hematological malignancy or autoimmune disease at the moment of harvest.
  • the individual may have a hematological disorder.
  • substantially full diversity may be achieved by harvesting 100 million or more naive T cells, e.g. 100 million to 1 billion naive T cells. In some instances, substantially full diversity may be achieved by harvesting 300 million or more total T cells, e.g. 300 million to 10 billion total T cells, i.e. 0.1 -3 or more of the total T cells, in some instances 0.01-10% of the total T cells, in certain instances, 2% of the total T cells.
  • the number of total or naive T cells in the obtained sample may be estimated by any convenient method, e.g. standard flow cytometry analysis using staining with the antibodies to any convenient characteristic surface markers.
  • Leukocytes may be harvested by any convenient method.
  • leukocytes may be harvested by automated blood collection, or "apheresis," i.e. leukocytapheresis.
  • apheresis a blood sample is passed through a machine that separates out certain components, e.g. platelets, erythrocytes, plasma, or leukocytes, and returns the remaining blood components to the blood stream.
  • leukocytapheresis leukocytes are selectively removed. Blood components acquired by apheresis are routinely used as a component of various therapies.
  • apheresis is commonly used for harvesting HSCs for autologous transplantation.
  • Equipment for apheresis allows for the collection of several billion leukocytes for one session, which will contain 100 million tol billion naive T cells.
  • a whole blood sample may be collected and fractionated by density gradient centrifugations, and the buffy coat (comprising the leukocytes) isolated for use.
  • Such amounts of T cells may be harvested by several harvests consecutive harvests of, for example, 100-500 milliliters blood, e.g. 20 harvests of 200 milliliters of blood.
  • the leukocytes are obtained from fresh blood, e.g. a whole blood sample freshly drawn from a patient, or drawn from a patient and stored refrigerated.
  • the leukocytes are obtained from frozen blood, e.g. a whole blood sample that is obtained and frozen, e.g. 1 week or more, e.g. 1 month or more, e.g. 1 year or more, e.g. 10 years or more.
  • multiple harvests e.g. 10-20 harvests of 500-200 milliliters may be performed to obtain the required amount of naive T cells.
  • no preconditioning of a subject is necessary before leukocyte harvest.
  • leukocytes are harvested from an individual that has not been preconditioned.
  • preconditioning it is meant that an agent is administered to the subject to promote the mobilization, i.e. proliferation and migration, of cells from tissues into the subject's blood.
  • HSCs hematopoietic stem cells
  • the subject receives injections of a preconditioning agent, e.g.
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage-colony stimulating factor
  • This procedure is not required to practice the subject methods; however it does not impede the subject methods.
  • the individual is not preconditioned. In other instances, the individual is preconditioned.
  • harvested leukocytes are preserved for transplantation at a future time.
  • Leukocytes may be preserved by any convenient technology for preserving viable cells, e.g. technologies for the preservation of peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), and the like.
  • PBMCs peripheral blood mononuclear cells
  • ULB umbilical cord blood
  • leukocytes may be suspended in medium with dimethyl sulfoxide (DMSO) and a high concentration of serum, slow cooled, and cryopreserved, i.e. stored in liquid nitrogen frozen at -190°C.
  • DMSO dimethyl sulfoxide
  • Such methods typically maintain the viability of the cells, e.g. 20% or more of the cells survive upon thaw, for example, 40% or more, 60% or more, 80% or more cells, in some instances, 90% or more, such as 95% or more, 97% or more, or 99% or more of the cells will be viable after removal of the preservative.
  • aliquots of preserved leukocytes are thawed after preservation in order to estimate the survival rate of naive T cells, and if the number is lower than desired, the collection procedure may be repeated to obtain a sufficient amount of naive T cells.
  • no specific or non-specific stimulation, exposure to specific antigens etc. is performed.
  • the T cells may be expanded in culture, e.g. by stimulating the cells to proliferate.
  • Leukocytes may be stored in this preserved state for extended lengths of time, e.g. 70 years or more.
  • leukocyte preparations may be preserved for one year or more, e.g. 1 year or more, 5 years or more, 10 years or more, in some instances 15 years or more, 20 years or more, 30 years or more, 35 years or more, or 40 years or more, such as 50 years or more, or 60 years or more, depending on the age of the individual at the time of leukocyte harvest and the age of the individual at the time of leukocyte reintroduction.
  • the preservative when reintroduction, i.e. transplantation, or restoration, of the preserved leukocytes to the blood stream of the individual is desired, the preservative, if present, may be replaced with any convenient solution for transplantation, e.g. any suitable buffer, and the leukocytes transplanted.
  • any convenient solution for transplantation e.g. any suitable buffer
  • the leukocytes transplanted e.g. the leukocytes are preserved by cryopreservation, they are thawed, e.g. by any convenient method for blood cell thawing used in cell transplantation (see, for example, Rubinstein et al. (1995) Proc. Nat. Acad. Sci. USA 92: 10119-10122), the preservative replaced, and the cells transplanted.
  • Transplantation of the leukocytes into the individual may be by any convenient technology for the engraftment of blood cells in a patient, e.g. for the engraftment hematopoietic stem cells; see, for example, Negrin et al. (2000) Biol Blood Marrow Transplant 6(3):262-71 ; McNiece et al. (2000) Blood 96(9):3001-7; and Balduzzi et al. (2001) Leukemia. 15(l):50-6.
  • Leukocytes may be transplanted using any convenient access device, e.g. needle for intravenous injection, compressor gun, peripheral cannula, central IV line, etc., e.g.
  • Implantable port tunneled line, central venous lines, peripherally inserted central catheters and the like. Transplantation may be through any vein typically used for engraftment, e.g. subclavian, internal jugular, femoral, superior vena cava, inferior vena cava, right atrium, etc.
  • the number of viable leukocytes is evaluated prior to reintroduction. In other instances, no such evaluation is performed.
  • the leukocytes may be genetically altered prior to transplanting to the individual, in order to introduce genes useful in the cell, e.g. repair of a genetic defect in an individual, to provide a selectable or traceable marker, etc.
  • Leukocytes may also be genetically modified to enhance survival, control proliferation, and the like.
  • Cells may be genetically altering by transfection or transduction with a suitable vector, homologous recombination, or other appropriate technique, so that they express a gene of interest, or with an antisense mRNA, siRNA or ribozymes to block expression of an undesired gene.
  • cells are transfected with genes encoding a telomerase catalytic component (TERT), often under a heterologous promoter that increases telomerase expression beyond what occurs under the endogenous promoter (see e.g. International Patent Application WO 98/14592).
  • TERT telomerase catalytic component
  • Various techniques are known in the art for the introduction of nucleic acids into target cells. To prove that one has genetically modified the leukocytes, various techniques may be employed. The genome of the cells may be restricted and used with or without amplification. The polymerase chain reaction; gel electrophoresis; restriction analysis; Southern, Northern, and Western blots; sequencing; or the like, may all be employed.
  • Various tests in vitro and in vivo may be employed to ensure that T cell phenotypes have been maintained.
  • a number of factors may be considered when determining whether transplantation of the leukocytes will be useful. For example, decreased diversity of the T cell receptor repertoire of naive T cells and/or all T cells and/or decrease the number of naive T-cells may be indicative of a need for transplantation.
  • a decrease in T cell diversity it is meant at least a 2-fold decrease or more in the types, i.e. sequences, of T cell receptors represented by T cells relative to the types, i.e. sequences, of T cell receptors represented by T cells at the cessation of normal T cell development at about 15-40 years of age, e.g.
  • a 5-fold decrease or more a 10-fold decrease or more, a 15 -fold decrease or more, a 20-fold decrease or more, a 25 -fold decrease or more, a 30-fold decrease or more, in some instances a 35-fold decrease or more, a 40-fold decrease or more, a 45-fold decrease or more, a 50-fold decrease or more, a 55-fold decrease or more, a 60- fold decrease or more, a 75 -fold decrease or more, or a 100-fold decrease or more.
  • Another indicator may be an increase in the oligoclonality of the T cell pool, i.e.
  • an increase in the number of cells in the T cell pool that are derived from a single naive T cell, in the aged individual's blood relative to the oligoclonality of the T cell pool when the individual was younger e.g. a 2-fold increase or more, a 5-fold increase or more, a 10-fold increase or more, a 15-fold increase or more, or a 20-fold increase or more, for example, a 25-fold increase or more, a 30-fold increase or more, in some instances a 35-fold increase or more, a 40-fold increase or more, a 45-fold increase or more, or a 50-fold increase or more.
  • Other factors to be considered include whether the individual is receiving a medical therapy that results in general immunosuppression, e.g.
  • a determination is made by evaluating at least one or more of the above factors. In some instances, a determination is made by evaluating at least 2 or more of the above factors, 3 or more of the above factors, or 4 or more of the above factors.
  • the subject methods and compositions are employed with a complete, or intact, population of leukocytes.
  • a complete or “intact”, population of leukocytes, it is meant the diverse population of leukocytes - including, but not limited to memory and effector T cells, B cells, NK cells, macrophages, neutrophils, eosinophils, and stem and progenitor cells thereof - that is obtained from the peripheral blood during harvesting.
  • all of the leukocytes that are obtained from the blood sample are preserved and transplanted back into an individual.
  • the complete leukocyte population is enriched for T cells to produce an enriched population of T cells, and the enriched population of T cells is transplanted into the individual.
  • the complete leukocyte population is enriched for naive T cells to produce an enriched population of naive T cells, and the enriched population of naive T cells is transplanted into the individual.
  • enriching it is meant selecting and isolating a desired cell type, e.g. T cells or naive T cells, from an initial, heterogeneous population of cells, i.e. the complete leukocyte population.
  • the complete leukocyte population is separated from the blood sample and stored frozen, e.g.
  • the complete leukocyte population may be stored in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such temperatures, and will be thawed and in some instances recultured by methods commonly known in the art and as described further below. In some instances the complete leukocyte population is subjected to T cell enrichment right after harvesting.
  • T cells may be separated from the complete leukocyte population directly, i.e. as the complete leukocyte population is harvested, e.g. by enriching for T cells directly from the whole blood sample.
  • the cells of the complete leukocyte population may be suspended in any appropriate solution that maintains cell viability, and the cells of the desired cell type selected there from.
  • solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.
  • T cells from the complete leukocyte population of cells may be by any convenient separation technique.
  • the T cells may be separated from the complete leukocyte population by affinity separation techniques.
  • Techniques for affinity separation may include fluorescence activated cell sorting, magnetic separation using magnetic beads coated with an affinity reagent, affinity chromatography, "panning" with an affinity reagent attached to a solid matrix, e.g. plate, or other convenient technique.
  • the cells may be selected against dead cells by employing dyes associated with dead cells (e.g. propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the T cells of interest.
  • the complete leukocyte population may be contacted with an affinity reagent that specifically recognizes and selectively binds a cell surface marker associated with T cells.
  • Affinity reagents for any convenient marker that is specific for T cells i.e. mature T cells, e.g. naive T cells, effector T cells, and memory T cells, may be used, e.g. CD3, CD5, CD7, CD4 and CD8.
  • affinity reagents for any convenient marker that is specific for naive cells may be used, e.g. CD62L, CD27, CD28, CCR7, and CD45RA.
  • non-T cells e.g. non-naive T cells
  • affinity reagents that are specific for cells other than naive T cells, e.g. CD45RO, CDl la, CD44, CD95, CXCR3, and CCR4 (Kohler, S. and Thiel, A. (2009) Blood 113(4): 769-774).
  • cells other than T cells may be depleted from the sample using antibodies that are specific for markers for B cells (e.g. anti- CD20 antibody), macrophages (e.g. anti-CDl lb antibody), etc, and/or the T cells positively selected for using antibodies specific for CD3, CD5, and/or CD7.
  • markers for B cells e.g. anti- CD20 antibody
  • macrophages e.g. anti-CDl lb antibody
  • CD7 antibodies specific for CD45RO, CDl la, CD44, CD95, CXCR3, CCR4, CD56, CD57, CD244, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD123, HLA-DR, and/or CD235a.
  • positive selection for the naive T cells can be performed using antibodies specific for markers of naive T cell, e.g. CD62L, CD27, CD28, CCR7, CD45RA. Additionally or alternatively, selection can be performed using antibodies specific for CD4 or CD8 antigens, to enrich for CD4 or CD8 T cells.
  • specific for is meant that the molecule binds preferentially to the target of interest or binds with greater affinity to the target than to other molecules.
  • an antibody will bind to a molecule comprising an epitope for which it is specific and not to unrelated epitopes.
  • the affinity reagent may be an antibody, i.e. an antibody that is specific for a marker of T cells.
  • the affinity reagent may be a specific receptor or ligand for marker for T cells, e.g. a peptide ligand and receptor; effector and receptor molecules, a B-cell receptor specific for a TCR, and the like.
  • multiple affinity reagents specific for a marker of T cells may be used.
  • multiple affinity reagents, each specific for a different marker of T cells may be used.
  • Antibodies and T cell receptors may be monoclonal or polyclonal, and may be produced by transgenic animals, immunized animals, immortalized human or animal B-cells, cells transfected with DNA vectors encoding the antibody or T cell receptor, etc.
  • the details of the preparation of antibodies and their suitability for use as specific binding members are well- known to those skilled in the art.
  • Of interest in some embodiments is the use of antibodies as affinity reagents. Conveniently, these antibodies are conjugated with a label for use in separation.
  • Labels include magnetic beads, which allow for direct separation; biotin, which can be removed with avidin or streptavidin bound to a support; fluorochromes, which can be used with a fluorescence activated cell sorter; or the like, to allow for ease of separation of the particular cell type.
  • Fluorochromes that find use include phycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluorescein and Texas red. Frequently each antibody is labeled with a different fluorochrome, to permit independent sorting for each marker.
  • the initial population of cells is contacted with the affinity reagent(s) and incubated for a period of time sufficient to bind the available cell surface antigens.
  • the incubation may be at least 5 minutes. In some instances, the incubation is less than 60 minutes. It is desirable to have a sufficient concentration of antibodies in the reaction mixture, such that the efficiency of the separation is not limited by lack of antibody.
  • the appropriate concentration is determined by titration, but will typically be a dilution of antibody into the volume of the cell suspension that is 1 :50 (i.e., 1 part antibody to 50 parts reaction volume), 1:100, 1: 150, 1:200, 1 :250, 1:500, 1: 1000, 1:2000, or 1:5000.
  • the medium in which the cells are suspended will be any medium that maintains the viability of the cells, e.g. phosphate buffered saline containing from 0.1 to 0.5% BSA or 1-4% serum.
  • Various media are commercially available and may be used according to the nature of the cells, including Dulbecco' s Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco' s phosphate buffered saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplemented with fetal calf serum, BSA, HSA, serum etc.
  • dMEM Dulbecco' s Modified Eagle Medium
  • HBSS Hank's Basic Salt Solution
  • dPBS Dulbecco' s phosphate buffered saline
  • RPMI Iscove's medium
  • PBS with 5 mM EDTA etc.
  • the cells in the contacted population that become labeled by the affinity reagent, i.e. the subject T cells, are selected for by any convenient affinity separation technique, e.g. as described above or as known in the art. Following separation, the separated cells may be collected in any appropriate medium that maintains the viability of the cells, in some instances having a cushion of serum at the bottom of the collection tube.
  • Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequently supplemented with serum or artificial supplements.
  • Populations that are enriched by selecting for the expression of one or more markers of the selected phenotype, i.e. T cells, or naive T cells, will have 40% or more cells of the selected phenotype, in some instances 50% or more, 60% or more, 70% or more, or 80% or more cells, in some such instances 90% or more cells of the selected phenotype, e.g. 99% or 100% of the cells of the selected phenotype.
  • the enriched population of T cells or naive T cells may be transplanted immediately, e.g. if the complete leukocyte population was preserved and stored, and the population is enriched for T cells post-storage.
  • the enriched population may be preserved and stored as described above for preserving and storing leukocytes, and transplanted at a later date.
  • the selection of T cells from the complete leukocyte population occurs prior to preservation.
  • the selection process occurs after preservation, i.e. after storage.
  • selection/enrichment may be performed both prior to and after thawing.
  • the methods described above find use in enhancing the diversity of T cells in individuals.
  • Enhancing T cell diversity may be useful for the treatment and/or prevention of aging-related disorders in an individual.
  • treatment used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., at least inhibiting its progression, i.e. arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient is of interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. In some instances, the method will be performed during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • the terms "individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • Individuals that would benefit from the methods described herein include those suffering from general immunosuppression resulting from the natural aging process. As such, an individual that is 50 years or older, e.g. 55 or older, 60 or older, 65 or older, 70 or older, may benefit from enhancing the diversity of T cells.
  • Individuals that would benefit from these methods also include those suffering from general immunosuppression resulting from a medical therapy, e.g. a medical treatment that kills dividing cells, e.g. chemotherapy.
  • a medical therapy e.g. a medical treatment that kills dividing cells, e.g. chemotherapy.
  • an individual that is receiving or has received an immunosuppressive medical treatment may benefit from the above methods.
  • Premature aging of the immune system occurs from medical conditions that impose a chronic stress on the immune system and essentially create immune system exhaustion, for example, chronic inflammation, and autoimmune-related diseases such as Rheumatoid Arthritis, Psoriasis, demyelinating diseases (Multiple Sclerosis), Acute disseminated encephalomyelitis (ADEM)), and Diabetes Mellitus (DM).
  • Chronic stress on the immune system may also occur in conditions in which extensive replication pressure is applied on leukocytes, e.g. following a bone marrow transplant.
  • Other individuals that may benefit from the above methods include those that may benefit from an enhanced adaptive immune response to antigen, for example, individuals with cancer, where an enhancement in T cell diversity may provide an enhanced tumor antigen-specific adaptive immune response, or, for example, individuals with an acute or chronic infection, where an enhancement in T cell diversity may provide an enhanced infectious antigen-specific adaptive immune response.
  • HSCs hematopoietic stem cells
  • transplantation is preceeded by high-dose chemotherapy.
  • such medical treatment is not required to practice the subject methods.
  • medical therapies such as this or any other, e.g. a therapy to treat an aging-associated disorder, generally will not hinder the efficacy of the methods to rejuvenate the aged immune system and restore its ability to mount an adaptive immune response.
  • the subject methods may enhance T cell diversity and prevent, inhibit the progression of, or reverse autoimmunity, chronic inflammatory processes, infections and cancer e.g. for 5 years or more, 7 years or more, 10 years or more, including 15 years or more, 20 years or more, etc. Additionally, multiple transplantations, i.e. 2 or more, 3 or more, 4 or more, 5 or more transplantations may be performed to achieve more pronounced affect and/or to prolong the therapeutic effect for more years.
  • reagents, devices and kits thereof e.g. for practicing one or more of the above-described methods.
  • Reagents, devices and kits thereof according to various aspects of the invention may vary greatly.
  • reagents are provided.
  • reagents of interest may include any reagents useful in the harvesting, preservation, storage, or transplantation of leukocytes.
  • reagents of interest may include buffers, e.g. HEPES, phosphate buffers, lactate buffers, Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS), etc.
  • Reagents may include media, e.g. Dulbecco's Modified Eagle Medium (dMEM), RPMI, Iscove's medium, etc.
  • Reagents useful for preserving cells e.g. DMSO and serum, may also be included.
  • reagents of interest may include reagents for the enrichment of T cells, or of naive T cells, from the harvested leukocyte population.
  • reagents include, but are not limited to, affinity reagents that are specific to markers of T cells or naive T cells, affinity reagents that are specific for cells other than T cells for depleting undesired cells, lysis reagents for lysing erythrocytes, and the like.
  • devices are provided.
  • devices may include devices for the acquisition of a blood sample from an individual, e.g. needles, tubes, apheresis machinery, etc.
  • devices of interest may include devices for the delivery, i.e. transplantation, of a leukocyte population into an individual, e.g. needles, syringes, pumps, peripheral cannula, central IV line, etc., e.g. implantable port, tunneled line, central venous lines, peripherally inserted central catheters and the like.
  • kits are provided.
  • kits of interest may include one or more collection receptacles, e.g. a tube, flask, or bag of sufficient volume, for the collection of the blood sample e.g. 100 cc, 250cc, or 500 cc, and one or more preservation receptacles, e.g. a tube of sufficient volume for the preservation of the leukocyte population, e.g. 5 cc, 10 cc, 25 cc, 50 cc, or lOOcc screw-cap cryopreservation tubes.
  • kits of interest may include a preservative, e.g. a cryopreservative, for the preservation of leukocytes.
  • kits of interest may include reagents for the enrichment of T cells, or of naive T cells, from the harvested leukocyte population.
  • reagents include, but are not limited to,affinity reagents that are specific to markers of T cells or naive T cells, affinity reagents that are specific for cells other than T cells for depleting undesired cells, lysis reagents for lysing erythrocytes, and the like.
  • kits in accordance with embodiments of the invention may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded.
  • Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
  • EXAMPLE 1 The ability of aged mice transplanted with young lymphocytes to mount an immune response against an implanted lymphoma xenograft (BM-185-GFP pre-B cell mouse lymphoma) and suppress tumorigenesis was analyzed. It was earlier demonstrated that young BALB/c mice (3 months old) generate a strong immune response to injected BM-185-GFP pre-B cell mouse lymphoma cells and suppress tumor growth in 100% cases (survival rate 100% 60 days after tumor cells injection). At the same time, aged animals (18-22 months) are incapable of tumor growth suppression and succumb in 100% cases within 30 days of tumor cells injection (Lustgarten et al. (2004) J Immunol 173:4510-4515). In this experiment, aged mice that were pre-injected with lymphocytes obtained from the young animals were tested for their ability to generate an immune response and suppress tumorigenesis.
  • mice 22 months old mice intravenously injected 30 days before the experiment with 3xl0 7 lymphocytes obtained from young (3-month-old) BALB/c mice.
  • mice were subcutaneously injected with 10 5 lymphoma cells (BM-185-GFP pre-B cell mouse lymphoma). After 30 days, all mice of group 1 were alive and tumor free, with a pronounced immune response to the tumor cells. Mice of group 2 were incapable to provide sufficient anti-tumor response and all mice succumbed due to the tumor tissue growth. Of group 3, only 1 mouse succumbed, and tumor rejection and pronounced anti-tumor response were observed in the remaining 9 mice. These data indicate that the transplantation of lymphocytes from a young mouse into an aged recipient suppresses tumor formation and/or tumor growth in the implanted lymphoma model of tumorigenesis.
  • mice The ability of young lymphocytes to suppress tumorigenesis in aged mice was studied.
  • MCA Methylcholanthrene
  • mice Group 2. aged mice (3 months old)
  • mice 22 months old intravenously injected 30 days before
  • mice of group 1 MCA treatment with 3xl0 7 lymphocytes obtained from young (3-month-old) mice of the same strain. Each mouse was subcutaneously injected with 400 ug MCA. The development of tumors was studied for 130 days thereafter. In mice of group 1, the first tumors were observed 100 days after MCA injection (2 mice), and 20% of the mice (4 mice) had developed tumors 130 days after injection. In mice of group 2, the first tumors were observed 85 days after MCA injection in 3 mice (15%), and 60% of the mice (12 mice) developed tumors after 130 days. In group 3, the rate of tumorogenesis was comparable to that of group 1 : the first tumors were observed 100 days after MCA injection, and 130 days after injection, only 25% animals (5 mice) developed tumors. These data indicate that the transplantation of lymphocytes from a young mouse into an aged recipient suppresses chemically-induced tumorigenesis.
  • the ability of grafted naive lymphocytes from young animals to restore an aging- impaired immune system is studied.
  • the lymphocytes isolated from the lymph nodes of the young animals are transferred intravenously to aged 20-month old mice.
  • Green fluorescent protein (GFP) -transgenic C57BL/6 mice are used, which allows for the fate of the transferred T cells to be tracked.
  • the recipient aged mice are housed under standard nonsterile conditions in a conventional animal facility to provide the most stringent environment in which to test the resulting efficiency of the rejuvenated adaptive immunity.
  • the recipient aged mice are lethally irradiated before transplantation. This avoids possible graft rejection, allows a major portion of native T cells to be replaced, and provides synchronous controls with mice transferred with the aged lymphocyte grafts.
  • BMSCs bone marrow cells
  • recipient aged mice Such transfer results in viable aged bone marrow chimeric mice with depleted lymphocytes. These chimeric mice are then used as recipients of the lymphocytes isolated from young versus aged animals or as control animals.
  • Target group (20 mice): irradiated aged mice, transplanted on the next day with senescent bone marrow cells and three days later with the GFP-positive lymphocytes from young donors;
  • mice non-irradiated aged mice, not transplanted;
  • mice 100 aged recipient mice are involved, of which 70 mice are irradiated. Additionally, 10 aged mice serve as donors of aged BMSC; and 5 young GFP-positive mice and 5 aged GFP- positive mice serve as lymphocyte donors. 20 unmanipulated young animals serve as a control group for the comparative analysis.
  • BMSC are harvested from 10 aged mice.
  • the cell suspension is injected into tail vein of mice in groups 1, 4 and 5 (3xl0 6 cells per mouse).
  • lymphocytes are isolated from the lymph nodes of 3-months old GFP-positive transgenic mice.
  • the single cell suspension is prepared and introduced into tail vein of mice in group 5 (20x10 6 cells/100 ml per mouse, in PBS). Typically, 50-60% of the cells obtained are GFP+CD3+.
  • the rejuvenation effect depends on efficiency of young T cell proliferation and their stable abundance for the long term period after transplantation.
  • a sample of blood is collected from the retro-orbital sinus of mouse eye once every 2 months.
  • the lymphocytes populations are analyzed by multicolor flow cytometry for surface lymphocyte markers such as CD3, CD19, CD4 and CD8, along with the GFP signal that indicates graft origin.
  • naive T cells Percentage of naive T cells. With aging, the proportion of the naive T cells declines, while effector subsets increase. The balance between the naive and effector lymphocytes is evaluated to determine how it is changed with time in our experimental mice groups. The percentage of naive T cells is examined among CD3+GFP+ and CD3+GFP- populations using antibodies specific for CD62, CD27 and CCR7. It is expected that the percentage of naive T cells is increased in the target group (group 1) as compared to groups 2, 4, and 5.
  • telomere shortening One hallmark of cellular senescence is telomere shortening, and it has been reported that the length of telomeres in T cells decreases with aging. Using fluorescence in situ hybridization (DAKO Telomere PNA Kit/FITC for Flow Cytometry), the relative telomere length of total CD3+ populations in different experimental mouse groups is compared. It is expected that the T cells of the target group have longer telomeres compared to all control groups, excluding the young animals control group. 5. Production of cytokines. It has been shown in humans and mice that there are age- related alterations in cytokine productions.
  • the levels of circulating pro-inflammatory cytokines including IL-1, IL-6, INF-gamma and TNF-alpha in response to administration of lipopoly saccharide (LPS) is examined. It has been shown that increased level of these factors is associated with aging (Tateda, 1996). Excluding the young animals control group, it is expected that the target group (group 1) has the lowest level of circulating proinflammatory cytokines.
  • TCR diversity analyzed by next generation sequencing Next generation sequencing (NGS) and advanced bioinformatics are used to compare TCR beta diversity for the target group of animals (group 1) and control groups. It is expected that TCR beta chains diversity in the target group 4 months after transplantation is the highest among all groups excluding the young animals control group.
  • NGS Next generation sequencing
  • advanced bioinformatics are used to compare TCR beta diversity for the target group of animals (group 1) and control groups. It is expected that TCR beta chains diversity in the target group 4 months after transplantation is the highest among all groups excluding the young animals control group.
  • Viral infections A standard panel of viral infections is monitored using serological profiles. It is expected that, excluding the young animals control group, the target group (group 1) have the lowest infection rate.
  • the ability of grafted naive lymphocytes from young animals to restore an aging- impaired immune system is studied.
  • the lymphocytes isolated from the lymph nodes of the young animals are transferred intravenously to aged 20-month old mice once per month.
  • CellTrackerTM Green CMFDA is used to label transferred cells, which allows for the fate of the transferred T cells to be tracked.
  • the recipient aged mice are housed under standard nonsterile conditions in a conventional animal facility to provide the most stringent environment in which to test the resulting efficiency of the rejuvenated adaptive immunity.
  • the recipient aged mice are not irradiated or treated any other way before the start of graft injections.
  • Target group 50 mice. These mice, starting from the age of 20 months, are transplanted with lymphocytes derived from young donors once per month.
  • Control group 50 mice: non-treated aged mice;
  • mice 100 aged recipient mice are involved, of which 50 mice are transplanted with lymphocytes derived from young donors. Additionally, 100 unmanipulated young animals serve as lymphocyte donors. Additionally, 30 unmanipulated young animals serve as a control group for the comparative analysis.
  • Lymphocytes are isolated from the lymph nodes of 3-5-months old mice.
  • the single cell suspension is prepared and introduced into tail vein of mice in target group 1 (20x10 6 cells/100 ml per mouse, in PBS).
  • naive T cells Percentage of naive T cells. With aging, the proportion of the naive T cells declines, while effector subsets increase. The balance between the naive and effector lymphocytes is evaluated to determine how it is changed with time in our experimental mice groups. The percentage of naive T cells is examined using antibodies specific for CD62, CD27 and CCR7. It is expected that the percentage of naive T cells is increased in the target group (group 1) as compared to group 2.
  • telomere shortening One hallmark of cellular senescence is telomere shortening, and it has been reported that the length of telomeres in T cells decreases with aging. Using fluorescence in situ hybridization (DAKO Telomere PNA Kit/FITC for Flow Cytometry), the relative telomere length of total CD3+ populations in different experimental mouse groups is compared. It is expected that the T cells of the target group have longer telomeres compared to the control group.
  • cytokines production of cytokines. It has been shown in humans and mice that there are age- related alterations in cytokine productions. 6 months after first T cell transfer, the levels of circulating pro-inflammatory cytokines including IL-1, IL-6, INF-gamma and TNF-alpha in response to administration of lipopoly saccharide (LPS) are examined. It has been shown that increased level of these factors is associated with aging (Tateda, 1996, Infect. Immun., 64, pp. 769-774). It is expected that the target group (group 1) has lower level of circulating proinflammatory cytokines.
  • LPS lipopoly saccharide
  • TCR diversity analyzed by next generation sequencing.
  • Next generation sequencing (NGS) and advanced bioinformatics are used to compare TCR beta diversity for the target group of animals (group 1) and control group 2. It is expected that TCR beta chains diversity in the target group 6 months after transplantation is higher compared to the control group.
  • a 35 year old male is concerned that he may develop an age-related disease later in life. He undergoes apheresis to have a pint of blood drawn and the leukocytes isolated. A sample of 1 billion leukocytes is harvested, preserved in a cryopreservation reagent, frozen on liquid nitrogen, and stored at -190°C.
  • massive sequencing of T cell receptors demonstrates that the T cell repertoire of his immune system has decreased more than 20-fold as compared to the repertoire at age of 35. His frozen leukocytes are retrieved and transferred into his blood stream. 9 months later, massive sequencing shows that the diversity of his T cells is enhanced by more than 5-fold. 5 years later, he is still alive and has developed no age-related diseases.
  • a 36 year old female is concerned that she may develop an aging-related disease later in life. She has a sample of 1 billion leukocytes harvested, preserved, and stored. At age 45, she develops multiple sclerosis, an autoimmune disease that affects the nerve cells in the brain and spinal cord. At age 55, after ineffectual therapy with chemical drugs, she undergoes autologous hematopoietic stem cell transplantation and chemotherapy following a standard EBMT (European Group for Blood and Marrow Transplantation) protocol. The therapy results in recovery from multiple sclerosis, however she begins suffer from herpes-virus-induced aphthous stomatitis with recurrences three times a year. At age 58, she receives the leukocyte sample that was frozen 22 years ago.

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Abstract

Des aspects de l'invention concernent des procédés, compositions et trousses pour l'augmentation de la diversité de lymphocytes T chez un individu. Ces procédés, compositions et trousses sont utiles dans un nombre d'applications, telles que l'amélioration de la protection immunitaire, l'inhibition de la lymphopénie, le traitement de l'épuisement des lymphocytes T, l'inhibition de l'immunosénescence et/ou la compensation de la perte de lymphocytes T due à l'immunosénescence, et la prévention de troubles liés à l'âge.
PCT/IB2012/001784 2011-07-22 2012-07-17 Procédés et compositions pour l'augmentation de la diversité des lymphocytes t WO2013014535A1 (fr)

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WO2017015490A1 (fr) * 2015-07-21 2017-01-26 City Of Hope Lymphocytes t pour l'expression de récepteurs d'antigènes chimères
WO2018185166A1 (fr) * 2017-04-05 2018-10-11 Adaptimmune Ltd Procédés d'isolement de cellules t régulatrices naïves
US11634497B2 (en) 2017-02-03 2023-04-25 Novartis Ag Anti-CCR7 antibody drug conjugates
WO2025026131A1 (fr) * 2023-07-28 2025-02-06 上海市第一人民医院 Procédé de préparation de lymphocytes tcd8 + naïfs et utilisation correspondante dans la préparation de médicaments destinés à retarder la sénescence

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104360050A (zh) * 2014-09-22 2015-02-18 重庆医科大学附属儿童医院 一种淋巴细胞免疫分型的方法和试剂盒
CN104360049A (zh) * 2014-09-22 2015-02-18 重庆医科大学附属儿童医院 一种t淋巴细胞免疫分型的方法和试剂盒
WO2017015490A1 (fr) * 2015-07-21 2017-01-26 City Of Hope Lymphocytes t pour l'expression de récepteurs d'antigènes chimères
US12115189B2 (en) 2015-07-21 2024-10-15 City Of Hope T cells for expression of chimeric antigen receptors and other receptors
US11634497B2 (en) 2017-02-03 2023-04-25 Novartis Ag Anti-CCR7 antibody drug conjugates
US12180291B2 (en) 2017-02-03 2024-12-31 Novartis Ag Anti-CCR7 antibody drug conjugates
WO2018185166A1 (fr) * 2017-04-05 2018-10-11 Adaptimmune Ltd Procédés d'isolement de cellules t régulatrices naïves
WO2025026131A1 (fr) * 2023-07-28 2025-02-06 上海市第一人民医院 Procédé de préparation de lymphocytes tcd8 + naïfs et utilisation correspondante dans la préparation de médicaments destinés à retarder la sénescence

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