Method for generating T cell lineage cells

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Patent Application No. 62 / 630,497, filed on 14 February 2018, which is incorporated herein by reference in its entirety.

[0002] field This application relates to a method, composition, and kit for generating T cell lineage cells, and to the use of said cells. Specifically, this application relates to a method, composition, and kit for generating primordial T cells and mature T cells, and to the use of said cells. [Background technology]

[0003] background T cells are crucial mediators in adaptive immunity and can be used as therapeutic agents against pathogens and in cancer immunotherapy. Hematopoietic stem cell transplantation (HSCT) offers 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 regimen required before treatment (Krenger et al., 2011). Unlike most other hematopoietic lineages that originate in the bone marrow (BM), T cell development requires BM-derived progenitor cells to migrate to the thymus, where they receive crucial signals that induce differentiation into T-lineage cells (Shah and Zuniga-Pflucker, 2014).

[0004] The primary signal delivered to incoming lymphocyte progenitor cells by thymic stromal cells is mediated by the Notch ligand, Delta-like-4, expressed by thymic cortical epithelial cells (Thompson and Zuniga-Pflucker, 2011). Notch receptors expressed by lymphocyte progenitor cells require mechanical traction induced by Delta-like-4 carrier cells for effective activation of the Notch receptor (D'Souza et al., 2010; Gordon et al., 2015; Meloty-Kapella et al., 2012). It is also known that T cell development requires a consistent and high level of Notch receptor activation (Schmitt et al., 2004). In the absence of Notch1 receptor signaling or Delta-like-4 induced signaling, T cell development does not occur in the thymus; instead, alternative lymphocyte lineages such as B cells develop. Thus, the development of T cells in the thymus is predicated on the Notch signaling pathway (Zuniga-Pflucker, 2004).

[0005] In the context of HSCT, thymic dysfunction or atrophy, resulting from conditioning and aging, respectively, coupled with the limited ability of transplanted HSCs to produce lymphocytes, restricts the degree of T cell development in the thymus (Porter and June, 2005). This leads to inadequate immune surveillance, making patients more susceptible to infections and / or cancer recurrence, which remains a major clinical challenge.

[0006] Adoptive transfer of primordial T (proT) cells is emerging as a promising strategy for enhancing T cell rearrangement, as it has been shown that human or mouse proT cells, regardless of their origin (heterogeneous or allogeneic), can 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. Therefore, ProT cells are restricted by the recipient's major histocompatibility complex (MHC), giving host-tolerant T cells that can evade the clinical challenges associated with graft-versus-host disease (GVHD). Importantly, proT cell transplantation restores thymic structure and improves subsequent thymic dissemination of HSC-derived primordial cells. In addition to their inherent regenerative medicine properties, proT cells can be modified in terms of their T cell receptor (TCR) and chimeric antigen receptor (CAR) to confer specificity to tumor-associated antigens (TAAs) for cancer treatment, and their synthetic gene circuits can also be modified to form individually tailored response programs.

[0007] An unresolved challenge in this field is the development of a clinically suitable system that can be easily scaled to generate large quantities of proT cells from various sources of human hematopoietic stem cells / primordial cells (HSPCs). Current methods rely on mouse-derived OP9 cells expressing Notch ligand Delta-like-1 (DL1) or Delta-like-4 (DL4), but this approach faces several challenges for clinical application (Awong et al., 2009; Awong et al., 2013). Most stromal cell-free approaches rely on two-dimensional (2D) tissue culture platforms, and therefore Notch ligand DL1 or DL4 is immobilized on the tissue culture plate (Gehre et al., 2015; Reimann et al., 2012; Simons et al., 2017). Further adhesion receptor ligands, such as vascular cell adhesion molecule-1 (VCAM-1), are also 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 such progress is encouraging, the application of these approaches to the generation of therapeutic proT cells is limited by the need for large-scale processing for clinical production and is not an effective method for routinely generating clinically applicable numbers of cells. Ideally, a truly scalable platform would enable the proliferation of proT cells in a closed, automated bioreactor system (Lipsitz et al., 2016). [Overview of the Initiative]

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

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

[0013] In another embodiment, the stem cells are selected from hematopoietic stem cells / 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

[0013] , or CD34 + CD38 - / lo HSPCs.

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

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

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

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

[0019] In another embodiment, the stem cells or progenitor cells are mouse cells, and optionally lineage - CD117 + Sca-1 + These are mouse cells, and proT cells have a phenotypic CD25 + or CD25 + CD90 + It holds.

[0020] In another embodiment, T cell lineage cells, CD4 + CD8 + double positive cells, CD4 + CD8 + CD3 + double positive cells, CD8 + CD3 + Single positive cell, or CD4 + CD3 + It is a single positive cell.

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

[0022] In another embodiment, stem cells 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 being optionally VCAM1.

[0023] The disclosure also provides T cell lineage cells, which are generated by a method for generating T cell lineage cells, comprising the steps of (a) culturing a sample containing stem cells or progenitor cells with Notch ligand conjugated on a suspension support, and (b) isolating the T cell lineage cells.

[0024] In one embodiment, the cells are primordial T cells, CD4 + CD8 + double positive cells, CD4 + CD8 + CD3 + Double-positive cells, or CD8 + CD3 + Single positive cell, CD4 + CD3 + It is a single positive cell.

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

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

[0027] This disclosure also provides the use of a suspension Notch ligand for generating T cell lineage cells.

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

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

[0030] In one embodiment of the kit, the suspended Notch ligand includes DL4 conjugated to microbeads.

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

[0032] This disclosure relates to a method for treating subjects having a condition requiring an increase in T cell count, (i) A step of generating T cell lineage cells, comprising (a) culturing a sample containing stem cells or progenitor cells with Notch ligand conjugated on a suspension support, and (b) isolating T cell lineage cells, and (ii) The step of administering an effective amount of cells of the T cell lineage to the subject. Further methods are provided, including the following.

[0033] In one embodiment, the cells of the T cell lineage are primordial T cells.

[0034] In another aspect, the cells of the T cell lineage are mature T cells.

[0035] In another embodiment, 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.

[0036] [Invention 1001] (a) A step of culturing a sample containing stem cells or progenitor cells together with Notch ligand conjugated on a suspension support, and (b) The step of isolating T cell lineage cells. A method for generating T cell lineage cells, including [specific cells]. [Invention 1002] The method of the present invention 1001, wherein the floating support is a particle. [Invention 1003] The method of the present invention 1001 or 1002, wherein the floating support is microbeads. [Invention 1004] A method according to any one of the present invention 1001 to 1003, wherein the stem cells or progenitor cells are cultured in suspension together with the Notch ligand. [Invention 1005] The method according to any one of items 1001 to 1004 of the present invention, wherein the stem cells are selected from hematopoietic stem cells / primordial cells (HSPCs), embryonic stem cells, or induced pluripotent stem cells (iPSCs). [Invention 1006] The aforementioned stem cells are CD34+ or CD34 + CD38 - / lo A method according to any of the present invention 1001 to 1005, which is HSPC. [Invention 1007] The aforementioned stem cells are CD34 + Hematopoietic progenitor cells, optionally differentiated from iPSCs, are CD34 + A hematopoietic progenitor cell, according to any of the methods described in 1001 to 1004 of this invention. [Invention 1008] The method according to any one of the present invention 1001 to 1007, wherein the Notch ligand is DL4. [Invention 1009] The method according to any one of the present invention 1001 to 1008, wherein the cells of the T cell lineage are primordial T (proT) cells. [Invention 1010] The stem cells or progenitor cells are human cells, and the proT cells have phenotype CD34 + CD7 + or CD7 + CD5 + CD1a - The method of the present invention 1009, comprising: [Invention 1011] The aforementioned stem cells or progenitor cells are mouse cells, optionally lineage-CD117+ Sca-1+ mouse cells, and the proT cells are phenotypic CD25 + or CD25 + CD90 + The method of the present invention 1010, comprising: [Invention 1012] The method according to any one of the present invention 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. [Invention 1013] The method according to any one of items 1001 to 1012 of the present invention, wherein the stem cells or progenitor cells are cultured in a medium that does not contain stromal cells. [Invention 1014] The method according to any one of items 1001 to 1013 of the present invention, wherein the stem cells 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. [Invention 1015] T cell lineage cells generated by any of the methods described in 1001 to 1014 of this invention. [Invention 1016] Cells according to the present invention 1015, which are primordial T cells, CD4+CD8+ double-positive cells, CD4+CD8+CD3+ double-positive cells, CD8+CD3+ single-positive cells, or CD4+CD3+ single-positive cells. [Invention 1017] A suspended Notch ligand comprising (a) a Notch ligand and (b) microbeads, wherein the Notch ligand is conjugated to the microbeads. [Invention 1018] (i) The microbeads have a diameter of 6.5 to 100 μm, and / or (ii) the C-terminus of the Notch ligand is conjugated to the microbeads, the suspended Notch ligand of the present invention 1017. [Invention 1019] Use of the suspension Notch ligand of the present invention 1017 or 1018 for generating T cell lineage cells. [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 the use of the suspension Notch ligand for generating T cell lineage cells A kit that includes this. [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 A kit that includes this. [Invention 1022] A kit according to the present invention 1020 or 1021, comprising the aforementioned suspended Notch ligand and DL4 conjugated to microbeads. [Invention 1023] (iii) At least one T cell costimulatory molecule attached to the suspended support. A kit according to any one of the inventions 1020 to 1022, further comprising, wherein the at least one T cell costimulatory molecule is optionally VCAM1. [Invention 1024] A method for treating a subject having a condition requiring an increase in the number of T cells, (i) A step of generating T cell lineage cells, comprising (a) culturing a sample containing stem cells or progenitor cells with Notch ligand conjugated on a suspension support, and (b) isolating T cell lineage cells, and (ii) The step of administering an effective amount of cells of the T cell lineage to the subject. Methods that include... [Invention 1025] The method of the present invention 1024, wherein the cells of the T cell lineage are primordial T cells. [Invention 1026] The method of the present invention 1023, 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. [Invention 1027] The method of the present invention 1023, wherein the cells of the T cell lineage are mature T cells. Other features and advantages of the present application will become apparent from the following detailed description. However, please understand that the detailed description and specific examples are provided for illustrative purposes only, as they illustrate preferred embodiments of the present application and will reveal to those skilled in the art various variations and modifications within the purpose and scope of the present application. [Brief explanation of the drawing]

[0037] The embodiments of the present invention will be described below with reference to the drawings.

[0038] [Figure 1] This shows the design and biotinylation of the DL4-Fc construct. A) The DL4-Fc fusion construct is illustrated with its component, the extracellular domain of human DL4, its purification purpose, 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] This demonstrates the activation of Notch reporter cells by first-generation DL4-μ beads. A) Chemically biotinylated DL4-Fc protein was conjugated onto SA-coated beads to form randomly oriented first-generation DL4-μ beads ranging from 50 nm gold nanoparticles (GNP) to 100 μm μ beads. B) 3 x 10⁴ 3T3-N1Cluc cells were incubated in plates pre-treated with hIgG or different concentrations of DL4-Fc as shown in the figure, as a negative control. Alternatively, cells were incubated in untreated plates with different numbers of DL4-μ beads. The total number of beads under each condition represented the same surface area and therefore the same total number of DL4-Fc molecules. Cells were lysed 24 hours after seeding and their luciferase activity was analyzed. [Figure 3] This study demonstrates the induction of T cell development from mouse hematopoietic stem cells (HSCs) using first-generation DL4-μbeads. Mouse fetal liver-derived HSPCs were incubated with either unconjugated μbeads or DL4-μbeads in a medium containing FBS, SCF, IL-7, and Flt3-L for 7 days. Co-cultures were collected and analyzed by flow cytometry for the presence of T-lineage (CD25+) cells, B-lineage (CD19+) cells, or myeloid (CD11b+) cells. The numbers in the plots represent the percentage of cells in each of the four fractional regions. [Figure 4]This shows site-directed biotinylation of DL4-Fc. A) The DL4-Fc fusion construct was redesigned to include a BirA recognition sequence (AviTag®) at the C-terminus, allowing the biotiny portion to be conjugated there 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] This study demonstrates the difference in Notch activation ability between first-generation and second-generation DL4-μ beads. A) BirA-biotinylated DL4-Fc protein was conjugated onto SA-coated beads to form directional second-generation DL4-μ beads. B) 3 x 10⁴ 3T3-N1Cluc cells were incubated on IgG- or DL4-Fc plate-bound controls, or with first- or second-generation DL4-μ beads (25 μm). Cells were lysed 24 hours after seeding, and luciferase activity was analyzed. [Figure 6] This study evaluates the effect of μbead size on Notch activation. DL4-μbeads ranging in size from 6.5 μm to 100 μm were incubated overnight with 3 x 10⁴ 3T3-N1 Cluc cells. Plate-bound (PB)IgG and DL4-Fc were included as negative and positive controls, respectively. The total number of beads under each condition represented the same surface area and therefore the same total number of DL4-Fc molecules. Cells were lysed 24 hours after seeding, and luciferase activity was analyzed. [Figure 7]This study demonstrates Notch activation induced by various concentrations of DL4-Fc and different compositions of μ-beads. A) DL4-μ-beads were prepared by conjugating a fixed number of 25 μm diameter SA-coated μ-beads with increasing amounts of DL4-Fc. 25 μm diameter magnetic μ-beads were included to evaluate the effect of changes in the core composition of the μ-beads on Notch activation ability. B) Biotinylated DL4-Fc was conjugated to equal-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. Cells were lysed 24 hours after seeding and luciferase activity was analyzed. [Figure 8] This study demonstrates the induction of T cell development from mouse fetal liver cells (HSPCs) using second-generation DL4-μ beads. 1x10³, 3x10³, or 8x10³ mouse fetal liver-derived HSPCs were incubated for 7 days in a medium containing FBS, SCF, IL-7, and Flt3-L with unconjugated μ beads or DL4-μ beads in a 10:1 (beads:cells) ratio. Co-cultures were collected and analyzed by flow cytometry for the presence of T-lineage (CD25+) cells, B-lineage (CD19+) cells, or myeloid (CD11b+) cells. The numbers in the plot represent the percentage of cells within each quadrant region. [Figure 9] This study describes the optimization of the HSPC to DL4-μbead ratio. Mouse fetal liver-derived HSPCs were cultured for 7 days under the following conditions: unconjugate μbeads, plate-bound DL4-Fc (PB-DL4), or DL4-μbeads titrated 3-fold. The cultures were analyzed by flow cytometry for inhibition of CD11b+ myeloid cells and CD19+ B-lineage cells, as well as for the emergence of proT(CD90+ CD25+) cells. [Figure 10A]This shows the progression of human T-lineage cell development from HSPCs co-cultured with DL4-μbeads. A) Human umbilical cord blood-derived CD34+ cells were cultured for 14 days in StemSpan® SFEM II supplemented with StemSpan® T Cell Progenitor Expansion Supplement, together with unconjugated μbeads, plate-bound DL4-Fc, or DL4-μbeads. Cells were collected every two days (arrows) and analyzed for CD34, CD5, CD1a, and CD7 surface expression using flow cytometry. [Figure 10B] This shows the progression of human T-lineage cell development from HSPCs co-cultured with DL4-μ beads. B) The number of cells was also counted, and the overall cell proliferation rate was assessed. The proliferation rate was calculated by dividing the total number of cells shown on each day by the initial seeding rate at the start of culture on day 0. [Figure 11] This report describes the analysis of the presence of mature human T cells induced 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 collected on days 28 and 47, and surface expression of CD34, CD5, CD1a, CD4, CD8, and CD3 was analyzed. Co-expression of CD3 is shown in SP-gated, DP-gated, or DN-gated cells, as indicated by the arrows. [Figure 12]This study demonstrates that DL4-μ beads induce T cell development from CD34+ cells derived from G-CSF and Plerixafor (PLX)-mobilized peripheral blood (mPB). 3 x 10³ CD34+ cells, treated with G-CSF and PLX for 5 days from umbilical cord blood and adult cells (n=3), were incubated with 9,000 DL4-μ beads. A) Progression to T cell development was analyzed on day 14 using flow cytometry to assess the expression of cell surface markers CD5, CD7, and CD34. B) Cell counts were measured on day 14 using a hemocytometer, and the proliferation rate was calculated based on the number of starting cells. The proliferation rate using umbilical cord blood (CB) was used as a comparison operator. [Figure 13] This study demonstrates the early and late stages of T cell development from induced pluripotent stem cells (iPSCs) using DL4-μ beads. Human iPSCs were induced to differentiate first into mesodermal fate, and then into CD34+ pre-hematopoietic fate. (A) 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 on days 6, 8, 10, and 12 using flow cytometry to examine the 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 cell surface markers for T cell coreceptors CD4 and CD8, as well as CD3. The inverted triangle indicates the day of analysis. RCN: Relative cell count. [Figure 14] This study demonstrates the induction of early and late stages of T cell development from T cell-derived iPSCs (T-iPSCs). T-iPSCs were induced to the mesoderm and then to a pre-hematopoietic 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 at day 12 (D12) and day 24 (D24) by flow cytometry, examining the cell surface expression of CD5, CD7 (early T cell development), and CD4, CD8, CD3, and TCRαβ (late T cell development). [Figure 15]This paper demonstrates the biotinylation of recombinant VCAM1-Fc. Recombinant VCAM1-Fc was genetically modified to consist of the VCAM-1 extracellular domain, IgG3 Fc domain, and Avi-tagged biotinylation site, similar to DL4-Fc. VCAM1-Fc was expressed in HEK293T cells, secreted into culture medium, purified using protein G conjugated beads, and then biotinylated in vitro with BirA enzyme. Samples of commercially available VCAM1-Fc from R&D Systems (R&D), laboratory-purified VCAM1-Fc(-Fc), and in vitro biotinylated VCAM1-Fc(biotin) were subjected to electrophoresis on polyacrylamide gel. Western immunoblotting was used to compare the size and concentration of VCAM-Fc samples using an anti-VCAM-1 antibody, and to detect the presence of biotinylated protein using an anti-biotin antibody. [Figure 16] This study demonstrates the combined effects of VCAM-1 and DL4 on human T cell development. Biotinylated VCAM1-Fc (VCAM) was conjugated to μbeads in different ratios as shown, and compared to unconjugated μbeads (UN) and DL4-μbeads. The amount of DL4-Fc was kept constant at 1 μg / 2x10⁵ SA-μbeads, while VCAM-Fc was added at 0.01 μg (100:1), 0.1 μg (10:1), 1 μg (1:1), and 10 μg (1:10). Next, DL4:VCAM1-μbeads were incubated with 3x10³ 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]This report describes the transplantation of primordial T (proT) cells into the thymus of immunodeficient NOD-SCID IL2rγnull (NSG) mice. Human CB-derived CD34+ cells were incubated with DL4-μ beads for 7 days. CD34+ CD7+ primordial T (proT) cells were sorted using flow cytometry, and 3 x 10⁵ cells were intravenously injected into immunodeficient NSG mice. As shown in the figure, IL-7 injection boosts were given at intervals of 3-4 days. After 3 weeks, the thymus was collected and processed. Live (DAPI-) human hematopoietic cells (CD45+) were identified using flow cytometry analysis. Furthermore, serial analysis was performed on electronically gated live CD45+ cells using cell surface markers CD19 (B cells), CD33 (myeloid cells), CD3, CD4, and CD8 (T cells). [Figure 18] This document describes the transplantation of proT cells into the thymus and their subsequent migration to the bone marrow, secondary lymphoid organs, and spleen. ProT cells sorted from CB-HSPC / DL4-μ bead cultures on day 7 were injected into NSG mice. Twelve weeks after injection, the thymus (T), bone marrow (BM), and spleen (S) were collected and processed, and engraftment of CD45+ human hematopoietic cells was assessed using flow cytometry (upper panel). Human CD45+ cells were electronically gated, and the presence of mature T cells was determined in each organ using cell surface markers CD3, CD4, CD8, and TCRαβ, as shown in the figure. [Figure 19] This shows the separation of magnetic DL4-μ beads from cellular components. 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, and magnetized particles were separated from the cellular components that did not bind. The negative fraction represents components that did not bind to the instrument's magnetization probe. The positive fraction represents the bound fraction. The content of beads and cells in each fraction was counted using a hemocytometer and compared to the original mixture (before sorting). The cells in each fraction are shown in the left bar, and the beads in the right bar. [Modes for carrying out the invention]

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

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

[0041] The term "T cell lineage cell" refers to a cell that exhibits at least one phenotypic feature of a T cell or its progenitor or progenitor cell that distinguishes it from other lymphocytes and cells of the erythrocyte or myeloid lineage. Such phenotypic features may include the expression of one or more T lineage-specific proteins in the cell or its progenitor or progenitor cell, or T cell-specific physiological, morphological, functional, or immunological characteristics.

[0042] T cell lineage cells include (a) progenitor cells or precursor cells that have been determined to become T cell lineage cells (as described herein as “progenitor T cells” or “proT cells”); (b) CD25+ immature T cells; (c) cells that have undergone differentiation determination to become CD4 or CD8 lineage cells (e.g., CD4+CD8). lo TCR int (d) cells characterized by TCR gene rearrangement; (e) progenitor thymocytes that are CD4+CD8+ double-positive (DP); (f) CD4-CD8+ or CD4+CD8- and optionally TCR hi(g)CD3+CD90+; (h)CD4-CD8+ or CD4+CD8-, TCR hi (i) TCR-αβ + and / or TCR-γδ + (j) Characterized by the expression of any of several Vβ chains (e.g., Vβ-3, -6, and 17a); or (k) TCR / CD3 hi These may be mature and functional or activated T cells, which can be characterized as CD4-CD8+ or CD4+CD8-.

[0043] In one embodiment, the cells of the T cell lineage are “primordial T cells” or “proT cells.” As used herein, the terms “primordial T cells” or “proT cells” mean T cells that can mature into mature T cells or mature lymphocytes.

[0044] In one embodiment, the primordial T cells are human primordial T cells. Phenotypes of human primordial T cells include CD34+CD7+ and CD7+CD5+CD1a-. In another embodiment, the primordial T cells are mouse primordial T cells. Phenotypes of mouse primordial T cells include CD25+.

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

[0046] As used herein, the term “suspension support” 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 suspend in the culture medium. Suspension supports can be made from a variety of materials and may be in various formats. Examples of supports that can be used as suspension 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 embodiment, the floating support is a particle. The particle may be any shape, but is not limited to spheres, ovals, rods, rectangles, etc. The particle may be made of a variety of materials, but is 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 terms “microbead” or “μbead,” as used herein, refer to a spherical or nearly spherical bead having a diameter of 0.01 μm (10 nm) to 500 μm, and 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 envisioned in this specification. In one embodiment, the microbeads are polymer, silica, or magnetic microbeads. In another embodiment, the microbeads are polystyrene microbeads, gold nanoparticles, or Dynabead. In yet another embodiment, the microbeads are a copolymer of lactic acid and glycolic acid (PLGA).

[0050] Various methods for conjugating proteins to a support are known in the art. Proteins can be conjugated directly or indirectly to a suspension support, such as microbeads.

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

[0052] As used herein, the term "Notch ligand" refers to a ligand capable of binding to Notch receptor polypeptides present on the membranes of numerous different mammalian cells, including hematopoietic stem cells / primordial 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-serate, and L-Lag2) containing 20–22 amino acids at their amino terminus and 3–8 EGF repeats on their extracellular surface.

[0053] The term Notch ligand also includes anti-Notch antibodies and aptamers (such as DNA aptamers) that can bind to and participate in Notch signaling.

[0054] A Notch ligand is selected that promotes and maintains the differentiation and proliferation of T cell lineage cells. The Notch ligand may be of human origin or of 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. The Delta family 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, mouse (M. This includes *Delta-like 3* (Genbank accession numbers NM_053666, N_446118, rat), *Delta-4* (Genbank accession numbers AF273454, BAB18580, mouse; Genbank accession number AF279305, AAF81912, Homo sapiens), and *Delta-like 4* (DL4; Genbank accession numbers Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank accession number NM019454, mouse). Notch ligands are commercially available or can be produced by recombinant DNA techniques and can be available in varying purities.

[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 to any known Notch ligand, either by amino acid sequence homology or nucleic acid sequence homology. A Notch ligand may be identical to its corresponding Notch ligand at the amino acid level by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, preferably 90%, more preferably 95%, and most preferably 98-99%.

[0057] In one embodiment, homologs of the Notch ligand contain a DSL domain at the N-terminus and have 3 to 8 EGF-like repeats on their extracellular surface. Preferred homologs can also bind to the Notch receptor. Binding to the Notch receptor can be determined by various methods known in the art, including in vitro binding assays.

[0058] The term "Notch ligand" includes variants or cultivars of known Notch ligands. The term "variant" refers to a polypeptide having a primary amino acid sequence different from the wild-type sequence due to the addition, substitution, or deletion of one or more amino acids. Preferably, the variant has at least 90% sequence identity with respect to the wild-type sequence. Preferably, the variant has 20 or fewer mutations with respect to the entire wild-type sequence. More preferably, the variant has 10 or fewer mutations with respect to the entire wild-type sequence, most preferably 5 or fewer mutations.

[0059] Optionally, a Notch ligand includes at least one protein tag. The protein tag is a peptide sequence attached to a protein of interest, such as a Notch ligand. This 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 number of purposes. In one embodiment, the Notch ligand includes 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 particular embodiment, the Notch ligand DL4 is fused with Fc (DL4-Fc). In another embodiment, the tag is a His tag. In a further embodiment, the tag is a molecule that facilitates the oligomerization of the Notch ligand. For example, a small domain of COMP (cartilage oligomer substrate protein) can be fused with a Notch ligand (e.g., DL4) to form a DL4 pentamer. Similarly, ferritin can be used to form a DL4 multimer.

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

[0061] The terms “hematopoietic stem cells / primordial cells,” “hematopoietic stem cells or primordial cells,” or “HSPCs,” as used herein, refer to undifferentiated hematopoietic cells that can differentiate into other cell types, such as T cell lineage cells. HSPCs can be obtained from numerous sources, but are not limited to, bone marrow, umbilical cord blood, and recruited 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 terms “embryonic stem cells” or “ESCs,” as used herein, refer to undifferentiated embryonic stem cells that have the ability to integrate into and become part of the germline of a developing embryo.

[0063] The terms “induced pluripotent stem cells” or “iPSCs,” as used herein, refer to cells derived from somatic cells, such as skin or blood cells, that have been retroactively reprogrammed into an embryonic pluripotent state. In one embodiment, iPSCs are derived from T cells with known or unknown TCR specificity (for example, T cells carrying a TCR specific to cancer).

[0064] Typically, non-stem cells or mature cells are first depleted from a sample containing stem cells or progenitor cells. Negative and positive selection methods known in the art may be used to enrich stem cells 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 having a specific cell surface antigen. Negative selection columns may be used to remove cells expressing lineage-specific surface antigens.

[0065] In one embodiment, a sample containing stem cells or progenitor cells is subjected to a series-negative (Lin - ) fraction and series positive (Lin + Divide into two fractions. - The fraction is CD34 + It can be sorted by cell type.

[0066] Progenitor cells or stem cells are cultured under the preferred conditions described herein to generate T cell lineage cells. 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 T cell lineage cells.

[0067] One advantage of the method described herein is that T cell lineage cells can be cultured in suspension. In one embodiment, progenitor cells or stem cells are cultured in suspension with Notch ligand conjugated to a suspension support such as microbeads. In suspension culture, cells grow while floating freely in the culture medium. In contrast, in adherent 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, together with Notch ligand conjugated to a suspension support. In one embodiment, the suspension support is microbeads having a diameter suitable for the bioreactor. Various bioreactors are known in the art, including batch, fed-batch, or continuous bioreactors. An example of a continuous bioreactor is the continuous agitated-tank reactor model.

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

[0070] In one embodiment, the ratio of Notch ligand conjugated to microbeads to progenitor cells 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 inventors have also shown that Notch signaling can be enhanced by specifying the orientation of the Notch ligand relative to the suspension support. Thus, in one embodiment, the C-terminus of the Notch ligand is conjugated to the suspension support. This can be done, for example, by adding a sequence that can be enzymatically conjugated to a biotin molecule to the end of the Notch ligand's C-terminus. 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 suspension support.

[0072] One or more positive cytokines that determine and promote the differentiation of T cell lineage cells may also be added to the culture. These cytokines may be of human origin or from other species. The concentration of cytokines in the culture is typically about 1–10 ng / ml. Representative examples of cytokines that may be used herein include: all members of Flt-3 ligands, as well as interleukin-7 (IL-7) and stem cell factors. In one embodiment, the cytokines used herein are Flt-3 ligands, IL-7, and stem cell factors. Cytokines may be used in combination with equimolar or larger amounts of glycosaminoglycans, such as heparin sulfate. Cytokines are commercially available or can be produced by recombinant DNA techniques and can be of varying purities. Some cytokines can be purified from cell line culture media by standard biochemical techniques.

[0073] One or more additional molecules may be added to the culture by conjugating each to a suspension support. In one embodiment, the additional molecules are molecules that promote T cell development (e.g., promote differentiation determination and differentiation of T cell lineage cells) and are also referred to herein as “T cell costimulatory molecules.” In one example, the inventors have shown that DL4 and VCAM1 conjugated to microbeads were cultured with HSPC and that differentiation into T cell lineage was accelerated. 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 surface antigen classification 106 (CD106), which is a protein encoded by the VCAM1 gene in humans. The term “VCAM1” also includes variants or cultivars 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 molecule (CD80, CD86). Optionally, the T cell costimulatory molecule includes at least one protein tag. Various protein tags are known in the art and can be used for numerous purposes. In one embodiment, the T cell costimulatory molecule includes 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 Iskov-modified Dulbecco's medium (IMDM), DMEM, or αMEM, or equivalent media. The culture medium may or may not contain serum (e.g., bovine serum, fetal bovine serum, calf serum, horse serum, human serum, or an artificial serum substitute). Other examples of media useful in this method include StemCell Technologies' media (StemSpan). (商標)Examples include SFEM II) or any other commercially available equivalent medium.

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

[0076] The method of this invention enables the generation of a large number of T cell lineage cells. Specifically, in one embodiment, cell proliferation rates exceeding 50, 75, 100, 125, 150, 175, or 200 times the initial number of stem cells or progenitor cells can be obtained after 14 days of culture.

[0077] As used herein, the term "isolated" means that a progenitor cell has been separated or purified from the cellular or biological material found in the cell in its natural environment. The cell is thereby distinguished from its natural form.

[0078] The terms "a cell" or "the cell" can also refer to multiple cells.

[0079] II. Suspended Notch Ligands The inventors have also developed novel suspension Notch ligands. As used herein, the term “suspension Notch ligand” refers to a Notch ligand used in suspension cell culture.

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

[0081] Specifically, the inventors have shown that directly conjugating DL4 to 25 μm diameter microbeads delivers a strong and sustained signal, inducing HSPCs to become T cell lineage cells. Therefore, in one embodiment, the suspension support is a microbead having a diameter of 10–100 μm, optionally 20–30 μm, 24–26 μm, or 25 μm.

[0082] The inventors have further demonstrated that Notch signaling can be enhanced by specifying the orientation of the Notch ligand relative to the suspension support. In another embodiment, the C-terminus of the Notch ligand is conjugated to the suspension support. As described above, this can be achieved, for example, by adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated to a biotin molecule.

[0083] The Notch ligand is optionally DL4 and may be fused with a tag, such as an Fc tag.

[0084] This specification also provides 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, wherein the T cell costimulatory molecule is conjugated to the suspension support.

[0085] The T cell costimulatory molecule is optionally VCAM1, which may be fused with a tag, such as an Fc tag.

[0086] III. Kit Suspended Notch ligands may be prepared and packaged as kits used to generate T cell lineage cells.

[0087] Accordingly, this specification also provides a kit comprising a suspension Notch ligand for producing T cell lineage cells, the suspension Notch ligand comprising (a) Notch ligand and (b) a suspension support, wherein the Notch ligand is conjugated to the suspension support. Optionally, the suspension 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 suspension Notch ligand.

[0088] In one embodiment, the kit further comprises a culture medium for culturing a sample containing stem cells and progenitor cells with a suspension Notch ligand. Examples of culture media include conditional media, unconditional media, or embryonic stem cell media. The culture medium may or may not contain serum (e.g., bovine serum, fetal bovine serum, calf serum, horse serum, human serum, or an artificial serum substitute). 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 suspension support. In one embodiment, the additional molecules are molecules that promote T cell development (e.g., promote differentiation determination and differentiation of cells of the T cell lineage) and are 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 differentiation determination and differentiation of T cell lineage cells. The cytokines may be of human origin or of other species. The concentration of cytokines in the culture is typically about 1–10 ng / ml. Representative examples of cytokines that may be used herein are: all members of Flt-3 ligands, as well as interleukin-7 (IL-7) and stem cell factors. In one embodiment, the cytokines used herein are Flt-3 ligands, IL-7, and stem cell factors. Cytokines may be used in combination with equimolar or larger amounts of glycosaminoglycans, such as heparin sulfate. Cytokines are commercially available or can be produced by recombinant DNA techniques and can be of varying purities. Some cytokines can be purified from cell line culture media by standard biochemical techniques.

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

[0092] In various embodiments, a printed instruction manual providing guidance on the use of the reagents may also be included in the kit. The terms “instruction manual” or “user manual” typically include clear and understandable language describing the concentration of the reagent or the amount of suspended Notch ligand, and / or at least one assay parameter, such as the relative amounts of suspended Notch ligand to cells to be mixed, culture time, temperature, medium conditions, etc. For example, in one embodiment, the instruction manual describes a method comprising the steps of (a) culturing a sample containing stem cells and progenitor cells with Notch ligand conjugated on a suspension support, and (b) isolating T cell lineage cells.

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

[0094] In one embodiment, the present disclosure provides “primordial T cells” or “proT cells” generated by the method described herein. In another embodiment, the primordial T cells are human primordial T cells, for example, human primordial T cells characterized by CD34+CD7+ or CD7+CD5+CD1a-.

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

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

[0097] In one embodiment, T cell lineage cells (e.g., primordial T cells or mature T cells) generated by the method described herein are modified with respect to T cell receptors (TCRs) or chimeric antigen receptors (CARs) to confer specificity to tumor-associated antigens (TAAs). Such modified cells may be useful in treating diseases such as cancer.

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

[0099] Suitable diluents and carriers are described, for example, in Remington's Pharmaceutical Sciences. Based on this, the composition comprises, but is not limited to, a solution of proT cells together with one or more pharmaceutically acceptable vehicles or diluents, housed 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 injection solutions or suspensions, which may further contain antioxidants, buffers, bacteriostatic agents, and solutes that substantially adapt the composition to 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) Examples include alcohols, polyols, glycerin, and vegetable oils. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrates or suspensions. The compositions may be supplied as lyophilized powders that are reconstituted with sterile water or saline before administration to the patient, for example, but are not limited to these.

[0101] The pharmaceutical composition also includes a cryopreservation solution. In one embodiment, T cell lineage cells generated by the method described herein are cryopreserved in a suitable medium, for example, a pharmaceutically acceptable or GMP-grade medium, and optionally formulated for administration to a subject requiring it.

[0102] Suitable pharmaceutically acceptable carriers include compositions that do not interfere with the efficacy of the biological activity of the pharmaceutical composition, are essentially chemically inert, and are non-toxic. Examples of suitable pharmaceutical carriers, but not limited to, include water, physiological saline, glycerol solution, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesyl-phosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions contain a therapeutically effective amount of the compound, along with a suitable amount of carrier to provide a form for direct administration to the patient.

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

[0104] Preferably, the T cell lineage cells are present in an amount effective for treating a disease condition in a subject requiring treatment of said condition. In one embodiment, the T cell lineage cells are present in an amount effective for enhancing hematopoietic cell transplantation in a subject requiring enhancement of said condition. Optionally, the composition further comprises transplanted T cell lineage cells or tissue. In one embodiment, the tissue includes the thymus. In another embodiment, the tissue includes an organ.

[0105] V. Therapeutic Applications Generating in vitro-derived human primordial T cells and testing their safety in human / mouse immunotransplantation models would pave the way for cell-based approaches to treat T-lineage immune-related disorders (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 hematological cancers, such as T-cell acute lymphoblastic leukemia (T-ALL), T cells proliferate, shutting out healthy immune cells and disrupting normal immune function (Ferrando et al., 2002; Weng et al., 2004). While chemotherapy can sometimes provide therapeutic benefits to cancer patients, it often results in immunodeficiency and susceptibility to opportunistic infections. Opportunistic infections also affect CD4 after HIV infection. +This poses a significant concern for AIDS patients whose T cells are depleted. While immunodeficiency remains a major concern in HIV / AIDS and cancer, hyperactive immune activity is equally problematic in autoimmune diseases where T cells, lacking proper regulatory control, trigger an immune response against the body's own tissues.

[0106] Therefore, the present invention relates to a method for treating a subject having a condition requiring an increase in the number of T cells, (i) A step of generating T cell lineage cells, comprising (a) culturing a sample containing stem cells or progenitor cells with a suspension support, optionally with Notch ligand conjugated to particles or microbeads, and (b) isolating T cell lineage cells, and (ii) The step of administering an effective amount of cells of the T cell lineage to a subject who needs it. Further methods are provided, including the following.

[0107] In one embodiment, the cells of the T cell lineage are primordial T cells.

[0108] In another aspect, the cells of the T cell lineage are mature T cells.

[0109] This disclosure also provides the use of T cell lineage cells, optionally primordial T cells or mature T cells, generated by the methods described herein, for treating subjects having conditions requiring an increase in T cell count.

[0110] This disclosure also provides the use of T cell lineage cells, optionally primordial T cells or mature T cells, generated by the methods described herein, for example, in regenerative medicine to replace and / or regenerate diseased or injured tissue.

[0111] As used herein, the terms “effective dose” or “therapeutic effective dose” mean an effective amount of dose and duration required to obtain the desired result. The effective dose may vary depending on factors such as the disease state of the subject, age, sex, and weight. The amount of a given cell preparation corresponding to such a dose may vary depending on various factors such as the pharmaceutical formulation, route of administration, type of disease or disorder, and the characteristics of the subject or host being treated, but can still be routinely determined by those skilled in the art. The “effective dose” is preferably an effective amount of T cell lineage cells to be transplanted into the subject being treated.

[0112] The terms “to treat” or “treatment,” as used herein and as is well known in the art, mean an approach to obtain beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, reduction or improvement of one or more symptoms or conditions, whether detectable or undetectable; a decrease in the severity of the disease; a stable (i.e., non-worsening) state of the disease; prevention of disease transmission; delay or slowing of disease progression; improvement or mitigation of the disease state; a decrease in the rate of disease recurrence; and (whether partial or total) remission. “To treat” and “treatment” may also mean an extension of life compared to the predicted lifespan without treatment. “To treat” and “treatment,” as used herein, also include preventive treatment.

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

[0114] "Conditions requiring an increase in T cell count" include, but are not limited to, any condition in which T cell levels are reduced compared to a healthy animal, such as immunodeficiency, cancer, genetic disorders (e.g., primary immunodeficiency disease (PID)), infectious diseases, immune disorders, and autoimmune conditions.

[0115] As described above, the T cell lineages described herein may be modified to express T cell receptors (TCRs) or chimeric antigen receptors (CARs) that specifically recognize tumor-associated antigens.

[0116] Therefore, this application also includes a method for treating the target cancer, and said method is (i) A step of generating T cell lineage cells, comprising (a) culturing a sample containing stem cells or progenitor cells with a suspension support, optionally with Notch ligand conjugated to particles or microbeads, and (b) isolating T cell lineage cells, and (ii) The step of administering an effective amount of cells of the T cell lineage to a subject who needs it. The cells of the T cell lineage are modified with respect to the T cell receptor (TCR) or chimeric antigen receptor (CAR) to confer specificity to tumor-associated antigens.

[0117] This disclosure also provides the use of T cell lineage cells, optionally primordial or mature T cells, generated by the methods described herein for the treatment of subjects with cancer, wherein the T cell lineage cells are modified with respect to a T cell receptor (TCR) or chimeric antigen receptor (CAR) to confer specificity to tumor-associated antigens. Optionally, iPSCs may be derived from T cells with known or unknown TCR specificity (e.g., T cells carrying a TCR specific to cancer), in which case these T-iPSCs may be used to generate T cells by the methods described herein.

[0118] The following non-limiting embodiments illustrate the present invention. [Examples]

[0119] Example 1 material and method: Hematopoietic stem cell source In accordance with approved guidelines established by the Research Ethics Board of Sunnybrook Health Sciences Centre, human umbilical cord blood (UCB) samples were collected by syringe from postpartum mothers who gave their consent and collected in blood pack units containing anticoagulant citrate phosphate dextrose (Baxter Healthcare, Deerfield, Illinois). Within 24 hours of collection, UCB mononuclear cells were isolated by Ficoll density centrifugation, and the EasySep Human CD34 Positive Selection Kit (Stemcell Technologies, Vancouver, British Columbia) was used according to the manufacturer's instructions to determine the series negative (Lin). - )CD34 + The cells were pre-concentrated. To isolate human HSPCs, Lin - Cells were stained with anti-human CD38-APC and anti-human CD34-PE mAb, and then sorted using a BD Biosciences FACSAria sorter (BD, San Jose, California). + CD38 - / lo The cells were sorted. Post-sorting analysis determined the sorted human HSPCs to have a purity of over 99%. A portion of the umbilical cord blood-derived CD34+ cells and all of the recruited 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 3-5 days before harvest) and Plerixafor (up to 0.24 mg / kg 1 day before harvest).

[0120] Design and production of the DL4-Fc The C-terminus of the coding sequence of the extracellular domain of human DLL4 (amino acid residues [aa]1-529) is separated by a linker sequence to contain a histidine (His) tag, followed by the Fc portion of human IgG3 (including the hinge region) and the Bir1A recognition sequence (Avitag). (商標)By fusing the above, Delta-like-4 was genetically modified to create the pDL4-Fc-His-B plasmid construct. Other constructs that can be used in these methods include: i) human DLL4 (aa1~524) fused with the Fc portion of IgG1; ii) signal sequences of alkaline phosphatase (aa1~17) human DLL4 (aa27~524) fused with the Fc portion of IgG1; iii) human DLL4 (aa1~524) fused with StreptagII and 6xHis at the C-terminus; iv) signal sequences of alkaline phosphatase (aa1~17) human DLL4 (aa27~524) fused with 10xHis at the C-terminus; and any combination thereof. This construct was inserted into the pIRESpuro2 mammalian expression plasmid (Clontech, Mountain View, California). The obtained plasmid was transfected into HEK-293T cells using a standard CaPO4 transfect method. Cells with stable plasmid integration were selected based on their resistance to 2 mg / mL puromycin added to standard DMEM [10% (v / v) FBS, 2 mM Glutamax, penicillin (100 U / ml) / streptomycin (100 mg / ml) (all products of Thermo Fisher Scientific, Rockford, Illinois), and 2 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, Michigan)]. The cells were grown and transferred to Freestyle 293 expression medium (Thermo Fisher Scientific). 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 automated chromatography system (GE Healthcare Life Sciences, Marlborough, Massachusetts).

[0121] Design and production of VCAM1-Fc Recombinant VCAM1-Fc, like DL4-Fc, contains the VCAM-1 extracellular domain, the IgG3 Fc domain, and Avitag (商標)The gene was modified to include a biotinylation site. The materials and methods used for the production and purification of the VCAM1-Fc fusion protein were the same as those used 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 DL4-Fc molecule. Unreacted NHS-biotin was removed from the mixture by dialysis in PBS or buffer exchange, and biotin incorporation was evaluated by the HABA (4'-hydroxyazobenzene-2-carboxylic acid) method (Pierce Biotin Quantitation Kit, product number 28005). The biotinylated DL4-Fc was then stored at 4°C.

[0123] To orient DL4-Fc to the streptavidin (SA) coated surface, use the BirA-500 Kit (Avidity) according to the manufacturer's instructions to direct one biotin molecule to the AviTag of the Fc region of DL4-Fc. (商標) The sequence was enzymatically conjugated. Briefly, 2.5 μg of BirA was added to 500 μL of PBS containing 500 μg of DL4-Fc and incubated at room temperature for 1 hour. Any remaining biotin was removed using a 40K MWCO Zeba (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. Avitag (商標) The contained VCAM1-Fc protein was biotinylated at its C-terminus using the same method and materials as described above.

[0124] Conjugation of biotinylated DL4-Fc onto streptavidin-coated microbeads 1 μg of biotinylated DL4-Fc was incubated with streptavidin (SA) coated polystyrene μbeads of various sizes ranging from 1 μm to 100 μm in diameter (Spherotech, Lake Forest, Illinois). DL4-Fc was also conjugated to SA-coated Dynabeads (Thermo-Fisher) and 50 nm nanogold particles, and each was incubated in 2 mL of PBS at room temperature for 30 minutes, mixed by vortexing every 10 minutes. In all cases, the surface area of ​​the particles was 2 x 10⁻⁶. 5 This was equivalent to 25 μm SA-μ beads. The DL4-Fc conjugate beads were washed with 4 mL of PBS and spun down at 3000 xg for 10 minutes. The supernatant was carefully collected to remove any unbound ligands from the mixture and to assess binding to the μ beads by assaying the DL4-Fc content. After a second wash, the DL4-μ beads were again suspended in various volumes of PBS to obtain the concentrations indicated in the text. Unconjugate μ beads were prepared in parallel as a negative control.

[0125] In the composite conjugation of DL4-Fc and VCAM1-Fc with SA-μ beads, the amount of DL4-Fc is 2x10 of SA-μ beads. 5 The amount of VCAM1-Fc added was kept constant at 1 μg per cell, but VCAM1-Fc was added at concentrations of 0.01 μg (100:1), 0.1 μg (10:1), 1 μg (1:1), and 10 μg (1:10).

[0126] Development of Notch-activated reporter cell line, 3T3-N1Cluc A Notch-responsive element (8x RBPJ consensus binding site) was inserted into a promoter-less pGL4.17[luc2 / Neo] plasmid (Promega). This created a plasmid (named pGL4.17-N1Rep) that conferred neomycin resistance while signaling Notch activation through luciferase enzyme expression. Next, NIH3T3 cells were transfected with pMIGR-NOTCH1 (donated by Dr. Warren Pear of the University of Pennsylvania) and the pGL4.17N1Rep plasmid. Cells resistant to neomycin treatment (1 μg / mL) were selected first, and then GFP-expressing cells were sorted by flow cytometry. Next, clones of NIH3T3 cells were isolated by single-cell collection into 96-well plates. Finally, the response of each clone to plate-bound DL4-Fc activation was measured (collected overnight at 20 μg / mL). A clone named 3T3N1CLuc, exhibiting the lowest background levels and the highest Notch activation response, was propagated and used to perform experiments measuring Notch receptor activation.

[0127] Luciferase assay of 3T3-N1CLuc to measure Notch activation 3 x 10 in a flat-bottomed 96-well plate treated with standard tissue culture (TC) 43T3-N1CLuc cells were treated with DL4-μ beads and incubated overnight in αMEM supplemented with 5% FBS. The cells were lysed, and luciferase activity was assayed using the Firefly Luciferase Assay Kit 2.0 (Biotium, Fremont, California) according to the manufacturer's instructions. Briefly, the growth medium was removed, the cells were washed with PBS, and then lysis buffer was added. The cells were lysed by freezing them at -80°C for 10 minutes and then thawing, and the lysates were transferred to 96-well flat-bottom opaque polystyrene plates (Corning). D-luciferin was prepared and added to each well using an automated dispenser, and analyzed using a Synergy H1 plate reader (BioTek Instruments Inc., Winooski, Vermont). Plate-bound controls for human IgG and DL4-Fc were prepared the day before by adsorbing 50 μL / well of protein (5-20 μg / mL) onto a flat-bottomed 96-well plate at 4°C overnight, washing the plate the following day, and seeding 3T3-N1CLuc cells.

[0128] Culture of coated DL4-Fc plates and DL4-μ beads with HSPC Different numbers of DL4-μ beads, 3x10 3 Individual mouse Lin - Sca-1 + cKit +The cells were added to HSCs (sorted by flow cytometry from the bone marrow of C57BL6 mice) to achieve cell-to-bead ratios of 1:1, 1:3, 1:9, and 1:27. Under each condition, the cell-bead combinations were 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, Minnesota)]. Plate-bound controls for human IgG and DL4-Fc were prepared the day before by adsorbing 50 μL / well of protein (20 μg / mL) onto a flat-bottom 96-well plate overnight at 4°C, washing the following day, and seeding the cells. Cells were collected on day 7 of co-culture, stained with anti-mouse antibodies CD45, CD25, CD44, CD90, CD11b, and CD19, and then analyzed using an LSR II cytometer (BD Biosciences).

[0129] In the case of human HSPCs, Lin is obtained from UCB by flow cytometry. - CD34 + CD38 - / lo Sort the cells and place 4x10 cells per well in a 96 round-bottom plate. 3 A single cell, along with 36,000 DL4-μ beads, is placed in StemSpan. (商標) 200 μL of StemSpan supplemented with T Cell Progenitor Expansion Supplement (Stemcell Technologies) (商標)Cultured in SFEM II (Stemcell Technologies) for 14 days, and the medium was changed by 50% on the 7th day. For cultures exceeding 14 days, the cells were collected, counted, and seeded with fresh DL4-μ beads. On the 14th day of co-culture, the number of cells was counted to determine the cell proliferation rate, and the cells were stained with antibodies against CD34, CD5, CD1a, and CD7 to assay for lineage progression. To assay for growth after the proT cell stage, at a later time point, the cells were also stained with antibodies against CD4, CD8, and CD3.

[0130] From human induced pluripotent stem cells (iPSCs) to CD34 + Differentiation from pre-hematopoietic cells or from hematopoietic endothelial cells to T cells Initialized hiPSC lines were derived from fibroblasts (AlStemBio Cat #iPSC11) and T cells (Harvard Stem Cell Science Cat # STiPS A3). These were cultured in mTeSR medium (StemCellTech) on Matrigel. To generate self-aggregating EBs, iPSCs were treated with collagenase B for 20 minutes followed by a short trypsin-EDTA step. Cells were gently scraped with a cell scraper to make 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 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). The cultures were maintained for 8 days in a low oxygen environment of 5% CO2 / 5% O2 / 90% N2. On day 8, the cells were enriched for CD34 + cells using MACS as described (Kennedy et al., 2012), and added to StemSpan along with DL4-μ beads (商標)StemSpan supplemented with T Cell Progenitor Expansion Supplement (Stemcell Technologies) (商標) Incubated as described above with SFEM II (Stemcell Technologies) to generate T cells.

[0131] Adoptive transfer of naive T cells into immunodeficient mice As preparation for adoptive transfer into immunodeficient mice, a large amount of human HPSC / DL4-μ bead cultures were prepared. 2x10 5 individual CD34 + HSPCs were incubated with 1.8x10 6 individual DL4-μ beads in a T25 flask (Thermo Scientific) for 7 - 10 days. At this time, naive T (proT) cells identified as CD34 + CD7 + cells were sorted using a FACSAria cell sorter (BD Biosciences, San Jose, CA). Alternatively, when culturing HSPCs with DL4-μ beads coated with iron oxide, magnetic separation of DL4-μ beads from the cell components was performed using an autoMACS-pro cell sorter (Miltenyi Biotec). proT cells were injected into the liver of 3 - 6-day-old immunodeficient NOD-Scid / IL2rγ null (NSG) neonates. Each mouse was given a total of 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 together with 3 - 5x10 5 individual proT cells. Mice were boosted with the IL-7 / M25 cocktail every 3 - 4 days. Lymphoid organs thymus, spleen, and bone marrow were collected at 3 or 12 weeks post transplantation. Single cell suspensions were prepared from each organ, stained, and analyzed using an LSR-II cytometer (BD Biosciences). Transplantation was assessed by electronically gating on live cells excluding the cell death marker 4'-6-diamidino-2-phenylindole (DAPI), and cells expressing human CD45.

[0132] result: DL4-μ beads induce strong Notch signaling. Our previous laboratory studies have shown that immobilizing the DL4 fusion protein, DL4-Fc, on the surface of standard TC-treated wells / plates can induce sufficient Notch signaling to induce T-lineage cell development in mouse and human HSPCs (Shukla et al., 2017). However, this 2D format has limitations in terms of scalability and the ability to deliver potent and consistent Notch signaling that promotes 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 stem cells and stromal cells, but allowed soluble factors essential for differentiation to pass through. Some CD90 was observed from the culture with DL4-functionalized μbeads. + (Thy1) cells appeared, but Thy1 expression is not a definitive T-series marker. Importantly, CD19 + B cells also appeared. There is a threshold for sustained Notch activation necessary to promote T cell development and inhibit B cell development. The fact that B cells were still present demonstrates that this system lacked the ability to activate and sustain Notch signaling. In fact, increasing the bead-to-cell ratio from 1:1 to 5:1 promoted B cell differentiation and inhibited Thy1 expression. Combined with the known requirements for cell-based or plate-bound Notch ligands to induce potent Notch signaling, the view that the soluble Notch ligands present in the μbead format could not induce the necessary level of Notch signaling suggests that this approach is unlikely to be applicable to T cell development from HSPCs. Furthermore, this type of study still lacks human CD34 +This is not yet at the stage where it can be implemented using cells. Thus, it remains uncertain 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 and applied to clinical use.

[0134] Therefore, the inventors investigated whether a higher-order DL4 multimer platform could be created by conjugating DL4-Fc onto μbeads, which can effectively bind to the Notch receptor, suspend and function together with HSPC, and thus be suitable for scaling up.

[0135] To facilitate the linkage 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 blotting (Figure 1B), and subsequent conjugation to μ-beads was assayed by protein quantification of supernatants collected before and after conjugation. To evaluate the effect of bead size on Notch signaling, unbiotinylated DL4-Fc was covalently linked to 50 nm NHS-activated gold nanoparticles, and biotinylated DL4-Fc was linked to 1 μm SA-coated Dynabeads. (商標) The DL4-Fc molecules were also conjugated onto 25 μm and 100 μm polystyrene beads (Figure 2). The functionalized beads were incubated with 3T3-N1Cluc, which signals Notch activation using the luciferase gene as a reporter. The total number of beads under 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). Furthermore, 25 μm diameter DL4-μ beads optimally enhanced Notch receptor activation compared to other bead sizes 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 inducing T cell differentiation. Next, we determined whether DL4-μ beads supplemented with IL-7, Flt3L, and SCF had the ability to induce T cell development by incubation with mouse HSCs for 7 days. The results showed that mouse HSCs readily developed proT cells when incubated with DL4-μ beads, as evidenced by CD25 expression in 41% of cells (Figure 3). In contrast, CD25 expression was induced in only 4% of cells with PB-DL4-Fc. DL4-μ beads also inhibited the expression of alternative B cells (CD19), which are known to be inhibited by Notch signaling. + ) and myeloid (CD11b + This hindered the performance of the ) series.

[0137] Directional conjugation of DL4-Fc to μbeads enhances Notch signaling induction. To improve the randomly oriented first-generation DL4-μ beads, the inventors investigated whether specifying the direction of DL4-Fc relative to the bead surface enhances Notch signaling. For this purpose, they investigated a BirA recognition sequence (AviTag) that can be enzymatically conjugated by a single biotin molecule. (商標)We designed DL4-Fc, which has ) at its C-terminus (Figure 4). Assessment by 3T3N1CLuc cell luciferase activity revealed that incubation of these second-generation DL4-μ beads significantly increased the level of Notch signaling (Figure 5). Using this directional conjugation method, we reassessed the effect of bead size on Notch transmission. The results showed that beads of 100 μm, 25 μm, 10 μm, and 6.5 μm in diameter all had the ability to activate Notch, but the 25 μm bead was the most effective (Figure 6). We also evaluated the optimal bead-to-cell ratio and determined that when the bead-to-cell ratio was 3:1, Notch activation increased tenfold compared to the plate-bound control (Figure 6).

[0138] Next, to determine the amount of DL4-Fc per bead that optimally activates Notch signaling, we investigated whether SA μ beads were saturated with biotinylated DL4-Fc. Different amounts of DL4-Fc (0.01, 0.1, 1, and 10 μg) were incubated with the same number of 25 μm SA beads. The beads were then incubated overnight with 3T3-N1 Cluc cells. The results were 2.25 x 10⁻⁵ 5 The study showed that 1 μg of DL4-Fc per SA-μ bead yielded the maximum response, suggesting that the activity of the DL4-μ beads was saturated (Figure 7A). It was also demonstrated that magnetized 25 μm polystyrene beads (coated with iron oxide) were just as effective in Notch activation as their unmagnetized counterparts (Figure 7A). Furthermore, replacing the SA-μ beads with protein G-μ beads of the same diameter did not significantly alter the ability of DL4-Fc to activate Notch (Figure 7B).

[0139] Thus, 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 stimulation of Notch activity and the determination of optimal conditions for T cell development. Furthermore, the DL4-Fc to bead ratio for obtaining maximum activity when loading DL4-Fc onto beads was determined, and magnetizing the beads had no effect on Notch activity. In addition, protein G-μ beads, which similarly bind to the Fc region of DL4-Fc, orient DL4-Fc in the same way as SA-μ beads.

[0140] DL4-μ bead conditions for maximizing T cell generation from HSPCs Based on the above results, the inventors then proceeded to determine whether the same factors affect T cell development. This was first tested using mouse HSPCs. As shown in Figure 8, DL4-μ beads induced mouse T cell development more effectively than PB-DL4.

[0141] To determine the optimal ratio of HSPC to DL4-μ beads to maximize proT cell development, 3 x 10 3 Each HSPC was incubated under different conditions, with the number of DL4-μ beads increasing threefold each time (Figure 9). Seven days after incubation, the proT cells (CD25) of the HSPC were incubated. + Regarding differentiation into ), different HSPC:DL4-μ bead ratios were analyzed. CD25 was approximately 1:9. + This ratio was considered optimal because it yielded the highest percentage of cells. Increasing the HSPC:bead ratio to 1:27 did not improve T-series differentiation.

[0142] Dynamics and expansion of human T cell development using DL4-μ beads Next, CD34 +The use of DL4-μ beads to induce human T cell development from UCB-derived HSPCs was investigated. For this purpose, the optimal HSPC:DL4-μ bead ratio of 1:9, determined above, was used. Cells were counted every two days, and flow cytometry analysis was performed. The results showed the emergence of human proT cells co-expressing CD34, CD7, and CD5 by day 4 of culture (Figure 10A). These results indicate that human HSPCs incubated with DL4-μ beads exhibit a robust proT cell phenotype (CD34). + CD7 + or CD7 + CD5 + CD1a - This was achieved with plate-bound DL4-Fc, but not with non-conjugated μbeads or PB-DL4-Fc, demonstrating that DL4-μbeads have a higher ability to activate Notch compared to plate-bound DL4-Fc.

[0143] One of the challenges in obtaining a clinically suitable yield of proT cells is the difficulty in obtaining a sufficient number of cells. To address this, we also assessed the cell proliferation rate during development and found that by day 14, the total cell proliferation rate exceeded 150 times the initial number (Figure 10B).

[0144] DL4-μ beads promote the development of mature human T cells. Next, we investigated whether the increased ability of DL4-μ beads to activate Notch could induce developing cells to differentiate into later stages of T cell development, and therefore whether a more mature phenotype would be expressed in later stages of culture (Figure 11). Analysis of cultures at both day 28 and day 47 revealed that CD4 + CD8 + It was shown that double-positive (DP) cells accounted for approximately 25% of the cells. Interestingly, the culture at day 47 showed CD4 + CD8 + CD3 + DP cells and CD8 + CD3 +This demonstrated the emergence of single-positive (SP) cells. These results indicate that robust Notch signaling induced by DL4-μ beads can overcome the developmental disorders of T cell maturation observed in previous attempts using PB-DL4-Fc.

[0145] When cultured with DL4-μ beads, recruited peripheral blood (mPB)-derived HSPCs differentiate into T-series cells. Adult mPBs are potentially a more readily available source of HSPCs than UCBs, because the number of HSPCs obtained from one adult is about 100 times greater than that from UCBs. To compare the ontogeny of T-series development, mPB-derived CD34 from three different individuals... + HSPCs were cultured with DL4-μ beads and compared with UBC-derived HSPC cultures (Figure 12A). On day 14, the progression of T cell development was very similar to that of CB-derived HSPCs, and proT cell population (CD34) + CD7 + Similar percentages were observed. However, the proliferation rate after 14 days was 110-fold for mPB-derived HSPCs, compared to nearly 190-fold for CB-derived cells (Figure 12B).

[0146] Induction of T cell development in iPSCs using DL4-μ beads By the method described herein, human iPSCs derived from fibroblasts are converted to CD34 + They were induced to differentiate 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 during early (Figure 13A) and late (Figure 13B) development. Early development shows normal acquisition of the CD7 cell surface marker, followed by CD5. Late T cell development shows CD4 on day 28. + This culture is characterized by the presence of immature single-positive (iSP) markers and the acquisition of single-positive CD8 cells at day 35. Notably, it also features the presence of the cell surface marker CD3, a T cell receptor (TCR) component, suggesting the presence of mature T cells in this culture.

[0147] When iPSCs originate from T cells (T-iPSCs), the TCRα and TCRβ gene loci have already undergone genetic rearrangement, and upon redifferentiation into the 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 + Pre-hematopoietic cells were incubated with DL4-μ beads. Cultures examined on day 12 showed that 40% of cells expressed TCRαβ and CD3 on the cell surface, and while mature T cell markers CD4 and CD8 were not yet expressed, 80% of cells expressed the early T cell marker CD7 (Figure 14, left panel). + When the cells were gated, over 70% expressed TCRαβ and CD3. By day 24, over 80% of the 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 can function as a modular base for attaching other biotinylated molecules, we investigated the functional effects of VCAM-1. Previous studies have shown that attaching VCAM-1 to plate-bound DLL4 accelerates the differentiation of HSPCs into T cells (Shukla et al., 2017). Here, we genetically designed, expressed, and biotinylated a novel fusion protein, VCAM1-Fc. Immunoblot analysis confirmed that the generated VCAM1-Fc was the same size as the commercially available VCAM1 product (Figure 15, left panel). Furthermore, VCAM1-Fc containing the target sequence for BirA enzyme biotinylation was demonstrated to be biotinylated (Figure 15, right panel). Next, biotinylated VCAM1-Fc, along with a fixed amount of DL4-Fc, was used to coat the surface of SA-μ beads and cultured with UBC-derived HSPCs. Analysis of the cultures on day 7 demonstrated that, generally, a higher VCAM1-Fc / DL4-Fc ratio was associated with accelerated differentiation (Figure 16). This demonstrated 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] Transplantation of primordial T cells into immunodeficient mice and subsequent migration to the surrounding area. CD34 derived from HSPC / DL4-μ bead cultures + CD7 + To assess the ability of primordial T (proT) cells to engraft in the thymus, an immunodeficient NSG mouse model was selected. ProT cell production was gradually increased, switching from 96-well plates to T25 flasks. ProT cells were sorted from the culture on day 7 and injected intrahepatically into NSG neonates. Early engraftment was examined at week 3, and human CD45 was found in the thymus. + The presence of cells was confirmed, and most of them were double-positive CD4 + CD8 + (DP) The cells were in the mature T cell stage (Figure 17). B (CD19) cells and myeloid (CD33) cells had not developed in the thymus.

[0150] Analysis at 12 weeks post-transplant revealed that almost all human CD45+ cells had differentiated and matured into CD4 and CD8 SP cells within the thymus, where proT cell renewal was absent (Figure 18). These mature CD4 and CD8 SP cells appeared to migrate from the thymus to the spleen and then to the bone marrow. This demonstrated that proT cells possessed the ability to mature within the thymus and then migrate normally to secondary lymphoid organs.

[0151] Isolation of μbeads from cellular components in cultures In preparation for large-scale proT cell production from HSPC / DL4-μbead co-cultures for clinical purposes, we assessed the ability of AutoMACS (Miltenyi) to isolate iron oxide-coated DL4-μbeads from co-cultured cells (Figure 19A). AutoMACS, functioning similarly to the clinically approved CliniMACS (Miltenyi), completely isolated the μbeads from the cellular components (Figure 19B), and no μbeads were detected in the cell fraction. In contrast, the bead fraction contained cells that were trapped or attached to the isolated μbeads.

[0152] summary This specification describes 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 suspension Notch ligands, such as DL4-μ beads, represent a unique strategy that enables effective T-lineage cell development, is readily achievable in large-scale bioreactor-based suspension cultures, and has the potential to overcome the developmental limitations, inefficiencies, and scalability associated with plate-bound approaches.

[0153] Previous studies have not demonstrated the development of human T-lineage cells after the immature proT cell stage in cell-free support systems. Furthermore, the results described herein are the first to demonstrate the development of proT cells and mature SP CD4 and CD8 cells from iPSCs using a cell-free support culture system. This specification shows that the suspension Notch ligand culture system described herein enables the emergence of mature SP T cells, and that Notch signaling obtained with DL4-μ beads described herein overcomes the developmental impairment of T cell maturation observed in plate-bound approaches.

[0154] References TIFF0007866537000001.tif190141TIFF0007866537000002.tif164141

Claims

1. The step involves culturing a sample containing stem cells or progenitor cells in a medium that does not contain stromal cells, together with Notch ligand conjugated onto a Notch ligand suspension support. A method for generating a cell population including T cell lineage cells, The T cell lineage cells are primordial T cells, CD8 + Single positive cell, CD4 + Single positive cell, and / or CD4 + CD8 + It is a double-positive cell, and The Notch ligand conjugated to the Notch ligand suspension support is CD19 + Inhibition of cell development, method.

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

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

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

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

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

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

8. The aforementioned CD34 + The method according to claim 7, wherein the hematopoietic progenitor cells are differentiated from iPSCs.

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

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

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

12. The stem cells or progenitor cells are mouse cells, and the proT cells have a phenotypic CD25 + or CD25 + CD90 + The method according to claim 11, comprising:

13. The method according to claim 12, wherein the mouse cells are lineage-CD117+ Sca-1+ mouse cells.

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

15. The method according to claim 14, wherein the at least one T cell costimulatory molecule is VCAM1.

16. (i) a floating Notch ligand comprising (a) a Notch ligand and (b) a Notch ligand floating support, wherein the Notch ligand is conjugated to the Notch ligand floating support, and (ii) Culture medium that does not contain stromal cells A kit for generating a cell population including T cell lineage cells under culture conditions in a medium that does not contain stromal cells, The T cell lineage cells are primordial T cells, CD8 + Single positive cell, CD4 + Single positive cell, and / or CD4 + CD8 + It is a double-positive cell, and The Notch ligand conjugated to the Notch ligand suspension support is CD19 + Inhibition of cell development, kit.

17. The kit according to claim 16, comprising DL4 conjugated to microbeads, wherein the suspended Notch ligand is present.

18. (iii) At least one T cell costimulatory molecule attached to the suspended support of T cell costimulatory molecules The kit according to claim 16 or 17, further comprising:

19. The kit according to claim 18, wherein the at least one T cell costimulatory molecule is VCAM1.

20. (a) the step of generating T cell lineage cells by the method according to any one of claims 1 to 15, and (b) The step of isolating cells of the T cell lineage. A method for isolating T cell lineage cells, including [specific cells].

21. (i) A floating Notch ligand comprising (a) a Notch ligand and (b) a Notch ligand floating support, (ii) Stem cells or progenitor cells, and (iii) Culture medium that does not contain stromal cells A system for generating a cell population that includes T cell lineage cells, The stem cells or progenitor cells are for suspension culture with Notch ligand, and the Notch ligand suspension support allows the Notch ligand to be suspended in the culture medium. The T cell lineage cells are primordial T cells, CD8 + Single positive cell, CD4 + Single positive cell, and / or CD4 + CD8 + It is a double-positive cell, and The Notch ligand conjugated to the Notch ligand suspension support is CD19 + Inhibition of cell development, system.

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