Classifications

• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • 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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/40—Regulators of development
  • C12N2501/42—Notch; Delta; Jagged; Serrate
• • 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
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
  • C12N2502/1114—T cells
• • 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
  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
• • 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
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
• • 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
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/52—Fibronectin; Laminin

Definitions

• the invention relates to the field of cell therapy and in particular hematopoietic stem cell transplants, transformed or not, and to immune reconstitution following such grafts.
• HSPC Hematopoietic Stem Cell Transplantation
• T lymphocyte differentiation pathway T cell progenitors
• T cell precursors are derived from a differentiation of HSPC CD34 + and in particular have the CD7 + marker, which is a differentiation marker in the T cell pathway. They may also have other markers.
• ETPs early thymic progenitors
• proT1 stage cells CD7 ++ / CD5-
• proT2 stage cells CD7 ++ / CD5 +
• preT stage cells CD7 ++ / CD5 + CD1 a +
• a concomitant T precursor graft at the HSPC graft would allow the rapid production of a mature and functional T lymphocyte compartment, and thereby prevent the risk of severe infections by allowing the patient to benefit from a minimum level of immunity before the total reconstitution of the immune system.
• Notch proteins are transmembrane receptors that regulate the cellular response to a large number of environmental signals.
• 4 Notch receptors (Notch 1-4) and five ligands (Delta-like-1, 3, and 4, Jaggedl, Jagged2) have been described (Weinmaster Curr Opin Genet Dev. 2000; 10: 363-369). .
• the Notch ligands have several denominations.
• the ligand Delta-like ligand 4 can be designated as:
• the Notch ligands Delta-like-1 and Delta-like-4 may be designated respectively by DL1 and DL4 or by DL-1 and DL-4.
• the DL-1 and DL-4 ligand sequences are specified as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
• Notch-1 The interaction of Notch-1 with DL-1 or DL-4 ligands has been shown to play an important role in early T lymphopoiesis.
• the aim is to develop a method for generating and increasing the number of CD7 + T lymphocyte precursors from stem cells hematopoietic CD34 + without using a cellular stroma.
• the hematopoietic stem cells have been isolated from an adult donor.
• T lymphoid potential of the progenitors generated in culture has been evaluated in vitro and in vivo.
• quantitation of the T-lymphoid potential under boundary dilution conditions in secondary cultures on an OP9 stroma expressing the DL-1 ligand demonstrated an increase of more than 200-fold over uncropped CD34 + HPSCs.
• this potential was determined by injection of these cells into 4 week irradiated NOD / SCID / vc - / - mice or neonates.
• the DL4 / Fc system makes it possible to obtain about 6.5 ⁇ 10 5 T progenitors from 10 6 CD34 + of cord blood. This number may be insufficient for an adult receiving a cord blood transplant.
• WO 201 1/068962 discloses methods of differentiating T or NK lymphocytes in vitro from hematopoietic stem cells or precursor cells.
• the first step of the T cell differentiation method allows the production of T cell progenitors by culturing the stem cells on a layer of transformed OP-9 nerve cells expressing the DLL Notch ligand
• the first example illustrates the generation of T cell progenitors from murine bone marrow stem cells.
• WO 2014/1 10353 relates to compositions comprising an osteoinductive Notch ligand bound to at least one biocompatible substrate. It also relates to methods of treating patients requiring bone formation by administering a composition comprising an osteoinductive Notch ligand bound to at least one biocompatible substrate. It is therefore necessary to develop a new method for improving the amount of T-cell precursors generated from CD34 + cells. Furthermore, this method should also make it possible to obtain T-cell precursors, including from transduced CD34 + HSPCs, which can be applicable to adult CD34 + cells for allogeneic transplants or autografts, thus making it possible to overcome the number relatively. weak cord blood cells.
• the inventors have shown that the amount of T cell precursors can be improved from CD34 + cells by exposing said CD34 + cells to a Notch ligand, in the presence of a fragment of fibronectin, containing an RDGS motif (SEQ ID No. 3, Arginine-Glycine-Aspartate-Serine), and / or a CS-1 motif and optionally a heparin-binding domain.
• a fragment of fibronectin containing an RDGS motif (SEQ ID No. 3, Arginine-Glycine-Aspartate-Serine), and / or a CS-1 motif and optionally a heparin-binding domain.
• said fibronectin fragment contains an RDGS motif, a CS-1 motif and a heparin binding domain.
• This joint exposure of CD34 + cells to the Notch ligand and to this fibronectin fragment also makes it possible to induce the differentiation of CD34 + cells tranducted towards the T lymphoid pathway, which allows the use of the method for gene therapy transplants.
• RGDS motif and / or the CS-1 motif in the fibronectin fragment.
• methods and methods described below are also applicable by using an RGDS peptide and / or a CS-1 peptide in place of the fibronectin fragment.
• use of RGDS and CS-1 peptides is preferred, particularly within the same protein.
• an RGDS peptide and / or a CS-1 peptide are used, they can thus be present as such within the culture medium, or present within a peptide or a protein present in said medium. .
• the culture medium contains only the RGDS peptide and / or the Cs-1 motif as such, we will speak of "free" peptide in said medium if this peptide is not immobilized on the bottom wall of the container. Indeed, the peptide may be present in solution, or immobilized on the bottom wall of the container in which exposure of CD34 + cells to the Notch ligand is performed.
• the invention relates to a method
• a method for generating T cell progenitors comprising the step of exposing CD34 + cells in a Notch ligand-containing medium in the presence of a fibronectin fragment.
• the Notch ligand is immobilized on a support.
• the fibronectin fragment contains an RDGS motif, a CS-1 motif, and a heparin binding domain.
• the process according to the invention is carried out in vitro, in a container (such as a cell culture dish (Petri dish, 24-well box, etc.)) on the bottom wall of which the Notch ligand is immobilized.
• a container such as a cell culture dish (Petri dish, 24-well box, etc.)
• the Notch ligand can also be immobilized on any other support present in the reaction medium, in particular the surface of microbeads.
• the immobilization of the Notch ligand is aimed essentially at stabilizing the ligand to allow activation of the Notch receptor.
• T cell progenitor any cell engaged in the differentiation pathway to the T lymphoid pathway from a HSPC CD34 +. This cell is therefore characterized in that it expresses the marker CD7, which is known to be one of the markers that appears most early during T cell lymphopoiesis. Depending on the differentiation state in the T lymphoid pathway, it can express or not the CD34 marker (loss of CD34 during differentiation).
• T-cell progenitors are cells that can be found in the post-natal thymus, ie early thymic progenitor (ETP) thymus cells (CD34 + / CD45RA + /). CD7 +), proT1 cells (CD34 + CD45RA + CD7 + CD5-CD1 +), proT2 cells (CD34 + CD45RA + CD7 + + CD5 + CD1 +), and preT cells (CD34 + CD7 + / CD5 +). CD1 a +). These cells are well known in the state of the art. They are cited by Reimann et al (2012, op cit), and by Awong et al (2009, op.cit).
• RGDS peptide any peptide or protein containing the RGDS motif, so that it can bind to VLA-5 integrin (see below.) Such a peptide or protein can be tested by its ability to suppress the interaction of fibronectin with this VLA-5 integrin, by methods widely documented in the art.
• the RGDS peptide binds to the VLA-5 integrin (Very Late Antigen-5), which is a dimer composed of CD49e (alpha5) and CD29 (betal).
• VLA-5 integrin Very Late Antigen-5
• heparin binding domain is particularly advantageous when also exposing CD34 + cells to a vector. retroviral for the purpose of transducing them, in order to obtain T-cell progenitors expressing a transgene.
• a CS-1 peptide or CS-1 motif is a 25 amino acid peptide (DELPQLVTLPHPNLHGPEILDVPST, SEQ ID NO: 6), which has been described by Wayner et al., 1989, J. Cell Biol. 109: 1321).
• This CS-1 motif binds to the VLA-4 (Very Late Antigen-4) receptor.
• This antigen is a dimeric integrin composed of CD49d (alpha 4) and CD29 (beta 1).
• the fragment of fibronectin is present in the culture medium or immobilized on the bottom wall of the container.
• Fibronectin is a protein, which in its natural form is a large v-shaped dimer of 100 nm in length and 460kDa. The two monomers are connected by two disulfide bridges at their C-terminus.
• fibronectin is meant the natural fibronectin protein (i.e. any isoform produced by alternative splicing), but also a monomer of this protein, or a fragment of this protein (but containing the peptide RGDS, as well as CS-1 peptide and heparin binding site).
• a fibronectin particularly suitable for the implementation of the process according to the invention is Retronectin®.
• This protein corresponds to a fragment of a human fibronectin (CH-296 fragment, Kimizuka et al., J. Biochem 1991 Aug; 1 (2): 284-91; Chono et al., J. Biochem. 2001 Sep; 130 (3 ): 331-4) and contains the three preferred functional domains in the implementation of the method (the C-cell binding domain containing the RGDS peptide, the heparin binding domain and the CS-1 sequence ).
• This protein is already used to improve transduction using retroviral vectors (Chono et al, op cif), by allowing colocalization of target cells (VLA-4 and VLA-5 integrin binding) and virions (which bind to the domain heparin binding).
• This protein is marketed by Takara Bio Inc. (Shiga, Japan).
• this protein can also improve the differentiation of CD34 + cells into T cell progenitors, including when it is desired to obtain such progenitors for viral vector-transduced cells as part of a gene therapy. as well as for adult cells.
• this improvement in the differentiation of CD34 + cells is probably related to the presence of the RGDS motif that binds to the VLA-5 integrin present on the cells and / or of the CS-1 motif that binds to the VLA-4 receptor.
• the fibronectin fragment is immobilized (i.e., bound to a solid support and is not present freely in solution (although it is possible that some elements may
• This solid support is preferably the bottom wall of the container in which the process is carried out, but it is also possible to envisage binding the fibronectin fragment to beads, such as polymer beads or magnetic beads (generally between 1 ⁇ and 5 ⁇ in diameter)
• beads such as polymer beads or magnetic beads (generally between 1 ⁇ and 5 ⁇ in diameter)
• the binding of the protein or peptide on these beads may be covalent or non-covalent.
• a peptide on a bead is well known in the art, and this fragment of fibronectin can also be introduced into a semi-solid medium, such as an agar or a gel.
• this immobilization can also be covalent or not.
• this immobilization is performed non-covalently by allowing the fibronectin fragment to be absorbed on the glass or plastic component of the bottom wall of the container.
• the inventors have been able to demonstrate that when previously transduced cells are exposed to the Notch ligand (in particular DL-4), the production of T lymphocytes in vivo is slower than when the transduction is performed at the same time as exposure to DL-4.
• the joint exposure thus makes it possible to accelerate the production of T cells in vivo (after transplantation).
• a second advantage of this joint exposure of the cells to the viral supernatant and the Notch ligand (in particular DL-4) is to shorten the culture period (7 days instead of 11 days).
• a joint exposure of the cells to the viral supernatant and to the Notch ligand (in particular DL-4) without the presence of the fibronectin fragment also makes it possible to accelerate the production of T cell progenitors, and is another aspect. of the invention, according to the same conditions as in the presence of the fibronectin fragment.
• cells are first exposed to the Notch ligand and fibronectin fragment for a certain time (preferably greater than 4 hours, more preferably greater than 6 hours, or greater than 8 hours or greater at 10h, but preferably less than 36 hours, more preferably less than 30 h, more preferably less than 24 h), in the presence of cytokines, which allows the cells to enter the division cycle (preactivation), then the viral supernatant is added for a certain time (preferably greater than 4 hours, more preferably greater than 6 hours, or greater than 8 hours or greater than 10 hours, but preferably less than 30 hours, more preferably less than 24 hours; h, more preferably less than 16 hours on average).
• transgene introduced by transduction with the viral supernatant is intended to express an absent or deficient protein in the patient in order to provide him with a therapeutic benefit.
• transgenes that can be used (without this list being exhaustive or exhaustive)
• immunodeficiency therapy especially the severe combined immunodeficiencies, SCID or not, CID
• HIV adrenoleukodystrophy linked to the X
• hemoglobinopathies including ⁇ -thalassemia or sickle cell anemia.
• Innate or acquired immunodeficiencies AIDS, due to HIV are therefore diseases that can be targeted by gene therapy strategies.
• the transduction is carried out preferentially with a retroviral supernatant allowing insertion of the transgene into the genome of the cell.
• the vectors, especially lentiviral vectors, present in said supernatant are particularly suitable for this type of application and have been widely described in the literature.
• the medium may also contain a viral vector for the transduction of said CD34 + cells, in the implementation of the method according to the invention.
• this viral vector is introduced after a period of preactivation of the cells in the medium (culture in the presence of cytokines allowing the cells to enter the dividing cycle), between 4 h and 36 h, preferably between 6 and 24 h.
• the viral vector is maintained in the medium in contact with the cells for a period of between 4 h and 30 h, preferably between 12 h and 24 h, more preferably for about 16 h, and is removed from the medium.
• To "remove" the viral vector from the medium the cells are harvested, washed and resuspended in the presence of the Notch ligand and the fibronectin fragment.
• said Notch ligand is the protein
• Delta-like-1 SEQ ID No. 1
• soluble domain SEQ ID No. 1
• the Notch ligand is Delta-like-4 protein (SEQ ID NO: 2).
• said Notch ligand is a fusion protein comprising the soluble domain of a natural Notch ligand fused to an Fc region of an IgG protein.
• the soluble domain of a Notch ligand represents the extracellular portion of said ligand.
• Varnum-Finney et al (Cell Sci 2000 Dec 1313 Pt 23: 4313-8) described a fusion protein of the soluble portion of DL-1 with a Fc portion of a IGg1.
• Reimann et al (op cit) have described a fusion protein of the soluble part of DL-4 (amino acids 1-526) with the Fc fragment of an IgG2b immunoglobulin. It is thus preferred when the IgG protein is IgG2.
• the culture medium used in the context of the present invention is any medium capable of culturing CD34 + cells and T cells.
• a perfectly adapted and preferred culture medium for carrying out the process according to the invention is X-VIVO TM medium (Lonza, Basel, Switzerland). This medium has been used in particular by Jonuleit et al (Eur J Immunol, 1997, 27, 12, 3135-42) and Lucas et al (J Immunol, 1998, 161, 4, 1947-53).
• a basal medium that is to say, which allows the growth of the cells without the need to add supplements
• serum as well as growth and cytokines
• fetal bovine serum (FBS) or fetal calf serum (FCS), autologous human serum or human serum AB are preferably added to the basal culture medium.
• this medium is supplemented with at least 15% of fetal serum, more preferably at least 20%.
• the FBS is particularly suitable for the implementation of this method.
• the defined FBS is a high quality serum that has been analyzed and filtered to prevent the presence of virus particles. It is sold as such by many suppliers, such as Thermo Scientific TM HyClone TM Defined Fetal Bovine Serum (FBS).
• the culture medium is also preferably complemented with cytokines and growth factors.
• cytokines and growth factors are especially chosen from the group consisting of SCF (stem cell factor), thrombopoietin (TPO, also called megakaryocyte growth and development factor, MGDF), Flt3-ligand (which is a growth factor). hematopoietic), interleukin 3 (IL-3), interleukin 7 (IL-7) and SCF (stem cell factor)
• the culture medium contains at least three, preferably at least four of these cytokines or growth factors.
• At least three or more cytokines are added.
• these three cytokines are N-interleukin-7 (IL-7), SCF (Stem Cell Factor) and Flt-3 Ligand (hematopoietic growth factor).
• IL-7 N-interleukin-7
• SCF Stem Cell Factor
• Flt-3 Ligand hematopoietic growth factor
• four cytokines i.e., the three cytokines mentioned above and TPO (thrombopoietin), are added.
• the nature of the cytokines and growth factors can be varied during the implementation of the method.
• the aforementioned cytokine and growth factor mixtures are sufficient to induce the differentiation of CD34 + cells into T-cell precursors, and generally the culture medium does not include any other cytokine or growth factor.
• the total exposure time of the CD34 + cells in the presence of the Notch ligand and of the protein or peptide having the RGDS motif is generally carried out for a duration preferably greater than 3 days, and less than 10 days.
• CD34 + cells are obtained from a donor blood sample, a bone marrow puncture, or from umbilical cord blood. Methods for screening CD34 + cells are known in the art. It is in particular possible to use magnetic beads having an antibody recognizing CD34 + on their surface to do this.
• the cell culture vessel is prepared by immobilizing the Notch ligand and the fibronectin fragment on the lower surface, before exposing the CD34 + cells.
• Retronectin® or another fibronectin naturally adheres to the plastic of the cell culture dish (petri dish or 24-well box, or other).
• the following protocol can be used to cover the lower surface with the Notch ligand fused with an Fc fragment, and the Retronectin®:
• Retronectin® 25 to 50 ⁇ g / ml
• PBS Phosphate Buffered Saline
• PBS + 2% BSA (bovine or human serum albumin) or HA (human hemagglutinin)) 0.5 ml for 24-well plates, 2ml for 6-well plates for 1 hour at 37 ° C
• the inventors have shown that at the concentration of 5 ⁇ g / ml, about 75% of DL-4 is fixed on the surface of the container. Different doses of DL-4 have been tested and it is preferable for the concentration to be greater than or equal to 1.25 ⁇ g / ml, an optimum concentration being between 2.5 and 5 ⁇ g / ml. With regard to the retronectin, the concentration of 25 ⁇ is perfectly adapted, although other concentrations (higher or lower) may also be suitable.
• the invention thus also relates to a cell culture vessel, characterized in that at least one of its surfaces is covered with a Notch ligand and a protein or peptide having the RGDS motif and or CS-1, and in particular a fragment of fibronectin as described above.
• the Notch ligand is the soluble part of the natural Notch ligand (in particular DL-1 or DL-4) and is fused with an Fc fragment of an immunoglobulin (such as IgG1 or IgG2).
• an immunoglobulin such as IgG1 or IgG2.
• the Fc fragment is derived from an IgG2.
• the Notch ligand immobilized on the surface of the culture dish (and used in the context of the process according to the invention) has the sequence SEQ ID No. 7.
• the protein having the RGDS motif is as described above. It is preferentially a fibronectin and in particular Retronectin®.
• the cell culture dish has at least one surface that has been coated with a DL-4 / Fc fragment fusion protein of IgG2 and Retronectin®.
• the concentrations are as mentioned above, and the container was obtained, in particular by the method described above, that is to say a step of adding a solution containing the Notch ligand, the protein or peptide having the RGDS and / or CS-1 motif, in particular the fragment of fibronectin in said container, a resting step allowing the proteins to settle and adhere to the walls of the container, followed by rinsing steps and preferentially blocking the walls of the container with a non-reactive protein (such as serum albumin).
• a non-reactive protein such as serum albumin
• the cells are added at a concentration of between 10 6 and 10 7 CD34 + / ml cells, in particular of the order of 2 ⁇ 10 6 CD34 + / ml cells in the culture vessel.
• a box ranging from 2 to 10 cm 2 (respectively, a plate ranging from 24 wells to 6 wells).
• each well is inoculated with 5 5 and 10 6 CD34 + cells per well, preferably of the order of 2.10 5 to 4 ⁇ 10 5 CD34 + cells per well.
• use is preferably made between 8 ⁇ 10 5 and 2 ⁇ 10 6 cells per well.
• the amount of cells to be inoculated can be easily adapted by those skilled in the art depending on the container that it uses.
• the cells are placed in the well in the basal medium chosen, preferably supplemented with cytokines or growth factors, as seen above.
• the concentrations of these cytokines or growth factors are between 2 and 300 ng / ml.
• the cytokines are added at a concentration generally greater than 40 ng / l and less than 300 ng / ml or 200 ng / l, more preferably at a concentration of the order of 100 ng / ml.
• T cell progenitors when it is desired to generate transduced T cell progenitors and the cells are pre-activated before bringing them into the presence of the viral vector, higher concentrations (of the order of 300-400) can be used. ng / ml).
• SCF and Flt3-L can be used at concentrations of the order of 300 ng / ml, TPO and IL-7 at concentrations of the order of 100 ng / ml, and IL-3 at about 40 ng / ml.
• the duration of exposure of the cells to the Notch ligand and to the fibronectin fragment is preferably greater than or equal to 3, or even greater than or equal to 5 days. It is generally less than 10 days, or even less than 8 days. An advantageous period is preferably between 5 and 8 days, that is to say about 7 days. If it is desired to exceed 7 days of culture, it is preferable to transfer the cells to new receptacles coated with Notch ligand and fibronectin fragment. This time depends in particular on the origin of the CD34 + cells. Thus the duration of exposure may be shorter for CD34 + cells obtained from an adult than for cells obtained from a child or cord blood. This is particularly surprising, given the prior art and the ability a priori less differentiation of adult stem cells. The transduced cells are generally exposed longer than the non-transduced cells.
• T cell progenitors After generating the T cell progenitors, by the method described above, it is possible to purify these T cell progenitors thus generated. This purification is carried out by washing the cells and suspending them in a suitable basal medium.
• T cell progenitors can also be packaged in a pouch so that they can be injected into a patient. In this case, these cells are reconditioned in a saline solution containing 5% SAH such as albunorm TM 5% 50 g / L (Octopharma, Lingolsheim, France).
• SAH 5% SAH
• These cells can also be frozen according to methods known in the art.
• the invention also relates to T cell progenitors for use in an immunocompromised patient, in particular to allow immune reconstitution in this patient and / or obtaining immune protection against infections in said patient during a period of the order of a few months (at least two months, preferably at least six months).
• the patient is an immunocompromised patient.
• the reasons for the deficiency can be multiple: inherited immunodeficiency, chemotherapy for leukemia, conditioning, transplant containing only stem cells, post-transplant treatment in prophylaxis of GVH (graft-versus-host reaction), age of the recipient, and complications of the type infections.
• it can be immunocompromised due to the depletion of its immune cells following therapy before bone marrow transplantation.
• the graft may be an allograft (in this case, the T cell progenitors are preferentially derived from an HLA donor partially compatible with the patient), or an autograft (in which case the T cell progenitors have preferentially have been transformed by a vector in order to express a gene and / or a protein making it possible to correct a genetic defect in said patient).
• an allograft in this case, the T cell progenitors are preferentially derived from an HLA donor partially compatible with the patient
• an autograft in which case the T cell progenitors have preferentially have been transformed by a vector in order to express a gene and / or a protein making it possible to correct a genetic defect in said patient.
• the T cell progenitors were obtained by exposure of CD34 + cells in the presence of a Notch ligand. (as described above) and a protein or peptide having the RGDS motif and / or the CS1 motif, in particular a fragment of fibronectin as described above, under the conditions mentioned above .
• the exposure be less than or equal to 10 days.
• the T cell progenitors envisaged in particular have the CD7 marker, and may or may not express the CD34 marker.
• the invention relates to a method of treating an immunocompromised patient, in particular to allow at least temporary immune reconstitution in this patient, comprising the step of administering to said patient T cell progenitors, such as than described earlier.
• This method may also include the step of obtaining such progenitors by exposing CD34 + cells to a Notch ligand (as described above) and a protein or peptide having the RGDS motif and / or the CS1 motif, in particular a fragment of fibronectin as described above, under the conditions mentioned above.
• a therapeutically effective amount that is to say of the order of 1 to 5 ⁇ 10 6 / kg progenitors, is administered, which makes it possible to provide the patient with cells capable of playing a protective role with respect to infections for a few months. month (of the order of 6 months).
• this administration of T cell progenitors is carried out just before, just after or concomitantly with a hematopoietic stem cell transplant in said patient.
• the injected cells can be transformed by a vector to allow the correction of a genetic defect in said patient.
• Figure 1 Comparison of the number and percentage of percentage and number of CD7 + CD34 +/- cells obtained after culture of CD34 + cells in absence (- RN) or presence (+ RN) of retronectin (RN).
• A flow cytometric analysis of the expression of CD34, CD7 markers.
• B Representation of the number of CD34-CD7 + cells (for six independent experiments).
• FIG. 2 comparison of the expression (with respect to the GAPDH control (glyceraldehyde-3-phosphate dehydrogenase)) of genes involved in the differentiation of CD34 + cells into Tcf-7 (transcription factor 7) and Lef-1 (lymphoid) lymphocyte lymphocytes enhancer-binding factor 1).
• Bcl1 genes 1b B-cell CLUlymphoma 11B
• LCK lymphocyte-specific protein tyrosine kinase
• CD34 + cells are isolated by Ficoll gradient mononuclear cell separation and immunomagnetic screening, as described in the prior art (Six et al., J Exp Med 2007 204: 3085). b) Preparation of Soluble Notch Ligand
• Soluble Notch ligand is a fusion protein generated by cloning the soluble portion of the Notch ligand and the human immunoglobulin constant portion as described in Reimann et al (2012, op cit).
• the sequence of the fusion protein is SEQ ID NO: 7.
• the container is composed of a culture well containing culture medium, on which the soluble Notch ligand has been immobilized according to the technique described in Reimann et al. (2012, op cit).
• the retronectin is immobilized together with the soluble Notch ligand in the culture well.
• the concentration of retronectin is 25 to 50 ⁇ g ml. It is operated at room temperature or at 4 ° C., under Microbiological Safety Positions (PSM). d) Exposure of CD34 + cells
• the CD34 + cells are cultured in the vessel at a concentration of between 2x10 4 and 1.5x10 5 cells / ml.
• the culture lasts between 3 and 10 days, as described in Reimann et al. (2012, op cit).
• e) Analysis of CD34 + cells comprises: a count of the living cells, a flow cytometric analysis of the expression of the markers CD34, CD7 and possibly CD5 and CD1a and by a secondary co-culture under conditions of limiting dilution on a stroma OP9 / DL-1 which quantifies T precursors generated by culture (Reimann C
• Patient bone marrow CD34 + cells are preactivated for 24h at a concentration of 2 ⁇ 10 6 cells / in an X-vivo medium containing SCF 300ng / ml, Flt3-L 300ng / ml, TPO 100ng / ml, IL-3 40ng / ml , IL-7 100ng / ml and 20% serum on a substrate obtained after having covered the culture plates with a solution of retronectin® 50 ⁇ g / ml and DL4 / Fc 5 ⁇ g / ml.
• the cells are transduced with the viral vector in the pre-activation medium enriched with protamine sulfate at 4 ⁇ g / ml.
• the cells are harvested, washed and resuspended until D7 on a DL4 / Fc protein substrate in serum-supplemented medium (20%), SCF (100 ng / ml), Flt3-L (100 ng / ml) , TPO (100 ng / ml) and IL-7 (100 ng / ml).
• SCF 100 ng / ml
• Flt3-L 100 ng / ml
• TPO 100 ng / ml
• IL-7 100 ng / ml
• CD34 + culture in the presence of DL4 / Fc and the retronectin fragment contains CD34 +/- CD7 + T lymphoid progenitors (14%).
• the grafting of the progenitors generated according to the defined protocol to NOD / SCID / vc ko mice shows the induction of human thymopoiesis, approximately 6 weeks after transplantation of transduced cells exposed to DL-4 / fc +. retronectine®.

Landscapes

• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biomedical Technology (AREA)
• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Cell Biology (AREA)
• Hematology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Veterinary Medicine (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• General Chemical & Material Sciences (AREA)
• Communicable Diseases (AREA)
• Oncology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (8)

Applications Claiming Priority (2)

Publications (1)

Family

ID=52102849

Family Applications (1)

Country Status (11)

Cited By (8)

Families Citing this family (3)

Citations (2)

Family Cites Families (14)

• 2014
  • 2014-10-06 FR FR1459539A patent/FR3026744B1/fr active Active
• 2015
  • 2015-10-05 ES ES15771979T patent/ES2914082T3/es active Active
  • 2015-10-05 WO PCT/EP2015/072889 patent/WO2016055396A1/fr not_active Ceased
  • 2015-10-05 HU HUE15771979A patent/HUE059468T2/hu unknown
  • 2015-10-05 JP JP2017537016A patent/JP6898240B2/ja active Active
  • 2015-10-05 DK DK15771979.0T patent/DK3204492T3/da active
  • 2015-10-05 PT PT157719790T patent/PT3204492T/pt unknown
  • 2015-10-05 CA CA2963756A patent/CA2963756C/fr active Active
  • 2015-10-05 US US15/516,731 patent/US12071635B2/en active Active
  • 2015-10-05 EP EP15771979.0A patent/EP3204492B1/fr active Active
  • 2015-10-05 EP EP22152734.4A patent/EP4036224A1/fr not_active Withdrawn
  • 2015-10-05 PL PL15771979.0T patent/PL3204492T3/pl unknown
• 2020
  • 2020-12-17 JP JP2020209370A patent/JP2021072779A/ja active Pending

Patent Citations (2)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events