RU2797260C2 - Method for production of t-cell precursors - Google Patents

Abstract

FIELD: cell biology; biotechnology; medicine.

SUBSTANCE: present invention relates to cell biology, biotechnology, and medicine, in particular to in vitro methods for the production of T-cell precursors, as well as modified and transformed T-cell precursors, a kit for implementation of methods, a population of CD7+ T-cell precursors, and to their use for increasing a number of T-cells in a subject. The specified methods provide a stage of cultivation of CD34+ cells in a medium containing TNF-alpha and/or an antagonist of an aryl-hydrocarbon/dioxin receptor, in particular StemRegenin 1 (SR1), in the presence of Notch ligand and optionally a fibronectin component. A population of CD7+ T-cell precursors is CD34- and CD1a-, or CD34- and CD5-, or CD34- and CD1a-, and CD5-, a fraction of such cells in this population exceeds 80%.

EFFECT: present invention allows for obtainment of a sufficiently large number of T-cell precursors, so that they can be effectively used for adults receiving stem cell transplantation.

19 cl, 9 dwg, 2 tbl, 14 ex

Description

The invention relates to the field of cell therapy, in particular to the transplantation of hematopoietic stem cells, transformed or not, and more specifically to the restoration of immunity after such transplantation.

Transplantation of progenitor cells and hematopoietic stem/progenitor cells (HSPCs) is considered the best therapy option for the treatment of the most severe hereditary immunodeficiencies, many malignant hemopathy, and a number of solid tumors.

Currently, in allotransplantation situations with partial HLA incompatibility, injections into previously conditioned recipients of increasing doses of sorted CD34+ HSPCs allow transplantation of donor cells with effective prevention of graft-versus-host disease (GVHD). However, it takes at least 4 months for new T-lymphocytes to differentiate from injected CD34+ cells, and it takes several months for these T-lymphocytes to become sufficient to play a protective role against infections.

This slow rate of immune recovery leads to numerous infectious complications, especially viral ones, as well as relapses that affect the long-term prognosis of transplant patients.

In addition, other therapeutic protocols use a gene therapy approach, namely autotransplantation of transduced HSPCs, which has been shown to be effective in the treatment of certain inherited immunodeficiencies. The advantage of this strategy over allogeneic CD34+HSPC transplantation in terms of survival and complication rate when there is no available HLA-matched donor is undeniable. However, clinical experience has shown that for some patients with severe infections, recovery of the T-lymphocyte compartment is slow and never reaches normal levels of circulating T-lymphocytes. The morbidity and mortality associated with this particular context are important.

Due to the high morbidity and mortality associated with this type of transplantation, the development of new therapeutic agents to reduce the period of immunodeficiency after transplantation is fully justified.

In particular, it is important to accelerate the production of T lymphocytes by administering to the patient progenitors already involved in the T lymphocyte differentiation pathway (T cell progenitors).

These T cell precursors are obtained by differentiation of CD34+ HSPCs, and they have, in particular, the CD7+ marker, which is a differentiation marker in the T cell pathway. They may also have other markers. Awong et al (Blood 2009; 114: 972-982) described the following progenitors of T lymphocytes: early thymic progenitor (ETP), which have markers (CD34+/CD45RA+/CD7+), proT1 progenitors (CD7++/CD5-) , precursor cells at the proT2 stage (CD7++/CD5+) and cells at the preT stage (CD7++/CD5+ CD1a+). HSPCs acquire these markers sequentially as they move from one stage to the next along the T-cell developmental pathway. It is also known that in humans, the CD1a antigen distinguishes the transition from a completely immature thymic progenitor to a progenitor clearly involved in the T-pathway (Cavazzana-Calvo et al, MEDECINE/SCIENCES 2006; 22: 151-9).

Transplantation of progenitor T cells, concurrently with transplantation (transplantation) of HSPC, would enable the rapid production of a mature and functional T lymphocyte compartment and thus help prevent the risk of severe infections, allowing the patient to benefit from some immunity until the immune system is fully restored. .

It is also important to be able to use adult cells rather than cord blood cells because adult cells are easier and cheaper to obtain than cord blood cells and because adult cells are more commonly used in allotransplantation.

However, data published in the literature from humans and mice show fundamental differences between fetal hematopoietic cells (including cord blood) and adult cells. These differences relate to survival, ability to repair DNA damage, proliferative ability, and ability to differentiate (see, for example, Yuan et al. adults compared to fetal cells, in terms of their ability to generate different cell types: Lansdorp et al (J Exp Med 1993 Sep 1; 178 (3): 787-91) Szilvassy et al (Blood, 2001 Oct. 1 , 98 (7): 2108-15), Frassoni et al. (Blood, 2003 Aug. 1, 102 (3): 1138-41), Liang et al. 106 (4): 1479-87), Six et al. . (J Exp Med 2007 Dec 24, 204 (13): 3085-93)).

WO 2016/055396 describes that it is possible to obtain T cell progenitors by culturing CD34+ cells in the presence of an immobilized Notch ligand (specifically, a soluble delta-like ligand domain fused to the Fc region of an IgG protein) and a fibronectin fragment containing RGDS domains ( SEQ ID NO: 3, arginine-glycine-aspartate-serine) and CS-1, as well as a heparin-binding domain (particularly in the presence of Retronectin®). The Notch ligand used may be referred to as DL4/FC.

Of note, this document discloses that the presence of both immobilized Notch ligand and fibronectin allows for increased generation of progenitor T lymphocytes (CD7+ cells), which is already an improvement in the art, but this percentage of CD7+ CD34 cells remains quite low, as shown in Figure 3 in WO 2016/055396. However, it is interesting and particularly important to increase this percentage in order to be able to administer more precursor to a patient in need thereof. On the other hand, it is also important that the cells remain at an early stage of differentiation so that they can provide proper immunity.

It should be remembered that Notch proteins are transmembrane receptors that regulate the cellular response to a wide range of environmental signals. In mammals, four Notch receptors (Notch 1-4) and five ligands (delta-like-1, 3 and 4, Jagged1, Jagged2) have been described (Weinmaster Curr Opin Genet Dev 2000: 10: 363-369).

Delta-like ligand 4 can be referred to as:

(ii) Delta-like ligand 4 (corresponding to the gene name DLL4)

(iii) Delta-like-4 or delta-ligand 4 (abbreviation DL-4).

In this application, delta-like-1 and delta-like-4 Notch ligand may be referred to as DL1 and DL4, or DL-1 and DL-4, respectively. The sequences of the DL-1 and DL-4 ligands are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Ohishi et al. (BLOOD, vol. 98, No. 5, 2001, p. 1402-1407) describes the effect of Notch signaling on the differentiation of monocytes into macrophages and dendritic cells. The monocyte cells used in the experiments disclosed herein were either negatively screened peripheral blood monocytes or monocytes derived from in vitro differentiation of CD34+ stem cells. Thus, the cells used in this document have entered the monocyte/macrophage/dendritic cell differentiation pathway, have lost the CD34 marker (which is a marker for hematopoietic stem cells), and present the CD14 marker. These cells are cultured in the presence of the delta-1 extracellular domain and GM-CSF, TNF-alpha (which is used to induce differentiation of CD1a-CD14+ cells derived from CD34+ cells into dendritic cells). This work does not use CD34+ cells in the presence of Notch ligand and TNF-alpha, and does not refer to the production of T cell progenitors (CD7+ T lymphocyte progenitors).

In SHUKLA et al. (NATURE METHODS, vol. 14, no. 5, 2017, 531-538) and WO 2017/173551 discloses a method for producing progenitor T cells from stem cells and/or progenitor cells, including exposure to stem cells and/or precursor cells with Notch ligand delta-like-4 (DL4) and vascular endothelial adhesion molecule 1 (VCAM-1).

The method described in this application allows obtaining and increasing the number (proliferation) of the precursors of CD7+ T-lymphocytes from CD34+ stem cells without using the cell stroma (which is not easy to imagine in a clinical context). In addition, this method is adapted, in particular, to be carried out with CD34+ cells obtained from adult donors.

In addition, cells obtained using the method described herein contain the Bcl11b marker, which is an important transcription factor turned on exclusively after T cell commitment (Kueh et al., 2016, Nat Immunol. 2016 Aug;17(8): 956-65 doi: 10.1038/ni.3514).

The inventors also showed no rearrangement of T-cell receptor loci (TCR-beta, TCR-gamma or TCR-delta) in the precursors obtained using the method disclosed herein.

In addition, for precursors obtained by the method described herein, a decrease in apoptosis markers was shown compared to the method described in WO 2016/055396.

Thus, the method described herein makes it possible to obtain a sufficiently large number of T cell progenitors so that they can be effectively used in adults receiving stem cell transplantation.

This method can also be used to generate transformed (transduced) T cell progenitors for gene therapy when a vector containing the gene of interest is used at some point in the process described herein.

Cells obtained by the method described herein can be obtained for adult CD34+ cells and can be used for allogeneic transplants or autografts, even when cord blood cells are used for such transplants.

It should be noted that this method, although very effective for adult CD34+ cells, can also be used with cord blood CD34+ cells.

Thus, in a particular embodiment, the method described herein accelerates in vivo generation of T cells following injection of HSPC-derived T-cell progenitors into an in vitro culture system combining an immobilized Notch ligand-derived fusion protein, delta-4, Retronectin ® and a combination of cytokines.

This system allows, within 7 days, to generate T-cell precursors that are phenotypically and molecularly similar to human thymus cell precursors. In addition, these T cell precursors give rise to mature and diverse human T cells in NSG mice with faster kinetics than HSPC.

The results presented herein were obtained with both umbilical cord blood (PC) HSPC and adult HSPC (mobilized peripheral blood (mPB) from adult donors).

The inventors have shown that T cell progenitors can be increased relative to CD34+ cells by exposing said CD34+ cells to Notch ligand and in the presence of soluble TNF-alpha (tumor necrosis factor alpha, Uniprot P01375, RefSeq NP_000585, SEQ ID NO:8) . Said exposure is carried out under conditions suitable for the generation of progenitor T cells. Optionally, cells are also exposed to a fibronectin fragment containing an RDGS motif and/or a CS-1 motif, and optionally a heparin-binding domain. Preferably, said fibronectin fragment contains an RDGS motif, a CS-1 motif, and a heparin-binding domain.

TNF is mainly produced as a type II transmembrane protein, 233 amino acids long, organized into stable homotrimers. The secreted form of human TNF-alpha takes the form of a triangular pyramid and weighs approximately 17 kDa.

As described in WO 2016/055396, the method described herein can be carried out using an RGDS peptide and/or a CS-1 peptide instead of a fibronectin fragment. The combined use of RGDS and CS-1 peptides, in particular fused in the same protein, is preferred. Thus, the RGDS and/or CS-1 peptides may be present as such in the culture medium or within a polypeptide or protein present in the culture medium. When the culture medium contains only the RGDS peptide and/or CS-1 per se, they can be referred to as "free" peptide(s) in the culture medium if such peptides are not immobilized on the inner surface of the culture vessel. Indeed, the peptide(s) may be in solution or immobilized on the inner surface of a vessel in which CD34+ cells are exposed to immobilized Notch ligand.

However, as stated above, it is preferable to use a fibronectin fragment that contains RGDS and CS-1 patterns as well as a heparin-binding domain. The fibronectin fragment may be free in solution or immobilized on the inner surface of the culture container.

The process is carried out in vitro in a container such as a cell culture plate (Petri dish, 24-well matrix or the like), preferably with Notch ligand immobilized on its inner surface. However, the Notch ligand can be immobilized on any other carrier present in the culture medium, for example on the surface of spheres (beads) (in particular microspheres). Immobilization of the Notch ligand is essentially intended to stabilize the ligand to allow activation of the Notch receptor on CD34+ cells.

The term "T cell precursor" is intended to refer to any cell involved in the differentiation pathway to the T lymphoid pathway from CD34+ HSPC. Accordingly, this cell is characterized in that it expresses the CD7 marker, which is known to be one of the earliest markers during T cell lymphopoiesis. Depending on the status of differentiation in the T-lymphoid pathway, it may or may not express the CD34 marker (loss of CD34 during differentiation). Such a T cell precursor may or may not express the CD5 marker as well.

"T cell progenitors" include those cells that can be found in the postnatal thymus, i.e. early thymic progenitor (ETR) (CD34+/CD45RA+/CD7+), proT1 cells (CD34+CD45RA+CD7++CD5-CD1a-), proT2 cells (CD34+CD45RA+CD7++CD5+CD1a-) and preT cells (CD34 -CD7++/CD5+CD1a+). The T cell receptor (TCR) loci are rearranged in a highly ordered manner (TCRδ-TCRγ-TCRβ-TCRα). It should be noted that the first functional TCR rearrangements occur at the CD34-CD7++/CD5+CD1a+ preT cell stage (Dik et al. J Exp Med 2005; 201: 1715-1723). T cell precursors are well known in the art. They are mentioned in particular by Reimann et al (STEM CELLS 2012;30:1771-1780.) and Awong et al (2009, op.cit.).

The term "RGDS peptide" is intended to refer to any peptide or protein that contains an RGDS pattern such that it can bind the VLA-5 integrin. Such a peptide or protein can be tested for its ability to bind the VLA-5 integrin by methods known and described in the art. The RGDS peptide binds to the VLA-5 (Very Late Antigen-5) integrin, which is a dimer consisting of CD49e (alpha5) and CD29 (beta1).

Heparin binding domains are known in the art and are present in numerous proteins that bind to heparin. Their sequence is usually XBBXBX or XBBBXXBX (B = basic amino acid; X = hydrophobic amino acid; Cardin and Weintraub, Arterioscler Thromb Vasc Biol. 1989; 9:21-32, SEQ ID NO: 4 and SEQ ID NO: 5).

The presence of such a heparin-binding domain is particularly useful when CD34+ cells are exposed to a viral (especially retroviral) vector to transduce them and produce transgene-expressing T cell precursors.

The CS-1 peptide or pattern CS-1 is a 25 amino acid peptide (DELPQLVTLPHPNLHGPEILDVPST, SEQ ID NO: 6) described by Wayner et al., 1989, J. Cell Biol. 109:1321). This CS-1 pattern 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).

In a particular embodiment, the fibronectin fragment is present in the culture medium or immobilized on the inner (in particular, on the bottom) wall of the container. Fibronectin is a protein that in its natural form is a V-shaped large dimer with a length of 100 nm and a mass of 460 kDa. The two monomers are connected by two disulfide bridges at their C-terminus. The term "fibronectin" or "fibronectin fragment" refers to the natural protein fibronectin (i.e. any isoform resulting from alternative splicing), but also a monomer of this protein or a fragment of this protein (but containing the RGDS peptide, as well as the CS-1 peptide and heparin-binding site).

A fibronectin particularly suitable for carrying out the method described herein is Retronectin®. This protein corresponds to a human fibronectin fragment (CH-296 fragment, Kimizuka et al., J Biochem., 1991 Aug. 110 (2): 284-91, Chono et al., J Biochem 2001 Sep 130 (3): 331-4 ) and contains three functional domains that are preferred for the implementation of this method (cell-binding C-domain containing the RGDS peptide, heparin-binding domain and CS-1 sequence). This protein is sold, in particular, by Takara Bio Inc. (Shiga, Japan).

In a particular embodiment, the fibronectin fragment is immobilized (i.e., bound to a solid support and not free in solution (although it is possible that certain elements may be present in solution)). This solid support is preferably the bottom wall of the container in which the process is carried out. However, it is also possible to envisage binding of the fibronectin fragment to microspheres such as polymeric or magnetic microspheres (typically 1 to 5 μm in diameter). The binding of a protein or peptide to these microspheres may or may not be covalent. Methods for attaching a protein or peptide to microspheres are known in the art. Also, the fibronectin fragment can be introduced into a semi-solid medium such as agar or gel.

When a fragment of fibronectin is immobilized on a carrier (in particular, on the bottom wall of the container in which the process is carried out), this immobilization can also be covalent or non-covalent. In a preferred embodiment, this immobilization is performed non-covalently, allowing the fibronectin fragment to be absorbed onto the glass or plastic constituting the bottom wall of the container.

In a particular embodiment, as shown above, differentiation of CD34+ cells into T cell precursors is carried out in conjunction with transduction or transfection (including nucleofection™, a specific electroporation system developed by Lonza) of CD34+ cells via a vector (such as a viral vector or fragment nucleic acid such as a plasmid or plasmid RNA or DNA sequences) to introduce the gene of interest (or gene editing system) into these cells. This means that cells that have been exposed to the Notch ligand and fibronectin fragment, with TNF-alpha, are also exposed to the viral supernatant for at least part of the time they are exposed to the Notch ligand and fibronectin fragment, with TNF-alpha.

The ideas of WO 2016/055396 regarding operating conditions for performing cell transduction are directly applicable to the present method and thus are considered to be explicitly described in this application. It can be cited, in particular:

(iv) Exposure of cells to Notch ligand, fibronectin fragment and TNF-alpha for some time (preferably more than 4 hours, more preferably more than 6 hours or more than 8 hours or 10 hours, but less than 36 hours, more preferably less than 30 hours or less 24 hours) with appropriate cytokines known in the art and disclosed in WO 2016/055396 and adding the viral supernatant for an appropriate amount of time (preferably more than 4 hours, more preferably more than 6 hours, 8 hours) or 10 hours, preferably less than 30 hours, more preferably less than 24 hours, more preferably about 16 hours).

(v) Second transduction, if required, as stated in WO 2016/055396

(vi) Application of this protocol for transplantation of autologous hematopoietic stem cells in gene therapy protocols so that the transgene can add a protein that is missing or deficient, thus providing a therapeutic benefit.

(vii) Suitable transgenes for use: genes that correct immunodeficiencies (in particular, severe combined immunodeficiencies SCID or non-SCID, CID), HIV, X-linked adrenoleukodystrophy, hemoglobinopathies, in particular β-thalassemia or sickle cell anemia. A gene encoding a chimeric antigen receptor (CAR), that is, a cell surface protein recognizing a cell surface protein that is specifically expressed by cancer cells, can also be used as a transgene to elicit an immune response against these cancer cells by the engineered cells.

(viii) The preferred use of the viral supernatant to allow the introduction of the transgene into the genome of the cell using, in particular, the known lentiviruses described in the art.

(ix) Introduction of the viral vector into the cell culture medium after pre-activation of CD34+ cells from 4 to 36 hours, preferably from 6 to 24 hours.

(x) Exposing cells to a viral vector for 4 to 30 hours, preferably 12 to 24 hours, more preferably about 16 hours, and removing the viral vector (collecting and washing the cells and resuspending these cells in the presence of Notch ligand, a fibronectin fragment and TNF- alpha).

As stated above, in another embodiment, CD34+ cells are exposed to a system that allows gene editing to be performed. Such systems are currently well known and described in the art and are primarily based on double nipped nucleic acid repair. Such a genome editing system may employ a nuclease selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas nucleases. The fact that CD34+ cells proliferate during exposure to the elements mentioned above, and therefore in the presence of TNF-alpha, makes it possible to use these gene editing systems that require cell proliferation.

In a particular embodiment of the invention, the Notch ligand is a delta-like protein 1 (SEQ ID NO: 1) or a fragment (soluble domain) thereof.

In another and preferred embodiment of the invention, the Notch ligand is delta-like protein 4 (SEQ ID NO: 2).

In a particular embodiment, said Notch ligand is a fusion protein comprising a soluble domain of the native Notch ligand fused to the Fc region of an IgG protein. As is known in the art, the soluble domain of a Notch ligand is the extracellular portion of said ligand. Varnum-Finney et al. (J Cell Sci., 2000 Dec; 113 Pt 23: 4313-8) described a fusion protein of the soluble portion of DL-1 with the Fc portion of IGg1 Reimann et al. (cit. cit.) described a fusion protein of the soluble portion of DL-4 (amino acids 1-526) with an IgG2b immunoglobulin Fc fragment. Thus, it is preferred that the IgG protein is IgG2. In a preferred embodiment, the sequence of the Notch ligand used in the method disclosed herein is SEQ ID NO: 7. A commercially available product (Sino Biologicals) containing the extracellular domain (Met 1-Pro 524) of human DLL4 (full-length DLL4, accession number (NP_061947.1), fused to the Fc region of human IgG 1 at the C-terminus, is a DL4 protein also suitable for use herein.

The culture medium used in the context of the method disclosed herein is any medium adapted for culturing CD34+ cells and T cells. In particular, mention may be made of α-MEM, DMEM, RPMI 1640, IMDM, BME, McCoy's 5A, StemSpan™, in particular SFII (StemCell Technologies) or Fisher's medium. StemSpan™ SFII media contains, inter alia, Ismove's MDM, bovine serum albumin, recombinant human insulin, human transferrin (rich in iron), 2-mercaptoethanol.

A suitable and preferred culture medium for carrying out the method described herein is X-VIVO™ medium (Lonza, Basel, Switzerland). This medium has been used, among others, by Jonuleit et al. (Eur J Immunol, 1997, 27, 12, 3135-42) and Luft et al. (J Immunol, 1998, 161, 4, 1947-53).

Preferably, a basal medium is used (i.e., a medium that allows cells to grow without additives), to which, however, it is preferable to add serum and/or growth factors and cytokines.

Thus, fetal bovine serum (FBS) or fetal calf serum (FCS), autologous human serum or human AV serum is preferably added to the basal culture medium. Preferably, at least 15% fetal serum is added to this medium, more preferably at least 20%. FBS is particularly suitable for implementing the method. In particular, it is preferable to use a standard FBS. Standard PBS is a high quality serum that has been examined and filtered to exclude the presence of viral particles. It is sold as such by many vendors, such as HyClone™ Standard Fetal Bovine Serum (FBS) from Thermo Scientific™.

In addition to TNF-alpha, the culture medium is also preferably supplemented with cytokines and growth factors. These cytokines and growth factors are specifically selected from the group consisting of SCF (stem cell growth factor), thrombopoietin (TPO, also called megakaryocyte growth and development factor, MGDF), Flt3 ligand (which is a hematopoietic growth factor), interleukin 3 ( IL-3), interleukin 7 (IL-7) and SCF (stem cell factor). In a particular embodiment, the culture medium contains at least three, preferably at least four of these cytokines or growth factors, in addition to TNF-alpha.

Preferably, and in particular to generate T cell precursors that are not transducible by the viral vector, at least or exactly three cytokines are added. Preferably these three cytokines are interleukin-7 (IL-7), SCF (stem cell growth factor) and Flt-3 ligand (hematopoietic growth factor).

In another preferred embodiment of the invention, four cytokines are added, i.e. the three cytokines mentioned above and TPO (thrombopoetin).

In another particular implementation, and in particular for the generation of progenitor T cells transduced with a viral vector, the nature of the cytokines and growth factors can be varied during the implementation of the method.

Thus, IL-3, IL-7, SCF, TPO, and Flt3-L can be applied to the medium at the stage of pre-activation of cells before adding the viral vector, and then supplement the medium with only IL-7, SCF, TPO, and Flt3-L after vector removal. .

The above mixtures of cytokines and growth factors are sufficient to induce differentiation of CD34+ cells into T cell progenitors, and typically the culture medium does not contain other cytokines or growth factors than TNF-alpha, which, as described in the examples, allows for an increase in T cell progenitors.

In the method provided herein, the total duration of exposure in the presence of Notch ligand to CD34+ cells of a protein or peptide exhibiting an RGDS motif and TNF-alpha is generally preferably greater than 3 days and less than 10 days.

This effect may vary depending on whether the cells are transduced. Thus, for non-transduced adult stem cells, an exposure time of three days may be sufficient, while for early-stage stem cells (approximately 7 days) or when transduced, it will usually be longer.

CD34+ cells are obtained by bone marrow aspiration, from cord blood or from peripheral blood from adult donors, after mobilization, in particular with G-CSF or any other mobilizing agent known in the art. Methods for sorting CD34+ cells are known in the art. In particular, magnetic microspheres having on their surface an antibody recognizing CD34 can be used for this purpose.

Preferably, the cell culture vessel is prepared by immobilizing Notch ligand and a fibronectin fragment on the inner (preferably bottom) surface prior to exposure to CD34+ cells. Retronectin® or other fibronectin naturally adheres to the plastic of the cell culture box (Petri dish or 24-well box or other). Similarly, if the Notch ligand is used in the form of a fusion protein with an immunoglobulin Fc fragment, this Fc fragment also adheres to the plastic. Therefore, it is sufficient to leave the container in the presence of these compounds for several hours to obtain an appropriate coating. A method for coating such a culture container with a Notch ligand and a fibronectin fragment is described in detail in WO 2016/055396 and can be used to implement the method disclosed herein.

It should be recalled that according to WO 2016/055396 approximately 75% of DL-4 will adhere to the surface of the container when a concentration of 5 μg/ml is applied; the optimum dose should be greater than or equal to 1.25 μg/ml and preferably 2.5 to 5 μg/ml.

With regard to the fibronectin fragment, a concentration of 25 μg/ml is particularly suitable (especially when using Retronectin®), although other concentrations (higher or lower) can be used.

In the method described herein, CD34+ cells are added at a concentration of 10 ⁶ to 10 ⁷ cells/ml, in particular about 2×10 ⁶ CD34+ cells/ml, into the culture vessel.

Depending on whether the cells are transduced and the concentration of CD34+ cells, a 2 to 10 cm ² plate (ie, a 24 to 6 well plate) may be used. When using a 24-well plate, 10 ⁴ to 10 ⁶ CD34+ cells are added to each well, preferably 2×10 ⁴ to 4×10 ⁵ CD34+ cells per well. When using a 6-well plate, add from 8×10 ⁴ to 2×10 ⁶ cells per well.

The number of added cells is selected by a specialist in accordance with the container used.

Cells are placed in a well, in a basal medium of choice, supplemented with TNF-alpha and preferably supplemented with growth factors and cytokines as above.

The concentration of cytokines or growth factors is from 2 to 300 ng/ml. Preferably the concentration is above 40 ng/l and below 300 ng/ml or 200 ng/l, more preferably about 100 ng/ml.

However, if transduced T cell progenitors are desired, and if the cells are pre-activated prior to exposure to the viral vector, higher concentrations (approximately 300-400 ng/mL) may be used. In this embodiment, SCF and Flt3-L can be used at concentrations in the 300 ng/mL range, TPO and IL-7 in the 100 ng/mL range, and IL-3 at approximately 40 ng/mL.

TNF-alpha is preferably used at a concentration equal to or greater than 5 ng/ml. Indeed, as shown in the examples, this low concentration is sufficient to obtain the proliferation of progenitor T cells without altering the differentiation pathway.

However, higher concentrations can also be used. In particular, the concentration of TNF-alpha can reach 300 ng/ml or 200 ng/ml. A suitable concentration is about 100 ng/ml. However, other concentrations are also suitable, such as 10 ng/ml, 20 ng/ml or 50 ng/ml.

Thus, the invention relates to an in vitro method for producing T cell precursors comprising the step of culturing CD34+ cells in a suitable medium containing TNF-alpha and in the presence of an immobilized Notch ligand and optionally a fibronectin fragment as described above.

Thus, the invention relates to an in vitro method for propagating T cell progenitors, comprising the step of culturing CD34+ cells in a suitable medium containing TNF-alpha and in the presence of an immobilized Notch ligand and optionally a fibronectin fragment, as described above.

The term "expansion of T cell progenitors" means that there are more T cell progenitors than without the use of TNF-alpha and/or that there are more T cell progenitors than the number of CD34+ cells introduced into the container.

In particular, T cell precursors obtained by the above methods are CD34-/CD7+/CD5- precursors.

Usually, these cells also do not contain the CD1a marker.

In a preferred embodiment, the T cell progenitors obtained by the above methods are progenitors expressing CD34-/CD7+/CD1a-.

This means that the T cell progenitor population generated by this method expresses the CD7 marker and that more than 80% of cells expressing this marker have the above phenotype (not expressing

- CD34 and CD1a, or

- CD34 and CD5, or

- CD34 and CD1a and CD5),

according to analysis by flow cytometry. This phenotype is usually obtained after 7 days of cultivation.

As indicated, the CD34+ cells are preferably not cord blood cells. However, this method can also be used with CD34+ umbilical cord blood cells, and in this case also gives improved results.

This method is of particular interest when implemented on CD34+ cells isolated from the body of an adult patient. The specified patient may be a healthy donor or a donor with a disease, in particular, for which the cells will be corrected by viral transduction.

The cells can be cultured for more than 3 days, and preferably for at least 5 days, more preferably for at least or exactly 6 days, most preferably for at least or exactly 7 days, although the culture may be longer.

In the method disclosed herein, TNF-alpha is preferably added to the culture medium from day 0 (i.e., at the start of CD34+ cell culture) and must remain in the culture medium for at least three days and preferably for the duration of the cell culture. (i.e. preferably about 7 days).

When implementing the method described herein, you can additionally add StemRegenin 1 (SR1, 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl )phenol, CAS 1227633-49-10) into a culture medium already containing TNF-alpha.

The invention also relates to a method for producing T cell precursors, comprising the steps

a. carrying out the method described above, and

b. purification of the resulting T cell precursors thus produced.

Purification can be done by washing the cells and resuspending in basal medium.

The method may also include the step of conditioning the T cell progenitor cells in the injection bag for the patient.

In this case it is preferable to reconstitute these cells in saline containing 5% HSA, eg Albunorm™ 5% 50 g/l (Octopharma, Lingolsheim, France).

These cells can also be frozen according to methods known in the art.

The invention also relates to an intravenous injection bag containing a population of T cell progenitors (which can be obtained or which are obtained by the method described above) for which the proportion of CD7+ cells in this population is greater than 80%, more preferably greater than 85%.

The invention also relates to an intravenous injection package containing a population of progenitor cells of CD7+ T cells (which can be obtained or which are obtained by the method described above), where the proportion of CD34- and CD5- cells (cells expressing CD7+ and both CD34-, and CD5-) in this population is above 80%, more preferably above 85%.

The invention also relates to an intravenous injection bag containing a population of CD7+ T cell precursors (which can be obtained or which are obtained by the method described above), where the proportion of CD34 cells (cells with CD7+ and CD34-) in this population is above 50%, more preferably above 60%. In addition, at least 80% and more preferably at least 85% of the cells in this population c are CD1a cells.

The invention also relates to an in vitro method for obtaining transformed T cell precursors, comprising the steps:

a. culturing CD34+ cells in a suitable medium containing TNF-alpha and in the presence of immobilized Notch ligand

b. exposing cells to a vector intended for transfection or transduction of CD34+ cells.

As described above, and after the steps of washing and reculturing the cells, transformed T cell progenitors (expressing the transgene integrated into their genome) are obtained.

The invention also relates to a method for producing modified T cell precursors in vitro, comprising the following steps:

a. cultivation of CD34+ cells in a medium containing TNF-alpha and in the presence of immobilized Notch ligand

b. exposing cells to a vector or nucleic acid sequence containing an element suitable for gene editing, at least for some time during cell culture.

In this way, progenitor T cells modified by gene editing are obtained, after possible washing and culturing steps.

The invention also relates to progenitors of T cells, in particular obtained by the method described herein, for their use in a patient with immunodeficiency, in particular to enable the restoration of immunity in this patient and/or to obtain immune protection against infections in said patient during a period of several months (at least two months, preferably at least six months).

In a particular embodiment, the patient is an immunocompromised patient. There can be several reasons for deficiency: hereditary immunodeficiency, chemotherapy for leukemia, conditioning, transplant containing only stem cells, post-transplant treatment to prevent graft-versus-host disease, age of the patient, and complications such as infections.

In particular, the patient may experience immunosuppression due to the depletion of his immune cells after therapy before hematopoietic stem cell transplantation. In this embodiment, the graft may be an allograft (in which case the T cell precursors are preferably obtained from a partially HLA-matched donor) or an autograft (in which case the T cell precursors are preferably transformed with a vector to express a gene and/or protein to correct the genetic defect in said patient).

The T cell progenitors are preferably a population of CD7+ cells (ie a population in which at least 75%, more preferably at least 80% of the cells in the population express the CD7 marker). This population is also part of the invention. In particular, it can be obtained by the method described herein. In a private embodiment, it is obtained by the method described herein.

More specifically, the T cell progenitors are preferably a population of CD7+ cells (i.e., a population in which at least 75%, more preferably at least 80% of the cells in the population express the CD7 marker), in which the proportion of CD34- and CD5- (cells expressing CD7+ and both CD34- and CD5-) in this population is greater than 80%, more preferably greater than 85%. This population is also part of the invention. In particular, it can be obtained by the method described herein. In a private embodiment, it is obtained by the method described herein.

In another embodiment, the T cell progenitors are preferably a population of CD7+ cells (ie, a population in which at least 75%, more preferably at least 80% of the cells in the population express the CD7 marker). In this population, more than 50%, more preferably, more than 60% of the cells do not express the CD34 marker. In this population, at least 80%, more preferably at least 85% of the cells do not express the CD1a marker. The proportion of CD7+ CD1a- cells in this population is preferably at least 80%. This population is also part of the invention. In particular, it can be obtained by the method described herein. In a private embodiment, it is obtained by the method described herein.

To determine whether a cell is positive or not for a surface marker (CD7, CD34, CD5, or CD1a), any method known in the art should be used, and in particular flow cytometry, after cells have been labeled with fluorescent antibodies directed against this surface antigen. The principle is that for each cell in the population, a signal will be emitted at a given intensity, and cells are considered positive for a given antigen if the signal is above a given threshold.

For CD34: A control population consisting of the HSPC population (such as those isolated from cord blood or from mobilized peripheral blood) is used to determine appropriate thresholds. In this cell population, the cells are CD34+CD7-CD5- or CD34+CD7-CD1a-. With this control, a threshold can be defined for each antigen, which will be used to determine whether cells in another population are positive or not for those antigens.

The control population typically contains approximately 90% CD34+CD7-CD5- or CD34+CD7-CD1a- hematopoietic stem cells. Threshold is the level of signal intensity for which cells in a population can be sorted in a 90/10 ratio.

For CD7 (respectively CD5 or CD1a): use a control population obtained by isolating peripheral blood mononuclear cells (PBMC) using any technique known in the art, and in particular a density gradient technique such as the Ficoll-Paque Ficoll-Paque PLUS (GE Healthcare Life Sciences). CD7 positive (respectively CD5 or CD1a) cells are isolated using any technique known in the art, such as the MACS® method (Magnetic Cell Isolation and Cell Separation, Miltenyi Biotec), which uses anti-CD7 magnetic microspheres (respectively anti-CD5 or anti-CD1a) antibody. In this population, cells will be predominantly CD7+ (respectively CD5+ or CD1a+) and this proportion is thought to be approximately 90/10.

Thresholds for assessing whether a cell is positive for a surface antigen are determined using a control population for that antigen and will correspond to an intensity for which more than 90% of the cells in the control population have a higher signal intensity.

Therefore, a cell is considered positive for an antigen if the intensity signal for that antigen is above the threshold defined above, and negative for an antigen if the intensity signal for that antigen is below the threshold defined above.

The fact that the vast majority of T cell progenitors in the population do not express the CD1a marker may be beneficial, as cells expressing this CD1a marker may not be very efficient in reaching the thymus and therefore may be as efficient as CD1a- cells, for repopulation in vivo. Since the recovery potential of CD1a+ cells is uncertain, it is preferable to reduce the number of such cells in the population.

The invention also relates to a method of treating an immunosuppressed patient, in particular to enable restoration of immunity, at least temporarily, in that patient, comprising the step of administering to said patients T cell precursors as described above.

This method may also include the step of obtaining such precursors by exposing CD34+ cells to a Notch ligand in the presence of TNF-alpha (as described above) and preferably a protein or peptide having an RGDS motif and/or a CS1 motif in a specific fragment of fibronectin as described above, under the conditions specified above.

In particular, the patient is administered a therapeutically effective amount, ie on the order of 1 to 5×10 ⁶ progenitors per kg, which allows the patient to be provided with cells capable of playing a protective role against infections for several months (about 6 months).

Preferably, this administration of T cell progenitors to a patient is performed immediately before, immediately after, or simultaneously with hematopoietic stem cell transplantation in said patient. As seen above, the injected cells can be transformed with a vector designed to correct a genetic defect in said patient.

In another embodiment, the T cell precursors are injected into a patient who does not require a hematopoietic stem cell transplant. Moreover, transformed or transduced T cell progenitors can be used to treat certain T cell-only immunodeficiencies, HIV patients, or cancer patients (using progenitors modified to generate CAR-T cells) without the need for debilitating chemotherapy. immune system.

It should be noted that the teachings of the invention described for the use of TNF-alpha can also be applied to the purine derivative StemRegenin 1 (SR1, described in Boitano et al., Science. 2010 Sep 10; 329 (5997): 1345-8). Thus, SR1 can be a substitute for TNF-alpha (ie, used instead of TNF-alpha) to generate T cell progenitors from CD34+ cells, with the onset of cell differentiation and multiplication (an increase in the number of cells). The SR1 concentration is preferably in the region of 750 nM (30 ng/ml); a higher concentration may also be provided, such as 1500 nM or 2500 nM (100 ng/mL), or even 5000 nM (200 ng/mL) or lower, such as 500 nM. (20 ng/ml). When used alone, but especially in combination with TNF-alpha, much lower concentrations can be used, down to 3 ng/mL or 10 ng/mL. Suitable concentrations also include 30 ng/ml or 100 ng/ml. Accordingly, any concentration greater than 3 ng/mL or greater than 10 ng/mL is suitable for enhancing differentiation of CD34+ cells into T cell progenitors.

SR1 is an aryl hydrocarbon/dioxin receptor (AhR) ligand (antagonist). Other AhR antagonists can be used alone or in combination with TNF-alpha to stimulate differentiation of CD34+ cells into a progenitor T cell lineage. Mention may be made of resveratrol, omeprazole, luteolin, alpha-naphthoflavone, mexiletine, tranilast, 6,2',4'-trimethoxyflavone, CH 223191 (1-methyl-N-[2-methyl-4-[2-(2-methylphenyl) diazenyl]phenyl-1H-pyrazole-5-carboxamide, CAS 301326-22-7). The amount of AhR antagonist is determined on a case-by-case basis by one skilled in the art, taking into account the antagonist's IC ₅₀ (SR1 has an IC ₅₀ of 127 nM while CH 223191 has an IC ₅₀ of 30 nM) and the fact that some products may have agonistic activity in against AhR when used in high concentrations (eg, alpha-naphthoflavone) or depending on the cellular context (eg, omeprazole). Given the difference in IC ₅₀ between SR1 and CH 223191, it is expected that the effective concentration of CH 223191 will be significantly lower than the effective concentration of SR1 disclosed herein.

Thus, the invention also relates to the following:

(i) an in vitro method for producing T cell precursors comprising the step of culturing CD34+ cells in a medium containing an aryl hydrocarbon/dioxin receptor antagonist, in particular StemRegenin 1 (SR1), and in the presence of an immobilized Notch ligand,

(ii) In vitro method as above, wherein the culture medium further comprises TNF-alpha.

(iii) In vitro method as above, wherein the aryl hydrocarbon/dioxin receptor antagonist, in particular SR1, is present in the culture medium from culture day 0.

(iv) An in vitro method according to any of the above, wherein CD34+ cells have been isolated from an adult donor or cord blood.

(v) An in vitro method according to any of the above, wherein the cells are cultured in the presence of an aryl hydrocarbon/dioxin receptor antagonist, in particular SR1, for no more than 10 days.

(vi) An in vitro method according to any of the above, wherein the cells are cultured in the presence of an aryl hydrocarbon/dioxin receptor antagonist, in particular SR1, for 3-7 days.

(vii) an in vitro method according to any of the above embodiments, wherein the aryl hydrocarbon/dioxin receptor antagonist, in particular SR1, is added to the culture medium at a concentration of 1 ng/mL to 300 ng/mL and preferably greater than or equal to 1 ng/mL, or greater than or equal to 3 ng/ml, or greater than or equal to 10 ng/ml and preferably less than 200 ng/ml or 150 ng/ml, and usually from 3 ng/ml to 100 ng/ml.

(viii) An in vitro method according to any of the above, wherein the Notch ligand is a soluble delta-like ligand 4 domain fused to the Fc region of an IgG protein, preferably an IgG2 protein.

(ix) An in vitro method according to any of the above, wherein the cells are also exposed to a fibronectin fragment, said fragment containing the RGDS and CS-1 structures, as well as a heparin-binding domain, preferably immobilized on the inner surface of the culture vessel.

(x) The in vitro method as above, wherein the fibronectin moiety is Retronectin® as described above.

(xi) An in vitro method according to any of the above, wherein the culture medium also contains a vector designed to transfect or transduce CD34+ cells for at least some time of exposing the CD34+ cells to a Notch ligand.

(xii) An in vitro method according to any of the above, wherein the culture medium contains at least three, and preferably all four, cytokines or growth factors selected from the group consisting of interleukin 7 (IL-7), SCF (factor stem cells), thrombopoietin (TPO) and Flt3 ligand (FLT3L).

(xiii) (In vitro) a method for producing T cell precursors, comprising the steps

a. carrying out the method according to any of the above options and

b. purification of the resulting T-cell precursors,

c. optionally conditioning T cell progenitor cells in a bag for injection to a patient.

(xiv) An in vitro method for producing transformed T cell progenitors, comprising the steps

a. culturing CD34+ cells in a medium containing an aryl hydrocarbon/dioxin receptor antagonist, in particular SR1, and in the presence of an immobilized Notch ligand

b. exposing cells to a vector intended for transfection or transduction of CD34+ cells.

(xv) An in vitro method for producing modified T cell progenitors, comprising the steps

a. culturing CD34+ cells in a medium containing an aryl hydrocarbon/dioxin receptor antagonist, in particular SR1, and in the presence of an immobilized Notch ligand

b. exposing cells to a vector or nucleic acid sequences containing an element suitable for gene editing.

The invention also relates to a kit for carrying out any method as described above, including:

(i) a coating medium containing a Notch ligand (in particular, a soluble delta-like ligand domain fused to the Fc region of an IgG protein, in particular an IgG2 protein) and optionally a fibronectin fragment;

(ii) a medium adapted for culturing (and/or expanding) CD34+ cells and T cells, such as α-MEM, DMEM, RPMI 1640, IMDM, BME, McCoy5A, StemSpan™ SFII (StemCell Technologies), X-VIVOTM medium or Fisher's medium;

(iii) progenitor propagation medium containing TNF-alpha and preferably three cytokines, specifically selected from SCF, TPO, Flt3L and IL-7. Preferably, the progenitor propagation medium contains TNF-alpha and all four cytokines SCF, TPO, Flt3L and IL-7.

In another embodiment, the invention relates to a kit containing the same elements (i) and (ii) as above and to a progenitor propagation medium (iii) which contains an aryl hydrocarbon/dioxin receptor antagonist, in particular SR1, and preferably three cytokines , in particular selected from SCF, TPO, Flt3L and IL-7. Preferably, the progenitor propagation medium contains SR1 and all four cytokines SCF, TPO, Flt3L and IL-7.

In another embodiment, the precursor propagation medium (iii) contains TNF-alpha and an aryl hydrocarbon/dioxin receptor antagonist, in particular SR1, and preferably three cytokines, in particular selected from SCF, TPO, Flt3L and IL-7. Preferably, the progenitor propagation medium contains TNF-alpha, SR1 and all four cytokines SCF, TPO, Flt3L and IL-7.

Such a kit is adapted and intended in particular for the implementation of the methods disclosed in this document.

First, a cover medium is used to (i) coat the walls of the culture vessel.

The medium (ii) is then used for culturing CD34+ cells (derived from cord blood or from mobilized peripheral blood, in particular from an adult). Such a medium is usually and preferably used at a concentration of 1x (ie it can be used without dilution).

Medium (iii) is usually used in a 10-fold dilution (ie, it must be diluted in medium (ii) for use). The reconstituted medium from media (ii) and (iii) is then used to promote differentiation of CD34+ cells into T cell progenitors, in a T cell line, as described above.

The invention also relates to a method for increasing the number of T cells in a subject in need thereof, the method comprising administering to the subject an effective amount of T cell precursors obtained by the method disclosed herein.

The invention also relates to T cell precursors, obtained by any method disclosed herein, for their use in the treatment of a subject in need of an increased number of T cells.

In particular, the subject is a person.

In particular, the introduced T cell precursors are autologous. In another embodiment, the T cell precursors administered are allogeneic.

In particular, a subject in need of an increased number of T cells has a disease that causes or results in lymphopenia, such as cancer, HIV infection, partial thymectomy, autoimmune disease, and/or organ transplant.

Description of figures

Figure 1: Total number of nucleated cells obtained from the initial 20,000 CD34+ cells from cord blood (PC, Figure 1.A) or mobilized peripheral blood (MPC, Figure 1.B), after 3 days (black bars) and 7 days (black and gray bars combined) cultivation. NC (normal control): no additives; SR1: addition of StemRegenin 1 (750 nM); TNF-alpha: addition of TNF-alpha (100 ng/ml); + (a), (b), (c): number of cells observed with supplementation after 0-3 days of culture (a), 0-7 days of culture (b) or 4-7 days of culture (c).

Figure 2: Number of CD7+ T progenitor cells obtained on day 7 from an initial 20,000 CD34+ cells from cord blood (PC, figure 2.A) or mobilized peripheral blood (mPC, figure 2.B). Black bars: CD34+ CD7+ cells; gray bars: CD34-CD7+ cells, (a), (b), (c): number of cells observed with supplementation after 0-3 days of culture (a), 0-7 days of culture (b), or 4-7 days cultivation (c).

Figure 3: Bcl11b expression was analyzed on live cells after 7 days of culture, derived from original CD34+ cells from umbilical cord blood (PC) or mobilized peripheral blood (MPC) cultured in the presence (grey bars) or absence (black bars) of TNF-alpha .

Figure 4: Combined effect of TNF-alpha and Notch DL4 ligand on the number of CD7+ cells obtained on day 7, from original cord blood CD34+ cells (PC, black bars, left) or mobilized peripheral blood (mPC, gray bars, right) . (+/- means the presence or absence of DL4 or TNF-alpha).

Figure 5: Frequency of myeloid cells obtained on day 7 from original CD34+ cells from umbilical cord blood (PC) or mobilized peripheral blood (MPC), in the presence (grey bars) or absence (black bars) of TNF-alpha ( mean ± SEM). Figure 6: Total number of CD7+ cells obtained from days 3 to 7 in a dose-response analysis of TNF-alpha from native CD34+ cells from umbilical cord blood (PC, Figure 6.A) or mobilized peripheral blood (mPC, Figure 6.B).

Figure 7: Proportion of CD34-CD7+ cells (gray bars) versus CD34+ CD7+ cells (black bars) in a dose-response analysis of TNF-alpha from native CD34+ cells from umbilical cord blood (PC, Figure 7.A) or mobilized peripheral blood ( IPC, Figure 7.B). Figure 8: Proportion of cells in different phases of the cell cycle. A. PC: cells differentiated from cord blood; B. cells differentiated from mobilized peripheral blood cells. NC: without complements; TNF-alpha: cultured in the presence of TNF-alpha (20 ng/ml); SR1: cultured in the presence of SR1 (30 ng/ml).

Figure 9: Percentage (A) and total (B) of CD5+ CD7+ cells cultured from original cord blood CD34+ cells in the presence of TNF-alpha and/or SR1 at various concentrations after 7 days of culture.

Examples

Example 1 - Material and Methods

human cells

Cord blood samples not bankable were used for research purposes after obtaining the informed consent of the mother of the child. Mobilized peripheral blood (MBC) samples were taken from healthy donors after mobilization with granulocyte colony stimulating factor (G-CSF). The samples were directly enriched in CD34+ cells. Informed consent was given by each donor (Department of Biotherapy, Necker Hospital, Paris).

Exposure of CD34+ Progenitor Cells to Notch Ligand DL-4

CD34+ cells from human CB samples or mobilized peripheral blood were cultured in 24-well plates or 6-well plates that were coated with recombinant human fibronectin (Retronectin, RetroNectin®, Clontech/Takara) and DL-4 (5 μg/ml, PX' Therapeutics, Grenoble, France). Coating was performed for 2 hours at 37°C, then DL-4 coated wells were blocked with 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 minutes at 37°C and washed with phosphate buffered saline . Cultivation was started at a concentration of 2×10 ⁴ cells/well or 1×10 ⁵ cells/well (for 24-well and 6-well plates, respectively) in α-MEM medium (Gibco, Life Technology), supplemented with NaHCO ₃ (7, 5%). (Gibco, Life Technology), and 20% standard fetal calf serum (Hyclone, Thermo Fisher Scientific, Illkirch, France) and recombinant human cytokines interleukin-7 (IL-7), Flt3 ligand (Flt-3), stem cell factor (SCF) and thrombopoietin (TPO) (all at 100 ng/mL and all purchased from PeproTech Inc, Rocky Hill, NJ) with or without TNF-α (R&D Systems, USA). After 3 days of cultivation, the cells were half replaced with fresh medium. Cultured cells were analyzed by fluorescent activated cell sorting (FACS) after 3 and 7 days of DL-4 culture, respectively, to exclude CD34-/CD7- myeloid cells from subsequent analyses.

In vitro T cell differentiation assay on OP9/DL1 cells

T-lymphoid potential of native CD34+ cells from PC and TNF-α-induced T cell precursors generated by DL-4 exposure was assessed in OP9/DL-1 co-cultures as previously described (Six et al, Blood Cells Mol Dis. 2011 Jun 15;47(1):72-8 and Six J Exp Med. 2007 Dec 24;204(13):3085-93).

Quantitative real-time polymerase chain reactions using the RT2 Profiler Matrix

CD7+ cells were sorted on an Ariall cytometer after 7 days of culture. Total RNA of sorted cell fractions from day 3 and day 7 was isolated using the Rneasy Micro Kit (Qiagen, Courtabeuuf, France). Assays on RT2 Profiler PCR Chips were performed according to the protocol detailed in the RT2 Profiler PCR Chip Manual (SA Biosciences, Frederick, MD).

Flow Cytometry Analysis and Cell Sorting

Monoclonal antibodies against human CD34 (AC136), CD3 (BW264/56), CD45 (5B1) were purchased from Miltenyi Biotech (Bergisch Gladbach, Germany), a CD4 (SK4), CD7 (M-T701), CD25 (M-A251 ), 7-aminoactinomycin D (7AAD) from BD Biosciences (San Jose, CA). The anti-human CD8 antibody (RPAT8) was purchased from Sony Biotechnology (San Jose, USA). An anti-human Ctip2 antibody (Bcl11b) was purchased from Abcam (Cambridge, UK).

Human cells were stained and analyzed using a Gallios analyzer (Beckman Coulter, Krefeld, Germany). Cells from xenogeneic recipients were analyzed on a MACSQuant® instrument (Miltenyi Biotech, Bergisch Gladbach, Germany). Data were analyzed using FlowJo software (Treestar, Ashland, OR) after selection of viable 7AAD-negative cells. Cell subpopulations were sorted using the ARIA II system.

Cell Proliferation Assays

For CD34+ cell proliferation assays, PK and PBM cells were labeled using the CellTrace™ CFSE kit (Life Technologies, Carlsbad, CA) prior to culturing with DL-4 and TNF-alpha (Life Technologies). Cell staining intensity was measured before cultivation daily from day 3 to day 7. CFSE positive cells were analyzed on a Gallios cytometer (Beckman Coulter).

Cell cycle analyzes

For cell cycle analysis, cells were stained with Hoechst33342 (Life technology) and Ki67-PC5 (BD Bioscience) after being fixed with the Fixative reagent from the PerFix-nc kit (Beckman Coulter) at room temperature for 15 minutes and the permeabilization reagent was added. Data were analyzed using FlowJo software (version 10.2, Treestar, Ashland, OR) after selection of viable 7AAD-negative cells.

Adoptive Transfer of In Vitro-Derived Adult HSPC-Derived T Cell Progenitors into Neonatal NSG Mice

All animal experiments and procedures were carried out in accordance with the rules of the French Ministry of Agriculture for animal experiments. Injection of in vitro generated human T cell progenitors into NSG mice has been approved by the Ministry of Higher Education and Research (APAFIS 2101-2015090411495178v4).

NSG mice (NOD-Scid(IL2Rg ^null )) (obtained from Jackson Laboratory, Bar Harbor, Maine, http://www.jax.org) were maintained under sterile conditions. Progeny derived from CD34+ HSPC mPCs in 7 day old DL-4 cultures with or without TNF-α (3×10 ⁵ or 1×10 ⁶ ) were injected intrahepatic to neonatal NSG mice (0-4 days). Control mice were injected with 3×10 ⁵ uncultivated CD34+ cells from mPC or 100 μl of PBS.

Mean graft engraftment rates in NSG mice were determined 4-12 weeks after transplantation. Flow cytometry analysis was performed on fresh cells harvested from femur, thymus, peripheral blood, and spleen. Cells were treated with 1x erythrocyte lysis buffer (Biolegend, USA) and washed before antibody staining.

T cell receptor diversity analysis

TCR gene rearrangement analysis was performed in duplicate and on two independently purified subpopulations (mean shown).

Quantitative determination of TCR-δ (D δ2-D δ3, D δ2-J δ1 and D δ3-J δ1) was performed using the listed sets of primers and probes.

The following primers and probe were used for the D δ2-D δ3 rearrangements:

D δ2, 5'-CAAGGAAAGGGAAAAAGGAAGAA-3' (SEQ ID NO: 9);

D δ3, 5'-TTGCCCCTGCAGTTTTTGTAC-3' (SEQ ID NO: 10);

and probe D'3, 5'-ATACGCACAGTGCTACAAAAACCTACAGAGACCT-3' (SEQ ID NO: 11).

The following primers and probe were used for D δ2-J δ1 rearrangements:

D δ2, 5'-AGCGGGTGGTGATGGCAAAGT-3' (SEQ ID NO: 12);

J δ1, 5'-TTAGATGGAGGATGCCTTAACCTTA-3' (SEQ ID NO: 13);

and probe J δ1, 5'-CCCGTGTGACTGTGGAACCAAGTAAGTAACTC-3' (SEQ ID NO: 14)

The following primers and probe were used for D δ3-J δ1 rearrangements:

D δ3, 5'-GACTTGGAGAAAACATCTGGTTCTG-3' (SEQ ID NO: 15);

Primer J δ1 and probe J δ1.

Analysis of TCR rearrangements by multiplex fluorescent PCR was performed by separating fluorochrome labeled single chain PCR products in a capillary sequencing polymer and detected by automated laser scanning.

Apoptosis assays

Cells were washed with cold PBS and resuspended in binding buffer at a concentration of 1 million cells/ml. After adding 5 µl of Annexin V-PE (BD Bioscience) and 2 µl of 7AAD, the cells were incubated in the dark at room temperature for 15 min. The cells were then washed with 500 µl of binding buffer and resuspended in 100 µl of binding buffer to perform the assay for 1 hour.

Example 2 Improving Proliferation and Differentiation of T Cell Progenitors

Figure 1 shows that when culturing CD34+ cells with DL-4, the addition of TNF-alpha to the cell culture medium allows a 10-fold increase in the total number of cells obtained on day 7 compared to culture without TNF-alpha, when using as initial CD34+ cells extracted from cord blood (Figure 1.A) or from RV (Figure 1.B).

The figure 2 shows that the addition of TNF-alpha to the cell culture medium makes it possible to increase the number of CD7+ cells by 20-40 times, when used as initial CD34+ cells extracted from cord blood (Figure 2.A) or from RV (Figure 2.B). This improvement is especially great for the CD34-CD7+ cell population.

Example 3 Analysis of Surface Markers of T Cell Progenitors

Surface markers present on the surface of cells obtained after 7 days of culture were determined by flow cytometry.

These tables show that the addition of TNF-alpha leads to an increase in the proportion of CD7+ cells without a significant increase in the proportion.

After 7 days of culture, HSPCs differentiate into CD34-CD7+ CD5- T cell progenitors.

Surface markers present on the cell surface obtained after 10 days of culture were also determined by flow cytometry.

No CD1a expression was detected (data not shown).

The kinetics of surface marker modification was studied and the presence of TNF-alpha in the culture medium was found to increase the proportion of CD7+ from day 4 to day 7 (data not shown).

Example 4: Rearrangement of T-cell receptors

DL-4 T cell precursors did not show any signs of TCR rearrangement with or without TNF-alpha or SR1 after 7 days of culture (data not shown).

A specific rearrangement analysis was performed:

Results of analysis of TCR-delta rearrangements

Detection of Dδ2-Dδ3 rearrangements in CB-NC and CB-SR1. No other TCR-delta rearrangements were found. Results were in line with RQ-PCR (Quantitative Real Time PCR) quantification

Results of analysis of TCR-gamma rearrangements

No TCR-gamma rearrangements were found.

Results of analysis of TCR-beta rearrangements

No TCR-beta rearrangements were found.

Example 5: T-commitment of TNFα-induced T cell progenitors

Bcl11b is an important transcription factor that is turned on exclusively after T cell commitment and is absolutely essential for T cell differentiation.

Intracellular staining on T-cell progenitors cultured with TNF-alpha showed Bcl11b positive expression for CD34+ cells derived from both cord blood and mPC. In FIG. 3 shows that TNF-alpha increases the proportion of total cells expressing the Bcl11b transcription factor. During cultivation with TNF-alpha, the proportion of cells expressing Bcl11b increased.

Example 6 Investigation of Differentiation in Other Cell Lines

The presence of other cell surface markers (CD14 and CD33) specific to other lines was assessed.

Figure 5 shows that culturing in the presence of TNF-alpha reduces the number of such cells below the detection level, while when culturing CD34+ cells without TNF-alpha, their proportion is less than 22%.

Example 7: TNF-alpha reduces cell apoptosis

Markers of apoptosis (7 AAD and annexin 5) were studied.

This table shows that cultivation in the presence of TNF-alpha reduces the presence of apoptotic markers. This is especially evident for the IPC.

Example 8 Dose-response analysis of TNF-alpha

Various doses of TNF-alpha have been used.

Figure 6 shows that TNFa can increase the number of CD7+ cells during culture at a concentration of more than 10 ng/ml, either for cells from PC (Figure 6.A) or for cells from MPC (Figure 6.B).

Dose-dependence kinetics showed that TNF-alpha increased T-cell differentiation only after 4 days of cultivation in DL-4. No difference was found between concentrations of 10, 50 and 100 ng/ml (not shown).

Lower concentrations (0.01-10 ng/mL) were analyzed to determine the effective concentration threshold.

The effect of TNF-alpha on PC and MPC T cell differentiation (percentage of CD34-CD7+ cells) was found to be concentration dependent at low concentration (FIG. 7). The total number of CD7+ progenitor T cells did not differ between 5 ng/mL and 100 ng/mL.

Example 9 Proliferation Assay During Culture

TNF-alpha was found to increase the proliferation of CD34+ CD7+ T cell progenitors from day 3 in culture with DL-4 compared to culture conditions without TNF-alpha (data not shown).

Example 10: Synergy between TNF-alpha and Notch ligand

Figure 4 shows that in the absence of DL4, neither cells from PCs nor from MPCs could differentiate into progenitors of CD7+ T cells. Even the addition of TNF-alpha medium could not change this situation.

When both TNF-alpha and Notch ligand are present in the medium, the observed effect is very high. Thus, there appears to be synergy between the two compounds and the effect of TNF-alpha on T cell differentiation is likely dependent on the presence of Notch.

Example 11 Addition of TNF-alpha Increases Proliferation of CD7+ Progenitors

After 7 days of cultivation in the presence of TNF-alpha, CD34+ CD7-, CD34+ CD7+ and CD34-CD7+ subpopulations were sorted, stained with CFSE (carboxyfluorescein succinimidyl ether) and monitored for dilution with CFSE (surrogate marker of cell proliferation) from days 8 to 10.

Only CD34+ cells CD7+ and CD34-CD7+ show increased proliferation when cultured with TNF-alpha. (Data not shown)

Example 12 Cell Cycle Analysis

Cell cycle analysis was performed. More cells were found to emerge from the G0 phase in the presence of TNF-alpha for CD7+ progenitors derived from both PC and MPC (Figure 8).

Example 13 Combination of SR1 and TNFa

SR1 accelerates T cell differentiation as evidenced by the presence of CD5+ CD7+ cells on day 7. The number of CD5+ CD7+ cells increases under the influence of both TNF-alpha and SR1 (figure 9).

Example 14: In vivo data

T cell precursors induced in the presence of TNF-alpha can significantly accelerate T-line recovery in vivo.

Indeed, 4 weeks after transplantation, recipient mice injected with T-cell mPC precursors produced in the presence of TNF-alpha had a larger thymus than mice injected with T-cell mPC precursors derived without TNF-alpha. alpha. T cell precursors induced in the presence of TNF-alpha can differentiate into activated TCRαβ T cells within 4 weeks in vivo. (Data not shown)

Thus, the addition of TNF-alpha from day 0 to a culture system containing DL4 results in an increase in the number of T cell progenitors (determined by CD7 surface expression) 40-fold for HSPC from mPC and 20-fold for HSPC from PC per day. 7.

The progenitors of CD7+ T cells derived from both PC and MPC were mostly CD34- and were CD1a-negative. Also, the cells were mostly CD5 negative.

They expressed Bcl11b, which is an important fine-tuning molecule for T-commitment and further T-cell differentiation.

These cells showed no evidence of T-cell receptor rearrangement.

Their phenotype and molecular characteristics were similar to those of CD34-CD7+ T cell progenitors derived without TNF-alpha.

Regarding the mechanisms involved in TNF-alpha activity, TNF-alpha reduces the expression of apoptotic markers and increases cell proliferation during culture. It also inhibits the production of myeloid cells.

The use of TNF-alpha in a culture system containing DL4 greatly increases the number of progenitor T cells produced from both adult HSPC and cord blood. Thus, its use can overcome the difficulty of obtaining large amounts of T cell progenitors from adult human HSPCs. It can also reduce the initial amount of HSPC needed in future clinical trials and the amount of GMP-quality and other reagents needed, thereby reducing the cost of producing these T cell precursors.

Claims (29)

1. An in vitro method for producing T cell precursors, comprising the step of culturing CD34+ cells in a medium containing TNF-alpha and optionally an aryl hydrocarbon/dioxin receptor antagonist, in the presence of an immobilized delta-like ligand 4. 2. In vitro method according to claim 1, wherein the delta-like ligand 4 is immobilized on the inner surface of the culture vessel or on the surface of the microspheres present in the culture medium. 3. The in vitro method of claim 1, wherein the aryl hydrocarbon/dioxin receptor antagonist is StemRegenin 1 (SR1), 4. The in vitro method of claim 1, wherein TNF-alpha and optionally the aryl hydrocarbon/dioxin receptor antagonist is present in the culture medium from culture day 0, wherein TNF-alpha is preferably present at a concentration greater than or equal to 3 ng/mL and optionally the aryl hydrocarbon/dioxin receptor antagonist is present in the culture medium at a concentration greater than or equal to 30 ng/mL. 5. In vitro method according to claim 1, wherein CD34+ cells have been isolated from an adult donor. 6. In vitro method according to claim 1, wherein the cells are cultured in the presence of TNF-alpha and optionally SR1 for no more than 10 days, preferably 3 to 7 days. 7. In vitro method according to claim 1, wherein delta-like ligand 4 is a soluble domain of delta-like ligand 4 fused to the Fc region of an IgG protein, preferably an IgG2 protein. 8. In vitro method according to claim 1, wherein the cells are additionally exposed to a fibronectin fragment, said fragment containing the RGDS and CS-1 structures, as well as a heparin-binding domain, preferably immobilized on the inner surface of the culture vessel or on microspheres, and preferably a fibronectin fragment is Retronectin®. 9. In vitro method according to claim 1, wherein the culture medium further comprises a vector designed to transfect or transduce CD34+ cells for at least some time of exposure to CD34+ cells with delta-like ligand 4. 10. The in vitro method of claim 9, wherein the vector for transfection or transduction of CD34+ cells is a transgene encoding a chimeric antigen receptor (CAR). 11. A method for obtaining T cell precursors, comprising the steps: a. implementation of the method according to claim 1 and b. purification of the resulting T-cell precursors, c. optionally conditioning T cell progenitors in a bag for injection to a patient. 12. In vitro method for obtaining transformed T-cell precursors, including the steps: a. culturing CD34+ cells in a medium containing TNF-alpha and in the presence of an immobilized delta-like ligand 4, b. exposing cells to a vector intended for transfection or transduction of CD34+ cells. 13. In vitro method for obtaining modified T-cell precursors, including the following steps: a. culturing CD34+ cells in a medium containing TNF-alpha and in the presence of an immobilized delta-like ligand 4; b. exposing cells to a vector or nucleotide sequences containing an element suitable for gene editing. 14. A population of CD7+ T cell precursors to increase the number of T cells in a subject in need thereof, wherein said precursors are obtained by the method of claim 1, wherein the proportion of cells that are CD34- and CD1a- or CD34- and CD5- or CD34- and CD1a- and CD5-, in this population exceeds 80%. 15. The population of CD7+ T cell precursors of claim 14, wherein said precursors express CAR. 16. A kit for implementing the method according to claim 1, containing: i. a cover medium containing delta-like ligand 4 and optionally a fragment of fibronectin, ii. medium adapted for culturing CD34+ cells and T cells, iii. T cell progenitor expansion medium containing TNF-alpha and optionally an aryl hydrocarbon/dioxin receptor antagonist and preferably three or four cytokines selected from the group consisting of SCF, TPO, Flt3L and IL-7. 17. Use of a CD7+ population of T cell precursors to increase the number of T cells in a subject in need thereof, said precursors being obtained by the method of claim 1, and wherein the proportion of cells that are CD34- and CD1a- or CD34- and CD5 -, or CD34- and CD1a- and CD5-, in this population exceeds 80%. 18. Use according to claim 17 for the treatment of lymphopenia. 19. Use according to claim 17 for the treatment of cancer, HIV infection, partial thymectomy, autoimmune disease and/or organ transplantation.

Images