Methods for generating Vdelta1+ T cells

Abstract

The present invention relates to a novel and efficient method for the large-scale selective generation of γδ T cells, preferably human V51+ γδ T cells, optimal for clinical application in adoptive immunotherapy against cancer. In this regard, taking into account that both human cord blood HPCs and human early thymic progenitor cells, the current source of stem cells of choice in the clinic, can efficiently generate de novo human γδ T cells in response to Notch signaling, most efficiently in response to the Notch ligand Jag2, the method comprises inducing the differentiation of cord blood CD34+ hematopoietic progenitor cells (HPCs) and / or human CD34+ early thymic progenitor cells by activating these progenitors with the Jag2 Notch ligand.

Description

[Technical field]

[0001] The present invention relates to the field of medicine, in particular the present invention relates to a novel and efficient method for the large-scale selective generation of human V51+ γδ T cells optimal for clinical application in adoptive immunotherapy against cancer. [Background technology]

[0002] The use of adoptive T cell therapy is often limited by barriers caused by MHC (major histocompatibility complex) mismatch. Thus, current clinical application of T cell products, such as chimeric antigen receptor (CAR)-expressing T cells (CAR-T), relies on individualized autologous T cell production. However, patient T cells are often functionally damaged due to the continuous administration of invasive drug therapies. In addition, the individualized, tailor-made autologous T cell production process narrows the broad application of T cells to specific tumor types, such as T cell tumors. Therefore, universal allogeneic T cells are needed to prepare T cell products that can serve as "off the shelf" ready-to-use therapeutic agents for large-scale clinical application.

[0003] Recently, γδ T cells have emerged as an alternative to αβ T cells for cellular immunotherapy, as they are not restricted by MHC presentation of tumor-associated peptides and exhibit limited alloreactive potential. Nevertheless, γδ T cells play a key role during viral infection and tumor progression, resulting in robust and long-lasting antitumor responses (Non-Patent Document 1, Non-Patent Document 2). In particular, Vδ1+ γδ T cells are highly attractive candidates for adoptive cell therapy of cancer, as they are usually predominant (vs. Vδ2+) in tumor infiltration, are less susceptible to activation-induced cell death, and can persist for long periods as tumor-reactive lymphocytes (Non-Patent Document 3). However, Vδ1+ γδ T cells, a Vδ1+ T cell subtype of γδ T cells prevalent at birth (Non-Patent Document 4), are scarce in peripheral blood, and their therapeutic use is hindered by the lack of suitable expansion / differentiation methods. In this regard, a clinical-grade method using TCR agonists and cytokines to selectively expand cytotoxic V51 T cells isolated from peripheral blood has recently been developed by the group of Bruno Silva-Santos (Non-Patent Document 5, Non-Patent Document 6). This cellular product, named Delta One T (DOT) cells, showed therapeutic potential in preclinical models of chronic lymphocytic leukemia (CLL), providing proof-of-principle for their clinical application in adoptive immunotherapy against cancer.

[0004] Nevertheless, the limited number of V51+ T cells that can be isolated from peripheral blood necessitates the development of complementary protocols for the robust generation / expansion of cytotoxic V51+ antitumor T cells. More importantly, the unfocused and diverse T cell receptor (TCR) repertoire of neonatal V51+ T cells is tightly restricted and focused to a few predominant clonotypes by adulthood due to clonal expansion in response to peripheral immune challenge, such as with CMV (Non-Patent Document 7). Clonal expansion of V51+ cells leads to the differentiation of naive V51 T cells into a V51 T cell effector / memory phenotype characterized by downregulation of CD27 (Non-Patent Document 9). Because human naive-derived effector T cells retain longer telomeres, they are more likely to expand in vitro and express T cell receptor transgenes, and have been associated with higher efficacy in clinical trials; it has been hypothesized that naive cells resist terminal differentiation or "exhaustion," maintain high replicative potential, and therefore may be a superior subset for use in adoptive immunotherapy (Non-Patent Document 10). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Vantourout and Hayday, Nat Rev Immunol., 2013 [Non-Patent Document 2] Silva-Santos et al., Nat. Rev. Immunol., 2015 [Non-Patent Document 3] Siegers et al., Mol. Ther. 2014 [Non-Patent Document 4] Morita et al., J. Immunol. 1994 [Non-Patent Document 5] Correia et al., Blood 2011 [Non-Patent Document 6] Almeida et al. Clin. Cancer Res. 2016

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

[0006] The object of the present invention, set forth to solve the above problems, relates to a novel and efficient method for the large-scale selective generation of human V51+ γδ T cells, preferably allogeneic human V51+ γδ T cells, optimal for clinical application in adoptive immunotherapy against cancer. In this regard, taking into account the present finding that both human umbilical cord blood CD34+ hematopoietic stem / progenitor cells (HPCs) and human CD34+ early thymic progenitor cells (ETPs), currently the source of hematopoietic stem cells of choice in the clinic, can efficiently generate de novo human V51+ γδ T cells in response to Notch signaling, the method of the present invention comprises inducing differentiation of human HPCs, preferably cord blood CD34+ HPCs, and / or human CD34+ ETPs, by activating these cells with a Jag2 Notch ligand, where said ligand is highly expressed on the surface of a bone marrow-derived stromal cell line. The method preferably comprises co-culturing human HPCs, preferably cord blood CD34+ HPCs, or human CD34+ ETPs, on stromal cells with high Jag2 expression, preferably supplemented with Flt3+SCF+IL-7, for up to 9 weeks. V51+ γδ T cells (STEP1), generated as described above in a Notch-dependent, TCR-independent manner, are then activated and expanded according to any method known in the art, for example by using anti-CD3 mAbs and cytokines including IL-4, IFN-γ and IL-15, to induce proliferation of V51+ γδ T cells upon TCR activation (Non-Patent Document 6) (STEP2).

[0007] The present invention provides an in vitro method for generating an expanded population of de novo Notch-induced differentiated CD1a-V51+ γδ T cells from a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or human ETPs, the method comprising: A first step of generating a cell composition comprising γδ T cells in greater amounts than αβ T cells, the first step comprising: a. Growing a cell population comprising HPCs and / or ETPs in an appropriate culture medium comprising a Jag2 Notch ligand or an agonist thereof; b. maintaining the cells in culture for a sufficient duration to generate γδ T cells; Preferably, the duration is between about 2 weeks and about 15 weeks, and a Jag2 Notch ligand or agonist should be present or added in sufficient amounts to the cell culture during and throughout the culture period; Including, The method includes a first step, characterized in that the cell composition resulting from the first step comprises CD1a-V51+ γδ T cells, A second step of activating and inducing proliferation of the CD1a-V51+ γδ T cells, the second step comprising: growing the cells obtained after the first step in a suitable culture medium, in the presence of a γδ TCR agonist and in the presence of at least IL21 and IL15, to obtain a cell population characterized in that at least 40% of the total γδ T cells are expanded and activated CD1a-V51+ γδ T cells, said population of CD1a-V51+ γδ T cells being At least 40%, preferably at least 50% or 60% of the total number of V51+ γδ T cells express the CD56 marker; At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp44 marker; At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp30 marker; At least 70%, preferably 80% to 100%, of the total number of Vδ1 + γδ T cells express the NKG2D marker; At least 80%, preferably 90% to 100%, of the total number of Vδ1 + γδ T cells express the DNAM-1 marker; It is characterized by: Preferably, the expression levels of the above markers are measured by flow cytometry. The method further comprises a second step.

[0008] Preferably, the second step comprises: growing the cells obtained after the first step for 5 days in a suitable culture medium in the presence of at least one γδ TCR agonist and at least IL21; On day 5, preferably day 7, IL15 is added to the culture medium and the cells are cultured in the presence of at least one γδ TCR agonist, IL21 and IL15 for at least 7 days; Includes.

[0009] Preferably, at least one γδ TCR agonist is added at a concentration between 0.5 μg / ml and 4 μg / ml, IL21 is added at a concentration between 7 ng / ml and 15 ng / ml, and IL15 is added at a concentration between 70 ng / ml and 150 ng / ml.

[0010] Preferably, the human HPCs are CD34+ cord blood HPCs.

[0011] Preferably, the activated CD1a-V51+ γδ T cells obtained after the second step are further characterized in that they express the CD25 and / or CD69 activation markers, but not the LAG3 and / or CTLA4 exhaustion markers.

[0012] Preferably, the activated CD1a-V51+ γδ T cells obtained after the second step are characterized by expressing the CD8 marker and having a T effector phenotype, which is characterized by the expression of the CD45RA marker and the lack of expression of the CD62L marker.

[0013] The present invention further provides a cell composition comprising de novo Notch-induced differentiated CD1a-V51+ γδ T cells obtained or obtainable after the second step of the method defined above. Preferably, the activated V51+ γδ T cells are At least 40%, preferably at least 50% or 60% of the total number of V51+ γδ T cells express the CD56 marker; At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp44 marker; At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp30 marker; At least 70%, preferably 80% to 100%, of the total number of Vδ1 + γδ T cells express the NKG2D marker; At least 80%, preferably 90% to 100%, of the total number of Vδ1 + γδ T cells express the DNAM-1 marker; It is characterized by: Preferably, the expression levels of the above markers are measured by flow cytometry.

[0014] The present invention further provides a cell composition obtained or obtainable after the first step of the method defined above, comprising γδ T cells in greater amounts than αβ T cells. Preferably, the cell composition is characterized in that the population of V51+ γδ T cells generated after the first step is a. a first cell population characterized by expression of the immature surface cell marker CD1a (CD1a+Vδ1+γδ T cells); b. A second cell population characterized by not expressing the immature surface cell marker CD1a (CD1a-Vδ1+γδ T cells); The present invention is characterized by comprising:

[0015] Preferably, the first cell population is characterized in that the cells do not express the surface cell markers CD25, CD27, NKp44, NKp30, and NKG2D. Preferably, the second cell population is characterized in that the cells express at least one, or at least a combination of two or more, preferably all, of the surface markers CD27, CD73, CD69, NKp44, NKp30, and NKG2D.

[0016] The present invention further provides a CAR T cell obtained or obtainable using the cell composition defined above.

[0017] The present invention further provides a pharmaceutical composition comprising the cell composition or CAR T cells defined above and further comprising a pharma- ceutically acceptable agent or carrier.

[0018] The present invention further provides a pharmaceutical composition as defined above for use in therapy, preferably in cell therapy, tumor or cancer treatment, tumor or cancer immunotherapy, and / or leukemia treatment.

[0019] The following drawings present preferred embodiments for the purpose of illustrating the description and should not be construed as limiting the scope of the invention. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a graph showing Notch-induced γδ (and αβ) T cell generation from CD34+ human early thymic progenitor cells (ETPs). Jag2 is the most efficient ligand for γδ T cell generation. Jag2 induces a 250- to 300-fold higher yield of γδ T cells from CD34+ human ETPs by day 30 of culture. [Diagram 2] Graph showing that Jag2-Notch signaling supports V51+ γδ T cell generation from ETPs. Jag2-induced V51+ γδ T cells (100-fold yield) generated from human CD34+ ETPs by day 30 of culture produce IFNγ but not IL-17. [Diagram 3] FIG. 1 shows the efficiency of Jag2-mediated in vitro V51+ γδ T cell generation from human CD34+ ETPs. [Figure 4] Graph showing Jag2-Notch induced V51+ γδ T cell generation from human CD34+ cord blood hematopoietic stem / progenitor cells (HPCs). Kinetics of Jag2 induced total cell expansion and γδ T cell generation from human CD34+ cord blood HPCs are shown. Dot plots show V51 expression in de novo generated γδ T cells by day 50. [Diagram 5]FIG. 13: Efficiency of generating total γδ T cells and Vδ1+ γδ T cells from human CD34+ cord blood HPCs upon Jag2-Notch signaling over 9 weeks of culture (STEP1). [Figure 6] Graphs showing percentages of total γδ T cells (left) and Vδ1+ γδ T cells (middle) within CD3+ T cells, and percentages of CD1a+ cells within Vδ1+ γδ T cells (right), generated from either human CD34+ CB HPCs subjected to human Jag2 signaling (CB-Jag2) or human CD34+ CB HPCs isolated ex vivo from umbilical cord blood (CB ex vivo) or from peripheral blood (PB ex vivo). Each dot represents one independent experiment or biological sample (n=4). [Figure 7] FIG. 1 is a graph showing Notch-induced γδ (and αβ) T cell generation from CD34+ human early thymic progenitor cells (ETPs). Stromal cells lacking human Notch ligands (OP9-GFP) are unable to support ETP cell expansion / differentiation. [Figure 8] Graph showing the phenotype of V51+ γδ T cells generated from human CD34+ CB HPCs subjected to human Jag2 signaling. (A) V51+ γδ T cells comprise a CD1a+ immature γδ T subset that displays either a CD4+, or a CD4+CD8+ double positive (DP) or CD4-CD8- double negative (DN) phenotype, and a CD1a- mature γδ T cell subset of DN or CD8+ cells. (B) Mature CD1a-V51+ γδ T cells are naive CD27+ expressing the γδ T cell differentiation marker CD73 and distinct levels of the NKp44, NKp30 and NKG2D cytotoxic NK receptors. [Figure 9] Graph showing that mature CD1a-V51+ γδ naive T cells generated from human CD34+ CB HPCs undergoing human Jag2 signaling differ from V51+ cells (CD1a-) present in PB with respect to expression of CD73 and cytotoxic NK receptors. Each dot represents one independent experiment or biological sample. [Figure 10] Graph showing increased expression (high expression) of Jag2 Notch ligand in transduced OP9 cells. Numbers indicate the mean fluorescence intensity of Jag2 expressed on the cell surface of either non-transduced OP9 stromal cells (left projection) or Jag2-transduced OP9 stromal cells (right projection). Cells were labeled with mAb against human Jag2 coupled to PE and analyzed by flow cytometry. [Figure 11] Graphs showing heterogeneity of Vδ subsets of γδ T cells present in the human thymus or generated in vitro from ETPs. (A) Bar graphs represent mean + / - SEM of the frequency of the indicated Vδ subpopulations relative to total γδ T cells present in vivo in the human thymus (left); right: mean + / - SEM of the frequency of CD1a+, CD1aint with CD1a low expression and CD1a- within Vδ1 and Vδ2 γδ T cell subsets in vivo in the human thymus. (B) Flow cytometry kinetic analysis of Vδ1 and Vδ2 expressed in human ETP thymocytes co-cultured with the indicated Notch ligand OP9 cell line. Numbers in quadrants represent mean + / - SEM of the frequency of the indicated cell subsets. (C) Kinetics of absolute numbers of Vδ1+, Vδ2+ and Vδ1-Vδ2- γδ T cells generated from ETPs cultured as in B for 31 days. Data represent the mean cell numbers + / - SEM from three independent experiments. [Figure 12] Figure 2: Two-step protocol for the generation (STEP1) and expansion (STEP2) of human V51+ γδ T cells from CB HPCs. CD34+ HPCs isolated from human CB samples were co-cultured on Jag2-expressing OP9 cells with Flt3 ligand, SCF and IL-7 for up to 8 weeks (STEP1). Cells obtained in STEP1 (CB-Jag2) and cell suspensions isolated ex vivo from either human CB or human peripheral blood (PB) and depleted of TCRαβ+ cells were expanded in vitro in suspension cultures supplemented with anti-TCR agonists and cytokines (STEP2). [Figure 13]Graph showing that the majority of CB-Jag2-STEP2 V51+ γδ T cells are CD8+CD1a- mature effector cells. Expression of CD8, CD1a, CD27, CD45RA and CD62L was analyzed in V51+ γδ T cell subsets contained in the indicated CB-Jag2-STEP2, CB-STEP2 and PB-STEP2 populations expanded using either the DOT protocol (upper panel) or the CSIC STEP2 protocol (lower panel). Expression of CD62L and CD45RA was used to determine the following T cell phenotypes (T effector memory (TEM): CD62L-CD45RA-; T central memory (TCM): CD62L+CD45RA-; T naive (TN): CD62L+CD45RA+; and T effector (Teff): CD62L-CD45RA+). Bars represent mean + / - SEM. Dots represent independent experiments or biological samples: CB-Jag2-STEP2, n = 3; CB-STEP2, n = 4; PB-STEP2, n = 6. One-way ANOVA, Kruskal-Wallis test (*p<0.05) was performed to assess statistical significance. [Figure 14] Graph showing that CB-Jag2-STEP2 Vδ1+ γδ T cells are activated T cells with a low exhaustion cell profile. Expression of activation (CD25 and CD69) and exhaustion-associated surface markers (LAG3 and CTLA4) in Vδ1+ gated γδ T cells in CB-Jag2-STEP2, CB-STEP2 and PB-STEP2 populations expanded using STEP2 DOT protocol (upper panel) or STEP2 CSIC protocol (lower panel). Each dot represents one independent experiment or biosample: CB-Jag2-STEP2, n=2-3; CB-STEP2, n=3-4; PB-STEP2, n=6. One-way ANOVA, Kruskal-Wallis test (*p<0.05) was performed to assess statistical significance. [Figure 15]Graph showing that the majority of CB-Jag2-STEP2 Vδ1+ cells display cytotoxicity-associated activating receptors. Expression of cytotoxicity-associated surface markers in Vδ1+ γδ T cell subsets contained in the indicated CB-Jag2-STEP2, CB-STEP2 and PB-STEP2 populations expanded using the STEP2 DOT protocol (upper panel) or STEP2 CSIC protocol (lower panel). Each dot represents one independent experiment or biological sample: CB-Jag2-STEP2, n=2-3; CB-STEP2, n=3-4; PB-STEP2, n=6. One-way ANOVA, Kruskal-Wallis test (*p<0.05) was performed to assess statistical significance. [Figure 16] Graph showing that CB-Jag2-STEP2 cells exhibit high in vitro cytotoxic potential against leukemia cell lines. CB-Jag2-STEP2, CB-STEP2 and PB-STEP2 populations expanded according to the STEP2 DOT protocol (upper panel) or STEP2 CSIC protocol (lower panel) were assayed for cytotoxicity against Jurkat and Molm13 leukemia cells in 48h assays at the indicated E:T ratios. (n=3), mean + / - SD data are shown. Statistical analysis was performed using the Holm-Sidak multiple comparison t-test. *p<0.05, **p<0.01. [Figure 17] Figure 1: Efficiency of generation of human Vδ1+CD1a-γδ T cells from CB cells after STEP1 (CB-Jag2) and STEP1+STEP2 (DOT or CSIC protocols) culture. Number of Vδ1+CD1a-γδ T cells (n=4) generated from CD34+ HPCs isolated from total CB cells (106) upon 8 weeks of culture on Jag2-expressing OP9 stroma (STEP1) and further expansion according to DOT or CSIC protocols (STEP2) (left panel). The percentage of γδTCR+ cells among T cells recovered after STEP2 DOT and CSIC protocols is shown in the middle panel and the percentage of Vδ1+ cells among γδTCR+ cells is shown in the right panel (n=3). [Figure 18]Schematic diagram of STEP2 DOT and CSIC extension protocols. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs at the time of filing. However, in case of any potential ambiguity, the definitions provided herein shall prevail over any other definitions. Further, unless otherwise required by context, singular terms shall include the plural, and similarly plural terms shall include the singular.

[0022] In this disclosure, "comprises," "comprising," "containing," "having," and the like shall have the meaning given them in European and U.S. patent law and may mean "includes," "including," and the like. "Consisting essentially of" or "consists essentially" shall likewise have the meaning given them in European and U.S. patent law, and the term is open-ended, permitting the presence of things other than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of the recited things are not changed by the presence of things other than what is recited.

[0023] The present invention relates to the de novo generation of Notch-induced V51+ γδ T cells differentiated from a cell population comprising primarily human HPCs, such as CD34+ cord blood HPCs, and / or CD34+ ETPs. "De novo" is the Latin expression for "new" or "ab initio", i.e. as used herein, de novo shall be understood as the creation of new Notch-induced differentiated V51+ γδ T cells, rather than based on pre-existing V51+ γδ T cells.

[0024] In the context of the present invention, a "Notch ligand" is understood as a protein that can bind to a surface Notch receptor, resulting in a cell signal that mediates cell fate decisions, including activation and differentiation of hematopoietic progenitor cells (Artavanis-Tsakonas et al., Science 1999). Thus, the term as used herein encompasses naturally occurring protein ligands, such as Delta and Serrate / Jagged family ligands, as well as engineered Notch ligands and Notch agonists, including antibodies, peptidomimetics and small molecules against Notch receptors, that have biological effects corresponding to those of the natural ligands. In some embodiments, the Notch ligand is a Jag2 Notch ligand. A preferred Notch ligand is selected from the group consisting of DLL1, DLL4, Jag1 or Jag2.

[0025] As used herein, "DLL1" is understood to be the naturally occurring human homolog of the Drosophila Notch Delta ligand (Delta-like 1). More preferably, the term may encompass engineered Notch ligands and Notch receptor agonists that have the biological effect of the native Delta-like 1 ligand.

[0026] As used herein, "DLL4" is understood to be the naturally occurring human homolog of the Drosophila Notch Delta ligand (Delta-like 4). More preferably, the term may encompass engineered Notch ligands and Notch receptor agonists that have the biological effect of the native Delta-like 4 ligand.

[0027] As used herein, "Jag1" is understood to be the naturally occurring human homolog of the Drosophila Notch Serrate / Jagged ligand (Jagged1). More preferably, the term may encompass engineered Notch ligands and Notch receptor agonists that have the biological effects of the native Jagged1 ligand.

[0028] As used herein, "Jag2" is understood to be the naturally occurring human homolog of the Drosophila Notch Serrate / Jagged ligand (Jagged2). More preferably, the term may encompass engineered Notch ligands and Notch receptor agonists that have the biological effect of the native Jagged2 ligand.

[0029] As used herein, a "V51+ γδ T cell" is understood as a T cell expressing a TCR composed of a γ chain and a δ chain expressing the V51 variable region in combination with a CD3 component. V51+ γδ T cells can be identified by phenotypic analysis using specific anti-V51 antibodies or by sequencing of the V5 region.

[0030] As used herein, "gamma delta TCR agonists" are understood to be antibodies, peptidomimetics and small molecules that specifically bind either to the TCR gamma delta heterodimer, or to the V delta 1 domain of TCR gamma delta, or to the CD3 components associated with the TCR, primarily the CD3 epsilon component, and induce cell activation and proliferation.

[0031] As used herein, "human hematopoietic stem / progenitor cells (HPCs)" are understood to be human CD34+ cells identified by phenotypic analysis using anti-CD34 antibodies and obtained ex vivo from human umbilical cord blood, placental blood, peripheral blood, bone marrow or fetal liver, or derived in vitro from pluripotent stem cells such as iPSCs (induced pluripotent stem cells).

[0032] As used herein, "CD34+ early thymic progenitor cells (ETPs)" are understood to be human CD34+ cells identified by phenotypic analysis using anti-CD34 antibodies and obtained ex vivo from the human fetal, neonatal or postnatal thymus.

[0033] In the context of the present invention, a "functional natural cytotoxicity receptor" (NCR) is to be understood as a surface receptor expressed by natural killer (NK) cells and also by human γδ T cells, almost exclusively by the Vδ1+ γδ T cell subset, after stimulation with potent TCR agonists or mitogens in the presence of IL-2 or IL-15 (Non-Patent Document 5). NCR induction plays a central role in cell activation, modulating cytotoxicity against primary leukemia cells and tumor cell lines, and enhancing IFN-γ expression.

[0034] The term "high expression" as used herein refers to a statistically significant increase in the expression of a Notch ligand in a cell compared to the basal expression level of said Notch ligand in a reference cell. The cell is preferably a mammalian cell, more preferably a stromal cell. Expression above basal level includes pharmacological and artificial upregulation and high expression of said Notch ligand. High expression of a Notch ligand or its agonist in a cell can be achieved by different means, for example by transfecting a cell with a plasmid encoding a gene of a Notch ligand operably linked to a promoter suitable for expression of the Notch ligand in said cell, or by introducing a gene encoding said Notch ligand into the genome of the cell by using genetic engineering techniques such as Crispr, integrative viral vectors, or homologous recombination. The term "reference cell" or "reference cell" refers to an untreated control cell, i.e. a cell that has not been genetically modified or artificially engineered to induce expression of said Notch ligand above the natural expression (i.e. basal expression) that the cell may have. The reference cell is preferably a reference stromal cell. Thus, the reference stromal cells do not express a Notch ligand or express a Notch ligand at a basal level. Notch ligand genes that are highly expressed in cells or have a statistically significant increase in expression compared to the basal expression level of the cells can be detected by RNA expression techniques (reverse transcription polymerase chain reaction, fluorescent in situ hybridization, Northern blotting, etc.) or protein expression techniques (western blotting, flow cytometry, etc.).

[0035] "Statistically significant increase in expression of Notch ligand" herein refers to the determination by an analyst that the increase in expression level cannot be explained by chance alone. The method by which a person skilled in the art makes this determination is statistical hypothesis testing. This test results in a p-value, which is the probability of observing an extreme result similar to that in the data, assuming that the result is truly attributable to chance alone. Herein, a p-value of 0.1 or less (preferably 0.05, 0.01, 0.001 or less) is considered to be statistically significant. For example, the increase in expression of Notch ligand in a cell, preferably a stromal cell, is statistically significant if a statistical test is performed to compare the expression of Notch ligand in a cell, preferably a stromal cell, with the basal expression level of a reference cell, preferably a reference stromal cell, as defined above, and the p-value obtained from said statistical test is 0.1 or less, preferably 0.05, 0.01, 0.001 or less.

[0036] As used herein, the term "suitable medium" is to be understood as any suitable mammalian cell culture medium. Any culture medium is preferred, preferably a serum-free culture medium (α-MEM) supplemented with 20% fetal bovine serum and preferably L-glutamine (i.e., at a concentration of about 2 mmol / l) and animal-derived component-free recombinant human (rh) cytokines such as IL-7 (200 IU / ml), Flt3L (100 IU / ml) and SCF (100 IU / ml) for STEP 1 of the method of the present invention; or a serum-free culture medium (OpTimizer-CTS) optionally supplemented with autologous plasma (i.e., 5% autologous plasma) or human AB serum and preferably L-glutamine and animal-derived component-free recombinant human (rh) cytokines such as rhIL-4 (preferably at a concentration of about 100 ng / ml), IFN-γ (preferably at a concentration of about 70 ng / ml), IL-21 (preferably at a concentration of about 7 ng / ml), and IL-1β (preferably at a concentration of about 15 ng / ml) for STEP 2 of the method of the present invention. However, a number of basal culture media suitable for use in the expansion of γδ T cells are available, including, inter alia, complete media such as AIM-V, Iscove's medium, and RPMI-1640 (Life Technologies). The media may be supplemented with other media factors, such as serum, serum proteins, and selection agents such as antibiotics. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1×MEM non-essential amino acids, Life Technologies). The basal medium may be supplemented with IL-2 and / or IL-15 at standard concentrations that can be readily determined by one of skill in the art through routine experimentation.

[0037] "Treating," "to treat," or "treatment" means, without limitation, arresting, limiting, reducing, stabilizing, or slowing the growth of a disease.

[0038] By "medicine" or "pharmaceutical product" is meant, as is widely accepted, any pharmaceutical or veterinary composition (also called a drug, medication or simply a medication) used to cure, treat or prevent disease in animals, including humans.

[0039] By "pharmaceutical composition" is meant an active substance or a combination of active substances intended to prepare a final medicinal product for prophylactic and / or therapeutic use.

[0040] "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject together with the compositions of the present invention without causing any undesirable biological effects or adversely interacting with any of the components of such composition. As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable vehicle" are synonymous and refer to a vehicle for containing the active substances of a pharmaceutical composition that can be administered to a subject and / or environment without adverse effects. Suitable pharmaceutically acceptable carriers include, but are not limited to, sterile water, purified water, saline, glucose, dextrose or buffer solutions. Carriers may contain auxiliary substances, including, but not limited to, diluents, stabilizers, preservatives, wetting agents, dispersants, emulsifiers, pH buffers (e.g., phosphate buffers), viscosity additives, and the like.

[0041] As used herein, "autologous" is understood to refer to a cell preparation in which the donor and recipient are the same individual. As used herein, "allogeneic" is understood to refer to a cell preparation in which the donor and recipient are not the same individual.

[0042] The term "isolated" indicates that the cell or cell population to which it refers is not in its natural environment. The cell or cell population is substantially separated from surrounding tissue.

[0043] The marker profile of the cell composition product referred to in the present invention may be further defined by the presence and / or absence of additional markers, or a particular profile of a combination of present and absent markers, in either case, a particular combination of markers may exist as a particular profile within a cell population and / or a particular profile of markers on individual cells within a population.

[0044] As used herein, the term "marker" encompasses any biomolecule whose presence, concentration, activity or phosphorylation state can be detected and used to identify a cellular phenotype.

[0045] In this case, the cells of the invention are positive for certain phenotypic markers and negative for others. By "positive" it is meant that the marker is expressed in the cell. To be considered expressed, the marker must be present at a detectable level.

[0046] The term "expressed" is used to refer to the presence of a marker on the surface of a cell or within the cell. To be considered expressed, a marker must be present at a detectable level. By "detectable level" is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting, immunofluorescence, ELISA or FACS analysis. "Expressed" may refer to, but is not limited to, the presence of a detectable protein, the phosphorylation state of the protein or the mRNA encoding the protein. A gene is considered to be expressed by a cell of the invention or a cell of a population of the invention if expression can be sufficiently detected after 25 PCR cycles, preferably after 30 PCR cycles (corresponding to an expression level in the cell of at least about 75 to 100 copies per cell). The terms "express" and "expression" have corresponding meanings. Below this threshold level of expression, the marker is not considered to be expressed.

[0047] A cell population as defined herein is considered to express a marker when at least about 60%, preferably about 80%, of the cells of the cell population exhibit detectable expression of the marker. It is preferred that at least about 85%, at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or more of the cells of the population exhibit detectable expression of the marker. In certain embodiments, at least about 99% or 100% of the cells of the population exhibit detectable expression of the marker. Expression can be detected by using any suitable means, such as RT-PCR, immunoblotting, immunofluorescence, ELISA, or by fluorescence activated cell sorting (FACS) or flow cytometry. It should be understood that this list is presented by way of example only and is not limiting. A cell population as defined herein is considered to express a marker when the expression level of the marker in the cells of the invention is higher than the expression level in control cells, such as cells isolated ex vivo or cells not generated de novo, such as ex vivo PB or CB, as shown in FIG. 13. In this context, "higher than" means that the level of expression of the marker in the cell population of the invention is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold higher than in the control cells.

[0048] Another way to characterize the population of cells defined herein is by the lack of detectable expression of a particular marker or combination or markers. As defined herein, these markers can be said to be negative markers. In some embodiments, the population of cells defined herein is considered not to express a marker when at least about 60%, preferably about 80%, of the cells of the population of cells do not show detectable expression of the marker. In other embodiments, at least about 85%, at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% of the cells of the population of cells do not show any detectable expression of the marker. Similarly, the lack of detectable expression can be demonstrated using RT-PCR, immunoblotting, immunofluorescence, ELISA, or using FACS or flow cytometry.

[0049] A marker described herein is considered to be not expressed by a cell if its expression cannot be reasonably detected at a level of 30 cycles of PCR, corresponding to an expression level in the cell of less than about 100 copies per cell, and / or if it cannot be readily detected by immunofluorescence, immunoblotting, ELISA or FACS.

[0050] The marker profile of a cell population as defined herein may be further defined by the presence and / or absence of markers, or by a particular profile of a combination of present and absent markers, in each case, a particular combination of markers may exist as a particular profile within a population of cells and / or as a particular profile of markers for individual cells within the population.

[0051] The term "cell population" refers to a group of cells. A cell population is heterogeneous if it comprises different groups of cells, each group being distinguished from the other groups by the presence of one or more distinguishing characteristics, such as the expression or absence of a particular marker, or the presence of a different function.

[0052] The term "stromal cells" refers to bone marrow derived stromal cell lines.

[0053] explanation Advantageous embodiments of the present invention will now be described in greater detail with reference to the accompanying drawings, in which: FIG.

[0054] The thymus is the primary organ for de novo generation of both the major αβ and the minor γδ T cell subsets from HPCs. Vδ1+ γδ T cells are the predominant γδ T cell subset in the postnatal thymus but the minor γδ T cell subset in peripheral blood. The generation of both αβ and γδ T cells in the thymus depends on Notch signaling, with Dll4 being essential for T cell commitment and development in vivo (Koch et al., J. Exp. Med. 2008;Hozumi et al., J. Exp. Med. 2008). In the present invention, it is shown that strong and sustained Notch signaling, specifically provided by high expression of the human Notch ligand Jag2, favors γδ T cell generation over αβ T cell generation, whereas high expression of Notch signaling using any of the other Notch ligands (Jag1, Dll4 and Dll1) favors αβ T cell generation over γδ T cell generation (see FIG. 1). Based on these evidence and on our knowledge of in vitro methods for de novo generation of human T cells from human thymic progenitor cells such as CD34+ ETPs, we have developed a culture method that supports preferential generation of human γδ T cells (particularly Vδ1+ γδ T cells) from CD34+ hematopoietic progenitor cells obtained from any biological sample, for example from thymic progenitor cells and from umbilical cord blood hematopoietic progenitor cells. The method involves co-culturing the CD34+ cells on a mouse OP9 stromal cell line transduced with human Jag2. First, we found that ETPs that receive strong Notch signals from four ligands (i.e., Dll1, Dll4, Jag1, and Jag2) expressed in the human thymus and their high expression in OP9 cells (Garcia-Leon et al. Development 2018) can differentiate into both αβ and γδ T cells, whereas ETPs cultured on non-transduced OP9 cells are unable to survive and / or differentiate in vitro and are lost from the culture (see Figure 7).However, we also found that human Jag2 signaling was the only Notch ligand that selectively induced a differentiation pattern of ETPs with very scarce αβ T cells and preferential differentiation into γδ T cells (enriched up to 300-fold by day 30) distinct from that observed in the presence of other Notch ligands (see Figure 1). As found in vivo in the postnatal human thymus (Figure 11), γδ T cells differentiated in vitro from ETPs in response to human Jag2 signaling highly expressed in OP9 cells showed predominant Vδ1 expression, and Vδ1+ cells expanded predominantly in response to Jag2 during culture (Figure 11). Approximately 35% of γδ T cells were Vδ1+ (Figure 2), indicating that high expression of Jag2 in OP9 cells resulted in a 100-fold increase in Vδ1+ T cell yield after 4 weeks of culture (Figure 3).

[0055] Moreover, surprisingly, as shown in Figure 4, it is demonstrated herein that human Jag2-Notch signaling also supports γδ T cell generation from human cord blood CD34+ HPCs in vitro with similar efficiency to γδ T cell generation from ETPs. Indeed, total γδ T cells originating from cord blood HPCs were enriched 210-250-fold after 9 weeks in OP9-Jag2 cultures, with up to 40% Vδ1+ γδ T cells (Figure 4). Thus, we further propose the use of cord blood as an optimal source of CD34+ progenitor cells to generate large numbers (up to approximately 100-fold yields) of human Vδ1+ T cells that can then be expanded for adoptive cell therapy (Figure 5). Phenotypic analysis of the cells showed that the resulting V51+ T cell population was a heterogeneous cell population, as found in vivo in the postnatal human thymus (Figure 11), including a major subset of immature CD1a+ cells that displayed either CD4+, double positive (DP) CD4+CD8+ or CD4-CD8- double negative (DN) phenotypes, non-activated CD25 naive γδ T cells (Figure 8), and a minor population of mature CD1a- γδ T cells, mostly composed of DN or CD8+ cells. Notably, mature CD1a-V51+ γδ T cells displayed a naive CD27+ phenotype and predominantly expressed the γδ T cell differentiation marker CD73 and distinct levels of several cytotoxic NK receptors (see Figure 8), thus more similar to naive V51+ fetal cells present in umbilical cord blood than to adult peripheral blood V51+ T cells (see Figure 9). The percentage and number of naive Vδ1+ γδ T cells isolated from single CB units and generated de novo from CD34+ HPCs that received human Jag2 signaling were significantly higher than those obtained ex vivo from single CB or peripheral blood units, or from the same number of starting CB or PB total cells (Table 2).

[0056] An object of the present invention is therefore a novel and efficient method for the large-scale selective generation of human γδ T cells, more preferably allogeneic human V51+ γδ T cells, optimal for clinical application in adoptive immunotherapy against cancer. In this regard, considering that both human cord blood HPCs and human ETPs, currently chosen as the clinically preferred source of stem cells, can efficiently generate de novo human γδ T cells in response to Notch signaling, the method comprises inducing differentiation of any biological source comprising hematopoietic stem / progenitor cells, such as cord blood CD34+ HPCs and / or human thymic ETPs, by Notch activation mediated by a Notch ligand, preferably DLL1, DLL4, Jag1 or Jag2 Notch ligand, more preferably Jag2. In particular, the method comprises co-culturing HPCs, preferably human CD34+ HPC cells or human ETPs, on cells highly expressing a Notch ligand, preferably stromal cells highly expressing human Jag2, preferably supplemented with Flt3, SCF and IL-7, preferably for up to 15 weeks, preferably for up to 10 weeks, up to 9 weeks, up to 8 weeks, up to 7 weeks, up to 6 weeks, up to 5 weeks or up to 4 weeks. The generated γδ T cells, comprising a population of Vδ1+ γδ T cells, obtained as described above in a TCR-independent Notch-dependent manner (referred to herein as STEP1), are then expanded according to any method known in the art, for example by using anti-CD3 mAb and cytokines including IL-4, IFNγ and IL-15 to activate and induce the proliferation of said Vδ1+ γδ T cells (referred to herein as STEP2) (Non-Patent Document 6).

[0057] Thus, the first aspect of the present invention (also referred to as STEP1) relates to the in vitro use of a Notch ligand, preferably a Jag2 Notch ligand, for generating a de novo cell composition comprising a higher amount of γδ T cells than αβ T cells from a cell population comprising human HPCs, such as CD34+ cord blood HPCs and / or human ETPs. A Notch ligand, preferably Jag2, is preferably expressed in a cell, preferably a stromal cell, wherein said cell shows a statistically significant increase in expression of said Notch ligand compared to a basal expression level of said ligand in a reference cell, preferably a reference stromal cell. Thus, provided herein is a Notch ligand, preferably Jag2, which is expressed above basal levels in a cell, preferably on its surface, and used to generate a de novo cell composition comprising a higher amount of γδ T cells than αβ T cells from a cell population comprising HPCs, such as CD34+ cord blood HPCs and / or CD34+ ETPs. The cells which highly express the Notch ligand are preferably stromal cell lines, most preferably OP9 cells. The terms "reference cells" and "statistically significant increase in expression" are defined above and apply throughout the patent description.

[0058] The degree of high expression of Notch ligand, preferably Jag2, in cells, preferably stromal cells, can be about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 10000-fold, 100000-fold increase in expression compared to the basal expression level of Notch ligand in reference cells, preferably reference stromal cells. Figure 10 shows 500-fold high expression in Jag2-transduced stromal cells compared to reference (non-transduced) stromal cells. Expression of Notch ligand, preferably Jag2, above basal level (i.e. high expression) can be achieved by any method known to those skilled in the art. As an example, expression above basal level can be induced by modulating the regulation of native genomic Notch ligand. Induction of expression above basal levels may be achieved by increasing transcription and / or translation of the Notch ligand, and / or by introducing heterologous regulatory sequences within or adjacent to the native regulatory region of the Notch ligand, and / or by replacing the native regulatory region of the Notch ligand with such heterologous regulatory sequences, for example by homologous recombination, and / or by interfering with or downregulating molecules that negatively regulate, block or downregulate the transcription, translation or function of said Notch ligand.

[0059] Transcription of a Notch ligand above basal levels can be increased by providing elevated levels of a transcriptional activator to a cell, preferably a stromal cell, for example, by contacting the cell with such an activator or by transforming the cell with a nucleic acid encoding the activator. Alternatively, transcription may be increased by transforming the cell with an antisense nucleic acid to a transcription inhibitor of the Notch ligand.

[0060] Alternatively or in addition to increasing the transcription and / or translation of endogenous Notch ligand, one or more additional copies of Notch ligand may be introduced into cells, preferably stromal cells, for example by transfecting or transducing a nucleic acid encoding the Notch ligand into the cells, resulting in expression of the Notch ligand above basal levels. The transforming Notch ligand may be contained on an extragenomic vector or may be integrated, preferably stably, into the genome. The transforming Notch ligand may be operably linked to a promoter that drives its expression above basal levels in the cell. "Operably linked" means joined as part of the same nucleic acid molecule and suitably positioned and oriented to initiate transcription from the promoter.

[0061] Methods for introducing genes into cells are known to those skilled in the art. A vector can be used to introduce Notch ligand into cells, whether the Notch ligand remains on the vector or is integrated into the genome. A suitable vector can be selected or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, and enhancer sequences. The vector can contain marker genes and other sequences as necessary. The regulatory sequence can drive the expression of the Notch ligand in the cell. For example, the vector can be an extragenomic expression vector, or the regulatory sequence can be integrated into the genome together with the Notch ligand. The vector can be a plasmid or a viral vector.

[0062] The nucleic acid comprising the coding sequence for the Notch ligand can be integrated into the genome of the cell, preferably the stromal cell. Integration can be facilitated by including sequences in the transducing or transfecting nucleic acid that facilitate genomic recombination, according to standard techniques. The nucleic acid to be integrated can include regulatory sequences that can drive expression of the Notch ligand above basal levels. The nucleic acid can include sequences that direct its integration into a site in the genome that is under the control of regulatory elements that can drive and / or control its expression in the cell. The nucleic acid to be integrated can be derived from a vector used to transduce or transfect the Notch ligand nucleic acid into the cell.

[0063] The introduction of a nucleic acid containing a Notch ligand, whether the nucleic acid is linear, branched or circular, is generally referred to as "transfection" or "transduction", without limitation. Any available technique can be used. Suitable techniques include mechanical techniques such as calcium phosphate transfection, DEAE-dextran, electroporation, microinjection, direct DNA uptake, receptor-mediated DNA transfer, transduction using retroviruses or other viruses, and liposome-mediated transfection. When introducing a selected genetic construct into a cell, certain considerations known to those skilled in the art must be taken into account.

[0064] Suitable vectors and techniques for in vivo transfection or transduction of Notch ligand into cells, preferably stromal cells, to produce cells, preferably stromal cells, that highly express the Notch ligand are known to those skilled in the art.Suitable vectors include adenovirus, papovavirus, vaccinia virus, herpes virus and retrovirus.Disabled viral vectors can be produced in helper cell lines in which genes required for the production of infectious viral particles are expressed.Suitable helper cell lines are known to those skilled in the art.

[0065] In a preferred embodiment, high expression of a Notch ligand in a cell, preferably a stromal cell, i.e. a statistically significant increase in the expression level of a Notch ligand, is achieved by transducing or transfecting said cell with a nucleic acid comprising a gene encoding said Notch ligand, preferably a human Jag2 Notch ligand, operably linked to a strong expression promoter such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an elongation factor (EF)-1 promoter, etc. Thus, in an embodiment of the first aspect of the invention, HPCs, such as CD34+ cord blood HPCs and / or CD34+ ETPs are cultured in a medium comprising a Notch ligand, preferably human Jag2, highly expressed in a cell line, preferably a stromal cell line, preferably OP9 cells, wherein said cell line has been transduced or transfected with a nucleic acid comprising a gene for said Notch ligand driving high expression of said Notch ligand in the cell.

[0066] A useful source containing CD34+ HPCs useful in the present invention is bone marrow and / or peripheral blood containing HPCs, preferably HPCs mobilized from bone marrow. Other sources containing HPCs include placental blood and fetal liver as well as CD34+ cells derived from pluripotent stem cells such as iPSCs (induced pluripotent stem cells), which may also be suitable for carrying out the present invention. Other sources containing HPCs include placental blood, fetal liver or CD34+ cells derived from pluripotent stem cells such as iPSCs (induced pluripotent stem cells), which may also be suitable for carrying out the present invention. The cell population containing human HPCs, such as CD34+ cord blood HPCs and / or CD34+ ETPs from which Notch-induced differentiated γδ T cells, preferably Vδ1+ γδ T cells, are preferably substantially pure or homogeneous populations. In the context of the present invention, a substantially pure population is a population in which HPCs, such as CD34+ cord blood HPCs and / or CD34+ ETPs may be substantially isolated cells. In one embodiment, HPCs, such as CD34+ cord blood HPCs, represent at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% of the cells in the composition. In another embodiment, human CD34+ ETPs represent at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% of the cells in the composition.

[0067] In another embodiment of the first aspect of the invention, HPCs, such as CD34+ cord blood HPCs, and / or CD34+ ETPs, are cultured in a medium containing a population of cells, preferably stromal cells, that highly express an engineered Notch ligand, preferably Jag2, or a Notch agonist, including antibodies, peptidomimetics, and small molecules against the Notch receptor that have the biological effect of the natural ligand, or are cultured with such compounds immobilized on a substrate (i.e., on a plastic surface or on microbeads). Preferably, HPCs, such as CD34+ cord blood HPCs, and / or CD34+ ETPs may be further cultured in a medium containing a Notch ligand, preferably Jag2, or a Notch agonist, including antibodies, peptidomimetics, and small molecules against the Notch receptor that have the biological effect of the natural ligand, immobilized on a cell line. Preferably, the Notch ligand is a human Notch ligand, preferably human Jag2. In some aspects of the invention, the Notch ligand or Notch agonist can be immobilized on a solid substrate suspended in the medium, thereby facilitating interaction of the Notch ligand or Notch agonist with HPCs, such as CD34+ cord blood HPCs and / or CD34+ ETPs. The method further comprises maintaining the cells in culture for a duration sufficient to generate V51+ γδ T cells. In some embodiments, the duration is between about 2 weeks and about 15 weeks, preferably between about 2 weeks and about 9 weeks.

[0068] In some embodiments, a cell-free system is used to immobilize the Notch ligand, preferably Jag2, or the Notch agonist. The cell-free system has the advantage of being easily scalable for clinical applications. The cell-free system preferably comprises a 3D scaffold. As used herein, "3D scaffold" refers to an artificial, biocompatible, malleable structure onto which cells can be embedded or seeded to support three-dimensional cell growth and differentiation. Furthermore, the scaffold can be used to deliver biochemical factors, such as differentiation-inducing ligands, growth factors, cell nutrients, to the body, to support and direct the growth of new cells in an organ or tissue. The scaffold can be of natural or synthetic materials, and can be permanent, biodegradable, or bioabsorbable. Examples of natural scaffold materials include agarose, collagen, some linear aliphatic polyesters, glycosaminoglycans such as chitosan and hyaluronic acid. Commonly used synthetic biodegradable scaffold materials include polylactic acid (PLA), polyglycolic acid (PGA); poly(D,L-lactide-co-glycolide) (PGLA) and polycaprolactone (PCL). Scaffolds generally have high porosity to facilitate cell seeding and diffusion throughout the structure. In some embodiments, the 3D scaffold contains or is conjugated with a Notch ligand, preferably Jag2, or an agonist thereof, so that the cultured cells are stimulated by said Notch ligand.

[0069] In another embodiment, the cell-free system comprises a floating support. The term "floating support" as used herein refers to any material that allows the Notch ligand, preferably Jag2, or the Notch ligand (or its agonist) to float in the culture medium when conjugated with an agonist. The floating support may be made of a wide variety of materials and may be in a variety of formats. Examples of supports that can be used as floating supports include, but are not limited to, particles, beads (including microbeads), proteins, lipids, nucleic acid molecules, filters, fibers, screens, meshes, tubes, hollow fibers, biological tissues, and any combination thereof. In one embodiment, the floating support is a particle. The particle may be of any shape, including, but not limited to, spheres, ovoids, rods, or squares. The particle may be of various materials, including, but not limited to, natural or synthetic polymers, natural or synthetic waxes, ceramics, metals, biological materials, or combinations thereof. In one embodiment, the floating support comprises microbeads. The term "microbeads" as used herein refers to spherical or substantially spherical beads having a diameter of 0.01 pm (10 nm) to 500 pm, optionally from 1 pm to 200 pm. In another embodiment, the diameter of the microbeads is from 6.5 pm to 100 pm, optionally from 20 pm to 30 pm, 24 pm to 26 pm or 25 pm.

[0070] Various types of microbeads are contemplated herein. In one embodiment, the microbeads are polymeric, silica, or magnetic, superparamagnetic, paramagnetic, or ferromagnetic microbeads. In another embodiment, the microbeads are polystyrene microbeads or gold nanoparticles. In another embodiment, the microbeads are cross-linked with polystyrene or iron oxide coated. In another embodiment, the microbeads are coated with poly(lactic acid-co-glycolic acid) (PLGA).

[0071] It is preferred to conjugate the Notch ligand, preferably Jag2, or its agonist to the 3D scaffold or floating support. Various means of conjugating proteins to supports are known in the art. "Conjugation" herein refers to the state in which two compounds, for example a microbead and a Jag2 Notch ligand, are linked. The protein may be directly or indirectly conjugated to the floating support or 3D scaffold, for example a microbead. In one embodiment, the Notch ligand is conjugated to the floating support or 3D scaffold using a biotin / streptavidin system. In that case, the Notch ligand is biotinylated and then conjugated to the streptavidin-coated floating support or 3D scaffold (for example, streptavidin-coated microbeads). In another embodiment, the Notch ligand is conjugated to the floating support or 3D scaffold via protein G or protein A. In suspension culture, cells are grown floating freely in the culture medium. In contrast, in 3D scaffold culture, cells are grown as a monolayer on an artificial substrate.

[0072] The cell population comprising human HPCs is cultured under suitable conditions as described herein to generate a population comprising γδ T cells. The cell population comprising human HPCs is preferably cultured in the presence of one Notch ligand or agonist, preferably in the presence of Jag2, where the Notch ligand or agonist is conjugated to a floating support or 3D scaffold, and the cells are cultured in contact with the Notch ligand for a sufficient time to form cells of the γδ T cell lineage. The Notch ligand, preferably Jag2, is conjugated to a floating support, preferably to microbeads contained in the floating support, and thus the cell population comprising human HPCs is cultured in suspension in contact with the Notch ligand, preferably Jag2. In another embodiment, the Notch ligand, preferably Jag2, is conjugated to a 3D scaffold, preferably to microbeads contained in the 3D scaffold, and thus the cell population comprising human HPCs is cultured in contact with the Notch ligand, preferably Jag2, on the artificial substrate of the scaffold.

[0073] In another embodiment, the cell population comprising human HPCs is cultured in a bioreactor, which may be a closed Gas Permeable Rapid Expansion (G-Rex) system bioreactor, or in a closed automated bioreactor, with a Notch ligand conjugated to a floating support or 3D scaffold. In one embodiment, the floating support or 3D scaffold comprises a Notch ligand conjugated to microbeads, preferably Jag2, where the diameter of the microbeads is adapted to the bioreactor. Various bioreactors are known in the art, and may include batch, fed-batch or continuous bioreactors. An example of a continuous bioreactor is a continuous stirred tank reactor model.

[0074] Notch signaling can be enhanced by directing the positioning of the Notch ligand relative to the floating support or 3D scaffold. Thus, in one embodiment, the C-terminus of the Notch ligand is conjugated to the floating support or 3D scaffold. This can be engineered, for example, by adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated with a biotin molecule. In another embodiment, the Fc segment present in the C-terminal region of the fusion protein Notch ligand-Fc can be directly bound to protein A or protein G conjugated to the floating support or 3D scaffold. One or more additional molecules, each conjugated to the floating support or 3D scaffold, can be added to the culture. In one embodiment, the additional molecule is a molecule that promotes T cell development (e.g., promotes the commitment and differentiation of cells of the T cell lineage), also referred to as a T cell costimulatory molecule.

[0075] The culture conditions involve culturing a cell population comprising human CD34+ HPCs in contact with a Notch ligand, preferably Jag2, for a sufficient period of time such that γδ T cells are generated in greater amounts than αβ T cells. It will be understood that the cells may be maintained for any suitable period of time necessary to achieve the desired cell composition described herein. The culture time is preferably 30 days or more, preferably 35 days or more, preferably 30-50 days.

[0076] The ratio of cells to microbeads (also referred to as the ratio of microbeads to cells) can be varied depending on the culture conditions and the stimuli provided for cell proliferation and differentiation. In one embodiment, a Notch ligand, preferably Jag2, or an agonist thereof, is conjugated to microbeads, where the ratio of Notch ligand conjugated to microbeads to human HPCs is between 1:1 and 27:1, optionally 5:1-15:1, 8:1-10:1 or 9:1. Those skilled in the art know how to establish the best ratio of microbeads to cells depending on the culture conditions.

[0077] Alternatively, the first aspect of the present invention also relates to an in vitro method for generating a cell composition comprising a higher amount of γδ T cells than αβ T cells from a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or CD34+ ETPs. Another source of HPCs useful for the method is bone marrow or peripheral blood, preferably peripheral blood comprising HPCs mobilized from bone marrow. Other sources of HPCs include placental blood, fetal liver or CD34+ cells from pluripotent stem cells, such as iPSCs, which may also be suitable for carrying out the method. The method preferably comprises growing a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or CD34+ ETPs in a suitable culture medium, preferably a medium comprising a Notch ligand (preferably DLL1, DLL4, Jag1 or Jag2 Notch ligand, more preferably Jag2) or a Notch receptor agonist immobilized on a substrate or highly expressed in a cell line. In some aspects of the invention, the Notch ligand or Notch agonist can be immobilized on a solid substrate suspended in the medium, thereby facilitating the interaction of the Notch ligand or Notch agonist with HPCs, such as CD34+ cord blood HPCs and / or CD34+ ETPs. In some other embodiments, the Notch ligand, preferably Jag2, is highly expressed in the stromal cell line. The method further comprises maintaining the cells in culture for a duration sufficient to result in a composition enriched for γδ T cells, preferably Vδ1+ γδ T cells. In some embodiments, the duration is between about 2 weeks and about 15 weeks, preferably between about 2 weeks and 9 weeks. The Notch ligand or Notch agonist can be present or added to the cell culture during and throughout the culture period. In a preferred embodiment, a Notch ligand, preferably Jag2, most preferably human Jag2, is highly expressed in a cell line, preferably stromal cells, preferably on their surface, and said cells are co-cultured and allowed to interact with a cell population comprising human HPCs, such as CD34+ cord blood HPCs and / or CD34+ ETPs. The term "highly expressed" has been explained above and applies here as well.

[0078] In view of the above, a first aspect of the invention provides a method and a use of a highly expressed Notch ligand, preferably Jag2, for generating a cell composition comprising more γδ T cells than αβ T cells, the cell composition being obtained from a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or CD34+ ETPs. As shown in the Examples, in particular in Example 1, and in Figures 1 to 3, the human Jag2 Notch ligand is the only ligand that, when highly expressed in a stromal cell line, selectively induces a differentiation pattern in which γδ T cells occur in higher proportions than αβ T cells. Thus, in an embodiment of the first aspect, which can be applied to both the uses and methods defined above, a highly expressed Jag2 Notch ligand is used in the invention to favor the generation of γδ T cells over αβ T cells from a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or CD34+ ETPs. A cell composition enriched for γδ T cells is a population of T cells comprising both γδ T cells and αβ T cells, and preferably after at least 30 days of culture, preferably after 60 days of culture according to the use and method of the first aspect, γδ T cells represent at least 80%-95%, preferably 90%-95% of the total T cells. In an embodiment, after at least 30 days of culture, preferably after 60 days of culture according to the use and method of the first aspect, αβ T cells represent less than 15%, preferably 5%-10% of the total T cells. It is preferred that at least 30%, preferably 30%-40% of the γδ T cells are Vδ1+ γδ T cells. It is preferred that the Vδ1+ γδ T cells are cognate Notch-induced differentiated Vδ1+ γδ T cells. A more detailed description of the γδ T cells obtained or obtainable according to the use and method of the first aspect is presented in the second aspect of the invention.

[0079] A second aspect of the present invention relates to a cell composition comprising a greater amount of γδ T cells than αβ T cells, obtained or obtainable by the use and method of the first aspect. It is preferred that after at least 30 days of culture, preferably after 60 days of culture according to the use and method of the first aspect, at least 80%-95%, preferably 90%-95% of the total number of T cells in the cell composition are γδ T cells. In one embodiment, after at least 30 days of culture, preferably after 60 days of culture according to the use and method of the first aspect, less than 15%, preferably 5%-10% of the total number of T cells contained in the cell composition are αβ T cells after at least 30 days of culture, preferably after 60 days of culture according to the use and method of the first aspect.

[0080] In an embodiment of the second aspect, the γδ T cells comprised in the cell composition are a heterogeneous cell population, i) at least 30%, preferably 30% to 40%, of the total number of γδ T cells are Vδ1+ γδ T cells; ii) at least 4%, preferably 4% to 7%, of the total number of γδ T cells are Vδ2+ γδ T cells; iii) Approximately 50% of the total number of γδ T cells are neither Vδ2+ nor Vδ1+ cells.

[0081] Furthermore, in a preferred embodiment, V51 γδ T cells, preferably V51 γδ T cells of i), are also identified herein as being from the following cell populations: a) a first cell population characterized by expression of the immature surface cell marker CD1a (hereinafter referred to as CD1a+Vδ1+γδ T cells); b) a second cell population characterized by not expressing the immature surface cell marker CD1a (hereafter referred to as CD1a-Vδ1+γδ T cells); It is a heterogeneous cell population containing

[0082] Heterogeneous V51+ γδ T cell populations comprising a) and b) are shown in the Examples, in particular in Figures 8 and 9.

[0083] In one embodiment, the first population of T cells (CD1a+V51+γ5 T cells) represents the majority of V51+γ5 T cells in the heterogeneous V51+γ5 T cell population. It is preferred that at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 85%-95%, of the total number of V51+γ5 T cells are CD1a+V51+γ5 T cells (first population). Thus, the population of V51+γ5 T cells is enriched for CD1a+V51+γ5 T cells. It is preferred that 8%-12% of the total number of V51+γ5 T cells are CD1a-V51+γ5 T cells (second population). It is preferred that at least 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more than 70% of the total number of V51+ γδ T cells are CD1a-V51+ γδ T cells (first population).

[0084] In a preferred embodiment, the cell composition obtainable or obtained from the method and use of the first aspect is a heterogeneous cell population enriched for γδ T cells, since after at least 30 days of culture, preferably after 60 days of culture, at least 80%-95%, preferably 90%-95% of the total number of T cells are γδ T cells, i) at least 30%, preferably 30% to 40%, of the total number of γδ T cells are Vδ1+ γδ T cells; ii) at least 4%, preferably 4% to 7%, of the total number of γδ T cells are Vδ2+ γδ T; iii) approximately 50% of the total number of γδ T cells are neither Vδ2+ nor Vδ1+ cells; The population i) is enriched for CD1a+Vδ1+ γδ T cells, as 85%-95% of the total Vδ1+ γδ T cells are CD1a+Vδ1+ γδ T cells and 8%-12% of the total Vδ1+ γδ T cells are CD1a-Vδ1+ γδ T cells.

[0085] V51+ γδ T cells preferably produce IFNγ but not IL-17, see Figure 2.

[0086] The method of measuring each of the above cell populations and their percentages is known in the art and is established in the description of the present invention.In particular, the method of measuring each of the T cell types is carried out using total cell counting and differential cell counting with an automated cell counter.The percentage of cells and the percentage of CD1a expressing cells can be determined using a flow cytometer.

[0087] In an embodiment of the second aspect, the first population of cells (CD1a+V51+γδ T cells) are further characterized in that they do not express at least one, and preferably all, of the following surface markers: CD25, CD27, NKp44, NKp30, and NKG2D. In an embodiment, the cells of the first population may express or not express CD4, CD8, or both, i.e., may be CD4+CD8-, CD4-CD8+, CD4+CD8+, or CD4-CD8-. See Figures 8 and 9.

[0088] In an embodiment of the second aspect, the second population of cells (CD1a-V51+ γδ T cells) are further characterized by expressing at least one, or a combination of at least, two or more, preferably all, of the following surface markers: CD27, CD73, CD69, NKp44, NKp30, and NKG2D (see Figures 8 and 9).

[0089] Preferably, the cell composition according to the second aspect comprises human allogeneic cytotoxic V51+ γδ cells obtained or obtainable according to the first aspect of the invention.

[0090] A third aspect of the invention relates to a cell composition comprising a first population of V51+ γδ T cells as defined in the second aspect. The cell composition according to the third aspect therefore comprises V51+ γδ T cells characterised in that they express at least the CD1a surface marker and preferably do not express at least one, preferably all, of the following surface markers: CD25, CD27, NKp44, NKp30 and NKG2D. A fourth aspect of the invention relates to a cell composition comprising a second population of V51+ γδ T cells as defined in the second aspect. The cell composition according to the fourth aspect therefore comprises V51+ γδ T cells characterised in that they do not express the CD1a surface marker and preferably express at least one, or at least a combination of two or more, preferably all, of the following surface markers: CD27, CD73, CD69, NKp44, NKp30 and NKG2D.

[0091] In a preferred embodiment, the cell population of the third and fourth aspects is a substantially pure or homogeneous population. In the context of the present invention, a substantially pure population is a population in which the cells may be substantially isolated cells. In one embodiment, the cells or cell population represent at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% of the cells in the composition. The term "substantially pure" encompasses and may be used interchangeably with "completely pure".

[0092] A fifth aspect of the invention relates to a heterogeneous cell population of autologous or allogeneic V51+ γδ T cells, said heterogeneous cell population comprising a first and a second subpopulation of V51+ γδ T cells, the first subpopulation comprising cells expressing the immature surface cell marker CD1a (CD1a+V51+ γδ T cells) and the second subpopulation comprising cells not expressing the immature surface cell marker CD1a (CD1a-V51+ γδ T cells). In an embodiment, the first subpopulation of cells (CD1a+V51+ γδ T cells) represents the majority of cells in the heterogeneous cell population. Preferably, the first subpopulation of cells represents at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the total V51+ γδ T cells in the heterogeneous cell composition. The method of measuring each of the above cell subpopulations and their percentages is known in the art and established in the description of the present invention. In particular, the method of measuring each of V51+ γδ T cells is performed using total and differential cell counting with an automated cell counter. The percentage of cells and the percentage of CD1a expressing cells can be determined using a flow cytometer.

[0093] In an embodiment of the fifth aspect, the cells of the first subpopulation (CD1a+V51+γδ T cells) are further characterized in that they do not express at least one, and preferably all, of the following surface markers: CD25, CD27, NKp44, NKp30, and NKG2D. In an embodiment, the cells of the first subpopulation may express or not express CD4, CD8, or both, i.e., may be CD4+CD8-, CD4-CD8+, CD4+CD8+, or CD4-CD8-. See Figure 8.

[0094] In an embodiment of the fifth aspect, the cells of the second subpopulation (CD1a-V51+ γδ T cells) are further characterized by expressing at least one, or a combination of at least, two or more, preferably all, of the following surface markers: CD27, CD73, CD69, NKp44, NKp30, and NKG2D (see Figure 8).

[0095] A sixth aspect of the invention relates to a cell composition comprising a subpopulation of first cells as defined in the fifth aspect. The cell composition according to the sixth aspect therefore comprises V51+ γδ T cells characterised in that they express at least the CD1a surface marker and preferably do not express at least one, preferably all, of the following surface markers: CD25, CD27, NKp44, NKp30 and NKG2D. A seventh aspect of the invention relates to a cell composition comprising a subpopulation of second cells as defined in the fifth aspect. The cell composition according to the fifth aspect therefore comprises V51+ γδ T cells characterised in that they do not express at least the CD1a surface marker and preferably express at least one, or at least a combination of two or more, preferably all, of the following surface markers: CD27, CD73, CD69, NKp44, NKp30 and NKG2D.

[0096] In a preferred embodiment, the population of cells of the sixth and seventh aspects is a substantially pure or homogeneous population. In the context of the present invention, a substantially pure population is a population in which the cells may be substantially isolated cells. In one embodiment, the cells or cell population represent at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% of the cells in the composition.

[0097] Thus, the method of the first aspect (STEP 1) can be used to generate a population of V51+ γδ T cells from CD34+ cells, preferably from cord blood CD34+ cells. Said population of V51+ γδ T cells is a heterogeneous population comprising CD1a+V51+ γδ T cells (first population) and CD1a-V51+ γδ T cells (second population), as defined herein in the second to seventh aspects. When the CD34+ progenitor cells are from cord blood, the cells obtained by the method or use of the first aspect are referred to herein as CB-Jag2 cells.

[0098] The second population comprising V51+ γδ T cells generated de novo according to the first embodiment (STEP 1), in particular CD1a-V51+ γδ T cells, can be further expanded in a second step (STEP 2) with the aim of activating them and inducing their proliferation. The complete two-step protocol is shown in Figure 12, in which CD34+ HPCs isolated from human umbilical cord blood are cultured in the presence of Jag2 Notch ligand for up to 8 weeks (STEP 1 or method of the first embodiment), resulting in CB-Jag2 cells, cells corresponding to the cell composition defined in the third, fourth, fifth, sixth and seventh embodiments. The CB-Jag2 cells are then expanded and activated (STEP 2) with the aim of resulting in an expanded population of highly cytotoxic V51+ γδ T cells, called CB-Jag2-STEP2 cells. It should be noted that CB ex vivo and PB ex vivo are Vδ1+ γδ T cells already present in the CB and PB, and therefore are not generated de novo from CD34+ hematopoietic progenitor cells. Therefore, Vδ1+ γδ T cells obtained from ex vivo CB or PB (referred to as CB ex vivo and PB ex vivo in Figure 12) are not Vδ1+ γδ T cells as defined in Aspects 1 to 7.

[0099] Results obtained with the two-step method showed that CD1a-V51+ γδ T cells in CB-Jag2-STEP2 cells (i.e. cells obtained de novo and subsequently expanded in STEP2 as defined in the first to seventh aspects) contained more CD8+ effector cells than CD1a-V51+ γδ T cells in populations of ex vivo isolated cells obtained from PB and CB and subsequently expanded in STEP2 (CB-STEP2 and PB-STEP2), see Figure 13. CD1a-V51+ CB-Jag2-STEP2 cells also showed a less exhausted cell profile as assessed by lower expression of LAG3 and CTLA-4 compared to CD1a-V51+ γδ T cells generated in CB-STEP2 and PB-STEP2, see Figure 14. CD1a-V51+CB-Jag2-STEP2 cells also displayed higher levels of cytotoxicity-associated activating receptors than CD1a-V51+CB-STEP2 and PB-STEP2 γδ T cells, as shown in Figure 15. Finally, CB-Jag2-STEP2 displayed higher in vitro cytolytic potential against leukemic cell lines than CB-STEP2 and PB-STEP2 cells obtained by STEP2 expansion of ex vivo cells isolated from CB and PB, respectively.

[0100] From these results, Use of hematopoietic progenitor cells (CD34+ cells), preferably derived from umbilical cord blood; De novo generation of CD1a-V51+ γδ T cells using STEP1 (the method or use of the first aspect); Expansion and activation of the de novo CD1a-Vδ1+γδT cells obtained in STEP1 using STEP2; We conclude that combining these results in a population of CD1a-Vδ1+ γδ T cells with higher cytotoxic potential and a reduced exhaustion profile.

[0101] Considering the above results, an eighth aspect of the invention relates to a method (also called STEP2) for activating and inducing the expansion of V51+ γδ T cells as defined in the second, third, fourth, fifth, sixth and seventh aspects of the invention. As highlighted above, V51+ γδ T cells (obtained after STEP1 (first aspect of the invention)) are a heterogeneous population of V51+ γδ T cells, some of which express the CD1a marker (CD1a+) (first population) and some of which are CD1a- (second population). STEP2 of the invention (i.e. the method of the eighth aspect) preferably relates to activating and expanding (i.e. expanding) a population of CD1a- V51+ γδ T cells in particular.

[0102] Thus, it is preferred that STEP2 activation and expansion is performed specifically on the subset of CD1a-V51+ γδ T cells (second population as defined above). The method of the eighth aspect may preferably comprise the use of an activating agent, such as a γδ TCR agonist, including but not limited to an anti-CD3 mAb, and a cytokine, including but not limited to IL-4, IFNγ and IL-15. To this end, and as shown in Example 1 or 2, a cell composition comprising V51+ γδ T cells obtained or obtainable according to the first aspect of the invention is preferably depleted of αβ T cells by any useful technique, such as magnetic cell sorting using an anti-TCRαβ mAb and magnetic beads (Miltenyi Biotec). The TCRαβ-depleted cell suspension is cultured for 7 days (2.5×10 cells) in RPMI1640 medium in the presence of an activating agent, such as anti-CD3 mAb OKT3, plus cytokines, such as IL-2, IL-4, IFN-γ, IL-21, IL-15 and / or IL-1β. 5cells / ml). The cells are then optionally washed and again cultured one or more times with an activator and one or more cytokines. The culture is usually stopped by day 10-30, preferably day 10-25, more preferably day 15-25, more preferably day 15-16, or the cells may be optionally diluted and subjected to a second round of expansion in the presence of an activator and one or more cytokines. It should be noted that the combination of the first and eighth aspects of the present invention (i.e., the combination of STEP 1 and STEP 2) results in an improved CD1a-Vδ1+ T cell yield (250×10 6 pieces~950×10 6 CB-Jag2-STEP2 cells / 10 6 A two-step method is constructed that is expected to allow for the isolation of 100 CD34+ CB HPCs.

[0103] It should be noted that the final subset of V51+ γδ T cells obtained from the eighth aspect of the invention should stably express natural functional cytotoxic receptors with enhanced cytotoxicity against lymphoid leukemia cells. Said subset is preferably cytotoxic expanded CD1a-V51+ γδ T cells, as further defined in the ninth aspect of the invention.

[0104] Another potentially useful method, merely exemplary, of activating and inducing the proliferation of V51+ γδ T cells, preferably of CD1a-V51+ γδ T cells (second population), as defined in the second, third, fourth, fifth, sixth and seventh aspects of the invention, is by growing these cells in a suitable culture medium in the presence of a γδ TCR agonist, preferably by adding said agonist, preferably soluble or immobilized, at regular intervals (more preferably continuously) and in the presence of at least one cytokine, such as a cytokine selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IFN-γ, IL-21, IL-15 and / or IL-1β, preferably by adding said cytokine(s) at regular intervals (more preferably continuously). The γδ TCR agonist and cytokines are added to the cell culture during and throughout the culture period, preferably every 3-6 days, so that the concentration of γδ TCR agonist and cytokines in the culture is generally always greater than zero. The addition of the γδ TCR agonist and cytokines can be carried out until at least 40%, more preferably at least 50%, 60%, 70%, 75%, 80%, 85%, 95% of the cells express the natural cytotoxic receptor. In a more preferred embodiment, the addition of the γδ TCR agonist and cytokines can be carried out until more than 50 million, more than 100 million, more than 200 million viable and functional cells expressing the natural cytotoxic receptor, i.e., the natural cytotoxic receptor comprising or consisting of the natural cytotoxic receptor NKp30, are achieved. It is preferred that the addition of said γδTCR agonist and cytokines can be carried out until at least 40%, more preferably at least 50%, 60%, 70%, 75%, 80%, 85%, 95%, 100% of the cells express NKp30.In a preferred embodiment of the disclosed method, said cytokine means a common cytokine, preferably an interleukin, i.e. IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-21, IFN-γ, IL-1β or a mixture thereof, especially preferably IL-7 or IL15. The interleukins used may be of human or animal origin, preferably of human origin. The interleukins used may be wild-type proteins or any fragments or variants that are biologically active, i.e. capable of binding to their receptor and inducing activation of γδ T cells under the conditions of the method according to the invention. More preferably, the cytokine may be in soluble form or fused or complexed with another molecule, such as a peptide, polypeptide or biologically active protein. It is preferred to use human recombinant cytokines. More preferably, the range of interleukin concentrations may vary between 1 U / ml and 10000 U / ml, even more preferably between 100 U / ml and 1000 U / ml. In another preferred embodiment of the disclosed method, the addition of said γδ TCR agonist and γc-cytokine at regular intervals can be carried out for 2 to 60 days, more preferably for 9 to 25 days, even more preferably for 15 to 25 days, i.e. for 15, 16, 17, 18, 19, 20 or 21 days. It is preferred to culture the cells at a temperature of 36°C to 38°C, preferably 37°C.

[0105] Most preferably, the method of the eighth aspect is a method for activating and inducing the proliferation of V51+ γδ T cells, preferably CD1a-V51+ γδ T cells (second population) as defined in the second, third, fourth, fifth, sixth and seventh aspects of the invention, comprising growing said V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, in a suitable culture medium in the presence of at least one γδ TCR agonist, preferably by regular intervals of addition (more preferably continuous addition) of said agonist, preferably soluble or immobilized, and in the presence of at least IL21 and IL15, to obtain an expanded and activated cell population comprising V51+ γδ T cells, preferably CD1a-V51+ γδ T cells. Preferably, V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, are first grown in the presence of at least one γδ TCR agonist and in the presence of at least IL21 for at least 5 days, preferably at least 7 days, and on day 5, preferably day 7 of culture, IL-15 is added to the culture medium while maintaining the presence of at least one γδ TCR agonist and at least IL21 in the culture medium. After addition of IL15, the cells are preferably cultured for a period of at least 7 days, preferably at least 9, 10, 11, 12, 13, 14, 15, 16 or more than 16 days to obtain an expanded and activated cell population comprising V51+ γδ T cells, preferably comprising CD1a-V51+ γδ T cells.

[0106] The method of the eighth aspect is a method for activating and inducing proliferation of V51+ γδ T cells, preferably CD1a-V51+ γδ T cells (second population) as defined in the second, third, fourth, fifth, sixth and seventh aspects of the invention, comprising growing said V51+ γδ T cells, preferably CD1a- V51+ γδ T cells, in a suitable culture medium in the presence of at least one γδ TCR agonist and in the presence of at least IL21 for at least 5 days; Preferably, said culturing is carried out by adding at regular intervals (more preferably by continuous addition) said agonists and cytokines, preferably soluble or immobilized; On day 5 of culture, preferably on day 7 of culture, adding IL-15 to the culture medium while maintaining the presence of at least one γδ TCR agonist and IL21 in the culture medium; Including, the culture time is carried out until at least 40% of the total γδ T cells are CD1a-Vδ1+ γδ T cells, and / or the culture time is carried out for at least 7 days, 8 days, preferably 9 days, 10 days, 11 days, 12 days, preferably 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days; Most preferably, it is a method.

[0107] In one embodiment, the method of the eighth aspect comprises: Culturing the cells obtained from the method of the first aspect or as defined in any of aspects 1 to 7 in a serum-free culture medium supplemented with plasma, preferably autologous plasma or human serum, in the presence of an anti-TCR γδ mAb and IL-21 for at least 7 days; On day 7 of culture, adding IL-15 to the culture medium and optionally replacing the IL-21 and anti-TCR γδ mAb with fresh anti-TCR γδ mAb and IL-21; culturing the cells for at least an additional 7 days (i.e., up to day 14 of culture), preferably up to day 15-16 of culture, to obtain activated, cytotoxic CD1a-Vδ1+γδ T cells; Optionally, performing a second round of expansion, culturing the cells in the presence of at least anti-TCRγδ mAb, IL-15 and IFN-γ from day 18 to day 21; Includes.

[0108] The method of the eighth aspect is a method for activating and inducing proliferation of CD1a-V51 + γδ T cells, preferably a method for activating and inducing proliferation of CD1a-V51 + γδ T cells (second population) as defined in the second, third, fourth, fifth, sixth and seventh aspects of the invention, comprising growing said V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, in a suitable culture medium in the presence of at least one γδ TCR agonist and in the presence of at least IL21, IL-4, IFN-γ, IL-21 and IL-1β for at least 5 days, preferably 7 days; Preferably, the culture is carried out by adding at regular intervals (more preferably continuously) the agonist and cytokine, preferably in soluble or immobilized form; On day 5 of culture, preferably on day 7 of culture, adding IL-15 to the culture medium while maintaining the presence of at least one γδ TCR agonist and IL21 in the culture medium; Including, the culture time is carried out until at least 40% of the total γδ T cells are CD1a-Vδ1+ γδ T cells, and / or the culture time is carried out for at least 7 days, 8 days, preferably 9 days, 10 days, 11 days, 12 days, preferably 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days; Most preferably, it is a method.

[0109] In another embodiment, the method of the eighth aspect is a method of activating and inducing the expansion of CD1a-V51+ γδ T cells as defined in the second, third, fourth, fifth, sixth and seventh aspects of the invention, comprising growing said V51+ γδ T cells in a suitable culture medium in the presence of at least one γδ TCR agonist, preferably an anti-CD3 mAb such as OKT3, and in the presence of at least the cytokines IL-4, IFN-γ, IL-21 and IL-1β; Adding new (fresh) culture medium containing at least one γδ TCR agonist, preferably anti-CD3 such as OKT3, and containing IL21 and IL15 on the fifth day of culture, preferably on the sixth or seventh day of culture; Adding new (fresh) culture medium containing at least one γδ TCR agonist, preferably anti-CD3 such as OKT3, and containing IL15 on the 10th day of culture, preferably on the 11th or 12th day of culture; Optionally, on day 15 of the culture, preferably on day 16 of the culture, adding new (fresh) culture medium containing at least one γδ TCR agonist, preferably anti-CD3 such as OKT3, and containing IL15 and IFN-γ; The method includes:

[0110] In an embodiment of the eighth aspect, at least one γδ TCR agonist is added at a concentration between 0.5 μg / ml and 4 μg / ml, preferably 2 μg / ml. In an embodiment of the eighth aspect, IL21 is added at a concentration between 7 ng / ml and 15 ng / ml, preferably 13 ng / ml. In an embodiment of the eighth aspect, IL15 is added at a concentration between 70 ng / ml and 150 ng / ml, preferably 100 ng / ml. In an embodiment of the eighth aspect, IFN-γ is added at a concentration between 30 ng / ml and 80 ng / ml, preferably 70 ng / ml. In an embodiment of the eighth aspect, IL-4 is added at a concentration between 50 ng / ml and 150 ng / ml, preferably 100 ng / ml. In an embodiment of the eighth aspect, IL-1β is added at a concentration between 5 ng / ml and 20 ng / ml, preferably 15 ng / ml. Optionally, the cells are cultured in serum-free culture medium, optionally supplemented with plasma or human serum, and / or glutamine.

[0111] In one embodiment, the population of cells obtained after STEP 2 (i.e. after the eighth aspect of the invention) by expansion of STEP 1 cells is more cytotoxic than cells obtained after STEP 2 from an isolated biological sample such as cord blood or peripheral blood, whereby cytotoxicity is preferably measured using Jurkat and / or MOLM13 target cells as shown in Figure 16. The cytotoxicity of the population of cells obtained after the method of the eighth aspect is preferably at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 8-fold or 10-fold higher than the cytotoxicity of cells obtained from an isolated biological sample, preferably cord blood or peripheral blood, activated and expanded according to the method of the eighth aspect, whereby cytotoxicity is preferably measured after at least 24 hours, preferably 48 hours, of co-culture using leukemic cells, preferably Jurkat or Molm13 cell lines, at an effector-target (E:T) ratio of 1:8 or 1:4.

[0112] Moreover, not only are the populations of cells obtained de novo more cytotoxic than cells obtained after STEP2 from an isolated biological sample, but also CD1a-V51+ γδ T cells (included in said populations generated de novo from CD34+ progenitor cells and obtained) have a better cytotoxic profile (measured by the presence of cytotoxic markers) than equivalent CD1a-V51+ γδ T cells expanded according to STEP2 from ex vivo isolated cells obtained from cord blood or peripheral blood (CB-STEP2, PB-STEP2). See FIG. 15. Thus, a ninth aspect of the invention relates to a composition comprising a population of activated and expanded V51+ γδ T cells obtained or obtainable according to the eighth aspect of the invention, preferably comprising a population of activated and expanded CD1a-V51+ γδ T cells. Preferably, the invention relates to a composition comprising a population of allogeneic activated and expanded V51+ γδ T cells, preferably a population of allogeneic activated and expanded CD1a-V51+ γδ T cells obtained or obtainable according to the eighth aspect of the invention. As shown in Figure 17, the cell population of the ninth embodiment preferably constitutes at least 30% to 90%, and preferably about 40% to 60%, of the total cells that are γδ T cells, of which at least 40%, preferably about 40% to 60%, and preferably more than 50% are CD1a- and Vδ1+ (i.e., Vδ1+ γδ T cells).

[0113] In a preferred embodiment, the population of activated and expanded CD1a-V51+ γδ T cells of the ninth aspect comprises CD8+ T effector cells, as depicted in Figure 13. In a preferred embodiment, at least 60%, 70%, 75%, 80%, 85% or 90% of the CD1a-V51+ γδ T cells of the ninth aspect express the CD8 marker. In a preferred embodiment, at least 50%, or at least 60%, preferably at least 70% or 80% of the CD1a-V51+ γδ T cells of the ninth aspect exhibit a T effector phenotype, characterized by the expression of the CD45RA marker and / or the lack of expression of the CD62L marker. In a preferred embodiment, less than 60%, preferably less than 50%, less than 40%, less than 30% or less than 20% of the V51+ γδ T cells of the ninth aspect express the CD27 marker.

[0114] In a preferred embodiment, the population of activated and expanded V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, of the ninth aspect is characterised by not having an exhaustion phenotype, where the exhaustion phenotype is measured by the expression of exhaustion associated surface markers such as LAG3 and / or CTLA4, as depicted in Figure 14. Preferably, at least about 80%, 85%, 90%, 95% of the activated and expanded CD1a-V51+ γδ T cells of the ninth aspect do not express the LAG3 and / or CTLA4 exhaustion markers at detectable levels. Preferably, less than 20%, less than 15%, less than 10% or less than 5% of the activated and expanded CD1a-V51+ γδ T cells of the ninth aspect express the LAG3 marker at detectable levels. Preferably, less than 20%, less than 15%, less than 10% or less than 5% of the activated and expanded CD1a-V51+ γδ T cells of the ninth aspect express the CTLA4 marker+ at detectable levels.

[0115] The population of activated and expanded V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, of the ninth aspect is further characterised by having an activated phenotype, where the activated phenotype is measured by expression of the surface activation markers CD25 and / or CD69, see figure 14. Preferably, at least 40%, 50%, 55%, 60% of the activated and expanded CD1a-V51+ γδ T cells of the ninth aspect express the CD25 marker at detectable levels. Preferably, at least 80%, 85%, 90%, 95% of the activated and expanded CD1a-V51+ γδ T cells of the ninth aspect express the CD69 marker at detectable levels.

[0116] In a preferred embodiment, the population of activated and expanded V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, of the ninth aspect is characterised in that it comprises a cytotoxicity associated activating receptor as depicted in Figure 15. Said V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, are further characterised in that they comprise at least one, or at least a combination of two or more, preferably all, of the cytotoxicity associated markers CD56, Nkp44, Nkp30, NkG2D and DNAM-1. In one embodiment, at least 80%, 85%, 90%, 95% of the activated and expanded CD1a-V51+ γδ T cells of the ninth aspect express detectable levels of at least one, or at least a combination of two or more, preferably all, of the NKp44, NKp30 and NKG2D markers. In a particular embodiment, at least 70%, 75%, 80%, 85%, 90% or 95% of the activated and expanded CD1a-V51+ γδ T cells of the ninth aspect express detectable levels of at least one, or at least a combination of two or more, preferably all, of the CD56, NKp44, NKp30, NKG2D and DNAM-1 markers.

[0117] In one embodiment, the population of activated and expanded V51 + γδ T cells, preferably CD1a − V51 + γδ T cells, of the ninth aspect comprises: At least 40%, preferably at least 50% or 60% of the total number of V51+ γδ T cells express CD56; or At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp44 marker, or At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp30 marker, or At least 70%, preferably 80% to 100%, of the total number of Vδ1 + γδ T cells express the NKG2D marker, or At least 80%, preferably 90% to 100%, of the total number of Vδ1+γδT cells express the DNAM-1 marker; It is characterized by: Here, the expression level of the surface marker is preferably measured by flow cytometry.

[0118] The expanded and activated CD1a-V51+ γδ T population of the ninth aspect comprises: At least 40%, preferably at least 50% or 60% of the total number of V51+ γδ T cells express the CD56 marker; At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp44 marker; At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp30 marker; At least 70%, preferably 80% to 100%, of the total number of Vδ1 + γδ T cells express the NKG2D marker; At least 80%, preferably 90% to 100%, of the total number of Vδ1 + γδ T cells express the DNAM-1 marker; Preferably, the composition is characterized by Here, the expression levels of the above markers are preferably measured by flow cytometry.

[0119] A tenth aspect of the invention relates to a composition comprising a population of de novo Notch-induced differentiated and expanded V51+ γδ T cells, preferably de novo Notch-induced differentiated and expanded CD1a-V51+ γδ T cells, as defined in the ninth aspect. Thus, the cell composition according to the tenth aspect comprises highly cytotoxic and activated V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, with low expression of exhaustion markers. The population of activated and expanded V51+ γδ T cells, preferably CD1a-V51+ γδ T cells, of the tenth aspect is preferably characterized by detectable expression of at least one, or at least a combination of two or more, preferably all, of the markers CD56, NKp44, NKp30, NKG2D and DNAM-1.

[0120] The population of activated and expanded V51 γδ T cells, preferably CD1a V51 γδ T cells, of the tenth aspect comprises: At least 40%, preferably at least 50% or 60% of the total number of V51+ γδ T cells express CD56; or At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp44 marker, or At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp30 marker, or At least 70%, preferably 80% to 100%, of the total number of Vδ1 + γδ T cells express the NKG2D marker, or At least 80%, preferably 90% to 100%, of the total number of Vδ1+γδT cells express the DNAM-1 marker; Preferably, the composition is characterized by Here, the expression level of the surface marker is preferably measured by flow cytometry.

[0121] The expanded and activated population of CD1a-V51+ γδ T cells of the tenth aspect comprises: At least 40%, preferably at least 50% or 60% of the total number of V51+ γδ T cells express the CD56 marker; At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp44 marker; At least 60%, preferably at least 70% or 80% of the total number of V51+ γδ T cells express the NKp30 marker; At least 70%, preferably 80% to 100%, of the total number of Vδ1 + γδ T cells express the NKG2D marker; At least 80%, preferably 90% to 100%, of the total number of Vδ1 + γδ T cells express the DNAM-1 marker; Preferably, the composition is characterized by Here, the expression levels of the above markers are preferably measured by flow cytometry.

[0122] In a preferred embodiment of the invention, the compositions of the second to seventh or ninth or tenth aspects are used to generate chimeric antigen receptor (CAR) T cells.

[0123] An eleventh aspect of the present invention relates to a composition comprising CAR T cells obtained or obtainable using any of the compositions of the second to seventh aspects or the ninth aspect or the tenth aspect of the present invention.

[0124] In a preferred embodiment of the invention, the composition of the second to seventh or ninth or tenth aspects is injectable. In a preferred embodiment, the injectable composition comprises a cell population composed of more than 80%, i.e. more than 80%, more than 85%, more than 90%, more than 95% functional V51+ γδ cells of the invention expressing a functional native cytotoxic receptor, where the injectable composition preferably comprises more than 100 million V51+ γδ cells of the invention expressing a functional native cytotoxic receptor. The composition preferably also comprises a pharma- ceutically acceptable agent or carrier, more preferably a stabilizer, in particular human serum albumin. The cells may be autologous, i.e. derived from the same biological preparation (or from the same donor), but more preferably the cells are allogeneic, i.e. not derived from the same biological preparation (or from the same donor). More preferably the cells are obtained by a method such as the method described by the disclosed subject matter. Another aspect of the disclosed subject matter is the use of a composition comprising a cell of the second to seventh, ninth or tenth aspects of the invention in medicine.

[0125] In a more preferred embodiment, the compositions disclosed in the second to seventh aspects or the ninth aspect or the tenth aspect can be used in autologous or allogeneic adoptive cell therapy, tumor or cancer treatment, tumor or cancer immunotherapy and / or leukemia treatment or for the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Burkitt's lymphoma, follicular lymphoma, T-cell lymphoma, breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, renal cell carcinoma, or cutaneous melanoma, among others. More preferably, the compositions disclosed in the second to seventh, ninth or tenth aspects can be used in autologous or allogeneic adoptive cell therapy, tumor or cancer treatment, tumor or cancer immunotherapy and / or leukemia treatment or for the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Burkitt's lymphoma, follicular lymphoma, breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, renal cell carcinoma or cutaneous melanoma, among others.

[0126] In a more preferred embodiment, the compositions disclosed in the present invention can be used for the treatment of viral infections.

[0127] The present invention further includes the following: 1. In vitro use of a Notch ligand to generate de novo Notch-induced differentiated V51+ γδ T cells, preferably allogeneic Notch-induced differentiated V51+ γδ T cells, from a cell population comprising human HPCs, such as CD34+ cord blood HPCs and / or CD34+ ETPs, wherein the Notch ligand is Jag2 (Jagged2). 2. An in vitro method for generating de novo Notch-induced differentiated V51+ γδ T cells, preferably cognate Notch-induced differentiated V51+ γδ T cells, from a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or human ETPs, the method comprising: a. growing a cell population comprising HPCs and / or ETPs in an appropriate culture medium containing a Jag2 Notch ligand or a Notch receptor agonist, preferably immobilized on a substrate or cell line; b. maintaining the cells in culture for a duration sufficient to generate V51+ γδ T cells; Preferably, the duration is between about 2 weeks and about 15 weeks, and the Notch ligand or agonist should be present or added in sufficient amounts to the cell culture during and throughout the culture period; A method comprising: 3. The method according to any of items 1 or 2, wherein the human HPCs are derived from CD34+ umbilical cord blood HPCs, CD34+ bone marrow HPCs, CD34+ peripheral blood HPCs, or are derived from CD34+ cells derived from pluripotent stem cells such as iPSCs. 4. The method according to any of items 1 or 3, wherein the human hematopoietic progenitor cells are human CD34+ ETPs. 5. A cell composition comprising human cytotoxic V51+ γδ cells obtained or obtainable according to any of items 1 to 4. 6. A method for activating and inducing proliferation of Vδ1+ γδ T cells obtained by any of the methods of items 1 to 5, the method comprising: a. growing a cell composition comprising V51+ γδ T cells obtained by any of the methods according to items 2 to 4 in a suitable culture medium in the presence of a γδ TCR agonist, preferably soluble or immobilized, preferably by adding said agonist at regular intervals (more preferably continuously) and in the presence of at least one cytokine, for example a cytokine selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IFN-γ, IL-21, IL-15 and / or IL-1β; b. adding said γδ TCR agonist and cytokine until at least 40% of the cells express the natural cytotoxic receptor, more preferably until at least 50%, 60%, 70%, 75%, 80%, 85%, 95% of the cells express the natural cytotoxic receptor; A method comprising: 7. A cell composition comprising human cytotoxic V51+ γδ cells obtained or obtainable according to item 6. 8. The composition of item 7, comprising a cell population composed of functional V51+ γδ cells expressing a functional native cytotoxic receptor, more than 80% of which is composed of functional V51+ γδ cells expressing a functional native cytotoxic receptor, and preferably comprising more than 100 million V51+ γδ cells expressing a functional native cytotoxic receptor. 9. The composition of any of items 7 or 8, which is a pharmaceutical composition that also comprises a pharma- ceutically acceptable agent or carrier, more preferably a stabilizer, in particular human serum albumin. 10. Any of the compositions according to items 7 to 9, for use in allogeneic adoptive cell therapy, tumor or cancer treatment, tumor or cancer immunotherapy, and / or leukemia treatment, or for the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Burkitt's lymphoma, follicular lymphoma, T-cell lymphoma, breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, renal cell carcinoma, or cutaneous melanoma. 11. Any of the compositions of items 7 to 9, for use in autologous adoptive cell therapy, tumor or cancer treatment, tumor or cancer immunotherapy, and / or leukemia treatment, or for the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Burkitt's lymphoma, follicular lymphoma, breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, renal cell carcinoma, or cutaneous melanoma. 12. Use of the composition of any of items 7 or 8 for the manufacture of CAR T cells. 13. A CAR T cell composition comprising human cytotoxic V51+ γδ cells obtained or obtainable according to item 12. 14. The composition of item 13, which is a pharmaceutical composition that also comprises a pharma- ceutically acceptable agent or carrier, more preferably a stabilizer, in particular human serum albumin. 15. The composition of any of items 13 or 14, for use in allogeneic adoptive cell therapy, tumor or cancer treatment, tumor or cancer immunotherapy, and / or leukemia treatment, or for the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Burkitt's lymphoma, follicular lymphoma, T-cell lymphoma, breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, renal cell carcinoma, or cutaneous melanoma. 16. The composition of any of items 13 or 14, for use in autologous adoptive cell therapy, tumor or cancer treatment, tumor or cancer immunotherapy, and / or leukemia treatment, or for the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Burkitt's lymphoma, follicular lymphoma, T-cell lymphoma, breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, renal cell carcinoma, or cutaneous melanoma.

[0128] The present invention further includes the following: 1. In vitro use of a Jag2 Notch ligand to generate a cell composition comprising a greater amount of γδ T cells than αβ T cells from a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or CD34+ ETPs, wherein the Jag2 Notch ligand is expressed on the surface of a stromal cell line, the stromal cell line exhibiting a statistically significant increase in expression of Jag2 Notch ligand compared to a reference stromal cell, the reference stromal cell being a stromal cell expressing a basal level of the ligand. 2. The use according to item 1, wherein the stromal cell line is the OP9 cell line. 3. An in vitro method for generating a cell composition comprising γδ T cells, preferably cognate Notch-induced differentiated γδ T cells in greater amounts than αβ T cells, from a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or human ETPs, the method comprising: a. growing a cell population comprising HPCs and / or ETPs in an appropriate culture medium comprising a cell population of a stromal cell line; wherein said stromal cell line exhibits a statistically significant increase in expression of a Jag2 Notch ligand compared to a reference stromal cell, said reference stromal cell being a stromal cell expressing a basal level of said ligand; b. maintaining the cells in culture for a sufficient duration to generate γδ T cells; Preferably, the duration is between about 2 weeks and about 15 weeks, and the Notch ligand or agonist should be present or added in sufficient amounts to the cell culture during and throughout the culture period; A method comprising: 4. The method according to item 3, wherein the human HPCs are derived from CD34+ umbilical cord blood HPCs, CD34+ bone marrow HPCs, CD34+ peripheral blood HPCs, or are derived from CD34+ cells derived from pluripotent stem cells such as iPSCs. 5. The method according to any of items 3 or 4, wherein the human hematopoietic progenitor cells are human CD34+ ETPs. 6. The method according to any of items 3 to 5, wherein at least 30% of γδ T cells are Vδ1+ γδ T cells. 7. A cell composition obtained or obtainable according to any of items 3 to 6, comprising γδ T cells in greater amounts than αβ T cells. 8. The cell composition according to item 7, characterized in that at least 30% of the γδ T cells are Vδ1+ γδ T cells. 9. Here, the Vδ1+γδT population is a. a first cell population characterized by expression of the immature cell surface marker CD1a (CD1a+Vδ1+γδ T cells); b. A second cell population characterized by not expressing the immature cell surface marker CD1a (CD1a-Vδ1+γδ T cells); 9. A cell composition according to item 8, comprising: 10. The cell composition according to item 8, wherein the first cell population is characterized in that the cells do not express the cell surface markers CD25, CD27, NKp44, NKp30, and NKG2D. 11. The cell composition according to item 9 or 10, characterized in that the second cell population expresses at least one, or at least a combination of two or more, preferably all, of the cell surface markers CD27, CD73, CD69, NKp44, NKp30, and NKG2D. 12. The cell composition according to any of items 7 to 11, wherein the first cell population represents at least 90% of the total V51+ γδ T cells contained in the cell composition. 13. A method for activating and inducing proliferation of V51+ γδ T cells obtained by any of the methods of items 3 to 6, the method comprising: a. growing a cell composition comprising V51+ γδ T cells obtained by any of the methods according to items 3 to 6 in a suitable culture medium in the presence of a γδ TCR agonist, preferably soluble or immobilized, preferably by adding said agonist at regular intervals (more preferably continuously) and in the presence of at least one cytokine, for example a cytokine selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IFN-γ, IL-21, IL-15 and / or IL-1β; b. adding said γδ TCR agonist and cytokine until at least 40% of the cells express the natural cytotoxic receptor, more preferably until at least 50%, 60%, 70%, 75%, 80%, 85%, 95% of the cells express the natural cytotoxic receptor; A method comprising:

[0129] The following examples serve merely to illustrate the invention and are not intended to be limiting thereof. EXAMPLES

[0130] Example 1. Generation of V51+ human T cells from human CD34+ early thymic progenitor cells (ETPs) In vitro culture conditions for de novo generation of V.DELTA.1+ γ.DELTA. T cells from human CD34+ ETPs activated with Jag2 (STEP1) Human postnatal thymocytes were isolated from thymic tissue removed during corrective cardiac surgery from patients aged 3 days to 4 years after obtaining informed consent in accordance with the Declaration of Helsinki, by mechanical disruption and Ficoll-Hypaque (Lymphoprep™; ATOM) centrifugation. Experiments were performed in accordance with the approved guidelines established by the Research Ethics Board of the Spanish Research Council (CSIC). CD34+ETPs were isolated from thymocyte cell suspensions by CD34-magnetic cell sorting (Dynal, CD34 Progenitor Cell selection System, Invitrogen) and further depletion of CD34+CD1a+ and CD34+CD123+ progenitor cells using anti-CD1a and anti-CD123 microbeads (AutoMACS, Miltenyi Biotec). Isolated CD34+ ETPs (>96% CD34+CD1a-CD123-) were cultured in p24 well plates seeded with OP9 stromal cells transduced with either GFP as a cell tracer and DLL1, DLL4, Jag1 or Jag2 Notch ligands, which were shown to express similar surface levels, or transduced with GFP alone as a control (10 cells). 5GFP, DLL1, DLL4, Jag1 or Jag2 transduction into OP9 cells with plasmids encoding the proteins results in high expression in the cells, especially on the cell surface in the case of the Notch ligands. Notably, the increase in expression of Jag2 in transduced OP9 cells was approximately 500-fold increased expression as measured by flow cytometry (Figure 10). Cultures were performed in α-MEM medium (Gibco) supplemented with 20% fetal calf serum (FCS), 2 mmol / l L-glutamine, 200 IU / ml recombinant human (rh)IL-7 (NIBSC) and 100 IU / ml rhFlt3L (PeproTech). Cultures were replated and analyzed by flow cytometry for the generation of γδ T cells every 3-4 days for up to 30 days. These analyses showed that Jag2 was the most efficient Notch ligand promoting γδ T cell differentiation (Figure 1), whereas expression of GFP alone in OP9 cells did not induce cell differentiation (Figure 7). Indeed, Jag2-mediated signaling preferentially generated γδ T cells over αβ T cells, with a 20-fold versus 300-fold higher yield over 30 days, respectively. Strikingly, flow cytometry analysis performed with anti-Vδ1 and anti-Vδ2 mAbs revealed that γδ T cells differentiated from ETPs in response to human Jag2 signaling were preferentially Vδ1+ cells and produced IFNγ but not IL-17 (Figure 2). Thus, from a single human ETP activated with Jag2, up to 100 Vδ1+ γδ T cells exhibiting characteristics of antitumor peripheral γδ T cells can be generated (Figure 3).

[0131] Example 2. Generation of V51+ human T cells from umbilical cord blood CD34+ hematopoietic progenitor cells (HPCs) In vitro culture conditions for de novo generation of V.DELTA.1+ γ.DELTA. T cells from human umbilical cord blood CD34+ HPCs activated with Jag2 (STEP1) Cord blood samples were obtained from the Centro de Transfusion de la Comunidad de Madrid, following the approved guidelines established by the Research Ethics Committee of the CSIC. HPCs were obtained from Ficoll Hypaque-isolated cell samples by immunomagnetic sorting using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec). Reanalysis demonstrated that the sorted population was >98% CD34+ and negative (Lin-) for CD3, CD4, CD8, CD13, CD14, CD19, and CD56 lineage markers. Isolated cord blood CD34+ HPCs were cultured in α-MEM medium (Gibco) supplemented with 20% fetal calf serum (FCS), 2 mmol / l L-glutamine, and 200 IU / ml recombinant human (rh)IL-7 (NIBSC), 100 IU / ml rhFlt3L (PeproTech), and 100 IU / ml rhSCF (PeproTech) in p24-well plates seeded with OP9 stromal cells expressing the human Jag2 Notch ligand (10 cells). 5 cells / well). The generation of differentiated γδ naive T cells was analyzed every 3-4 days for up to 9 weeks, revealing a 4000-fold total cell expansion and a 210- to 250-fold increase in γδ T cell yield (Figure 4). Up to 40% of these Jag2-differentiated γδ naive T cells were Vδ1+ (Figure 4). Thus, from 1 million human umbilical cord blood HPCs activated with Jag2, up to 100 million Vδ1+ γδ T cells can be generated (Figure 5).

[0132] Example 3. Generation of non-activated naive V51+ human T cells from umbilical cord blood CD34+ hematopoietic progenitor cells (HPCs), distinct from V51+ γδ T cells present in adult peripheral blood V51+ γδ T cells generated from CD34+ HPCs isolated from umbilical cord blood and cultured with OP9 cells highly expressing Jag2 represent a heterogeneous cell population containing phenotypically immature CD1a+ and mature CD1a-V51+ cells. The mature CD1a-V51+ γδ T cell subset is largely composed of non-activated naive CD25-CD27+ cells (Figure 8), whereas V51+ γδ T cells isolated from adult peripheral blood express the cytotoxic NK receptors NKp30 and NKG2D (Figure 9).

[0133] Table 1 below and Figure 6 show the comparative cell yields and phenotypes of V51+ γδ T cells generated from human CD34+ CB HPCs subjected to human Jag2 signaling (CB-Jag2) or V51+ γδ T cells isolated ex vivo from the CB (CB ex vivo) or V51+ γδ T cells isolated ex vivo from peripheral blood (PB ex vivo). Mean ± SD percentages of TCRγδ+ and V51+ cells among T cells recovered from CB-Jag2 cells from a single CB unit (n=4), or from CB ex vivo cells isolated from a single CB or PB unit, respectively (n=4), or PB ex vivo cells (n=3).

[0134] [Table 1]

[0135] Furthermore, Table 2 below shows the comparative cell yields in absolute numbers of V51+ γδ T cells generated from human CD34+ CB HPCs undergoing human Jag2 signaling (CB-Jag2) or V51+ γδ T cells isolated ex vivo from the CB (CB ex vivo) or V51+ γδ T cells isolated ex vivo from peripheral blood (PB ex vivo) per bag (unit) of blood.

[0136] It can be seen that due to generation from CD34+ precursor cells, the yield of V51+ γδ T cells generated de novo after STEP 1 of the method of the invention (CB-Jag2) is higher than the yield of V51+ γδ T cells obtained ex vivo from PB for the same number of total starting cells from either CB or PB (Table 2).

[0137] [Table 2]

[0138] Each bag or unit of CB contains approximately 1 million CD34+ precursor cells, from which an average of 71.75 million V51+ γδ T cells are generated. On the other hand, each bag or unit of PB contains an average of 300 million total cells, from which an average of 0.51% are V51+ γδ T cells, which corresponds to 1.53 million V51+ γδ T cells. Thus, the method of producing V51+ γδ T cells from cord blood (CB) and OP9, which highly expresses Jag2, is highly efficient, with an average of 46 times more V51+ γδ T cells being generated from cord blood precursors than from peripheral blood (PB) cells.

[0139] Importantly, Vδ1+ γδ T cells isolated from PB are predominantly CD1a- (see Table 1 and Figure 6), whereas only 0.4% of cells from PB ex vivo were found to be CD1a+. However, as shown in Table 1 and Figure 6, approximately 9%-10% of cells arising from CB co-cultured with OP9-Jag2 cells are CD1a-. This was also observed in the following representative experiment (from a total of four experiments) in which the percentages of CD1a+ and CD1a- from Vδ1+ γδ T cells obtained from one unit of CB were measured: TIFF2025502150000004.tif21170

[0140] Taking the above into consideration, it can be concluded that from one bag of PB approximately 1.53 million CD1a-V51+ γδ T cells are obtained, whereas from one bag of CB, according to the method claimed herein, an average of 7 million CD1a-V51+ γδ T cells and 63 million CD1a+V51+ γδ T cells are obtained.

[0141] TCR-dependent activation and expansion of generated Vδ1+γδ naive T cells reported for DOT (STEP2) V51+ naive γδ T cells generated de novo in a TCR-independent, Jag2-Notch-dependent manner (STEP1) from cord blood HSCs as described above are activated and expanded in vitro using anti-CD3 mAb and cytokines according to a protocol referred to herein as STEP2, as shown for peripheral blood V51+ T cells (Non-Patent Document 6). For this purpose, cells differentiated from cord blood CD34+ HSCs (STEP1) are depleted of αβ T cells by magnetic cell sorting using anti-TCRαβ mAb and magnetic beads (Miltenyi Biotec). TCRαβ-depleted cell suspensions are cultured for 7 days (2.5 × 10 cells) in serum-free culture medium (OpTimizer-CTS) supplemented with autologous plasma (i.e., 5% autologous plasma) or human AB serum and 2 mmol / l L-glutamine in the presence of 1 μg / ml anti-CD3 mAb OKT3 + 100 ng / ml rhIL-4, 70 ng / ml IFN-γ, 7 ng / ml IL-21, and 15 ng / ml IL-1β (Peprotech). 5On day 7, medium containing 2 μg / ml OKT3+13 ng / ml IL-21 and 70 ng / ml IL-15 is added and the cells are cultured for another 4 days with 2 μg / ml OKT3+13 ng / ml IL-21 and 70 ng / ml IL-15. By day 11, the cells are diluted 1 / 6 and cultured for another 4-5 days in the presence of 2 μg / ml OKT3+100 ng / ml IL-15. The culture is either stopped by day 15-16 or the cells are diluted 1 / 3 and subjected to a second round of expansion in the presence of 1 μg / ml OKT3+70 ng / ml IL-15 and 30 ng / ml IFN-γ from day 18-21. The above STEP2 expansion and activation protocol is referred to herein as the DOT protocol.

[0142] Furthermore, an additional expansion and activation protocol, referred to herein as the CSIC protocol, was tested in which V51+ naive γδ T cells generated de novo from cord blood HSCs in a TCR-independent, Jag2-Notch-dependent manner (STEP1) as described above are activated and expanded in vitro using anti-TCRγδ mAb and cytokines (STEP2) as shown in Figure 18. For this purpose, cells differentiated from cord blood CD34+ HSCs (STEP1) are depleted of αβ T cells by magnetic cell sorting using anti-TCRαβ mAb and magnetic beads (Miltenyi Biotec). The TCRαβ-depleted cell suspension is cultured for 7 days (2.5 × 10 cells) in serum-free culture medium (OpTimizer-CTS) supplemented with autologous plasma (i.e., 5% autologous plasma) or human AB serum and 2 mmol / l L-glutamine in the presence of 2 μg / ml anti-TCRγδ mAb + 7 ng / ml IL-21 (PeproTech). 5On day 7, medium containing 2 μg / ml anti-TCR gamma delta mAb + 13 ng / ml IL-21 and 70 ng / ml IL-15 is added and cells are cultured for another 7 days. Culture is either stopped by day 15-16 or cells are diluted 1 / 3 and subjected to a second round of expansion in the presence of 2 μg / ml anti-TCR gamma delta mAb + 70 ng / ml IL-15 and 30 ng / ml IFN-γ from day 18-21.

[0143] Both the CSIC and DOT expansion and activation protocols are shown in FIG.

[0144] Therefore, CD1a-V51+ T cells obtained after STEP 1 were activated and expanded according to STEP 2, in particular according to the activation / expansion protocols referred to herein as DOT and CSIC. The resulting activated and expanded CD1a-V51+ T cells were compared to those derived from ex vivo isolated PB and CB cells expanded with STEP 2, in order to elucidate whether the combination of CD34+ progenitor cells with the STEP 1 and STEP 2 methods results in an improved population of CD1a-V51+ γδ T cells. The results are shown in Figures 13 to 15, where it can be seen that the de novo activated and expanded CD1a-V51+ γδ T cells obtained after the method of the invention show: They have more of a T cell effector phenotype and express more CD8 than cells from CB / PB-STEP2. See FIG. 13. They have a more activated phenotype and a less exhausted profile than CB / PB-STEP2 cells, see FIG. CB / PB-STEP2 cells have higher levels of cytotoxicity-associated activating receptors than CB / PB-STEP2 cells. See FIG.

[0145] The results above demonstrate that the methods described herein result in a superior population of CD1a-V51+ γδ T cells than those obtained from ex vivo samples. The results above also demonstrate that both STEP2 protocols, i.e., the CSIC and the DOT protocols, are suitable for reaching a superior population of CD1a-V51+ γδ T cells.

Claims

1. 1. An in vitro method for generating an expanded population of de novo Notch-induced differentiated CD1a-Vδ1+ γδ T cells from a cell population comprising human HPCs, such as CD34+ cord blood HPCs, and / or human ETPs, the method comprising: A first step of generating a cell composition comprising γδ T cells in greater amounts than αβ T cells, the first step comprising: a. growing the cell population comprising the HPCs and / or ETPs in an appropriate culture medium containing a Jag2 Notch ligand or an agonist thereof; b. maintaining the cells in culture for a duration sufficient to generate the γδ T cells; Including, The method includes a first step, wherein the cell composition resulting from the first step comprises CD1a-Vδ1+ γδ T cells, a second step of activating the CD1a-Vδ1+ γδ T cells and inducing their proliferation, the second step comprising: growing the cells obtained after the first step in a suitable culture medium in the presence of a γδ TCR agonist and in the presence of at least IL21 and IL15 to obtain a cell population characterized in that at least 40% of total γδ T cells are expanded and activated CD1a-Vδ1+ γδ T cells, wherein the population of CD1a-Vδ1+ γδ T cells is at least 40% of the total number of V51 γδ T cells express the CD56 marker; and at least 60% of the total number of V51 γδ T cells express the NKp44 marker; at least 60% of the total number of V51 γδ T cells express the NKp30 marker; at least 70% of the total number of V51 γδ T cells express the NKG2D marker; At least 80% of the total number of Vδ1 γδ T cells express the DNAM-1 marker; The method further comprises a second step,

2. The second step comprises: growing the cells obtained after the first step for 5 days in a suitable culture medium in the presence of at least one γδ TCR agonist and at least IL21; On day 5, adding IL15 to the culture medium and culturing the cells in the presence of at least one γδ TCR agonist, IL21 and IL15 for at least 7 days; The method of claim 1 , comprising:

3. 3. The method of claim 1 or 2, wherein the at least one γδ TCR agonist is added at a concentration between 0.5 μg / ml and 4 μg / ml, IL21 is added at a concentration between 7 ng / ml and 15 ng / ml, and IL15 is added at a concentration between 70 ng / ml and 150 ng / ml.

4. The method of any one of claims 1 to 3, wherein the human HPCs are derived from CD34+ umbilical cord blood HPCs.

5. 5. The method according to claim 1, further characterized in that the activated CD1a-Vδ1+ γδ T cells obtained after the second step express the CD25 and / or CD69 activation markers, but do not express the LAG3 and / or CTLA4 exhaustion markers.

6. 6. The method according to any one of claims 1 to 5, wherein the activated CD1a-Vδ1+ γδ T cells obtained after the second step are characterized by expressing the CD8 marker and having a T effector phenotype, wherein the T effector phenotype is characterized by the expression of the CD45RA marker and the lack of expression of the CD62L marker.

7. A cell composition comprising de novo Notch-induced differentiated CD1a-Vδ1+ γδ cells obtained or obtainable after the second step of the method according to any one of claims 1 to 6.

8. A cell composition comprising de novo Notch-induced differentiated CD1a-Vδ1+ γδ T cells, wherein the activated Vδ1+ γδ T cells are: at least 40% of the total number of V51 γδ T cells express the CD56 marker; and at least 60% of the total number of V51 γδ T cells express the NKp44 marker; at least 60% of the total number of V51 γδ T cells express the NKp30 marker; at least 70% of the total number of V51 γδ T cells express the NKG2D marker; At least 80% of the total number of Vδ1 γδ T cells express the DNAM-1 marker; It is characterized by Preferably, the expression level of the marker is measured by flow cytometry. Cell composition.

9. A cell composition comprising more γδ T cells than αβ T cells, obtained or obtainable after the first step of the method of any one of claims 1 to 6.

10. The population of Vδ1 γδ T cells generated after the first step is herein a. a first cell population characterized by expression of the immature surface cell marker CD1a (CD1a V51 γδ T cells); b. A second cell population characterized by not expressing the immature surface cell marker CD1a (CD1a-Vδ1+ γδ T cells); The cell composition of claim 9, comprising:

11. The cell composition of claim 10, wherein the first cell population is characterized in that the cells do not express the surface cell markers CD25, CD27, NKp44, NKp30, and NKG2D.

12. The cell composition of claim 9 or 10, wherein the second cell population is characterized in that the cells express at least one, or at least a combination of two or more, of the surface markers CD27, CD73, CD69, NKp44, NKp30, and NKG2D.

13. CAR T cells obtained or obtainable using the cell composition of any one of claims 7 to 12.

14. 14. A pharmaceutical composition comprising the cell composition of any one of claims 7 to 12 or the CAR T cells of claim 13, and further comprising a pharmaceutically acceptable agent or carrier.

15. 15. A pharmaceutical composition according to claim 14 for use in therapy.

16. 16. The pharmaceutical composition of claim 15 for use in cell therapy, tumor or cancer treatment, tumor or cancer immunotherapy, and / or leukemia treatment.

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