Method for generating cells of the t cell lineage

Abstract

To provide methods, compositions, and kits for generating progenitor and mature T cells, and uses of the cells.SOLUTION: A method of generating cells of the T cell lineage is provided comprising: (a) culturing a sample comprising stem and progenitor cells with a Notch ligand conjugated to a suspension support; and (b) isolating cells of the T cell lineage. In one aspect, the cell of the T cell lineage is a primary T cell or a mature T cell. Compositions, kits, and uses of the cells are also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 630,497, filed February 14, 2018, which is incorporated herein by reference in its entirety.

[0002] Field This application relates to methods, compositions, and kits for generating cells of the T cell lineage, and uses of the cells. Specifically, this application relates to methods, compositions, and kits for generating primitive and mature T cells, and uses of the cells. [Background technology]

[0003] background T cells are key mediators of adaptive immunity and can be used as therapeutic agents against pathogens and in cancer immunotherapy. Hematopoietic stem cell transplantation (HSCT) provides effective treatment for a wide range of malignant and non-malignant disorders, but T cell recovery is significantly delayed as a result of the preconditioning regimens required prior to treatment (Krenger et al., 2011). Unlike most other hematopoietic lineages, which arise in the bone marrow (BM), T cell development requires the migration of BM-derived progenitor cells to the thymus, where lymphoid progenitor cells receive key signals that induce their differentiation into T lineage cells (Shah and Zuniga-Pflucker, 2014).

[0004] The primary signal delivered by thymic stromal cells to incoming lymphoid progenitors is mediated by the Notch ligand, Delta-like-4, expressed by thymic cortical epithelial cells (Thompson and Zuniga-Pflucker, 2011). Notch receptors expressed by lymphoid progenitors require mechanical traction induced by Delta-like-4-bearing cells to effectively induce Notch receptor activation (D'Souza et al., 2010; Gordon et al., 2015; Meloty-Kapella et al., 2012). It has also been shown that T cell development requires consistent and high levels of Notch receptor activation (Schmitt et al., 2004). In the absence of Notch1 receptor signaling or Delta-like-4-inducible signaling, T cell development in the thymus fails, and alternative lymphoid lineages, such as B cells, develop instead. Thus, the development of T cells within the thymus is predicated on the Notch signaling pathway (Zuniga-Pflucker, 2004).

[0005] In the context of HSCT, thymic dysfunction or atrophy, a consequence of conditioning and aging, respectively, combined with the limited capacity of transplanted HSCs to produce lymphocytes, limits the extent of thymic T cell development (Porter and June, 2005). This leads to inadequate immune surveillance, predisposing patients to infections and / or cancer recurrence, and remains a major clinical challenge.

[0006] Adoptive transfer of progenitor T (proT) cells has emerged as a promising strategy to enhance T cell reconstitution, as human or mouse proT cells, whether of xenogeneic or allogeneic origin, have been shown to engraft in the thymus of immunodeficient mice (Awong et al., 2009; Awong et al., 2013; Zakrzewski et al., 2006; Zakrzewski et al., 2008). ProT cells are developmentally immature and undergo positive and negative selection within the host thymus. Thus, proT cells are restricted to the recipient's major histocompatibility complex (MHC), providing host-tolerant T cells that can circumvent the clinical challenges associated with graft-versus-host disease (GVHD). Importantly, proT cell transplantation restores thymic architecture and improves subsequent thymic seeding of HSC-derived progenitor cells. In addition to their inherent regenerative medicine properties, proT cells can be engineered with T cell receptors (TCRs) and chimeric antigen receptors (CARs) to confer specificity for tumor-associated antigens (TAAs) for cancer therapy, and with synthetic gene circuits to generate customized response programs.

[0007] An unsolved challenge in this field is the development of a clinically relevant system that can be easily scaled to generate large numbers of proT cells from human hematopoietic stem / progenitor cells (HSPCs) from various sources. Previous methods rely on mouse-derived OP9 cells expressing the Notch ligands Delta-like-1 (DL1) or Delta-like-4 (DL4), but several challenges exist before this approach can be applied clinically (Awong et al., 2009; Awong et al., 2013). Most stromal cell-free approaches rely on two-dimensional (2D) tissue culture platforms, whereby the Notch ligands DL1 or DL4 are immobilized on tissue culture plates (Gehre et al., 2015; Reimann et al., 2012; Simons et al., 2017). Additional adhesion receptor ligands, such as vascular cell adhesion molecule-1 (VCAM-1), have also been included in this format (Shukla et al., 2017). Human proT cells produced using these strategies have been shown to successfully reconstitute the thymus of immunodeficient mice. While these advances are encouraging, the use of these approaches for generating therapeutic proT cells is limited by the need for large-scale processing for clinical manufacturing, making them ineffective methods for routinely generating large numbers of clinically applicable cells. Ideally, a truly scalable platform would enable the expansion of proT cells in a closed, automated bioreactor system (Lipsitz et al., 2016). Summary of the Invention

[0008] overview We have developed a cell-free, bead-based system for generating T-lineage cells from mouse or human hematopoietic stem / progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). Non-plate-bound suspensions of Notch ligands (e.g., DL4-μ beads) enable efficient generation of T-lineage cells, including primitive and mature T cells.

[0009] Accordingly, the present disclosure provides a method for generating cells of a T cell lineage, comprising: (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support; and (b) isolating cells of a T cell lineage.

[0010] In one embodiment, the floating support is a particle.

[0011] In another embodiment, the floating support is a microbead.

[0012] In one embodiment, stem or progenitor cells are cultured in suspension with a Notch ligand.

[0013] In another embodiment, the stem cells are selected from hematopoietic stem / progenitor cells (HSPCs), embryonic stem cells, or induced pluripotent stem cells (iPSCs).

[0014] In another embodiment, the stem cells are human stem cells, optionally CD34 + or CD34 + CD38 - / lo It is HSPC.

[0015] In another embodiment, the stem cells are CD34 + CD34 hematopoietic progenitor cells, optionally differentiated from iPSCs + Hematopoietic progenitor cells.

[0016] In another embodiment, the Notch ligand is DL1 or DL4.

[0017] In another embodiment, the cell of the T cell lineage is a progenitor T (proT) cell.

[0018] In another embodiment, the stem or progenitor cells are human cells and the proT cells have the phenotype CD34 + CD7 + or CD7 + CD5 + CD1a - It has.

[0019] In another embodiment, the stem or progenitor cells are mouse cells, optionally of lineage. - CD117 + Sca-1 + mouse cells, proT cells, phenotype CD25 + or CD25 + CD90 + It has.

[0020] In another embodiment, the cells of the T cell lineage are CD4 + CD8 + double positive cells, CD4 + CD8 + CD3 + double positive cells, CD8 + CD3 + Single positive cell, or CD4 + CD3 + Single positive cell.

[0021] In another embodiment, the stem or progenitor cells are cultured in a medium that does not contain stromal cells.

[0022] In another embodiment, the stem or progenitor cells are cultured with at least one T cell costimulatory molecule attached to a suspension support, the at least one T cell costimulatory molecule optionally being VCAM1.

[0023] The present disclosure also provides cells of a T cell lineage, the cells being generated by a method of generating cells of a T cell lineage, comprising: (a) culturing a sample comprising stem or progenitor cells with a Notch ligand conjugated to a suspension support; and (b) isolating cells of the T cell lineage.

[0024] In one embodiment, the cells are primary T cells, CD4 + CD8 + double positive cells, CD4 + CD8 + CD3 + Double positive cells, or CD8 + CD3 + Single positive cells, CD4 + CD3 + Single positive cell.

[0025] The present disclosure also provides a floating Notch ligand comprising (a) a Notch ligand and (b) a floating support, optionally a microbead, wherein the Notch ligand is conjugated to the floating support.

[0026] In one embodiment, the floating support is a microbead, (i) the microbead has a diameter of 6.5 to 100 μm, optionally a diameter of 20 to 30 μm, and / or (ii) the C-terminal region of the Notch ligand is conjugated to the microbead.

[0027] The present disclosure also provides the use of suspended Notch ligands to generate cells of the T cell lineage.

[0028] The present disclosure also provides a kit comprising: (i) a suspended Notch ligand comprising (a) a Notch ligand and (b) a suspension support, wherein the Notch ligand is conjugated to the suspension support; and (ii) instructions for using the suspended Notch ligand to generate cells of a T cell lineage.

[0029] The present disclosure further provides a kit comprising: (i) a floating Notch ligand, the kit comprising (a) a Notch ligand and (b) a floating support, wherein the Notch ligand is conjugated to the floating support; and (ii) a culture medium.

[0030] In one embodiment of the kit, the free-floating Notch ligand comprises DL4 conjugated to microbeads.

[0031] In another embodiment, the kit further comprises (iii) at least one T cell costimulatory molecule attached to the floating support, wherein the at least one T cell costimulatory molecule is optionally VCAM1.

[0032] The present disclosure provides a method of treating a subject having a condition requiring an increase in T cell numbers, comprising: (i) generating cells of a T cell lineage, comprising: (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support; and (b) isolating cells of a T cell lineage; and (ii) administering to said subject an effective amount of cells of said T cell lineage. Further provided is a method comprising:

[0033] In one embodiment, the cell of the T cell lineage is a primitive T cell.

[0034] In another embodiment, the cell of the T cell lineage is a mature T cell.

[0035] In another embodiment, the cells of the T cell lineage are CD4+CD8+ double positive cells, CD4+CD8+CD3+ double positive cells, CD8+CD3+ single positive cells, or CD4+CD3+ single positive cells.

[0036] [The present invention 1001] (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support; and (b) isolating cells of the T cell lineage 2. A method for generating cells of the T cell lineage, comprising: [The present invention 1002] 1001. The method of claim 1001, wherein said floating support is a particle. [The present invention 1003] 1003. The method of claim 1001 or 1002, wherein said floating support is a microbead. [The present invention 1004] The method according to any one of claims 1001 to 1003, wherein said stem cells or progenitor cells are cultured in suspension together with said Notch ligand. [The present invention 1005] The method of any of claims 1001 to 1004, wherein said stem cells are selected from hematopoietic stem / progenitor cells (HSPCs), embryonic stem cells, or induced pluripotent stem cells (iPSCs). [The present invention 1006] The stem cells are CD34+ or CD34 + CD38 - / lo The method of any one of 1001 to 1005, wherein the subject is an HSPC. [The present invention 1007] The stem cells are CD34 + CD34 hematopoietic progenitor cells, optionally differentiated from iPSCs + The method according to any one of claims 1001 to 1004, wherein the cells are hematopoietic progenitor cells. [The present invention 1008] The method of any one of claims 1001 to 1007, wherein said Notch ligand is DL4. [The present invention 1009] The method of any one of claims 1001 to 1008, wherein the T cell lineage cells are proT cells. [The present invention 1010] The stem or progenitor cells are human cells, and the proT cells have the phenotype CD34 + CD7 + or CD7 + CD5 + CD1a - The method of the present invention 1009, comprising: [The present invention 1011] The stem or progenitor cells are murine cells, and optionally are lineage-CD117+ Sca-1+ murine cells, and the proT cells have the phenotype CD25 + or CD25 + CD90 + The method of the present invention 1010, comprising: [The present invention 1012] The method of any one of claims 1001 to 1008, wherein the T cell lineage cells are CD4+CD8+ double positive cells, CD4+CD8+CD3+ double positive cells, CD8+CD3+ single positive cells, or CD4+CD3+ single positive cells. [The present invention 1013] The method of any of claims 1001 to 1012, wherein said stem or progenitor cells are cultured in a medium that does not contain stromal cells. [The present invention 1014] Any of the methods of claims 1001 to 1013, wherein the stem or progenitor cells are cultured together with at least one T cell costimulatory molecule attached to a floating support, and the at least one T cell costimulatory molecule is optionally VCAM1. [The present invention 1015] A cell of the T cell lineage generated by any of the methods of the present inventions 1001 to 1014. [The present invention 1016] The cell of the present invention 1015, which is a primary T cell, a CD4+CD8+ double positive cell, a CD4+CD8+CD3+ double positive cell, a CD8+CD3+ single positive cell, or a CD4+CD3+ single positive cell. [The present invention 1017] A floating Notch ligand comprising: (a) a Notch ligand; and (b) a microbead, wherein the Notch ligand is conjugated to the microbead. [The present invention 1018] 1017. The floating Notch ligand of the present invention, wherein (i) said microbeads have a diameter of 6.5 to 100 μm, and / or (ii) the C-terminus of said Notch ligand is conjugated to said microbeads. [The present invention 1019] Use of the free-floating Notch ligand of the present invention 1017 or 1018 to generate cells of the T cell lineage. [The present invention 1020] (i) a floating Notch ligand comprising (a) a Notch ligand and (b) a floating support, wherein the Notch ligand is conjugated to the floating support; and (ii) instructions for using the suspension Notch ligand to generate cells of the T cell lineage; Includes a kit. [The present invention 1021] (i) a floating Notch ligand comprising (a) a Notch ligand and (b) a floating support, wherein the Notch ligand is conjugated to the floating support; and (ii) Culture medium Includes a kit. [The present invention 1022] 1022. The kit of claim 1020 or 1021, wherein said free-floating Notch ligand comprises DL4 conjugated to microbeads. [The present invention 1023] (iii) at least one T cell costimulatory molecule attached to a floating support; The kit of any of claims 1020 to 1022, further comprising: wherein the at least one T cell costimulatory molecule is optionally VCAM1. [The present invention 1024] 1. A method of treating a subject having a condition requiring an increase in T cell numbers, comprising: (i) generating cells of a T cell lineage, comprising: (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support; and (b) isolating cells of a T cell lineage; and (ii) administering to said subject an effective amount of cells of said T cell lineage. A method comprising: [The present invention 1025] 1025. The method of claim 1024, wherein said cell of the T cell lineage is a primitive T cell. [The present invention 1026] 1024. The method of claim 1023, wherein said T cell lineage cells are CD4+CD8+ double positive cells, CD4+CD8+CD3+ double positive cells, CD8+CD3+ single positive cells, or CD4+CD3+ single positive cells. [The present invention 1027] The method of claim 1023, wherein said cell of the T cell lineage is a mature T cell. Other features and advantages of the present application will become apparent from the following detailed description, although it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present application, are given for purposes of illustration only, since various changes and modifications within the spirit and scope of the present application will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0037] Aspects of the present invention will now be described with reference to the drawings.

[0038] [Figure 1] Design and biotinylation of the DL4-Fc construct. A) The DL4-Fc fusion construct is illustrated with its components: the extracellular domain of human DL4, purification purposes, and the Fc region of human IgG3 (IgG3Fc). B) Western blot analysis of chemically biotinylated DL4-Fc using anti-biotin (top) and anti-human IgG (bottom) under non-reducing conditions. [Figure 2] Activation of Notch reporter cells by first-generation DL4-μ beads is shown. A) Chemically biotinylated DL4-Fc protein was conjugated to SA-coated beads to form randomly oriented first-generation DL4-μ beads, ranging from 50 nm gold nanoparticles (GNPs) to 100 μm μbeads. B) 3 x 10 3T3-N1Cluc cells were incubated in plates pretreated with hIgG as a negative control or with different concentrations of DL4-Fc as indicated. Alternatively, cells were incubated with different numbers of DL4-μ beads in untreated plates. The total number of beads in each condition represented the same surface area and therefore the same total number of DL4-Fc molecules. 24 hours after plating, cells were lysed and analyzed for luciferase activity. [Figure 3] This figure shows the induction of T cell development from mouse HSCs using first-generation DL4-μBeads. Mouse fetal liver-derived HSPCs were incubated with unconjugated μBeads or DL4-μBeads in medium containing FBS, SCF, IL-7, and Flt3-L for 7 days. Cocultures were collected and analyzed by flow cytometry for the presence of T-lineage (CD25+), B-lineage (CD19+), or myeloid (CD11b+) cells. The numbers in the plots represent the percentage of cells within each quadrant. [Figure 4]Site-specific biotinylation of DL4-Fc is shown. A) The DL4-Fc fusion construct was redesigned to contain a BirA recognition sequence (AviTag™) at the C-terminus, allowing a biotin moiety to be conjugated thereto by the enzyme BirA. B) Western blot analysis of chemically biotinylated DL4-Fc using anti-human IgG (top) and anti-biotin (bottom) under non-reducing conditions. [Figure 5] Figure 1 shows the difference in Notch activation ability between first- and second-generation DL4-μ beads. A) BirA-biotinylated DL4-Fc protein was conjugated to SA-coated beads to form directed second-generation DL4-μ beads. B) 3x10 3T3-N1Cluc cells were incubated on IgG- or DL4-Fc plate-bound controls or with first- or second-generation DL4-μ beads (25 μm). 24 hours after plating, cells were lysed and analyzed for luciferase activity. [Figure 6] The effect of μ-bead size on Notch activation is shown. DL4-μ-beads, ranging in size from 6.5 μm to 100 μm, were incubated overnight with 3 x 10 3T3-N1Cluc cells. Plate-bound (PB) IgG and DL4-Fc were included as negative and positive controls, respectively. The total number of beads in each condition represented the same surface area and therefore the same total number of DL4-Fc molecules. 24 hours after plating, cells were lysed and analyzed for luciferase activity. [Figure 7]Figure 1 shows Notch activation induced by varying concentrations of DL4-Fc and μ-beads of different compositions. A) DL4-μ-beads were prepared by conjugating increasing amounts of DL4-Fc to a fixed number of 25 μm diameter SA-coated μ-beads. 25 μm diameter magnetic μ-beads were included to assess the effect of varying μ-bead core composition on Notch activation capacity. B) Biotinylated DL4-Fc was coupled to equivalent sized SA-μ-beads or protein G-μ-beads to determine whether binding to the Fc region was equally effective in Notch activation. DL4-μ-beads were incubated overnight with 3 x 104 3T3-N1Cluc cells. Plate-bound IgG was included as a negative control. 24 hours after plating, cells were lysed and analyzed for luciferase activity. [Figure 8] Figure 1 shows the induction of T cell development from mouse HSPCs using second-generation DL4-μBeads. 1 x 10, 3 x 10, or 8 x 10 mouse fetal liver-derived HSPCs were incubated with unconjugated μBeads or DL4-μBeads at a 10:1 (beads:cells) ratio in medium containing FBS, SCF, IL-7, and Flt3-L for 7 days. Cocultures were collected and analyzed by flow cytometry for the presence of T-lineage (CD25+), B-lineage (CD19+), or myeloid (CD11b+) cells. Numbers in the plot indicate the percentage of cells within each quadrant. [Figure 9] Optimization of the HSPC to DL4-μ-bead ratio is shown. Mouse fetal liver-derived HSPCs were cultured for 7 days with unconjugated μ-beads, plate-bound DL4-Fc (PB-DL4), or a 3-fold titration of DL4-μ-beads. Cultures were analyzed by flow cytometry for the inhibition of CD11b+ myeloid cells and CD19+ B-lineage cells, as well as the appearance of proT (CD90+ CD25+) cells. [Figure 10A]Progression of human T-lineage cell development from HSPCs cocultured with DL4-μ-Beads. A) Human cord blood-derived CD34+ cells were cultured with unconjugated μ-Beads, plate-bound DL4-Fc, or DL4-μ-Beads in StemSpan™ SFEM II supplemented with StemSpan™ T Cell Progenitor Expansion Supplement for 14 days. Cells were harvested every 2 days (arrows) and analyzed for surface expression of CD34, CD5, CD1a, and CD7 using flow cytometry. [Figure 10B] Progression of human T-lineage cell generation from HSPCs co-cultured with DL4-μ-beads is shown. B) Cell numbers were also counted to assess overall cell expansion. Fold expansion was calculated by dividing the total number on the indicated day by the initial seeding amount at the start of culture on day 0. [Figure 11] Analysis of the presence of mature human T cells generated using DL4-μ beads. Human umbilical cord blood-derived CD34+ cells were cultured with DL4-μ beads in StemSpan™ SFEM II supplemented with StemSpan™ T Cell Progenitor Expansion Supplement. Cells were harvested on days 28 and 47 and analyzed for surface expression of CD34, CD5, CD1a, CD4, CD8, and CD3. Co-expression of CD3 is indicated by the arrow in SP-, DP-, or DN-gated cells. [Figure 12]DL4-μBeads induce T cell development from CD34+ cells derived from G-CSF and Plerixafor (PLX)-mobilized peripheral blood (mPB). 3 x 103 CD34+ cells from umbilical cord blood and adults (n=3) treated with G-CSF and PLX for 5 days were incubated with 9,000 DL4-μBeads. A) Progression toward T cell development was analyzed on day 14 for expression of cell surface markers CD5, CD7, and CD34 using flow cytometry. B) Cell numbers were counted on day 14 using a hemocytometer, and fold expansion was calculated based on the starting cell number. The expansion rate using umbilical cord blood (CB) was used as a comparative operator. [Figure 13] Figure 1 shows the induction of early and late T cell development from induced pluripotent stem cells (iPSCs) using DL4-μ beads. Human iPSCs were induced to differentiate first toward a mesodermal fate and then toward a CD34+ prehematopoietic fate. (A) 3 x 103 CD34+ cells enriched using MACS were incubated with 27 x 103 DL4-μ beads, and their progression toward T cell development was analyzed by flow cytometry on days 6, 8, 10, and 12 for cell surface expression of CD5, CD7, and CD34. B) Cultures on days 14, 28, and 35 were analyzed for the presence of mature T cells using the T cell coreceptors CD4 and CD8, as well as the cell surface marker CD3. Inverted triangles indicate the day of analysis. RCN; relative cell count. [Figure 14] Figure 1 shows the induction of early and late T cell development from T cell-derived iPSCs (T-iPSCs). T-iPSCs were induced to mesoderm and then to a prehematopoietic fate. 3 x 103 CD34+ cells enriched using MACS were incubated with 27 x 103 DL4-μ beads, and their progression to T cell development was analyzed by flow cytometry at days 12 (D12) and 24 (D24) for cell surface expression of CD5, CD7 (early T cell development), and CD4, CD8, CD3, and TCRαβ (late T cell development). [Figure 15]Biotinylation of recombinant VCAM1-Fc is shown. Similar to DL4-Fc, recombinant VCAM1-Fc was genetically engineered to consist of the VCAM-1 extracellular domain, IgG3 Fc domain, and Avi-tag biotinylation site. VCAM1-Fc was expressed in HEK293T cells, secreted into the culture medium, purified using protein G-conjugated beads, and biotinylated in vitro with BirA enzyme. Samples of commercially available VCAM1-Fc (available from R&D Systems), laboratory-purified VCAM1-Fc (-Fc), and in vitro biotinylated VCAM1-Fc (biotin) were electrophoresed on a polyacrylamide gel. Western immunoblot analysis was used to compare the size and concentration of VCAM-Fc samples with each other using an anti-VCAM-1 antibody (A), and to detect the presence of biotinylated protein using an anti-biotin antibody (B). [Figure 16] The combined effect of VCAM-1 and DL4 on human T cell development is shown. Biotinylated VCAM1-Fc (VCAM) along with DL4-Fc (DL4) was conjugated to μ-beads at different ratios as shown, and compared with unconjugated μ-beads (UN) and DL4-μ-beads. The amount of DL4-Fc was kept constant at 1 μg per 2 x 105 SA-μ-beads, but VCAM-Fc was added at 0.01 μg (100:1), 0.1 μg (10:1), 1 μg (1:1), and 10 μg (1:10). DL4:VCAM1-μ-beads were then incubated with 3 x 103 CB-derived CD34+ cells, and the progression of T cell development was analyzed on day 7 using CD34, CD7, and CD5 cell surface markers. [Figure 17]Transplantation of progenitor T (proT) cells into the thymus of immunodeficient NOD-SCID IL2rγnull (NSG) mice is shown. Human CB-derived CD34+ cells were incubated with DL4-μ beads for 7 days. CD34+ CD7+ progenitor T (proT) cells were sorted using flow cytometry, and 3 x 105 cells were injected intravenously into immunodeficient NSG mice. IL-7 boost injections were given at 3-4 day intervals, as shown. After 3 weeks, thymi were harvested and processed. Flow cytometry analysis was used to identify live (DAPI-) human hematopoietic cells (CD45+). Lineage analysis was also performed on electronically gated live CD45+ cells using the cell surface markers CD19 (B cells), CD33 (myeloid cells), CD3, CD4, and CD8 (T cells). [Figure 18] Engraftment of proT cells into the thymus and their subsequent migration to the bone marrow and secondary lymphoid organs, the spleen, is shown. ProT cells sorted from day 7 CB-HSPC / DL4-μ-bead cultures were injected into NSG mice. Twelve weeks after injection, the thymus (T), bone marrow (BM), and spleen (S) were collected and processed to assess engraftment of CD45+ human hematopoietic cells using flow cytometry (upper panel). Human CD45+ cells were electronically gated, and the presence of mature T cells was determined in each organ as indicated using the cell surface markers CD3, CD4, CD8, and TCRαβ. [Figure 19] Separation of magnetic DL4-μ beads from cellular components is shown. 2 x 105 CD34+ CB-derived HSPCs were cultured in a T25 flask with 1.8 x 106 iron oxide-coated DL4-μ beads. A) On day 5 of culture, the percentage of CD34+ CD7+ proT cells was determined. B) This culture was then subjected to separation via AutoMACS to separate the magnetized particles from unbound cellular components. Negative fractions represent components that did not bind to the instrument's magnetic probe. Positive represents the bound fraction. The bead and cell content of each fraction was counted using a hemocytometer and compared to the original mixture (before sorting). Cells in each fraction are shown in the left bar, and beads in the right bar. DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description As described above, the present inventors have developed a cell-free, bead-based system for generating cells of the T cell lineage from stem or progenitor cells, such as mouse or human hematopoietic stem / progenitor cells (HSPCs) or induced pluripotent stem cells (iPSCs). Non-plate-bound suspensions of Notch ligands (e.g., DL4-μ beads) enable efficient generation of T lineage cells, including progenitor and mature T cells.

[0040] I. Methods for generating cells Accordingly, the present disclosure provides a method for generating cells of a T cell lineage, comprising: (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support; and (b) isolating cells of a T cell lineage.

[0041] The term "cell of the T-cell lineage" refers to a cell that exhibits at least one phenotypic characteristic of a T-cell or its precursor or progenitor cells that distinguishes the cell from other lymphoid cells and from cells of the erythroid or myeloid lineages. Such phenotypic characteristic may include the expression by the cell or its precursor or progenitor cells of one or more T-lineage-specific proteins, or a physiological, morphological, functional, or immunological characteristic specific to T cells.

[0042] Cells of the T cell lineage include: (a) progenitor or precursor cells that are committed to the T cell lineage ("progenitor T cells" or "proT cells" as described herein); (b) CD25+ immature T cells; and (c) cells that have undergone commitment to the CD4 or CD8 lineage (e.g., CD4+CD8 lo TCR int (d) characterized by TCR gene rearrangement; (e) CD4+CD8+ double-positive (DP) progenitor thymocytes; (f) CD4-CD8+ or CD4+CD8- and optionally TCR hi(g) CD3+CD90+; (h) CD4-CD8+ or CD4+CD8- and TCR hi Single positive (SP) cells that are TCR-αβ + and / or TCR-γδ + (j) characterized by expression of any of multiple Vβ chains (e.g., Vβ-3, -6, and 17a); or (k) TCR / CD3 hi , mature and functional or activated T cells, which may be characterized as CD4-CD8+ or CD4+CD8-.

[0043] In one embodiment, a cell of the T cell lineage is a "primordial T cell" or "pro T cell." The terms "primordial T cell" or "pro T cell," as used herein, refer to a T cell that can mature into a mature T cell or a mature lymphocyte.

[0044] In one embodiment, the primary T cells are human primary T cells. The phenotype of human primary T cells includes CD34+CD7+ and CD7+CD5+CD1a-. In another embodiment, the primary T cells are mouse primary T cells. The phenotype of mouse primary T cells includes CD25+.

[0045] In another embodiment, cells of the T cell lineage are CD4 and CD8 double positive (DP) cells and are characterized by CD4+CD8+ and CD4+CD8+CD3+ phenotypes. In another embodiment, cells of the T cell lineage are CD4 or CD8 single positive (SP) cells and are characterized by CD4-CD8+, CD4+CD8-, or CD4-CD8+CD3+, CD4+CD8-CD3+.

[0046] The term "floating support" as used herein refers to any material that, when conjugated to a Notch ligand or other T cell costimulatory molecule, allows the Notch ligand (or costimulatory molecule) to float in a culture medium. Floating supports can be made from a variety of materials and can 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.

[0047] In one aspect, the floating support is a particle. The particle can be of any shape, including but not limited to, a sphere, an oval, a rod, a rectangle, etc. The particle can be made of a variety of materials, including but not limited to, natural or synthetic polymers, natural or synthetic waxes, ceramics, metals, biological materials, or combinations thereof.

[0048] In one embodiment, the floating support is a microbead. The term "microbead" or "μ-bead," as used herein, refers to a spherical or nearly spherical bead having a diameter of 0.01 μm (10 nm) to 500 μm, optionally 1 to 200 μm. In another embodiment, the microbead has a diameter of 6.5 to 100 μm, optionally 20 to 30 μm, 24 to 26 μm, or 25 μm.

[0049] Various types of microbeads are contemplated herein. In one embodiment, the microbeads are polymeric, silica, or magnetic microbeads. In another embodiment, the microbeads are polystyrene microbeads, gold nanoparticles, or Dynabeads. In another embodiment, the microbeads are copolymers of lactic and glycolic acid (PLGA).

[0050] Various means for conjugating proteins to supports are known in the art. Proteins can be conjugated directly or indirectly to a floating support, such as a microbead.

[0051] In one embodiment, the Notch ligand described herein is conjugated to a floating support using a biotin / streptavidin system. In this case, the Notch ligand is biotinylated and then conjugated to a streptavidin-coated floating support (e.g., streptavidin-coated microbeads). In another embodiment, the Notch ligand described herein is conjugated to a floating support via protein G or protein A.

[0052] The term "Notch ligand," as used herein, refers to a ligand capable of binding to a Notch receptor polypeptide present in the membrane of many different mammalian cells, including hematopoietic stem / progenitor cells. Notch receptors identified in human cells include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a characteristic DSL domain (D-delta, S-serrate, and L-Lag2) containing 20-22 amino acids at their amino termini and 3-8 EGF repeats on the extracellular surface.

[0053] The term Notch ligand also includes anti-Notch antibodies and aptamers (eg, DNA aptamers) that are capable of binding to and participating in Notch signaling.

[0054] Notch ligands are selected that promote and maintain the differentiation and proliferation of cells of the T cell lineage. Notch ligands can be derived from humans or from other species, including mammalian species such as rodents, dogs, cats, pigs, sheep, cattle, goats, and primates.

[0055] Specific examples of Notch ligands include the Delta family, which includes Delta-1 (Genbank accession number AF003522, Homo sapiens), Delta-3 (Genbank accession number AF084576, rat (Rattus norvegicus)), Delta-like 1 (DL1; Genbank accession numbers NM_005618 and NP_005609, Homo sapiens; Genbank accession numbers X80903, 148324, house mouse (M. musculus), Delta-like 3 (Genbank accession numbers NM_053666, N_446118, rat), Delta-4 (Genbank accession numbers AF273454, BAB18580, house mouse; Genbank accession numbers AF279305, AAF81912, Homo sapiens), and Delta-like 4 (DL4; Genbank accession numbers Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank accession number NM019454, house mouse). Notch ligands are commercially available or can be produced by recombinant DNA techniques and can be of various purity levels.

[0056] The term "Notch ligand" also includes homologs of known Notch ligands that can be identified by standard techniques. A "homolog" refers to a gene product that exhibits sequence homology, either amino acid or nucleic acid sequence homology, to any known Notch ligand. A Notch ligand can be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, preferably 90%, more preferably 95%, and most preferably 98-99% identical at the amino acid level to the corresponding Notch ligand.

[0057] In one embodiment, a homolog of a Notch ligand contains an N-terminal DSL domain and 3 to 8 EGF-like repeats on the extracellular surface. Suitable homologs are also capable of binding to a Notch receptor. Binding to a Notch receptor can be determined by various methods known in the art, including in vitro binding assays.

[0058] The term "Notch ligand" also includes mutants or variants of known Notch ligands. The term "mutant" refers to a polypeptide having a primary amino acid sequence that differs from the wild-type sequence by one or more amino acid additions, substitutions, or deletions. Preferably, the mutant has at least 90% sequence identity to the wild-type sequence. Preferably, the mutant has 20 or fewer mutations relative to the entire wild-type sequence. More preferably, the mutant has 10 or fewer mutations relative to the entire wild-type sequence, and most preferably 5 or fewer mutations.

[0059] Optionally, the Notch ligand comprises at least one protein tag. A protein tag is a peptide sequence added to a protein of interest, such as a Notch ligand. The protein tag can be directly or indirectly linked to the protein of interest. Various protein tags are known in the art and can be used for a variety of purposes. In one embodiment, the Notch ligand comprises an Fc tag (also known as an Fc fusion protein). As used herein, the term "Fc" refers to the Fc domain of IgG. In a specific embodiment, the Notch ligand DL4 is fused to Fc (DL4-Fc). In another embodiment, the tag is a His tag. In a further embodiment, the tag is a molecule that promotes oligomerization of the Notch ligand. For example, a small domain of COMP (cartilage oligomeric matrix protein) can be fused to a Notch ligand (e.g., DL4) to form a DL4 pentamer. Similarly, ferritin can be used to form DL4 multimers.

[0060] In the methods described herein, cells of the T cell lineage are generated by culturing a sample containing stem and progenitor cells. The stem or progenitor cells can be obtained from any suitable source, including, but not limited to, umbilical cord blood, embryos, embryonic tissue, fetal tissue, bone marrow, and blood. In one embodiment, the stem or progenitor cells are hematopoietic stem or progenitor cells (HSPCs). In another embodiment, the stem cells are embryonic stem cells (ESCs). In a further embodiment, the stem or progenitor cells are induced pluripotent stem cells. In another embodiment, the stem or progenitor cells are CD34 + CD34 hematopoietic progenitor cells, optionally differentiated from ESCs or iPSCs + CD34 hemogenic endothelial progenitor cells, or differentiated from ESCs or pluripotent stem cells (PSCs) + Pre-hematopoietic cells. CD34 + A variety of differentiation protocols for obtaining cells are known in the art. In therapeutic applications, stem or progenitor cells used to generate cells of the T cell lineage may be obtained from the patient to be treated.

[0061] The terms "hematopoietic stem / progenitor cells," "hematopoietic stem or progenitor cells," or "HSPCs," as used herein, refer to undifferentiated hematopoietic cells that can differentiate into other cell types, such as cells of the T cell lineage. HSPCs can be obtained from numerous sources, including, but not limited to, bone marrow, umbilical cord blood, and mobilized peripheral blood (mPB). HSPCs can also be obtained from several fetal and embryonic sites, such as the liver, yolk sac, or dorsal aorta. HSPCs can also be obtained by inducing differentiation of ESCs or iPSCs in culture.

[0062] The term "embryonic stem cell" or "ESC" as used herein refers to an undifferentiated embryonic stem cell that has the potential to integrate into and become part of the germline of the developing embryo.

[0063] The term "induced pluripotent stem cells" or "iPSCs," as used herein, refers to cells derived from somatic cells, such as skin or blood, that have been retroactively reprogrammed to an embryonic-like pluripotent state. In one aspect, iPSCs are derived from T cells with known or unknown TCR specificity (e.g., T cells bearing a TCR with specificity for cancer).

[0064] Typically, a sample containing stem or progenitor cells is first depleted of non-stem or mature cells. Negative and positive selection methods known in the art can be used to enrich for stem or progenitor cells. For example, cells can be sorted based on cell surface antigens using a fluorescence-activated cell sorter or magnetic beads that bind to cells with specific cell surface antigens. A negative selection column can be used to remove cells that express lineage-specific surface antigens.

[0065] In one embodiment, the sample containing stem or progenitor cells is lineage negative (Lin - ) fraction and lineage-positive (Lin + ) fraction and Lin - The fraction was then purified to identify CD34 + Cells can be sorted.

[0066] The progenitor or stem cells are cultured under suitable conditions as described herein to generate cells of the T cell lineage. Preferably, the cells are cultured in the presence of one or more Notch ligands conjugated to a suspension support for a time sufficient to form cells of the T cell lineage.

[0067] One advantage of the method described herein is that it can culture T cell lineage cells in suspension.In some embodiments, progenitor cells or stem cells are cultured in suspension with the Notch ligand that is conjugated to a suspension support such as microbeads.In suspension culture, cells grow floating freely in culture medium.In contrast, in adhesion culture, cells grow as a monolayer on an artificial substrate.

[0068] In another embodiment, progenitor cells or stem cells are cultured in suspension in a bioreactor, optionally in a closed or closed automated bioreactor, with a Notch ligand conjugated to a suspension support. In one embodiment, the suspension support is a microbead with a diameter that fits into the bioreactor. Various bioreactors are known in the art, and can include batch, fed-batch, or continuous bioreactors. An example of a continuous bioreactor is a continuous stirred tank reactor model.

[0069] Various concentrations of progenitor or stem cells in the culture are contemplated, for example, the concentration of progenitor or stem cells in the culture can be anywhere from one cell per ml to several million cells per ml of medium.

[0070] In one embodiment, the ratio of Notch ligand conjugated to microbeads to progenitor or stem cells is 1:1 to 27:1, optionally 5:1 to 15:1, 8:1 to 10:1, or 9:1. This ratio is also referred to herein as the "microbead to cell ratio."

[0071] The present inventors have also shown that Notch signaling can be enhanced by specifying the orientation of the Notch ligand relative to the floating support. Thus, in one embodiment, the C-terminus of the Notch ligand is conjugated to the floating support. This can be achieved, for example, by adding a sequence that can be enzymatically conjugated to a biotin molecule to the C-terminal end of the Notch ligand. In another embodiment, the Fc segment of the fusion protein present in the C-terminal region, Notch ligand-Fc, can be directly bound to protein A or protein G conjugated to the floating support.

[0072] One or more positive cytokines that promote lineage commitment and differentiation of T cell lineage cells can also be added to the culture. The cytokines can be derived from humans or other species. The concentration of cytokines in the culture is typically about 1-10 ng / ml. Representative examples of cytokines that can be used herein include the entire Flt-3-ligand family, as well as interleukin-7 (IL-7) and stem cell factor. In one embodiment, the cytokines used herein are Flt-3-ligand, IL-7, and stem cell factor. Cytokines can be used in combination with equimolar or higher amounts of glycosaminoglycans, such as heparin sulfate. Cytokines are commercially available or can be produced by recombinant DNA techniques and can be produced to various degrees of purity. Some cytokines can be purified from the culture medium of cell lines using standard biochemical techniques.

[0073] One or more additional molecules may be added to the culture by conjugating each to a floating support. In one embodiment, the additional molecule is a molecule that promotes T cell development (e.g., promotes lineage commitment and differentiation of cells of the T cell lineage), also referred to herein as a "T cell costimulatory molecule." In one example, the inventors cultured DL4 and VCAM1 conjugated to microbeads with HSPCs and demonstrated accelerated differentiation into the T cell lineage. Thus, in one embodiment, the T cell costimulatory molecule is VCAM1. As used herein, the term "VCAM1" refers to vascular cell adhesion protein 1, also known as vascular cell adhesion molecule 1 (VCAM1) or cluster of differentiation 106 (CD106), which in humans is a protein encoded by the VCAM1 gene. The term "VCAM1" also includes mutants or variants of VCAM1. In another embodiment, the T cell costimulatory molecule is a cytokine or chemokine (stem cell factor, IL-7, CCL25, or CXCR4), a major histocompatibility complex (MHC) class I or class II, or a costimulatory (CD80, CD86) molecule. Optionally, the T cell costimulatory molecule comprises at least one protein tag. A variety of protein tags are known in the art and can be used for numerous purposes. In one embodiment, the T cell costimulatory molecule comprises an Fc tag (also known as an Fc fusion protein).

[0074] Progenitor cells and stem cells can be cultured in culture media including conditioned media, unconditioned media, or embryonic stem cell media. Examples of suitable conditioned media include IMDM, DMEM, or αMEM, or equivalent media, conditioned with embryonic fibroblasts (e.g., human embryonic fibroblasts or mouse embryonic fibroblasts). Examples of suitable unconditioned media include Iscove's Modified Dulbecco's Medium (IMDM), DMEM, or αMEM, or equivalent media. Culture media may contain serum (e.g., bovine serum, fetal bovine serum, calf serum, horse serum, human serum, or artificial serum substitutes) or may be serum-free. Other examples of media useful in the present method include StemCell Technologies' medium (StemSpan (商標)SFEM II) or any other commercially available equivalent medium.

[0075] In one embodiment, the culture conditions involve culturing the progenitor or stem cells for a sufficient time to cause the cells in the preparation to form pro-T cells. In another embodiment, the culture conditions involve culturing the progenitor or stem cells for a sufficient time to cause the cells in the preparation to form mature T cells, e.g., mature SP T cells. It will be understood that the cells may be maintained for any appropriate time necessary to obtain the desired cell composition. Optionally, the progenitor or stem cells are cultured for at least 6, 8, 10, 12, 14, 21, 28, 35, or 42 days. In one example, the progenitor or stem cells are cultured with a Notch ligand described herein for 4-21 days, 6-18 days, or 7-14 days to generate pro-T cells. In another example, the progenitor or stem cells are cultured with a Notch ligand described herein for at least 21, 28, 35, or 42 days to generate mature T cells.

[0076] The methods of the present application allow for the generation of large numbers of T cell lineage cells, specifically, in one embodiment, cell expansion rates of greater than 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 175-fold, or 200-fold over the initial starting number of stem or progenitor cells after 14 days of culture.

[0077] The term "isolated," as used herein, means that the primary cells have been separated or purified from cellular or biological material found with the cells in their natural environment, thereby distinguishing the cells from the way they exist in nature.

[0078] The term "a cell" or "the cell" includes a plurality of cells.

[0079] II. Floating Notch Ligands The present inventors have also developed a novel suspension Notch ligand. As used herein, the term "suspension Notch ligand" refers to a Notch ligand used in suspension cell culture.

[0080] Accordingly, the present disclosure also provides a floating Notch ligand as described herein, which comprises (a) a Notch ligand and (b) a floating support, wherein the Notch ligand is conjugated to the floating support.

[0081] Specifically, the inventors have shown that direct conjugation of DL4 to 25 μm diameter microbeads delivers a strong and sustained signal to induce HSPCs to become T cell lineage cells. Thus, in one embodiment, the floating support is a microbead having a diameter of 10-100 μm, optionally 20-30 μm, 24-26 μm, or 25 μm.

[0082] The present inventors have further demonstrated that Notch signaling can be enhanced by specifying the orientation of Notch ligand relative to the floating support.Therefore, in another embodiment, the C-terminus of Notch ligand is conjugated to the floating support.As mentioned above, this can be produced, for example, by adding a sequence that can be enzymatically conjugated to biotin molecule to the C-terminus end of Notch ligand.

[0083] The Notch ligand is optionally DL4 and may be fused to a tag, for example an Fc tag.

[0084] Also provided herein are suspension T cell costimulatory molecules. As used herein, the term "suspension T cell costimulatory molecule" refers to a T cell costimulatory molecule used in suspension cell culture. The suspension T cell costimulatory molecule comprises (a) a T cell costimulatory molecule and (b) a suspension support, and the T cell costimulatory molecule is conjugated to the suspension support.

[0085] The T cell costimulatory molecule is optionally VCAM1 and may be fused to a tag, for example an Fc tag.

[0086] III. Kit The free Notch ligand may be prepared and packaged as a kit for use in generating cells of the T cell lineage.

[0087] Therefore, also provided herein is a kit for producing cells of a T cell lineage, comprising a suspended Notch ligand, the suspended Notch ligand comprising (a) a Notch ligand and (b) a suspension support, the Notch ligand being conjugated to the suspension support. Optionally, the suspended Notch ligand is contained in a preservative and / or buffer, and the kit further comprises a device, such as a vial or syringe, for dispensing the suspended Notch ligand.

[0088] In one embodiment, the kit further comprises a culture medium for culturing a sample containing stem and progenitor cells with a suspension of Notch ligand. Examples of culture medium include conditioned medium, unconditioned medium, or embryonic stem cell medium. The culture medium may contain serum (e.g., bovine serum, fetal bovine serum, calf serum, horse serum, human serum, or artificial serum substitute) or may be serum-free. Other examples of useful culture media include StemCell medium or any other commercially available equivalent medium.

[0089] In another embodiment, the kit further comprises one or more additional molecules, each conjugated to a floating support. In one embodiment, the additional molecules are molecules that promote T cell development (e.g., promote lineage commitment and differentiation of cells of the T cell lineage), also referred to herein as "T cell costimulatory molecules." In another embodiment, the T cell costimulatory molecule is VCAM1.

[0090] The culture medium optionally contains one or more cytokines that promote lineage commitment and differentiation of T-cell lineage cells. The cytokines may be of human or other species origin. The concentration of cytokines in the culture is typically about 1-10 ng / ml. Representative examples of cytokines that can be used herein include the entire family of Flt-3-ligands, as well as interleukin-7 (IL-7) and stem cell factor. In one embodiment, the cytokines used herein are Flt-3-ligand, IL-7, and stem cell factor. Cytokines may be used in combination with equimolar or higher amounts of glycosaminoglycans, such as heparin sulfate. Cytokines are commercially available or can be produced by recombinant DNA technology and can be produced to various degrees of purity. Some cytokines can be purified from cell line culture media using standard biochemical techniques.

[0091] In one embodiment, the kit includes one or more containers for the reagents described herein.

[0092] In various embodiments, the kit may also include printed instructions providing guidance for use of the reagents. The term "instructions" or "instructions" typically includes easy-to-understand language describing the concentration of the reagent or the amount of suspension Notch ligand, and / or at least one assay parameter, such as the relative amounts of suspension Notch ligand and cells to be mixed, incubation time, temperature, media conditions, etc. For example, in one embodiment, the instructions describe a method including: (a) culturing a sample containing stem and progenitor cells with a Notch ligand conjugated to a suspension support, and (b) isolating cells of the T cell lineage.

[0093] IV. Cells of the T cell lineage The present disclosure further provides cells of the T cell lineage generated by the methods, systems, and kits described herein, or mitotic or differentiated cells that are progeny of said cells.

[0094] In one aspect, the present disclosure provides "primordial T cells" or "pro T cells" generated by the methods described herein. In another aspect, the primitive T cells are human primitive T cells, e.g., human primitive T cells characterized by CD34+CD7+ or CD7+CD5+CD1a-.

[0095] In another embodiment, the primitive T cells are mouse primitive T cells, for example, mouse primitive T cells characterized as CD25+.

[0096] The present disclosure also provides double positive (DP) T cells characterized as CD4+CD8+ or CD4+CD8+CD3+. The present disclosure further provides cells of the T cell lineage that are single positive (SP) cells characterized as CD4-CD8+, CD4+CD8-, or CD8+CD3+, CD4+CD3+.

[0097] In one embodiment, cells of the T cell lineage (e.g., primitive T cells or mature T cells) generated by the methods described herein are modified with a T cell receptor (TCR) or chimeric antigen receptor (CAR) to confer specificity for a tumor-associated antigen (TAA). Cells modified in this manner may be useful in treating diseases such as cancer.

[0098] In another aspect, the present disclosure provides a pharmaceutical composition comprising isolated cells of a T cell lineage generated by the methods described herein and a pharmaceutically acceptable diluent or carrier.

[0099] Suitable diluents and carriers are described, for example, in Remington's Pharmaceutical Sciences, whereby compositions include, but are not limited to, a solution of pro-T cells together with one or more pharmaceutically acceptable vehicles or diluents, contained in a buffer having a suitable pH and isotonic with physiological fluids.

[0100] Pharmaceutical compositions include, but are not limited to, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats, and solutes that render the composition substantially compatible with the tissue or blood of the intended recipient. Other components that may be present in such compositions include, for example, water, surfactants (e.g., Tween (商標) ), alcohols, polyols, glycerin, and vegetable oils. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. Compositions may be supplied, for example and without limitation, as a lyophilized powder to be reconstituted with sterile water or saline prior to administration to a patient.

[0101] Pharmaceutical compositions also include cryopreservation solutions. In one embodiment, cells of the T cell lineage generated by the methods described herein are cryopreserved in an appropriate medium, e.g., a pharmaceutically acceptable or GMP-grade medium, and optionally formulated for administration to a subject in need thereof.

[0102] Suitable pharmaceutically acceptable carriers include those that do not interfere with the effectiveness of the biological activity of pharmaceutical compositions, are essentially chemically inert, and are non-toxic.Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline, glycerol solution, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), dioleyl-phosphotidyl-ethanolamine (DOPE) and liposomes.Such compositions contain a therapeutically effective amount of compound together with a suitable amount of carrier to provide a form that can be directly administered to patients.

[0103] The composition can be administered, for example, parenterally, intravenously, subcutaneously, intramuscularly, intracranially, intraorbitally, ophthalmically, intraventricularly, intraarticularly, intraspinally, intracisternally, intraperitoneally, intranasally, by aerosol, or orally. For parenteral administration, a solution of the pro-T cells described herein can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof with or without alcohol, and in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. Those skilled in the art will know how to prepare suitable formulations.

[0104] Preferably, the cells of the T cell lineage are present in an amount effective to treat a disease state in a subject in need thereof. In one embodiment, the cells of the T cell lineage are present in an amount effective to enhance hematopoietic progenitor cell transplantation in a subject in need thereof. Optionally, the composition further comprises T cell lineage cells or tissue to be transplanted. In one embodiment, the tissue comprises the thymus. In another embodiment, the tissue comprises an organ.

[0105] V. Therapeutic Applications The ability to generate in vitro-derived human progenitor T cells and test their safety in human / mouse immune transplantation models paves the way for cell-based approaches to treat immune-related disorders of the T lineage (Legrand et al., 2006; van den Brink et al., 2004). T cells are the primary effector arm of the adaptive immune system, recognizing and eliminating viral and bacterial pathogens. In certain rare hematologic cancers, such as T-cell acute lymphoblastic leukemia (T-ALL), T cells proliferate and crowd out healthy immune cells, disrupting normal immune function (Ferrando et al., 2002; Weng et al., 2004). Chemotherapy can sometimes provide therapeutic benefit to cancer patients, but it often results in immunodeficiency and susceptibility to opportunistic infections. Opportunistic infections also occur after HIV infection, resulting in CD4 +This poses a major concern for AIDS patients who have depleted T cells. While immune deficiency remains a major concern in HIV / AIDS and cancer, immune hyperactivity is equally problematic in autoimmune diseases, where T cells lack proper regulatory control and mount an immune response against self-tissues.

[0106] Accordingly, the present application provides a method of treating a subject having a condition requiring an increase in T cell numbers, comprising: (i) generating cells of a T cell lineage, comprising: (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support, optionally conjugated to particles or microbeads; and (b) isolating cells of a T cell lineage; and (ii) administering an effective amount of cells of the T cell lineage to a subject in need thereof. Further provided is a method comprising:

[0107] In one embodiment, the cell of the T cell lineage is a primitive T cell.

[0108] In another embodiment, the cell of the T cell lineage is a mature T cell.

[0109] The present disclosure also provides for the use of cells of the T cell lineage, optionally primitive or mature T cells, generated by the methods described herein to treat a subject having a condition requiring an increase in T cell numbers.

[0110] The present disclosure also provides for the use of cells of the T cell lineage, optionally primitive or mature T cells, generated by the methods described herein for use in regenerative medicine, for example, to replace and / or regenerate tissue affected by disease or trauma.

[0111] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired result. An effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject. The amount of a given cell preparation corresponding to such an amount will vary depending on various factors, such as the pharmaceutical formulation, route of administration, type of disease or disorder, and the individuality of the subject or host being treated, but can nevertheless be routinely determined by one of ordinary skill in the art. An "effective amount" is preferably an amount effective for transplanting cells of a T cell lineage into the subject being treated.

[0112] The term "treat" or "treatment," as used herein, and as known in the art, refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, stable disease (i.e., not worsening), prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, reduced disease recurrence rate, and remission (whether partial or total). "Treating" and "treatment" can also mean an increase in lifespan compared to the expected lifespan if not treated. "Treating" and "treatment," as used herein, also include prophylactic treatment.

[0113] The term "subject", as used herein, means any member of the animal kingdom, preferably a human.

[0114] A "condition requiring increased numbers of T cells" includes any condition in which T cell levels are reduced compared to a healthy animal, including, but not limited to, immunodeficiency, cancer, genetic diseases (e.g., primary immunodeficiency diseases (PIDs)), infectious diseases, immune disorders, and autoimmune conditions.

[0115] As mentioned above, the cells of the T cell lineage described herein can be engineered to express a T cell receptor (TCR) or chimeric antigen receptor (CAR) that specifically recognizes a tumor-associated antigen.

[0116] Accordingly, the present application also includes a method of treating cancer in a subject, the method comprising: (i) generating cells of a T cell lineage, comprising: (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support, optionally conjugated to particles or microbeads; and (b) isolating cells of a T cell lineage; and (ii) administering an effective amount of cells of the T cell lineage to a subject in need thereof. wherein the cells of the T cell lineage are engineered with a T cell receptor (TCR) or chimeric antigen receptor (CAR) to confer specificity for tumor-associated antigens.

[0117] The present disclosure also provides the use of cells of a T cell lineage, optionally primitive or mature T cells, generated by the methods described herein to treat a subject with cancer, wherein the cells of the T cell lineage are modified with a T cell receptor (TCR) or chimeric antigen receptor (CAR) to confer specificity for a tumor-associated antigen. Optionally, iPSCs can be derived from T cells with known or unknown TCR specificity (e.g., T cells bearing a TCR with specificity for cancer), in which case these T-iPSCs can be used to generate T cells by the methods described herein.

[0118] The following non-limiting examples illustrate the present application. [Example]

[0119] Example 1 material and method: Hematopoietic stem cell source Human umbilical cord blood (UCB) samples were collected by syringe withdrawal from consenting postpartum mothers and collected in anticoagulant citrate phosphate dextrose blood pack units (Baxter Healthcare, Deerfield, IL) according to approved guidelines established by the Research Ethics Board of Sunnybrook Health Sciences Centre. UCB mononuclear cells were isolated within 24 hours of collection by Ficoll density centrifugation and subjected to lineage-negative (Lin) selection using the EasySep Human CD34 Positive Selection Kit (Stemcell Technologies, Vancouver, BC) according to the manufacturer's instructions. - )CD34 + To isolate human HSPCs, Lin - Cells were stained with anti-human CD38-APC and anti-human CD34-PE mAbs and analyzed for CD34 using a BD Biosciences FACSAria sorter (BD, San Jose, CA). + CD38 - / lo Cells were sorted. Sorted human HSPCs were determined to be >99% pure by post-sort analysis. A portion of the cord blood-derived CD34+ cells and all of the mobilized peripheral blood (mPB)-derived CD34+ cells were purchased from Stemcell Technologies. For mPB, volunteers were treated with a combination of G-CSF (up to 10 μg / kg / day G-CSF 3–5 days before collection) and Plerixafor (up to 0.24 mg / kg 1 day before collection).

[0120] Design and production of DL4-Fc The coding sequence for the extracellular domain of human DLL4 (amino acid residues [aa] 1 to 529) is C-terminally linked with a linker sequence, followed by a histidine (His) tag, followed by the Fc portion of human IgG3 (including the hinge region) and a Bir1A recognition sequence (Avita). (商標)Delta-like-4 was genetically engineered to generate the pDL4-Fc-His-B plasmid construct by fusing human DLL4 (aa 1-524) with the Fc portion of IgG1; ii) the signal sequence of alkaline phosphatase (aa 1-17) and human DLL4 (aa 27-524) fused with the Fc portion of IgG1; iii) human DLL4 (aa 1-524) fused at the C-terminus with StreptagII and 6xHis; iv) the signal sequence of alkaline phosphatase (aa 1-17) and human DLL4 (aa 27-524) fused at the C-terminus with 10xHis; and any combination thereof. This construct was inserted into the pIRESpuro2 mammalian expression plasmid (Clontech, Mountain View, CA). The resulting plasmid was transfected into HEK-293T cells using standard CaPO4 transfection methods, and cells with stable integration of the plasmid were selected based on resistance to 2 mg / mL puromycin in standard DMEM [supplemented with 10% (v / v) FBS, 2 mM Glutamax, penicillin (100 U / mL) / streptomycin (100 mg / mL) (all from Thermo Fisher Scientific, Rockford, IL), and 2 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MI)]. Cells were expanded and transferred to Freestyle 293 Expression Medium (Thermo Fisher Scientific) for growth. The DL4-Fc fusion protein secreted into the medium was purified using a HiTrap Protein G affinity column (GE Healthcare) attached to an AKTAprime plus (GE Healthcare Life Sciences, Marlborough, MA) automated chromatography system.

[0121] Design and production of VCAM1-Fc Like DL4-Fc, recombinant VCAM1-Fc contains the VCAM-1 extracellular domain, IgG3 Fc domain, and Avitag (商標)It was genetically modified to contain a biotinylation site. The materials and methods used for the production and purification of the VCAM1-Fc fusion protein were the same as those for the DL4-Fc fusion protein described above.

[0122] Biotinylation of DL4-Fc and VCAM1-Fc NHS-activated biotin was added to purified DL4-Fc at an optimal molar ratio of two biotin molecules per one DL4-Fc molecule. Unreacted NHS-biotin was removed from the mixture by dialysis in PBS or buffer exchange, and biotin incorporation was assessed by the HABA (4'-hydroxyazobenzene-2-carboxylic acid) method (Pierce Biotin Quantitation Kit, Product No. 28005). Biotinylated DL4-Fc was then stored at 4°C.

[0123] To orient DL4-Fc onto a streptavidin (SA)-coated surface, a single biotin molecule was attached to the AviTag in the Fc region of DL4-Fc using the BirA-500 Kit (Avidity) according to the manufacturer's instructions. (商標) The sequences were enzymatically conjugated. Briefly, 2.5 μg of BirA was added to 500 μg of DL4-Fc in a 500 μL reaction volume of PBS and incubated at room temperature for 1 hour. To remove any residual biotin, a 40K MWCO Zeba filter was used. (商標) 350 μL of purified DL4-Fc protein was desalted using a Spin Desalting Column (Thermo-Fisher) according to the manufacturer's instructions. The biotinylated DL4-Fc was then stored at 4°C. (商標) The containing VCAM1-Fc protein was biotinylated at the C-terminus using the same methods and materials as above.

[0124] Conjugation of biotinylated DL4-Fc to streptavidin-coated microbeads One microgram of biotinylated DL4-Fc was incubated with streptavidin (SA)-coated polystyrene μ-beads (Spherotech, Lake Forest, IL) of various sizes ranging from 1 μm to 100 μm in diameter. DL4-Fc was also conjugated to SA-coated Dynabeads (Thermo-Fisher) and 50 nm nanogold particles, each incubated in 2 mL of PBS for 30 minutes at room temperature and mixed by vortexing every 10 minutes. In all cases, the particle surface area was 2x10 5 The total volume of the DL4-Fc-conjugated beads was equivalent to 25 μm SA-μBeads. The DL4-Fc-conjugated beads were washed with 4 mL of PBS and spun down at 3000 × g for 10 minutes. The supernatant was carefully collected to remove any unbound ligand from the mixture and to assay the DL4-Fc content to assess binding to the μBeads. After the second wash, the DL4-μBeads were resuspended in various volumes of PBS to obtain the indicated concentrations described in the text. Unconjugated μBeads were prepared in parallel and served as a negative control.

[0125] For combined conjugation of DL4-Fc and VCAM1-Fc with SA-μ beads, the amount of DL4-Fc was 2 x 10 SA-μ beads. 5 The concentration of VCAM1-Fc was kept constant at 1 μg per cell, but VCAM1-Fc was added at 0.01 μg (100:1), 0.1 μg (10:1), 1 μg (1:1), and 10 μg (1:10).

[0126] Generation of a Notch-activated reporter cell line, 3T3-N1Cluc A Notch-responsive element (8x RBPJ consensus binding site) was inserted into the promoterless pGL4.17[luc2 / Neo] plasmid (Promega). This resulted in a single plasmid (designated pGL4.17-N1Rep) that conferred neomycin resistance while signaling Notch activation through the expression of luciferase. NIH3T3 cells were then transfected with pMIGR-NOTCH1 (a gift from Dr. Warren Pear, University of Pennsylvania) and the pGL4.17N1Rep plasmid. Cells resistant to neomycin treatment (1 μg / mL) were first selected, and then GFP-expressing cells were sorted by flow cytometry. NIH3T3 cell clones were then isolated by single-cell harvesting into 96-well plates. Each clone was then measured for its response to activation with plate-bound DL4-Fc (harvested overnight at 20 μg / mL). The clone designated 3T3N1CLuc, which had the lowest amount of background and the highest Notch activation response, was expanded and used to perform experiments measuring Notch receptor activation.

[0127] Luciferase assay in 3T3-N1CLuc to measure Notch activation 3 x 10 in a standard tissue culture (TC) treated flat bottom 96 well plate 4DL4-μ beads were added to 3T3-N1C Luc cells and incubated overnight in α-MEM supplemented with 5% FBS. Cells were lysed and assayed for luciferase activity using the Firefly Luciferase Assay Kit 2.0 (Biotium, Fremont, CA) according to the manufacturer's instructions. Briefly, growth medium was removed, cells were washed with PBS, and lysis buffer was added. Cells were lysed by freezing at -80°C for 10 minutes and then thawing. The lysate was then transferred to a 96-well flat-bottom opaque polystyrene plate (Corning). D-luciferin was prepared and added to each well using an automated dispenser. Analysis was performed using a Synergy H1 plate reader (BioTek Instruments Inc., Winooski, VT). Plate-binding controls for human IgG and DL4-Fc were prepared the day before by adsorbing 50 μL / well of protein (5–20 μg / mL) onto flat-bottom 96-well plates overnight at 4°C, followed by washing the next day and seeding with 3T3-N1CLuc cells.

[0128] Culturing HSPCs with coated DL4-Fc plates and DL4-μ beads Different numbers of DL4-μ beads were added, 3x10 3 Mouse Lin - Sca-1 + cKit +HSCs (sorted by flow cytometry from the bone marrow of C57BL6 mice) were added to the beads at cell-to-bead ratios of 1:1, 1:3, 1:9, and 1:27. For each condition, the cell-bead combination was incubated in a single well of a round-bottom 96-well plate in 200 μL of IMDM supplemented with 20% BIT (STEMCELL Technologies), 1% Glutamax (Thermo), 50 ng / mL SCF, 10 ng / mL Flt3L, and 10 ng / mL IL-7 (R&D Systems, Minneapolis, MN). Plate-binding controls for human IgG and DL4-Fc were prepared the day before by adsorbing 50 μL / well of protein (20 μg / mL) to a flat-bottom 96-well plate overnight at 4°C, followed by washing and seeding the cells the next day. On day 7 of coculture, cells were collected and stained with anti-mouse antibodies CD45, CD25, CD44, CD90, CD11b, and CD19, and then analyzed on an LSR II cytometer (BD Biosciences).

[0129] For human HSPCs, Lin was isolated by flow cytometry from UCB. - CD34 + CD38 - / lo Sort the cells and plate them at 4x10 per well of a 96 round-bottom well plate. 3 cells with 36,000 DL4-μ beads in StemSpan (商標) 200 μL of StemSpan supplemented with T Cell Progenitor Expansion Supplement (Stemcell Technologies) (商標)Cells were cultured in SFEM II (Stemcell Technologies) for 14 days, with a 50% medium change on day 7. For cultures longer than 14 days, cells were harvested, counted, and seeded with new DL4-μ beads. On day 14 of coculture, cells were counted to determine cell proliferation rates and assayed for lineage progression by staining with antibodies against CD34, CD5, CD1a, and CD7. At later time points, cells were also stained with antibodies against CD4, CD8, and CD3 to assay for growth beyond the proT cell stage.

[0130] CD34 from human induced pluripotent stem cells (iPSCs) + Differentiation of prehematopoietic cells or hemogenic endothelial cells into T cells Reprogrammed hiPSC lines were derived from fibroblasts (AlStemBio Cat # iPSC11) and T cells (Harvard Stem Cell Science Cat # STiPS A3). They were cultured on Matrigel in mTeSR medium (StemCellTech). To generate self-aggregating EBs, iPSCs were treated with collagenase B for 20 minutes, followed by a brief trypsin-EDTA step. Cells were gently scraped with a cell scraper to form small aggregates. EBs were generated during the first 24 hours of culture in StemPro-34 (Invitrogen) in the presence of BMP-4, then cultured for an additional 24 hours in the presence of BMP-4 and bFGF, and then cultured for an additional 48 hours (days 2–4) in the presence of BMP-4, bFGF, and SB. On day 4, BMP-4 and SB were removed and replaced with VEGF, IL-6, IL-11, IGF-1, SCF, EPO, TPO, Flt-3, IL-3, and DKK1 (all cytokines from Miltenyi Biotec, Auburn, CA, or R&D Systems). Cultures were maintained in a hypoxic environment of 5% CO2 / 5% O2 / 90% N2 for 8 days. On day 8, cells were purified using MACS to identify CD34 + Enriched for cells and added to DL4-µ beads with StemSpan (商標)StemSpan supplemented with T Cell Progenitor Expansion Supplement (Stemcell Technologies) (商標) T cells were developed by incubation with SFEM II (Stemcell Technologies) as described above.

[0131] Adoptive transfer of primary T cells into immunodeficient mice Large-scale human HPSC / DL4-μ-bead cultures were prepared in preparation for adoptive transfer into immunodeficient mice. 2x10 5 CD34 + HSPC, 1.8x10 6 The cells were incubated with 1000 DL4-μ beads in a T25 flask (Thermo Scientific) for 7–10 days, at which time the CD34 + CD7 + Identified progenitor T (proT) cells were sorted using a FACSAria cell sorter (BD Biosciences, San Jose, CA). Alternatively, when HSPCs were cultured with iron oxide-coated DL4-μ beads, magnetic separation of the DL4-μ beads from cellular components was performed using an autoMACS-pro cell sorter (Miltenyi Biotec). ProT cells were sorted from 3- to 6-day-old immunodeficient NOD-Scid / IL2rγ mice. null (NSG) were injected intrahepatically into neonatal mice. Each mouse received 3-5x10 5 Mice were given 50 μl of hIL-7 (0.5 μg / mouse) and anti-IL-7 monoclonal antibody (mAb), clone M25 (2.5 μg / mouse), in α-MEM along with proT cells. Mice were boosted every 3–4 days with the IL-7 / M25 cocktail. At 3 or 12 weeks post-transplant, lymphoid organs (thymus, spleen, and bone marrow) were collected. Single-cell suspensions were prepared from each organ, stained, and analyzed using an LSR-II cytometer (BD Biosciences). Engraftment was assessed by electronic gating on live cells that excluded the cell death marker 4'-6-diamidino-2-phenylindole (DAPI) and cells expressing human CD45.

[0132] result: DL4-μ-beads induce strong Notch signaling Previous work from our laboratory has shown that immobilizing a DL4 fusion protein, DL4-Fc, on standard TC-treated well / plate surfaces can induce Notch signaling in mouse and human HSPCs sufficient to induce T-lineage cell development (Shukla et al., 2017). However, this 2D format is limited in its scalability and ability to deliver strong and consistent Notch signaling to promote T-cell development.

[0133] Taqvi et al. (2006) attempted to functionalize 2.8 μm magnetic beads with DL4 and co-cultured them with mouse HSCs. This mixture was placed on OP9 cells with a permeable insert in between. This prevented physical contact between the stem cells and stromal cells, but allowed the passage of soluble factors essential for promoting differentiation. Some CD90 expression was observed from the culture with DL4-functionalized μ-beads. + Although Thy1 (Thy1) cells emerged, Thy1 expression is not a definitive T lineage marker. Importantly, CD19 + B cells also emerged. There is a threshold of sustained Notch activation required to promote T cell development and inhibit B cell development. The fact that B cells were still present demonstrates that this system was incapable of activating and sustaining Notch signaling. Indeed, increasing the bead-to-cell ratio from 1:1 to 5:1 promoted B cell differentiation and prevented Thy1 expression. The observation that soluble Notch ligand present in the μ-bead format was unable to induce the necessary level of Notch signaling, coupled with the known requirement for cell-based or plate-bound Notch ligands to induce strong Notch signaling, suggests that this approach is unlikely to be applicable to T cell development from HSPCs. Furthermore, studies of this type have yet to be performed using human CD34 +Thus, it remains to be determined whether DL4-functionalized μBeads can provide the necessary, consistent, and high levels of Notch signaling required to induce T cell development from HSPCs and whether they can be easily scaled up for clinical use.

[0134] Therefore, we investigated whether conjugating DL4-Fc to μ-beads could generate a higher-order DL4 multimeric platform that could effectively bind Notch receptors and function in suspension with HSPCs, making it suitable for scale-up.

[0135] To facilitate the coupling of DL4-Fc to μ-beads, DL4-Fc was chemically biotinylated in preparation for conjugation to SA-coated polystyrene beads (Figure 1A). Biotin incorporation into DL4-Fc was confirmed by Western blot (Figure 1B), and subsequent conjugation to μ-beads was assayed by protein quantification of supernatants collected before and after conjugation. To assess the effect of bead size on Notch signaling, non-biotinylated DL4-Fc was covalently attached to 50 nm NHS-activated gold nanoparticles, and biotinylated DL4-Fc was attached to 1 μm SA-coated Dynabeads. (商標) DL4-Fc beads were conjugated to 25 μm and 100 μm polystyrene beads (Figure 2). The functionalized beads were incubated with 3T3-N1Cluc, which uses the luciferase gene as a reporter to signal Notch activation. The total number of beads in each condition represented the same surface area and therefore the same total number of DL4-Fc molecules. Bead size was shown to affect the ability of DL4-Fc beads to activate Notch signaling, as indicated by the level of luciferase activity (Figure 2B). Note that 25 μm diameter DL4-μ beads optimally enhanced Notch receptor activation compared to other sized beads of the same material, or compared to 1 μm Dynabeads and 50 nm nanoparticles.

[0136] The level of Notch signaling obtained with 25 μm DL4-μ beads was higher than the known level equivalent to 20 μg / mL plate-bound (PB) DL4-Fc, which is known to be necessary for the induction of T-lineage differentiation. Next, we determined whether DL4-μ beads supplemented with IL-7, Flt3L, and SCF had the ability to induce T-cell development by incubating with mouse HSCs for 7 days. The results showed that when incubated with DL4-μ beads, mouse HSCs readily generated pro-T cells, as evidenced by CD25 expression in 41% of the cells (Figure 3). In contrast, PB-DL4-Fc induced CD25 expression in only 4% of the cells. DL4-μ beads also induced the expression of an alternative B cell type (CD19), which is known to be blocked by Notch signaling. + ) and myeloid (CD11b + ) hindered the performance of the series.

[0137] Directional conjugation of DL4-Fc to μ-beads enhances Notch signaling induction To improve on the random orientation of the first-generation DL4-μ beads, we investigated whether specifying the orientation of DL4-Fc relative to the bead surface would enhance Notch signaling. To this end, we used a BirA recognition sequence (AviTag) to which a single biotin molecule could be enzymatically conjugated. (商標)We designed DL4-Fc, a 2-bead fusion protein (DL4-Fc) with a C-terminal nucleotide sequence (Figure 4). Incubation of these second-generation DL4-μ beads significantly increased the level of Notch signaling, as assessed by luciferase activity in 3T3N1CLuc cells (Figure 5). Using this directed conjugation method, we again assessed the effect of bead size on Notch transduction. Results showed that beads with diameters of 100 μm, 25 μm, 10 μm, and 6.5 μm all had the ability to activate Notch, but beads with a diameter of 25 μm were the most effective (Figure 6). The optimal bead-to-cell ratio was also evaluated, and it was determined that a 3:1 bead-to-cell ratio resulted in a 10-fold increase in Notch activation compared to plate-bound controls (Figure 6).

[0138] Next, we investigated whether SA μ-beads were saturated with biotinylated DL4-Fc to determine the amount of DL4-Fc per bead that optimally activated Notch signaling. Different amounts of DL4-Fc (0.01, 0.1, 1, and 10 μg) were incubated with an equal number of 25 μm SA-beads. The beads were then incubated overnight with 3T3-N1Cluc cells. The result was a 2.25x10 5 We demonstrated that 1 μg of DL4-Fc per SA-μ beads gave the maximal response, suggesting that the activity of DL4-μ beads was saturated (Figure 7A). We also demonstrated that magnetized 25 μm polystyrene beads (coated with iron oxide) were as effective as their non-magnetized counterparts in activating Notch (Figure 7A). Furthermore, replacing SA-μ beads with protein G-μ beads of the same diameter did not significantly affect the ability of DL4-Fc to activate Notch (Figure 7B).

[0139] These results established the following parameters: i) bead size, ii) orientation of the DL4 molecule, and iii) bead-to-cell ratio. Optimization of these parameters influenced the determination of optimal conditions for stimulating Notch activity and T cell development. Furthermore, the DL4-Fc-to-bead ratio for maximal activity was determined for loading DL4-Fc onto beads, and magnetization of the beads had no effect on Notch activity. Furthermore, Protein G-μ beads, which also bind to the Fc region of DL4-Fc, oriented DL4-Fc in a manner similar to SA-μ beads.

[0140] DL4-μ-bead conditions that maximize T cell development from HSPCs Based on the above results, we next set out to determine whether the same factors influence T cell development. This was first tested using mouse HSPCs. As shown in Figure 8, DL4-μBeads induced mouse T cell development more effectively than PB-DL4.

[0141] To determine the optimal ratio of HSPCs to DL4-µ beads to maximize proT cell generation, 3 x 10 3 HSPCs were incubated with increasing numbers of DL4-μ beads in three-fold increments (Figure 9). After 7 days of incubation, pro-T cells (CD25 + We analyzed different HSPC:DL4-μ bead ratios for differentiation into CD25 cells at approximately 1:9 ratio. + This appeared to be the optimal HSPC:DL4-μ bead ratio, as it resulted in the highest percentage of cells. Increasing the HSPC:bead ratio to 1:27 did not improve T-lineage differentiation.

[0142] Kinetics and expansion of human T cell development using DL4-μ beads Next, CD34 +We validated the use of DL4-μ beads to induce human T cell development from UCB-derived HSPCs. To do so, we used the optimal HSPC:DL4-μ bead ratio of 1:9, as determined above. Cells were counted every two days and flow cytometry analysis was performed. Results showed the emergence of human proT cells co-expressing CD34, CD7, and CD5 by day 4 of culture (Figure 10A). These results suggest that human HSPCs incubated with DL4-μ beads exhibited a robust proT cell phenotype (CD34 + CD7 + or CD7 + CD5 + CD1a - ) was achieved with plate-bound DL4-Fc but not with unconjugated μ-beads or PB-DL4-Fc, confirming the enhanced ability of DL4-μ-beads to activate Notch compared to plate-bound DL4-Fc.

[0143] One challenge to achieving clinically relevant proT cell yields is the difficulty in obtaining sufficient cell numbers. To address this, we also assessed cell proliferation rates during development and found that by day 14, total cell proliferation was >150-fold greater than the initial starting number (Figure 10B).

[0144] DL4-μ beads promote the development of mature human T cells We next investigated whether the increased ability of DL4-μ beads to activate Notch could induce developing cells to differentiate toward later stages of T cell development, thus expressing a more mature phenotype later in culture (Fig. 11). Analysis of both day 28 and day 47 cultures revealed that CD4 + CD8 + It was shown that double-positive (DP) cells accounted for approximately 25% of the cells. Interestingly, at day 47, the cultures were CD4 + CD8 + CD3 + DP cells and CD8 + CD3 +The emergence of single positive (SP) cells was observed. These results indicate that the strong Notch signaling induced by DL4-μ beads can overcome the developmental defect in T cell maturation observed in previous attempts using PB-DL4-Fc.

[0145] Mobilized peripheral blood (mPB)-derived HSPCs differentiate into T-lineage cells when cultured with DL4-μ beads Adult mPB is potentially a more readily accessible source of HSPCs than UCB, because the number of HSPCs obtained from an individual is approximately 100 times higher than that from UCB. To compare the ontogeny of T lineage development, we used mPB-derived CD34 cells from three different individuals. + HSPCs were cultured with DL4-μ beads and compared with UBC-derived HSPC cultures (Figure 12A). At day 14, the progression of T cell development was very similar to CB-derived HSPCs, with a pro-T cell population (CD34 + CD7 + ) were observed. However, after 14 days, the proliferation rate was 110-fold for mPB-derived HSPCs, whereas that for CB-derived cells was nearly 190-fold (Figure 12B).

[0146] Induction of T cell development in iPSCs using DL4-μ beads The methods described herein allow for the generation of fibroblast-derived human iPSCs, which are expressed as CD34 + Induced differentiation into pre-hematopoietic progenitor cells. CD34 + Cells were incubated with DL4-μ beads to determine their ability to induce pluripotent cells into the T lineage at early (FIG. 13A) and later (FIG. 13B) stages of development. Early development is indicated by the normal acquisition of CD7 cell surface markers followed by CD5. Late T cell development is indicated by the normal acquisition of CD4 at day 28. + These were characterized by the immature single positive (iSP) marker and the acquisition of single positive CD8 cells on day 35. Notably, the presence of the cell surface marker CD3, a component of the T cell receptor (TCR), suggests the presence of mature T cells in these cultures.

[0147] When iPSCs are derived from T cells (T-iPSCs), the TCRα and TCRβ loci are already genetically rearranged, and upon redifferentiation to a T lineage fate, the cells will express the already rearranged TCRαβ. To determine at what stage of development TCRs are expressed by T-iPSC-derived cells, CD34 + Prehematopoietic progenitor cells were incubated with DL4-μ beads. Cultures examined on day 12 showed that 40% of the cells expressed TCRαβ and CD3 on the cell surface, did not yet express mature T cell markers CD4 and CD8, and 80% of the cells expressed the early T cell marker CD7 (Figure 14, left panel). + Cells were gated and showed greater than 70% expression of TCRαβ and CD3. By day 24, greater than 80% of cells expressed TCRαβ and CD3, and many of these cells had acquired CD4 and CD8 expression (Figure 14, right panel).

[0148] VCAM-1 accelerated the differentiation of HSPCs into T cells To determine whether SA-μ beads could serve as a modular base for the addition of other biotinylated molecules, we investigated the functional effect of VCAM-1. It has previously been shown that the addition of VCAM-1 to plate-bound DLL4 accelerates the differentiation of HSPCs into T cells (Shukla et al., 2017). Here, we genetically engineered, expressed, and biotinylated a new fusion protein, VCAM1-Fc. Using immunoblot analysis, we determined that this VCAM1-Fc was the same size as a commercially available VCAM1 product (Figure 15, left panel). Furthermore, we demonstrated that VCAM1-Fc, which contains the target sequence for BirA enzyme biotinylation, was biotinylated (Figure 15, right panel). Next, biotinylated VCAM1-Fc was used to coat the surface of SA-μ beads along with a fixed amount of DL4-Fc and then cultured with UBC-derived HSPCs. Analysis of day 7 cultures demonstrated that, in general, higher VCAM1-Fc / DL4-Fc ratios accelerated the rate of differentiation (Figure 16), demonstrating the activity of VCAM-1 and the flexibility of μBeads as a platform for the controlled addition of costimulatory molecules involved in T cell development.

[0149] Engraftment of primary T cells into immunodeficient mice and their subsequent migration to the periphery. CD34 derived from HSPC / DL4-μ-bead cultures + CD7 + To assess the ability of progenitor T (proT) cells to engraft in the thymus, we chose an immunodeficient NSG mouse model. We scaled up proT cell production stepwise, switching from 96-well plates to T25 flasks. ProT cells were sorted from day 7 cultures and injected intrahepatically into NSG pups. Early engraftment was assessed at week 3, revealing the expression of human CD45 T cells in the thymus. + The presence of cells was demonstrated, most of which were double-positive CD4 + CD8 + (DP) were at the mature T cell stage (Figure 17). B (CD19) cells and myeloid (CD33) cells had not yet developed in the thymus.

[0150] Analysis at 12 weeks after transplantation revealed that almost all human CD45+ cells had differentiated and matured into CD4 and CD8 SPs in the thymus, where proT cell renewal did not occur (Figure 18). Mature CD4 and CD8 SPs appeared to migrate from the thymus to the spleen and bone marrow. This demonstrated that proT cells had the ability to mature in the thymus and migrate normally to secondary lymphoid organs.

[0151] Separation of μ-Beads from cellular components in culture In preparation for large-scale production of proT cells from HSPC / DL4-μ-bead cocultures for clinical purposes, we assessed the ability of the AutoMACS (Miltenyi) to separate iron oxide-coated DL4-μ-beads from cocultured cells (Figure 19A). The AutoMACS, which performed similarly to the clinically approved CliniMACS (Miltenyi), completely isolated μ-beads from cellular components (Figure 19B), with no μ-beads detectable in the cell fraction. In contrast, the bead fraction contained cells that were either captured or remained attached to the isolated μ-beads.

[0152] summary Here, we describe the development of a cell-free, bead-based system for generating T cells from both mouse and human HSPCs and iPSCs. Non-plate-bound or floating Notch ligands, such as DL4-μ beads, represent a unique strategy that enables efficient generation of T-lineage cells, is easily achieved in large-scale bioreactor-based suspension cultures, and has the potential to overcome the developmental obstacles, as well as the inefficiencies and scalability drawbacks, associated with plate-bound approaches.

[0153] Previous studies have not demonstrated the generation of human T-lineage cells beyond the immature proT cell stage in a cell-free support system. Furthermore, the results herein are the first to demonstrate the generation of T-lineage cells, including proT and mature SP CD4 and CD8 cells, from iPSCs using a cell-free support culture system. We demonstrate that the suspension Notch ligand culture system described herein enables the emergence of mature SP T cells, and that Notch signaling achieved by the DL4-μ-beads described herein overcomes the developmental impediment to T-cell maturation seen with plate-bound approaches.

[0154] References TIFF2026031647000002.tif190141TIFF2026031647000003.tif164141

Claims

1. (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support; and (b) isolating cells of the T cell lineage 2. A method for generating cells of the T cell lineage, comprising:

2. The method of claim 1 , wherein the floating support is a particle.

3. 3. The method of claim 1 or 2, wherein the floating support is a microbead.

4. The method of any one of claims 1 to 3, wherein the stem or progenitor cells are cultured in suspension together with the Notch ligand.

5. 5. The method of any one of claims 1 to 4, wherein the stem cells are selected from hematopoietic stem / progenitor cells (HSPCs), embryonic stem cells, or induced pluripotent stem cells (iPSCs).

6. The stem cells are CD34 + or CD34 + CD38 - / lo The method of any one of claims 1 to 5, wherein the cells are HSPCs.

7. The stem cells are CD34 + CD34 hematopoietic progenitor cells, optionally differentiated from iPSCs + The method of any one of claims 1 to 4, wherein the cells are hematopoietic progenitor cells.

8. The method of any one of claims 1 to 7, wherein the Notch ligand is DL4.

9. The method of any one of claims 1 to 8, wherein the cells of the T cell lineage are progenitor T (proT) cells.

10. The stem or progenitor cells are human cells, and the proT cells have the phenotype CD34 + CD7 + or CD7 + CD5 + CD1a - 10. The method of claim 9, comprising:

11. The stem or progenitor cells are murine cells, and optionally are lineage-CD117+ Sca-1+ murine cells, wherein the proT cells are of the phenotype CD25 + or CD25 + CD90 + 11. The method of claim 10, comprising:

12. 9. The method of any one of claims 1 to 8, wherein the T cell lineage cells are CD4+CD8+ double positive cells, CD4+CD8+CD3+ double positive cells, CD8+CD3+ single positive cells, or CD4+CD3+ single positive cells.

13. The method of any one of claims 1 to 12, wherein the stem or progenitor cells are cultured in a medium that does not contain stromal cells.

14. 14. The method of any one of claims 1 to 13, wherein the stem or progenitor cells are cultured with at least one T cell costimulatory molecule attached to a suspension support, and the at least one T cell costimulatory molecule is optionally VCAM1.

15. A cell of the T cell lineage generated by the method of any one of claims 1 to 14.

16. 16. The cell of claim 15, which is a primitive T cell, a CD4+CD8+ double positive cell, a CD4+CD8+CD3+ double positive cell, a CD8+CD3+ single positive cell, or a CD4+CD3+ single positive cell.

17. A suspended Notch ligand comprising: (a) a Notch ligand; and (b) a microbead, wherein the Notch ligand is conjugated to the microbead.

18. 18. The floating Notch ligand of claim 17, wherein (i) the microbead has a diameter of 6.5 to 100 μm, and / or (ii) the C-terminus of the Notch ligand is conjugated to the microbead.

19. 19. Use of the floating Notch ligand of claim 17 or 18 for generating cells of the T cell lineage.

20. (i) a floating Notch ligand comprising (a) a Notch ligand and (b) a floating support, wherein the Notch ligand is conjugated to the floating support; and (ii) instructions for using the suspension Notch ligand to generate cells of the T cell lineage; Includes a kit.

21. (i) a floating Notch ligand comprising (a) a Notch ligand and (b) a floating support, wherein the Notch ligand is conjugated to the floating support; and (ii) Culture medium Includes a kit.

22. 22. The kit of claim 20 or 21, wherein the free-floating Notch ligand comprises DL4 conjugated to microbeads.

23. (iii) at least one T cell costimulatory molecule attached to a floating support; 23. The kit of any one of claims 20 to 22, further comprising: wherein the at least one T cell costimulatory molecule is optionally VCAM1.

24. 1. A method of treating a subject having a condition requiring an increase in T cell numbers, comprising: (i) generating cells of a T cell lineage, comprising (a) culturing a sample containing stem or progenitor cells with a Notch ligand conjugated to a suspension support, and (b) isolating cells of a T cell lineage; and (ii) administering to the subject an effective amount of cells of the T cell lineage. A method comprising:

25. 25. The method of claim 24, wherein the cell of the T cell lineage is a primitive T cell.

26. 24. The method of claim 23, wherein the T cell lineage cells are CD4+CD8+ double positive cells, CD4+CD8+CD3+ double positive cells, CD8+CD3+ single positive cells, or CD4+CD3+ single positive cells.

27. 24. The method of claim 23, wherein the cell of the T cell lineage is a mature T cell.

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