Methods for generating T cell lineage populations from stem / progenitor cells

Abstract

The present disclosure provides a method for generating a T cell lineage population from progenitor T cells. The method includes culturing progenitor cells in the presence of a Notch signaling ligand, such as Delta-like-4 (DL4), in a volume of at least 7 square centimeters (7 cm) per milliliter of culture volume. 2 A population of cells produced using this method and methods of using the population are provided. The progenitor T cells are provided in a surface area of ​​0.78 to 4.7 cm. 2 Further provided is a method of differentiating a progenitor T cell population enriched for CD4-CD8+TCRγδ+ cells, comprising culturing in the presence of a Notch signaling ligand, e.g., DL4, provided at a surface area of ​​1 / mL.

Description

[Technical Field]

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 441,718, filed January 27, 2023, and U.S. Provisional Patent Application No. 63 / 466,465, filed May 15, 2023, which are incorporated by reference herein as if set forth in their entireties.

[0002] Field The present invention relates generally to in vitro methods for generating T cell lineage populations from progenitor cells and uses thereof. [Background technology]

[0003] Background to the disclosure A variety of feeder cell-based methods are available for the in vitro generation of T cell lineage cells.However, the expression of cell surface proteins by supporting stromal cells in feeder cell-based systems is heterogeneous, leading to various results.In addition, feeder cell-based systems cannot be easily scaled up to meet the needs of clinical production.

[0004] Immobilized Notch signaling ligands, such as DL4, in combination with VCAM-1 have been shown to promote the in vitro generation of precursor T cells in feeder-free and serum-free culture systems (Shukla et al., 2017). Microbeads modified to display DL4 have also been shown to support the in vitro differentiation of T cell lineage cells, although progression to mature lineages such as CD4-CD8+ cells is limited (Trotman-Grant et al., 2021). TCR stimulation of T cell precursors with anti-CD3 antibodies in the absence of Notch signaling has been shown to promote maturation into CD4-CD8αβ+ T cells (Iriguchi et al., 2021). Chimeric antigen receptor (CAR) engagement in the absence of Notch signaling has also been shown to induce the generation of CD4-CD8αβ+ cells from CD4+CD8+ cells in the TRAC- / - cell line (Sjoukje et al., 2022). Currently, there are no reported methods for controlling the in vitro budding of mature T cell lineage populations via Notch signaling. There are also no reports of the specific phenotype of cells emerging from such processes. Summary of the Invention

[0005] Disclosure Overview In a first aspect of the present disclosure, a method for generating a T cell lineage cell population from progenitor T cells is provided. The method includes providing a population of progenitor T cells and culturing the progenitor T cells in a culture volume of at least 7 square centimeters (7 cm) per milliliter. 2 The method comprises culturing precursor T cells in the presence of a Notch signaling ligand provided to a surface area of ​​100 μg / mL.

[0006] In one embodiment, the T cell lineage cell population is enriched for CD4-CD8+ cells.

[0007] In one embodiment, culturing precursor T cells with increasing concentrations of a Notch signaling ligand increases the absolute and / or relative numbers of CD4-CD8+ cells in the cell population.

[0008] In one embodiment, the Notch signaling ligand is 7 to 56 cm 2 / mL, 7.8-55.2cm 2 / mL, or 15.7–55.2 cm 2 / mL of surface area provided.

[0009] In one embodiment, the Notch signaling ligand is provided on a three-dimensional substrate.

[0010] In one embodiment, the three-dimensional substrate is one or more beads.

[0011] In one embodiment, the one or more beads are made of a material selected from the group consisting of polystyrene, iron oxide, and gold.

[0012] In one embodiment, one or more of the beads are made of polystyrene.

[0013] In one embodiment, the Notch signaling ligand is covalently attached to one or more beads.

[0014] In one embodiment, the Notch signaling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

[0015] In one embodiment, the concentration of DL4 is 7.89 x 10 10 ~1.66×10 13 molecules / mL.

[0016] In one embodiment, the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

[0017] In one embodiment, DL4 and VCAM-1 are administered intramuscularly between 7.8 and 55.2 cm 2 / mL of surface area provided.

[0018] In one embodiment, the concentration of DL4 is 7.89 x 10 10~1.66×10 13 molecules / mL, and the concentration of VCAM-1 is 7.89 x 10 10 ~1.66×10 13 molecules / mL.

[0019] In one embodiment, the cell density is 5×10 5 ~2×10 6 cells / mL.

[0020] In one embodiment, the precursor T cells are cultured for at least 3 days, at least 7 days, at least 11 days, or at least 14 days.

[0021] In one embodiment, the precursor T cells are cultured for at least 14 days.

[0022] In one embodiment, the progenitor T cells are re-cultured at least once during or after being cultured in the presence of a surface-bound Notch signaling ligand for at least 3 days, at least 7 days, at least 11 days, or at least 14 days.

[0023] In one embodiment, the CD4-CD8+ cells are CD8αβ+ cells.

[0024] In one embodiment, the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

[0025] In one embodiment, the CD8αβ+ cells are T cell receptor (TCR)− cells, surface CD3 negative (sCD3−) cells, or TCR− / sCD3− cells.

[0026] In one embodiment, the progenitor T cells are derived from pluripotent stem cells.

[0027] In one embodiment, the progenitor T cells are derived from induced pluripotent stem cells (iPSCs).

[0028] In one embodiment, the progenitor T cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

[0029] In one embodiment, the pluripotent stem cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

[0030] In a second aspect of the present disclosure, there is provided a population of CD4-CD8+ cells produced according to the method of the first aspect.

[0031] In one embodiment, the CD4-CD8+ cells are CD8αβ+ cells.

[0032] In one embodiment, the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

[0033] In one embodiment, the CD8αβ+ cells are T cell receptor (TCR)− cells, surface CD3 negative (sCD3−) cells, or TCR− / sCD3− cells.

[0034] In one embodiment, the CD4-CD8+ cells express a chimeric antigen receptor (CAR).

[0035] In one embodiment, the CD4-CD8+ cells express an exogenous T cell receptor (TCR).

[0036] In a third aspect of the present disclosure, there is provided a pharmaceutical composition comprising CD4-CD8+ cells and a pharmaceutically acceptable carrier, wherein the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3-negative (sCD3-) cells, or TCR- / sCD3- cells.

[0037] In one embodiment, the CD4-CD8+ cells are grown in a density of at least 7 square centimeters (7 cm) per milliliter of culture volume. 2They are derived in vitro from precursor T cells by culturing them in the presence of surface-bound Notch signaling ligands at a surface area of ​​1000 kJ / mL.

[0038] In one embodiment, the progenitor T cells are derived in vitro from pluripotent stem cells.

[0039] In a fourth aspect of the present disclosure, a method of treating a disease or condition in a subject is provided. The method comprises culturing at least 7 square centimeters (7 cm) per milliliter of culture volume. 2 The method includes culturing a cell population comprising progenitor T cells in the presence of a surface-bound Notch signaling ligand provided to a surface area of ​​1000 μg / mL, and administering an effective amount of the T cell lineage cell population to a subject in need of treatment for a disease or condition.

[0040] In one embodiment of the methods of treating a disease or condition in a subject provided herein, the T cell lineage cell population is enriched for CD4-CD8+ cells.

[0041] In one embodiment of the methods of treating a disease or condition in a subject provided herein, the CD4 − CD8 + cells are CD8αβ + cells.

[0042] In one embodiment of the methods of treating a disease or condition in a subject provided herein, the CD4-CD8+ cells are T cell receptor (TCR) cells, CD3- cells, or TCR- / CD3- cells.

[0043] In one embodiment of the methods of treating a disease or condition in a subject provided herein, the CD4-CD8αβ+ cells are T cell receptor (TCR) cells, CD3- cells, or TCR- / CD3- cells.

[0044] In one embodiment of the methods of treating a disease or condition in a subject provided herein, the T cell lineage cell population comprises a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

[0045] In one embodiment of the methods of treating a disease or condition in a subject provided herein, a Notch signaling ligand is provided on a three-dimensional substrate.

[0046] In one embodiment of the methods of treating a disease or condition in a subject provided herein, the Notch signaling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

[0047] In one embodiment of the method of treating a disease or condition in a subject provided herein, the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

[0048] In one embodiment of the methods of treating a disease or condition in a subject provided herein, the disease is cancer.

[0049] In a fifth aspect of the present disclosure, there is provided a use of a T cell lineage cell population in the manufacture of a medicament for treating a disease or condition. The T cell lineage cell population is cultured in a volume of at least 7 square centimeters (7 cm) per milliliter of culture volume. 2 The method is produced by a method comprising culturing a cell population comprising progenitor T cells in the presence of a surface-bound Notch signaling ligand provided to a surface area of ​​100 μg / mL.

[0050] In one embodiment of the use of a T cell lineage cell population in the manufacture of a medicament for treating a disease or condition provided herein, the T cell lineage cell population is enriched for CD4-CD8+ cells.

[0051] In one embodiment of the use of a population of T cell lineage cells in the manufacture of a medicament for the treatment of a disease or condition provided herein, the CD4-CD8+ cells are CD8αβ+ cells.

[0052] In one embodiment of the T cell lineage cell population in the manufacture of a medicament for the treatment of a disease or condition provided herein, the CD4-CD8+ cells are T cell receptor (TCR) cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

[0053] In one embodiment of the use of a T cell lineage cell population in the manufacture of a medicament for treating a disease or condition provided herein, the CD4-CD8αβ+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

[0054] In one embodiment of the use of a T cell lineage cell population in the manufacture of a medicament for treating a disease or condition provided herein, the T cell lineage cell population comprises a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

[0055] In one embodiment of the use of a T cell lineage cell population in the manufacture of a medicament for treating a disease or condition provided herein, the Notch signaling ligand is provided on a three-dimensional substrate.

[0056] In one embodiment of the use of the T cell lineage cell population in the manufacture of a medicament for treating a disease or condition provided herein, the Notch signaling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

[0057] In one embodiment of the use of a T cell lineage cell population in the manufacture of a medicament for treating a disease or condition provided herein, the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

[0058] In one embodiment of the use of a T cell lineage cell population in the manufacture of a medicament for treating a disease or condition provided herein, the disease is cancer.

[0059] In a sixth aspect of the present disclosure, a method is provided for differentiating a population of progenitor T cells to generate a differentiated cell population enriched for CD4-CD8+TCRγδ+ cells. The method includes providing a population of progenitor T cells and culturing a population of progenitor T cells that is 0.78 to 4.7 cm 2 The method comprises culturing precursor T cells in the presence of a surface-bound Notch signaling ligand provided to a surface area of ​​1 / mL.

[0060] In one embodiment of the methods provided herein for differentiating a progenitor T cell population to generate a differentiated cell population enriched for CD4-CD8+TCRγδ+ cells, a Notch signaling ligand is provided on a three-dimensional substrate.

[0061] In one embodiment of the methods provided herein for differentiating a progenitor T cell population to generate a differentiated cell population enriched for CD4-CD8+TCRγδ+ cells, the three-dimensional substrate is one or more beads.

[0062] In one embodiment of the method of differentiating a progenitor T cell population to generate a differentiated cell population enriched for CD4-CD8+TCRγδ+ cells provided herein, the one or more beads are comprised of a material selected from the group consisting of polystyrene, iron oxide, and gold.

[0063] In one embodiment of the methods provided herein for differentiating a progenitor T cell population to generate a differentiated cell population enriched for CD4-CD8+TCRγδ+ cells, the one or more beads are made of polystyrene.

[0064] In one embodiment of the methods provided herein for differentiating a progenitor T cell population to generate a differentiated cell population enriched for CD4-CD8+TCRγδ+ cells, the Notch signaling ligand is covalently attached to one or more beads.

[0065] In one embodiment of the methods provided herein for differentiating a progenitor T cell population to generate a differentiated cell population enriched for CD4-CD8+TCRγδ+ cells, the Notch signaling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

[0066] In one embodiment of the methods provided herein for differentiating a progenitor T cell population to generate a differentiated cell population enriched for CD4-CD8+TCRγδ+ cells, the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

[0067] In a seventh aspect of the present disclosure, there is provided a population of CD4-CD8+TCRγδ+ cells generated in vitro according to the method of the sixth aspect.

[0068] In an eighth aspect of the present disclosure, a method for generating a cell population enriched for CD4-CD8+ cells is provided, the method comprising providing a population of progenitor T cells and selecting at least 7 cm 2 The method comprises culturing precursor T cells in the presence of a surface-bound Notch signaling ligand provided on a surface area of ​​1 / mL to generate a cell population enriched for CD4-CD8+ cells.

[0069] In a ninth aspect of the present disclosure, a method of differentiating a population of progenitor T cells is provided, the method comprising providing a population of progenitor T cells and differentiating the progenitor T cells to at least 7 cm 2 The method comprises culturing precursor T cells in the presence of surface-bound Notch signaling ligands provided at a surface area of ​​1000 / mL, and the differentiated cell population is enriched for CD4-CD8+ cells.

[0070] In a tenth aspect of the present disclosure, a method of generating a T cell lineage cell population from progenitor T cells is provided, the method comprising providing a population of progenitor T cells and culturing the progenitor T cells in the presence of a Notch signaling ligand provided on a substrate, wherein the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is at least 1.77 to 1.

[0071] In one embodiment, the T cell lineage cell population is enriched for CD4-CD8+ cells.

[0072] In one embodiment, culturing precursor T cells while increasing the ratio of substrate surface to culture surface area of ​​the culture vessel increases the absolute and / or relative numbers of CD4-CD8+ cells in the cell population.

[0073] In one embodiment, the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is between 1.77:1 and 14:1, between 2:1 and 14:1, or between 4:1 and 14:1.

[0074] In one embodiment, the Notch signaling ligand is provided on a three-dimensional substrate.

[0075] In one embodiment, the three-dimensional substrate is one or more beads.

[0076] In one embodiment, the one or more beads are made of a material selected from the group consisting of polystyrene, iron oxide, and gold.

[0077] In one embodiment, the one or more beads are made of polystyrene.

[0078] In one embodiment, the Notch signaling ligand is covalently attached to one or more beads.

[0079] In one embodiment, the Notch signaling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

[0080] In one embodiment, the concentration of DL4 is 7.89 x 10 10 ~1.66×10 13 molecules / mL.

[0081] In one embodiment, the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

[0082] In one embodiment, DL4 and VCAM-1 are provided on a substrate, and the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is 1.77:1 to 14:1, 2:1 to 14:1, or 4:1 to 14:1.

[0083] In one embodiment, the concentration of DL4 is 7.89 x 10 10 ~1.66×10 13 molecules / mL, and the concentration of VCAM-1 is 7.89 x 10 10 ~1.66×10 13 molecules / mL.

[0084] In one embodiment, the cell density is 5×10 5 ~2×10 6 cells / mL.

[0085] In one embodiment, the precursor T cells are cultured for at least 3 days, at least 7 days, at least 11 days, or at least 14 days.

[0086] In one embodiment, the precursor T cells are cultured for at least 14 days.

[0087] In one embodiment, the progenitor T cells are re-cultured at least once during or after being cultured in the presence of a surface-bound Notch signaling ligand for at least 3 days, at least 7 days, at least 11 days, or at least 14 days.

[0088] In one embodiment, the CD4-CD8+ cells are CD8αβ+ cells.

[0089] In one embodiment, the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

[0090] In one embodiment, the CD8αβ+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells. In one embodiment, the progenitor T cells are derived from pluripotent stem cells.

[0091] In one embodiment, the progenitor T cells are derived from induced pluripotent stem cells (iPSCs).

[0092] In one embodiment, the progenitor T cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

[0093] In one embodiment, the pluripotent stem cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

[0094] In an eleventh aspect of the present disclosure, a method is provided for generating a T cell lineage cell population from progenitor T cells, the method comprising providing a population of progenitor T cells and culturing the progenitor T cells in the presence of a Notch signaling ligand and in the absence of a T cell receptor stimulator, wherein the T cell lineage cell population comprises CD4-CD8+ cells.

[0095] In one embodiment, the CD4-CD8+ cells are surface CD3 negative (sCD3-).

[0096] In one embodiment, the CD4-CD8+ cells are T cell receptor negative (TCR-) cells.

[0097] In one embodiment, the progenitor T cells comprise a nucleic acid encoding a CAR, and the step of culturing the progenitor T cells further comprises culturing in the absence of a CAR activator.

[0098] In one embodiment, the Notch signaling ligand is surface-bound, and the surface-bound Notch signaling ligand is present in an amount of at least 7 square centimeters (7 cm) per milliliter of culture volume. 2 / mL) surface area.

[0099] In one embodiment, the Notch signaling ligand is surface-bound, and the surface-bound Notch signaling ligand is present in an amount of 7 square centimeters (7 cm) per milliliter of culture volume. 2 / mL) ~ 56cm 2 / mL of surface area provided.

[0100] In one embodiment, the Notch signaling ligand is provided on a three-dimensional substrate, wherein the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is at least 1.77 to 1 (1.77:1).

[0101] In one embodiment, the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is between 1.77 to 1 (1.77:1) and 14 to 1 (14:1).

[0102] In one embodiment, the three-dimensional substrate is one or more beads.

[0103] In a twelfth aspect of the present disclosure, a method is provided for generating a T cell lineage cell population from hematopoietic stem and / or progenitor cells, the method comprising providing a population of hematopoietic stem and / or progenitor cells and culturing the hematopoietic stem / progenitor cells in the presence of a Notch signaling ligand and in the absence of a T cell receptor stimulator;

[0104] In one embodiment, the CD4-CD8+ cells are surface CD3 negative (sCD3-).

[0105] In one embodiment, the CD4-CD8+ cells are T cell receptor negative (TCR-) cells.

[0106] In one embodiment, the hematopoietic stem and / or progenitor cells comprise a nucleic acid encoding a CAR, and the step of culturing the hematopoietic stem and / or progenitor cells further comprises culturing in the absence of a CAR activating agent.

[0107] In one embodiment, the Notch signaling ligand is surface bound.

[0108] In one embodiment, the Notch signaling ligand is provided on a three-dimensional substrate.

[0109] In one embodiment, the three-dimensional substrate is one or more beads.

[0110] In one embodiment, the Notch signaling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

[0111] In a thirteenth aspect of the present disclosure, a method for generating a population of T cell lineage cells from progenitor T cells is provided, the method comprising providing a population of progenitor T cells and culturing the progenitor T cells in the presence of surface-bound Notch signaling ligand, wherein the Notch signaling ligand is present in an amount of 7 square centimeters (7 cm) per milliliter of culture volume. 2 / mL) ~ 56cm 2 / mL surface area, and the T cell lineage cell population comprises CD4-CD8+ cells.

[0112] In a fourteenth aspect of the present disclosure, there is provided a method for generating a T cell lineage cell population from progenitor T cells, the method comprising providing a population of progenitor T cells and culturing the progenitor T cells in a culture vessel in the presence of a Notch signaling ligand, wherein the Notch signaling ligand is provided on a three-dimensional substrate, the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is between 1.77 to 1 (1.77:1) and 14 to 1 (14:1), and the T cell lineage cell population comprises CD4-CD8+ cells. [Brief explanation of the drawings]

[0113] In order that the subject matter may be more readily understood, embodiments are illustrated by way of non-limiting examples in the accompanying drawings, in which: FIG.

[0114] [Figure 1A]Figure 1A is a schematic diagram of the induced pluripotent stem cell (iPSC) to T cell maturation process, highlighting progenitor T (proT) cell to CD4-CD8+ (CD8 single positive, CD8SP) cell progression resulting from exposure of iPSC to a three-dimensional engineered thymic niche (3D ETN) in a concentration-responsive manner.

[0115] [Figure 1B] Figure 1B is a graph showing the percentage of cells comprising four cell populations: CD4-CD8+ (CD8SP), CD4+CD8+ (double positive, DP), CD4+CD8- (CD4 immature single positive, CD4ISP), and CD4-CD8- (double negative, DN). Precursor T cells were cultured for 7 days in two-dimensional engineered thymic niches (2D ETNs) and then cultured with StemSpan™ lymphocyte differentiation coating material (SCT or commercial coating; STEMCELL Technologies) and 3D ETNs (increasing bead concentrations: 0.27 x 107 beads / mL - 8.1 x 107 beads / mL (0.5x - 1.5x bead concentrations)).

[0116] [Figure 1C] Figure 1C is a graph showing the percentage of CD4-CD8+ cells that are CD8αα+ or CD8αβ+ after culturing precursor T cells for 7 days with 2D ETNs, SCTs, and 3D ETNs at increasing bead concentrations (0.27 × 107 beads / mL to 8.1 × 107 beads / mL (0.5 × bead dose)).

[0117] [Figure 2A] Figure 2A is a graph showing cell proliferation as a function of 3D ETN bead concentration after 7 days of culture. The two input populations (A and C) are plotted separately. Cell numbers were measured using a Cellaca® automated cell counter.

[0118] [Figure 2B]Figure 1B shows the viability of the four cell populations shown in Figure 1B: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4ISP), and CD4-CD8- (DN) after culturing precursor T cells with increasing 3D ETN bead concentrations (0.27 x 107 beads / mL to 8.1 x 107 beads / mL (0.5x bead dose)) for 7 days. Cell viability was measured by acridine orange / propidium iodide using a Cellaca® automated cell counter.

[0119] [Figure 3A] Figure 3A is a graph showing the percentage of cells comprising the four cell populations described in Figure 1B. Progenitor T cells were cultured for 7 days with 2D ETNs, SCTs, and 3D ETNs at increasing bead concentrations (0.02x-2x bead concentration, or 1.08 x 106 beads / mL to 10.8 x 107 beads / mL (expressed as 0.1-10 x 107 beads / mL)) to determine the percentage of CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4ISP), and CD4-CD8- (DN) as measured by flow cytometry.

[0120] [Figure 3B] Figure 3B is a graph showing the percentage of cells comprising the four cell populations described in Figure 3A. Progenitor T cells were cultured for 14 days with 2D ETNs, SCTs, and 3D ETNs at increasing bead concentrations (0.02x-2x bead concentration, or 1.08 x 106 beads / mL-10.8 x 107 beads / mL (shown as 0.1-10 x 107 beads / mL)) as measured by flow cytometry: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4ISP), and CD4-CD8- (DN).

[0121] [Figure 4A]Figure 4A is a graph showing the percentage of cells that are CD3-TCRαβ-, CD3+TCRαβ-, or CD3+TCRαβ+, as measured by flow cytometry, after culturing precursor T cells with increasing bead concentrations (0.02 x 2x bead dose, or 1.08 x 106 beads / mL to 10.8 x 107 beads / mL (shown as 0.1 to 10 x 107 beads / mL)) with 2D ETNs, SCTs, and 3D ETNs for 14 days.

[0122] [Figure 4B] Figure 4B is a graph showing TCRαβ and TCRγδ expression in cell populations, as measured by flow cytometry, after culturing precursor T cells with increasing bead concentrations (0.02 × 2 × bead dose, or 1.08 × 106 beads / mL to 10.8 × 107 beads / mL (shown as 0.1 to 10 × 107 beads / mL)) for 14 days with 2D ETNs, SCTs, and 3D ETNs.

[0123] [Figure 4C] Figure 4C is a graph showing the expression of TCRαβ and TCRγδ in CD8+CD4+ (DP) cells, as determined by flow cytometry, after 14 days of culturing precursor T cells in 2D ETNs, SCTs, and 3D ETNs at increasing bead concentrations (0.02×–2× bead dose, or 1.08×106 beads / mL–10.8×107 beads / mL (shown as 0.1–10×107 beads / mL)).

[0124] [Figure 4D] Figure 4D is a graph showing TCRαβ and TCRγδ expression in CD4-CD8+ (CD8SP) cells, as measured by flow cytometry, after culturing precursor T cells with increasing bead concentrations (0.02 × 2 × bead dose, or 1.08 × 106 beads / mL to 10.8 × 107 beads / mL (shown as 0.1 to 10 × 107 beads / mL)) for 14 days with 2D ETNs, SCTs, and 3D ETNs.

[0125] [Figure 5A] Figure 5A is a graph showing the percentage of viable cells as a function of 3D ETN bead concentration after 7 days of culture. Cell viability was measured by acridine orange + / propidium iodide using a Cellaca® automated cell counter.

[0126] [Figure 5B] Figure 5B is a graph showing the percentage of viable cells as a function of 3D ETN bead concentration after 14 days of culture. Cell viability was measured by acridine orange + / propidium iodide using a Cellaca® automated cell counter.

[0127] [Figure 5C] Figure 5C is a graph showing the cell viability of the four cell populations shown in Figure 3A. Progenitor T cells were cultured for 7 days with increasing 3D ETN bead concentrations (1.08 x 10 beads / mL to 10.8 x 10 beads / mL (0.02x to 2x bead dose)) to determine the viability of CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4ISP), and CD4-CD8- (DN).

[0128] [Figure 5D] Figure 5D is a graph showing the cell viability of the four cell populations shown in Figure 3A. Progenitor T cells were cultured for 14 days with increasing 3D ETN bead concentrations (1.08 x 10 beads / mL to 10.8 x 10 beads / mL (0.02x to 2x bead dose)) to determine the viability of CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4ISP), and CD4-CD8- (DN).

[0129] [Figure 6A] Figure 6A is a graph showing cell number over time for CD19+ A549 cells ("target cells") co-cultured with primary CD8+ CAR-T cells (left) or iPSC-derived CAR+ CD4- CD8+ (CD8SP) cells (right) at various effector:target (E:T) cell ratios.

[0130] [Figure 6B] FIG. 6B is a graph that expands the data from FIG. 6A, omitting the control cell-only control.

[0131] [Figure 7] Figure 7 is a graph showing cell proliferation (left axis) and viability (right axis) of cells cultured with 2D ETNs and 3D ETNs in 12-well cell culture plates (wp) and with 3D ETNs in stirred tank reactors (STRs). 3D ETN bead concentrations of 0.54 x 107 beads / mL (0.1 x bead dose) and 2.7 x 107 beads / mL (0.5 x bead dose) were utilized in both the 12-well cell culture plates and STRs.

[0132] [Figure 8A] Figure 8A is a graph showing quantification of flow cytometry data showing the percentage of CD4+CD8- (CD4ISP), CD4-CD8- (DN), CD4-CD8+ (CD8SP), and CD4+CD8+ (DP) cells after culturing cells with 2D ETNs and 3D ETNs in 12-well cell culture plates (wp), and with 3D ETNs in STRs. 3D ETN bead concentrations of 0.54 x 107 beads / mL (0.1 x bead dose) and 2.7 x 107 beads / mL (0.5 x bead dose) were utilized in both the 12-well cell culture plates and STRs.

[0133] [Figure 8B] Figure 8B is a graph showing quantification of flow cytometry data showing the percentage of cells expressing CD8αα and / or CD8αβ after culturing cells with 2D ETNs and 3D ETNs in 12-well cell culture plates (wp), and with 3D ETNs in STRs. 3D ETN bead concentrations of 0.54 x 107 beads / mL (0.1 x bead dose) and 2.7 x 107 beads / mL (0.5 x bead dose) were utilized in both the 12-well cell culture plates and STRs.

[0134] [Figure 9A] FIG. 9A is a flow cytometry plot of CD5 and CD7 expression in untransduced (top, NTD) and TCR-transduced precursor T cells (bottom).

[0135] [Figure 9B] FIG. 9B is a flow cytometry plot of CD4 and CD8α (CD8A) expression in untransduced (top, NTD) and TCR-transduced precursor T cells (bottom).

[0136] [Figure 9C] FIG. 9C is a flow cytometry plot of CD3 and TCR expression in untransduced (top, NTD) and TCR-transduced precursor T cells (bottom).

[0137] [Figure 10A] Figure 10A is a flow cytometry plot of CD5 and CD7 expression in untransduced (top, NTD) and TCR-transduced (bottom) cells after culturing the precursor T cell population for 7 days at a 3D ETN bead concentration of 1.08 x 10 beads / mL (2x bead dose).

[0138] [Figure 10B] Figure 10B is a flow cytometry plot of CD4 and CD8α (CD8A) expression in untransduced (top, NTD) and TCR-transduced (bottom) cells after 7 days of culture of precursor T cell populations at a 3D ETN bead concentration of 1.08 x 10 beads / mL (2x bead dose).

[0139] [Figure 10C]Figure 10C is a flow cytometry plot of CD3 and TCRαβ (TCRab) expression in untransduced (top, NTD) and TCR-transduced (bottom) cells after 7 days of culture of a progenitor T cell population with a 3D ETN bead concentration of 1.08 x 10 beads / mL (2x bead dose).

[0140] [Figure 10D] FIG. 10D is a flow cytometry plot of CD8β (CD8B) and CD8α (CD8A) expression in untransduced (top, NTD) and TCR-transduced (bottom) cells after 7 days of culture of a progenitor T cell population with a 3D ETN bead concentration of 1.08×108 beads / mL (2× bead dose).

[0141] [Figure 11A] FIG. 11A is a flow cytometry plot of CD5 and CD7 expression in untransduced (top, NTD), TCR-transduced (middle), and CD8β-enriched TCR-transduced (bottom) cells after 11 days of culture of a progenitor T cell population with a 3D ETN bead concentration of 1.08×10 beads / mL (2× bead dose).

[0142] [Figure 11B] Figure 11B is a flow cytometry plot of CD4 and CD8α (CD8A) expression in untransduced (top, NTD), TCR-transduced (middle), and CD8β-enriched, TCR-transduced (bottom) cells after 11 days of culture of precursor T cell populations at a 3D ETN bead concentration of 1.08 x 108 beads / mL (2x bead dose).

[0143] [Figure 11C]Figure 11C is a flow cytometry plot of CD3 and TCRαβ (TCRab) expression in untransduced (top, NTD), TCR-transduced (middle), and CD8β-enriched, TCR-transduced (bottom) cells after 11 days of culture of precursor T cell populations at a 3D ETN bead concentration of 1.08 x 108 beads / mL (2x bead dose).

[0144] [Figure 11D] Figure 11D is a flow cytometry plot of CD8β (CD8B) and CD8α (CD8A) expression in untransduced (top, NTD), TCR-transduced (middle), and CD8β-enriched, TCR-transduced (bottom) cells after 11 days of culture of precursor T cell populations at a 3D ETN bead concentration of 1.08 x 108 beads / mL (2x bead dose).

[0145] [Figure 12A] Figure 12A is a graph showing the cumulative fold proliferation over time of non-transduced (NTD, squares) and TCR-transduced (TCR, circles) cells cultured at a 3D ETN bead concentration of 1.08 x 10 beads / mL (2x bead dose). D17 and D21 correspond to 7 and 11 days of culture with 3D ETNs, respectively.

[0146] [Figure 12B] Figure 12B is a graph showing cell viability over time for non-transduced (NTD, squares) and TCR-transduced (TCR, circles) cells cultured at a 3D ETN bead concentration of 1.08 x 10 beads / mL (2x bead dose). Days D17 and D21 correspond to 7 and 11 days of culture with 3D ETN, respectively. TCR-transduced cells were also analyzed after CD8β selection (post-CD8B).

[0147] [Figure 12C]Figure 12C is a graph showing the ratio of cell input / output over time for non-transduced (NTD, squares) and TCR-transduced (TCR, circles) cells cultured at a 3D ETN bead concentration of 1.08 x 10 beads / mL (2x bead dose). D17 and D21 correspond to 7 and 11 days of culture with 3D ETNs, respectively.

[0148] [Figure 13A] Figure 13A is a graph showing the percentage of cells comprising four cell populations: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4ISP), and CD4-CD8- (DN) after 7 days of culture of progenitor T cells with increasing bead concentrations (0.27 x 107 beads / mL to 8.1 x 107 beads / mL (0.5x bead dose)) in 2D ETNs, SCTs (commercially coated), and 3D ETNs.

[0149] [Figure 13B] Figure 13B is a graph showing the expression of genes DTX1, TCF7, BCL11B, and GATA3 after culturing precursor T cells for 3 days with 2D ETNs, SCT (commercially coated), and 3D ETNs at increasing bead concentrations (0.27 x 107 beads / mL to 8.1 x 107 beads / mL (0.5x to 1.5x bead dose)).

[0150] [Figure 13C] Figure 13C is a uniform manifold approximation and projection (UMAP) plot of a 13-color flow cytometry dataset of precursor T cells cultured for 7 days in 2D ETNs, SCT (commercially coated), and 3D ETNs at increasing bead concentrations (0.27 × 10 beads / mL to 8.1 × 10 beads / mL (0.5 × 1.5 × bead dose)).

[0151] [Figure 13D]Figure 13D is a UMAP plot showing cells within six clusters identified by unsupervised machine learning using FlowSOM (Van Gassen et al., 2015). Each cluster is defined by a distinct combination of expressed surface proteins, as identified by flow cytometry.

[0152] [Figure 13E] FIG. 13E is a bar graph showing the relative levels of cell surface proteins expressed in each cluster shown in FIG. 13D (e.g., cluster 1 was identified as describing CD4+CD8+ (DP) cells).

[0153] [Figure 13F] Figure 13F is a bar graph showing the percentage of each cluster identified in Figure 13D as a function of cell culture with 2D ETNs, SCT (commercially coated), and 3D ETNs at increasing bead concentrations (0.27 x 10 beads / mL to 8.1 x 10 beads / mL (0.5 x to 1.5 x bead dose)).

[0154] [Figure 13G] Figure 13G is a flow cytometry plot of CD5 and CD7 expression, CD4 and CD8α (CD8A) expression, and CD8β (CD8B) and CD8α (CD8A) expression for cells cultured for 14 days in 3D ETN at a concentration of 5.4 x 107 beads / mL (1 x bead dose).

[0155] [Figure 14A] Figure 14A is a UMAP plot of single-cell RNA sequencing (scRNAseq) data showing the developmental trajectory of T cell maturation stages in the human thymus. Data obtained from Park et al., 2020.

[0156] [Figure 14B]Figure 14B is a UMAP plot of T cell maturation in the human thymus. Selected single-cell RNA-seq data from the Thymic Atlas (Park et al., 2020) were represented as a UMAP and annotated by cell type, including the NK-T cell ("NKT") cell type.

[0157] [Figure 15A] Figure 15A is a UMAP plot showing the enrichment of Notch and TCR signaling through T cell maturation in the thymus, respectively. Data taken from Park et al., 2020.

[0158] [Figure 15B] Figure 15B is a UMAP plot showing the enrichment of Notch and TCR signaling through T cell maturation in the thymus, respectively. Data taken from Park et al., 2020.

[0159] [Figure 15C] Figure 15C is a graph showing the enrichment of Notch and TCR signaling at various T cell developmental stages in the thymus (single sample gene set enrichment analysis, ssGSEA). The developmental stages are early DN cells, DNI; proliferating DN cells, DN(p); resting DN cells, DN(q); proliferating DP cells, DP(p); resting DP cells, DP(q); early stage alpha-beta T cells, αβT; and CD8+ T cells, CD8+ T cells. Data and cell labels were obtained from Park et al., 2020.

[0160] [Figure 15D] Figure 15D is a UMAP plot of single-cell RNAseq data (Park et al., 2020) annotated by collective central Notch gene regulatory network ("Notch GRN", Figure 15D) and TCR signaling ("TCR signaling", Figure 15E) transcriptional signals (ssGEA scores).

[0161] [Figure 15E]Figure 15E is a UMAP plot of single-cell RNAseq data (Park et al., 2020) annotated by collective central Notch gene regulatory network ("Notch GRN", Figure 15D) and TCR signaling ("TCR signaling", Figure 15E) transcriptional signals (ssGEA scores).

[0162] [Figure 16A] Figure 16A shows a UMAP plot of primary unstimulated CD8αβ T cells (CD8ab UnStim) and iPSC-derived CD4-CD8+ cells cultured at a 3D ETN bead concentration (ipsD CD8ab UnStim) of 5.4 x 107 beads / mL (1x bead dose). Cell annotation was performed with the SingleR package, referencing the Thymic Cell Atlas (Parket et al., 2020).

[0163] [Figure 16B] Figure 16B is a graph quantifying the percentage of cells corresponding to T cell maturation stages in the thymus (Park et al., 2020) for primary unstimulated primary CD8αβ T cells (CD8ab UnStim) and iPSC-derived CD4-CD8+ cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of 5.4 x 107 beads / mL (1 x bead dose).

[0164] [Figure 17A] Figure 17A is a UMAP plot showing the T cell status (Andreatta et al., 2021) derived from ProjecTIL for primary unstimulated CD8αβ+ T cells (CD8ab UnStim) and iPSC-derived CD4-CD8+ cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of 5.4 x 107 beads / mL (1x bead dose).

[0165] [Figure 17B]Figure 17B is a graph quantifying the percentage of T cells in each condition from Figure 17A in primary unstimulated CD8αβ+ T cells (CD8ab UnStim) and unstimulated iPSC-derived CD4-CD8+ T cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of 5.4 x 107 beads / mL (1 x bead dose).

[0166] [Figure 18A] Figure 18A is a graph showing the expression of selected genes in primary unstimulated CD8αβ T cells (CD8ab UnStim) and iPSC-derived CD4− CD8+ cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of 5.4×107 beads / mL (1× bead dose) as determined by scRNAseq.

[0167] [Figure 18B] Figure 18B is a graph illustrating the enrichment of gene sets related to Notch signaling, TCR signaling, and downstream TCR signaling in primary unstimulated CD8αβ+ T cells (CD8ab UnStim) and iPSC-derived CD4− CD8+ cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of 5.4×107 beads / mL (1× bead dose) as determined by scRNAseq.

[0168] [Figure 19A] Figure 19A is a flow cytometry plot of CD5 and CD7 expression for unmodified iPSC-derived cells after 10 days of culture of an iPSC-derived hematopoietic stem / progenitor cell population containing CD34+ cells with a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose).

[0169] [Figure 19B]Figure 19B is a flow cytometry plot of CD34 and CD7 expression for unmodified iPSC-derived cells after 10 days of culture of an iPSC-derived hematopoietic stem / progenitor cell population containing CD34+ cells with a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose).

[0170] [Figure 19C] Figure 19C is a flow cytometry plot of CD4 and CD8α (CD8A) expression for unmodified iPSC-derived cells after 10 days of culture of an iPSC-derived hematopoietic stem / progenitor cell population containing CD34+ cells with a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose). [Figure 19D] Figure 19D is a flow cytometry plot of CAR expression and CD7 expression for unmodified iPSC-derived cells after 10 days of culture of an iPSC-derived hematopoietic stem / progenitor cell population containing CD34+ cells with a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose).

[0171] [Figure 20A] Figure 20A is a flow cytometry plot of CD5 and CD7 expression for CAR-modified iPSC-derived cells after 10 days of culture of an iPSC-derived hematopoietic stem / progenitor cell population containing CD34+ cells with a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose).

[0172] [Figure 20B] Figure 20B is a flow cytometry plot of CD34 and CD7 expression for CAR-modified iPSC-derived cells after 10 days of culture of an iPSC-derived hematopoietic stem / progenitor cell population containing CD34+ cells with a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose).

[0173] [Figure 20C]Figure 20C is a flow cytometry plot of CD4 and CD8α (CD8A) expression for CAR-modified iPSC-derived cells after 10 days of culture of an iPSC-derived hematopoietic stem / progenitor cell population containing CD34+ cells with a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose).

[0174] [Figure 20D] Figure 20D is a flow cytometry plot of CAR expression and CD7 expression for CAR-modified iPSC-derived cells after 10 days of culture of an iPSC-derived hematopoietic stem / progenitor cell population containing CD34+ cells with a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose).

[0175] [Figure 21A] Figure 21A is a graph quantifying the percentage of CD4-CD8α+ (CD8SP) cells relative to unmodified iPSC-derived cells ("4A1") or CAR-modified iPSC-derived cells ("4B3") after culturing precursor T cells at a density of 2x106 cells / mL or 5x105 cells / mL with a 3D ETN bead concentration of 1.08x108 beads / mL (2x bead dose) for 7 days (after 17 days of culture on 3D ETNs from a hematopoietic stem / progenitor cell starting population).

[0176] [Figure 21B] Figure 21B is a graph quantifying the percentage of CD8αβ+ (CD8ab+) cells relative to unmodified iPSC-derived cells ("4A1") or CAR-modified iPSC-derived cells ("4B3") after culturing precursor T cells at a density of 2×106 cells / mL or 5×105 cells / mL with a 3D ETN bead concentration of 1.08×108 beads / mL (2× bead dose) for 7 days (after 17 days of culture on 3D ETNs from a hematopoietic stem / progenitor cell starting population).

[0177] [Figure 21C]Figure 21C is a graph quantifying cell viability of unmodified iPSC-derived cells ("4A1") or CAR-modified iPSC-derived cells ("4B3") after culturing precursor T cells at a density of 2 x 106 cells / mL or 5 x 105 cells / mL with a 3D ETN bead concentration of 1.08 x 108 beads / mL (2x bead dose) for 7 days (after 17 days of culture on 3D ETNs from a hematopoietic stem / progenitor cell starting population).

[0178] [Figure 21D] Figure 21D is a graph quantifying the percentage of CAR+ cells relative to unmodified iPSC-derived cells ("4A1") or CAR-modified iPSC-derived cells ("4B3") after culturing precursor T cells at a density of 2 x 106 cells / mL or 5 x 105 cells / mL with a 3D ETN bead concentration of 1.08 x 108 beads / mL (2x bead dose) for 7 days (after 17 days of culture on 3D ETNs from a hematopoietic stem / progenitor cell starting population).

[0179] [Figure 22A] Figure 22A is a graph quantifying the percentage of CD4-CD8α+ (CD8SP) cells before and after CD8α enrichment of unmodified iPSC-derived cells ("4A1") and CAR-modified iPSC-derived cells ("4B3") after 11 days of culture of cells at a density of 2×106 cells / mL or 5×105 cells / mL at a 3D ETN bead concentration of 1.08×108 beads / mL (2× bead dose)

[0180] [Figure 22B]Figure 22B is a graph quantifying the percentage of CD8αβ+ (CD8ab+) cells before and after CD8α enrichment of unmodified iPSC-derived cells ("4A1") and CAR-modified iPSC-derived cells ("4B3") after 11 days of culture of cells at a density of 2×106 cells / mL or 5×105 cells / mL at a 3D ETN bead concentration of 1.08×108 beads / mL (2× bead dose) [Figure 22C] Figure 22C is a graph quantifying the percent cell viability before and after CD8α enrichment of unmodified iPSC-derived cells ("4A1") and CAR-modified iPSC-derived cells ("4B3") after 11 days of cell culture (after 21 days of culture with 3D ETNs from hematopoietic stem / progenitor cell starting populations) at a density of 2x106 cells / mL or 5x105 cells / mL at a 3D ETN bead concentration of 1.08x108 beads / mL (2x bead dose).

[0181] [Figure 22D] Figure 22D is a graph quantifying the percentage of CAR expression before and after CD8α enrichment of unmodified iPSC-derived cells ("4A1") and CAR-modified iPSC-derived cells ("4B3") after 11 days of cell culture (after 21 days of culture with 3D ETNs from hematopoietic stem / progenitor cell starting populations) at a density of 2x106 cells / mL or 5x105 cells / mL at a 3D ETN bead concentration of 1.08x108 beads / mL (2x bead dose).

[0182] [Figure 23A] FIG. 23A is a graph showing cell viability over time for unmodified iPSC-derived cells ("4A1").

[0183] [Figure 23B] Figure 23B is a graph showing cell viability over time for CAR-modified iPSC-derived cells ("4B3").

[0184] [Figure 24A] FIG. 24A is a graph showing fold expansion over time for unmodified iPSC-derived cells ("4A1").

[0185] [Figure 24B] Figure 24B is a graph showing fold expansion over time for CAR-modified iPSC-derived cells ("4B3").

[0186] [Figure 25] Figure 25 is a schematic diagram showing standard (gray arrows) and non-physiological (black arrows) differentiation of pluripotent stem cells (e.g., induced pluripotent stem cells, iPSCs) into CD8+CD4- cells. HSPC, hematopoietic stem / progenitor cells; DN, double-negative cells; ISP, intermediate single-positive cells; DP, double-positive cells; SP, single-positive cells.

[0187] [Figure 26A] FIG. 26A is a graph quantifying the percentage of progenitor T cells (CD5+CD7+) and myeloid cells (CD33+CD7-) after differentiation of iPSC-derived HSPCs with various concentrations of ETN.

[0188] [Figure 26B] Figure 26B is a representative flow cytometry plot of differentiation of iPSC-derived HSPCs at ETN doses of 5.4 x 106 beads / mL (0.1x bead dose) or 5.4 x 107 beads / mL (1x bead dose), outlining the progenitor T cell population (CD5+CD7+) and myeloid cell population (CD33+CD7-).

[0189] [Figure 27A]Figure 27A is a graph showing quantification of flow cytometry data showing the percentage of CD4-CD8- (double negative, DN), CD4+CD8+ (double positive, DP), CD4+CD8- (CD4ISP), and CD4-CD8+ (CD8 single positive, CD8SP) cells after culturing two cell lines: TRAC-deficient (TRAC- / -, left plot) and TRAC-deficient CD19-CAR-operated (TRAC- / -CD19-CAR+, right plot) with 3D ETN at a range of bead doses.

[0190] [Figure 27B] Figure 27B is a flow cytometry plot of CD4 and CD8α (CD8a) expression in TRAC-deficient CD19-CAR engineered cells after culture with 3D ETN at three doses. The annotated boxes in the plot on the left indicate the populations as quantified in Figure 26A.

[0191] [Figure 28A] Figure 28A is a graph showing changes in gene expression after 2 days of culture with 3D ETNs that are positively or negatively associated with the CD8SP phenotype, as identified by a machine learning workflow (see Figure 28B).

[0192] [Figure 28B] Figure 28B is a graph showing the accuracy distribution of models used in the machine learning workflow.

[0193] [Figure 28C] Figure 28C is a graph showing the correlation between expression of the gene DTX1, identified as the most positive predictive feature, and the percentage of CD4-CD8a+ (CD8SP) cells for both TRAC-deficient (TRAC KO, circles) and TRAC-deficient CD19-CAR-operated (TRAC KO+CAR, x numbers) cell lines.

[0194] [Figure 28D]Figure 28D is a graph showing the expression of selected genes in TRAC-deficient (TRAC- / -) and TRAC-deficient, CD19-CAR-engineered (TRAC- / -CD19-CAR+) cell lines across a range of bead doses (z-score normalized mean expression values ​​shown, n=3).

[0195] [Figure 28E] Figure 28E is a graph showing changes in gene expression after 2 days of culture with 3D ETNs that are positively or negatively associated with the CD8SP phenotype, as identified by the machine learning workflow (see Figure 28F). The average weighted regression coefficients of the top 12 predictive features are shown.

[0196] [Figure 28F] Figure 28F is a graph showing the accuracy distribution of models used in the machine learning workflow. Day 17 CD8αSP phenotype is predicted with good accuracy (85% cross-validation) using a machine learning workflow trained on day 12 gene expression, independent of cell line.

[0197] [Figure 28G] Figure 28G is a graph showing the correlation between expression of NOTCH1, the gene identified as the most positive predictive feature, and bead dose for wild-type (WT, squares), TRAC-deficient (TRAC- / -, circles), and TRAC-deficient CD19-CAR-operated (TRAC- / -CD19-CAR+, x) cell lines.

[0198] [Figure 28H] Figure 28H is a graph showing expression of select genes in wild-type (WT, left), TRAC-deficient (TRAC- / -, center), and TRAC-deficient CD19-CAR-engineered (TRAC- / -CD19-CAR+, right) cell lines across a range of bead doses (z-score normalized mean expression is shown, n=3 or 6).

[0199] [Figure 28I]FIG. 28I is a graph showing a variance partitioning analysis showing that both bead dose and the interaction between day and cell line effect account for a large portion of the transcriptional variation.

[0200] [Figure 28J] Figure 28J is a graph showing differentially expressed pathways (using ssGSEA scores) across low and high bead doses and culture time (days 12 and 17) in a TRAC-deficient CD19-CAR engineered cell line (TRAC- / -CD19-CAR+).

[0201] [Figure 29A] Figure 29A is a UMAP plot of the single-cell transcriptomes of iPS cell-derived T cells, peripheral blood-derived CD8+ T cells (PBMC-T), and cord blood-derived CD8+ T cells (CB-T).

[0202] [Figure 29B] Figure 29B is a graph showing cell populations for iPSC-derived T cells, CD8+ T cells from peripheral blood (PBMC-T) and umbilical cord blood (CB), sorted using an algorithm derived from ProjecTIL (Haradhvala et al., 2022).

[0203] [Figure 29C] Figure 29C is a graph showing gene expression of T cell markers, Notch-responsive genes, innate lymphoid cells (ILCs), and transcripts associated with T cell activation and exhaustion in iPS cell-derived T cells, peripheral blood-derived CD8+ T cells (PBMC-T), and cord blood-derived CD8+ T cells (CB-T).

[0204] [Figure 29D] Figure 29D is a graph showing the differential enrichment of selected gene signatures in each cell cluster for iPSC-derived T cells (iPSC-T, numbers 1-4), peripheral blood-derived CD8+ T cells (PBMC-T), and umbilical cord blood-derived CD8+ T cells (CB-T) by single sample gene set enrichment analysis (GSEA).

[0205] [Figure 29E] Figure 29E is a graph showing 1,473 gene signatures differentially enriched in each cell cluster for iPSC-derived T cells (iPSC-T, numbers 1-4), peripheral blood (PBMC-T), and umbilical cord blood (CB-T) derived CD8+ T cells by single sample gene set enrichment analysis (GSEA).

[0206] [Figure 29F] Figure 29F is a UMAP of single-cell transcriptomes for iPSC-derived T cells through differentiation (CD34+, ProT, CD8+ SP stage and after CD19 antigen stimulation) versus primary lymphocytes (CD4+ T cells, CD8+ T cells, CD56+ NK cells, and T cells activated by Dynabeads (CD3 / CD28 stimulation).

[0207] [Figure 29G] Figure 29G is a bubble plot of selected genes associated with blood progenitors, T cell lineage commitment, canonical T cell function, innate innate lymphoid cells, cytotoxic T cells, exhausted T cells, and Notch response elements.

[0208] [Figure 29H] Figure 29H is a graph showing the expression of the transcriptional pathway signatures (ssGSEA scores) shown across samples.

[0209] [Figure 29I] Figure 29I is a UMAP plot of single-cell transcriptomes focused on iPSC-CD8+ cells, iPSC-CD8+ cells after antigen stimulation, and activated T cells, annotated by transcriptional similarity to developing thymocytes (DN proliferating, DN early, DN quiescent, CD8+ T, NKT, gamma delta T (GDT), DP proliferating, and Treg).

[0210] [Figure 29J]Figure 29J is a plot of the emergence of different cell phenotypes in CAR-iPSC-derived cells as determined by single-cell lineage inference and pseudotime organization.

[0211] [Figure 29K] Figure 29K is a plot of transcriptional pathway emergence in CAR-iPSC-derived cells as determined by single-cell lineage inference and pseudotime organization.

[0212] [Figure 30A] Figure 30A is a graph showing target cell (CD19-expressing A549) kinetics across four in vitro cytotoxicity assays using primary CAR-Ts versus iPSC-CD8 cells. Effector T cells were added at an E:T ratio of 2:1 (mean ± standard deviation, n=3), and cell density readout using Incucyte® live cell imaging.

[0213] [Figure 30B] Figure 30B is a graph showing in vitro cytotoxicity versus effector cell expansion for primary CARTs versus iPSC-CARTs (n=2 replicates). Cumulative cytotoxicity is the sum of the relative change in AUC from the target cell-only control for each stimulus (not shown).

[0214] [Figure 30C] Figure 30C is a graph showing secretion of the inflammatory cytokines IFN-γ, granzyme B, and tumor necrosis factor alpha (TNFα) by iPSC-derived T cells and primary CAR-T cells when co-cultured with antigen-expressing target cells.

[0215] [Figure 31A] Figure 31A is a schematic diagram of the 3D engineered thymic niche (ETN) platform for scalable T cell manufacturing using iPSC-derived CD34+ HPCs.

[0216] [Figure 31B]Figure 31B is a graph showing quantification of flow cytometry data showing the percentages of CD4-CD8- (double negative, DN), CD4+CD8- (CD4ISP), CD4+CD8+ (double positive, DP), and CD4-CD8+ (CD8 single positive, CD8SP) cells following microplate culture differentiation of iPSC-derived CD34+ HPCs generated in microplate culture, with differentiation of iPSC-derived HPCs generated in microplate culture shown as a reference (mean ± SEM, 3 independent STR runs, n = 6 bioreactors).

[0217] [Figure 31C] Figure 31C is a graph showing quantification of flow cytometry data showing the percentage of CD8α (CD8a)- and CD8β (CD8b)-expressing cells after microplate culture differentiation of iPSC-derived CD34+ HPCs (mean ± SEM, 3 independent STR runs, n = 6 bioreactors).

[0218] [Figure 32A] Figure 32A is a graph showing quantification of flow cytometry data showing the percentage of CD4-CD8- (double negative, DN), CD4+CD8- (CD4ISP), CD4+CD8+ (double positive, DP), and CD4-CD8+ (CD8 single positive, CD8SP) cells after 7 days of microplate culture of iPSC-derived ProT cells with a 30-fold range of ETN doses (0.1x to 3x bead dose, or 5.4 x 106 beads / mL to 1.62 x 108 beads / mL).

[0219] [Figure 32B] Figure 32B is a graph showing expression of CD8A, IL7R, CD4, and BCL11A genes over 7 days after culture with ETN at 2x or 3x (1.08x108 beads / mL or 1.62x108 beads / mL) bead dose.

[0220] [Figure 33A]Figure 33A is a graph showing quantification of flow cytometry data showing CD7 and CD5 expression and percentage of total ProT cell population after 10 days of culture of iPSC-derived CD34+ cells in microplates and STRs with 3D ETNs. Two CD34+ cell banks were seeded in duplicate at 1 x 104 and 5 x 104 cells / mL in DASbox® bioreactors (Eppendorf) and 24-well plates.

[0221] [Figure 33B] Figure 33B is a graph showing quantification of flow cytometry data showing CD7 and CD5 expression and percentage of the total ProT cell population after 10 days of culture of CD34+ cells with 3D ETNs. One CD34+ cell bank was seeded at 5 x 104 cells / mL into eight bioreactors.

[0222] [Figure 34A] Figure 34A is a graph showing quantification of flow cytometry data showing the percentage of CD4-CD8- (double negative, DN), CD4+CD8- (CD4ISP), CD4+CD8+ (double positive, DP), and CD4-CD8+ (CD8 single positive, CD8SP) cells, as well as the yield of CD8+ cells after culturing ProT cells with ETN at 2.5x bead dose (1.35x108 beads / mL) for 11 days in eight bioreactors.

[0223] [Figure 34B] Figure 34B is a graph showing quantification of flow cytometry data showing the percentage of CD8α+, CD8αβ+, and CD8β+ cells at day 8 matured with ETN at 2.5x bead dose (1.35x108 beads / mL) in eight bioreactors.

[0224] [Figure 35A]Figure 35A is a graph showing the expression of CD8A, IL7R, CD4 and BCL11A genes over 7 days of culture with ETN at 2.5x bead dose (1.35x108 beads / mL) in the STR system.

[0225] [Figure 35B] Figure 35B is a graph showing the correlation between gene expression and 3D ETN (Pearson's r=0.71, p<0.001) for a panel of 24 genes related to leukocyte development after culture in microplate ("microwell") or stirred tank bioreactor ("STR") systems.

[0226] [Figure 36A] Figure 36A is a graph showing a serial restimulation assay to measure the cytotoxic activity of iPSC-derived CD8+ CAR-T cells ("iPSCCD8CAR") or primary CD8+ CAR-T cells ("primary CD8CAR") using GFP-expressing CD19+ cells as target cells (n=3 technical replicates).

[0227] [Figure 36B] Figure 36B is a graph showing the fold expansion of iPSC-derived CD8+ CAR-T cells ("iPSCCD8CAR") or primary CD8+ CAR-T cells ("primary CD8CAR") at the end of each round of target exposure. The cumulative fold expansion of iPSC-derived CD8+ CAR-T cells is shown in bold.

[0228] [Figure 36C] Figure 36C is a graph showing target cell-specific secretion of the effector molecules perforin (far left), interferon-γ (IFNγ, center left), granzyme B (center right), and tumor necrosis factor alpha (TNFα, far right) by iPSC-derived CD8+ CAR-T cells ("iPSCCD8CAR") or primary CD8+ CAR-T cells ("primary CD8CAR").

[0229] [Figure 36D]Figure 36D is a graph showing the percentage of T cell subsets in iPSC-derived CD8+ CAR-T cells ("iPSCCD8CAR") or primary CD8+ CAR-T cells ("primary CD8CAR") before (baseline) and after ("Stim1") one ("Stim1") or four ("Stim4") serial restimulation assays. T cell subsets were classified based on expression of CD45RA, CD62L, and CD95 as measured by flow cytometry.

[0230] [Figure 36E] Figure 36E is a graph showing the expression of T cell exhaustion markers PD1, TIM3, LAG3, TIGIT, and CD39 in primary CD8+ CAR-T cells ("Primary CD8CAR") or iPSC-derived CD8+ CAR-T cells ("iPSCCD8CAR"). Sections indicate the percentage of cells expressing none ("0"), one ("1"), two ("2"), three ("3"), or four ("4") of these markers.

[0231] [Figure 37A] Figure 37A is a flow cytometry plot of iPSC-derived CAR+CD8+ cells after CD8α enrichment.

[0232] [Figure 37B] Figure 37B is a flow cytometry plot of iPSC-derived CAR+CD8+ cells after CD8β enrichment.

[0233] [Figure 38A] Figure 38A is a graph showing the viability of unmodified ("6A1") and TCR-modified ("172", "174") iPSC-derived cells during differentiation.

[0234] [Figure 38B] Figure 38B is a graph showing the pre- and post-enrichment viability of unmodified ("6A1") and TCR-modified ("172", "174") iPSC-derived cells after differentiation.

[0235] [Figure 38C] Figure 38C is a graph showing cumulative fold expansion for unmodified iPSC-derived cells ("6A1") and TCR-modified iPSC-derived cells ("172", "174") during differentiation.

[0236] [Figure 39A] Figure 39A is a graph showing co-expression of CD5 and CD7 for unmodified ("6A1") and TCR-modified ("172", "174") iPSC-derived cells at the indicated time points.

[0237] [Figure 39B] Figure 39B is a graph depicting CD8 expression for unmodified ("6A1") and TCR-modified ("172", "174") iPSC-derived cells at the indicated time points.

[0238] [Figure 39C] Figure 39C is a graph showing CD8α and CD8β expression for unmodified ("6A1") and TCR-modified ("172", "174") iPSC-derived cells after differentiation and enrichment.

[0239] [Figure 40] Figure 40 is a flow cytometry plot of cells differentiated from unmodified or TCR-modified iPSCs ("unmodified (6A1)," "TCR(172)," or "TCR(174)").

[0240] [Figure 41A] Figure 41A is a graph showing TCR expression for unmodified iPSC-derived cells ("6A1") and TCR-modified iPSC-derived cells ("172", "174") at the indicated time points.

[0241] [Figure 41B]Figure 41B is a flow cytometry plot of TCR (MAGE-A4) expression for TCR-modified ("172", "174") iPSC-derived cells after differentiation and enrichment, as determined by tetramer staining.

[0242] [Figure 42A] Figure 42A is a graph showing the fold change in proliferation for primary T cells and unmodified ("6A1") and TCR-modified ("172", "174") iPSC-derived CD8+ cells during an in vitro serial restimulation assay.

[0243] [Figure 42B] Figure 42B is a graph showing the fold change in proliferation for primary T cells and TCR-modified ("172", "174") iPSC-derived CD8+ cells during an in vitro serial restimulation assay.

[0244] [Figure 43] Figure 43 is a graph showing the in vitro cytotoxicity of primary T cells and TCR-modified ("172", "174") iPSC-derived CD8+ cells against A375 target cells at various effector-to-target (E:T) ratios, as indicated.

[0245] [Figure 44A] Figure 44A is a graph showing the cytotoxicity of primary T cells and TCR-modified ("174") iPSC-derived CD8+ cells in an in vitro serial restimulation assay with A375 target cells compared to target cells and unmodified iPSC-derived cell controls.

[0246] [Figure 44B] Figure 44B is an enlargement of Figure 44A and is a graph showing the cytotoxicity of primary T cells and TCR-modified ("174") iPSC-derived CD8+ cells.

[0247] [Figure 44C]Figure 44C is a graph showing the cytotoxicity of primary T cells and TCR-modified ("174") iPSC-derived CD8+ cells in an in vitro serial restimulation assay using A375 target cells compared to target cells and unmodified iPSC-derived cell controls.

[0248] [Figure 44D] Figure 44D is an enlargement of Figure 44C and is a graph showing the cytotoxicity of primary T cells and TCR-modified ("174") iPSC-derived CD8+ cells.

[0249] [Figure 45] Figure 45 is a graph showing the in vitro cytotoxicity of primary T cells and TCR-modified ("174") iPSC-derived CD8+ cells against various target cells, as indicated.

[0250] [Figure 46A] Figure 46A is a flow cytometry plot of iPSC-derived cells during differentiation at the indicated time points. Cells were analyzed for intracellular ("ICS") TCRβ and CD3.

[0251] [Figure 46B] Figure 46B is a flow cytometry plot of iPSC-derived cells at day 24 of differentiation. Cells were analyzed for intracellular ("ICS") TCRβ and CD3.

[0252] [Figure 46C] Figure 46C is a graph showing viability and expression of CD4 and / or CD8α for differentiating iPSC-derived cells.

[0253] [Figure 47] Figure 47 is a flow cytometry plot of differentiating iPSC-derived cells at the indicated time points.

[0254] [Figure 48]Figure 48 is a flow cytometry plot of differentiating iPSC-derived cells at the indicated time points.

[0255] [Figure 49A] Figure 49A is a graph showing viability during differentiation (Figure 50A) for the following cell lines: unmodified iPSC-derived cells ("Unmodified"), iPSC-derived cells TCR-transduced on day 7 ("Unmodified; TCR-transduced"), CAR-iPSC-derived cells ("CARSSI"), and TCR-iPSC-derived cells ("TCRSSI").

[0256] [Figure 49B] Figure 49B is a graph showing fold expansion during differentiation (Figure 50B) for the following cell lines: unmodified iPSC-derived cells ("unmodified"), iPSC-derived cells transduced with a TCR on day 7 ("unmodified; TCR-transduced"), CAR-iPSC-derived cells ("CARSSI"), and TCR-iPSC-derived cells ("TCRSSI").

[0257] [Figure 49C] Figure 49C is a graph showing CAR and / or TCR expression for the cell lines of Figures 49A and 49B, as described above.

[0258] [Figure 50A] FIG. 50A is a graph showing CD5 and / or CD7 expression for the cell lines of FIGS. 49A and 49B, as described above (FIG. 50A).

[0259] [Figure 50B] FIG. 50B is a graph showing CD56 and / or CD7 expression for the cell lines of FIGS. 49A and 49B, as described above (FIG. 50B). [Figure 51A] FIG. 51A is a graph showing CD4 and / or CD8α expression for the cell lines of FIGS. 49A and 49B, as described above.

[0260] [Figure 51B] Figure 51B is a flow cytometry plot of CD8α and CD8β expression at differentiation day 21. Cells were subgated on CD4−CD8α+.

[0261] [Figure 52A] FIG. 52A is a graph showing CD8α and / or CD8β expression for the cell lines of FIGS. 49A and 49B, as described above.

[0262] [Figure 52B] Figure 52B is a flow cytometry plot of CD4 and CD8β expression at differentiation day 21. Cells were subgated as CD8α+CD8β+.

[0263] [Figure 53] Figure 53 is a flow cytometry plot of CAR modified cells ("SSI CAR") after differentiation, CD8α enrichment, cryopreservation and thawing.

[0264] [Figure 54A] Figure 54A is a graph showing the fold expansion of CAR-modified cells ("SSI CARs") during an in vitro serial restimulation assay with RajiCD19+ target cells.

[0265] [Figure 54B] Figure 54B is a graph showing the percentage of CD8α+CD8β+, CD8α+CD8β−, CD8α−CD8β−, and CD8α−CD8β+ cells for CAR-modified cells (“SSI CAR”) during an in vitro serial restimulation assay with RajiCD19+ target cells.

[0266] [Figure 54C] Figure 54C is a graph showing the cytotoxicity of CAR-modified cells ("SSI CARs") during an in vitro serial restimulation assay using RajiCD19+ target cells.

[0267] [Figure 55A]Figure 55A is a graph showing the fold expansion of CAR-modified cells ("SSI CARs") during an in vitro serial restimulation assay with A549 CD19+ target cells.

[0268] [Figure 55B] Figure 55B is a graph showing the fold change in proliferation of CAR-modified cells ("SSI CARs") during an in vitro serial restimulation assay with A549 CD19+ target cells.

[0269] [Figure 55C] Figure 55C is a graph showing the percentage of CD8α+CD8β+, CD8α+CD8β−, CD8α−CD8β−, and CD8α−CD8β+ cells for CAR-modified cells (“SSI CAR”) during an in vitro serial restimulation assay using A549 CD19+ target cells.

[0270] [Figure 56] Figure 56 is a graph showing cytokine secretion of CAR-modified cells ("SSI CAR") after in vitro serial restimulation assays with A549 or Raji CD19+ / + target cells or CD19- / - controls.

[0271] [Figure 57A] Figure 57A is a graph showing the viability of CAR-modified cells ("SSI CARs") after an in vitro serial restimulation assay with Raji (Figure 58) CD19+ target cells.

[0272] [Figure 57B] Figure 57B is a graph showing the viability of CAR-modified cells ("SSI CAR") after an in vitro serial restimulation assay using A549 (Figure 58) CD19+ target cells.

[0273] [Figure 58] Flow cytometry plots of CAR-iPSC-derived cells after differentiation and CD8 enrichment.

[0274] [Figure 59A] Figure 59A is a graph showing the inhibition of in vivo Raji tumor growth of CAR-iPSC-derived cells ("iPSC CD8") and primary CAR+ T cells ("primary CD8") in a mouse model. Statistical differences between the tumor-only and CAR-iPSC-derived groups were assessed by Welch's two-sample t-test (two-tailed, *P<0.05; **P<0.01).

[0275] [Figure 59B] Figure 59B is a graph showing survival for untreated (tumor only), CAR-iPSC-derived CD8+ cell-treated, and primary CAR+ T cell-treated groups. Statistical differences between tumor only and CAR-iPSC-derived groups were assessed by Log-rank (Mantel-Cox) test (*P<0.05; **P<0.01).

[0276] [Figure 59C] Figure 59C is a graph showing in vivo toxicity by % body weight change of CAR-iPSC-derived T cells and primary CAR+ T cells.

[0277] [Figure 60A] Figure 60A is a graph showing the in vivo cell kinetics of CAR-iPSC-derived T cells and primary CAR+ T cells as detected in peripheral blood.

[0278] [Figure 60B] Figure 60B is a flow cytometry plot of CAR-iPSC-derived CD8+ cells detected in peripheral blood 8 days after infusion.

[0279] [Figure 61A] Figure 61A shows bioluminescence images over time of untreated (tumor only), CAR-iPSC-derived CD8+ cell-treated, and primary CAR+CD8+ T cell-treated mice in an A549 CD19 tumor model.

[0280] [Figure 61B]Figure 61B is a graph showing the growth inhibition of A549-CD19 tumors in vivo of CAR-iPSC-derived and primary CAR+CD8+ T cells.

[0281] [Figure 61C] Figure 61C is a graph showing survival for untreated (tumor only), CAR-iPSC-derived CD8+ cell-treated, and primary CAR+CD8+ T cell-treated groups. Statistical differences between tumor only and CAR-iPSC-derived groups were assessed by Log-rank (Mantel-Cox) test (*P<0.05; **P<0.01).

[0282] [Figure 61D] Figure 61D is a graph showing the in vivo toxicity by % body weight change of CAR-iPSC-derived T cells and primary CAR+CD8+ T cells.

[0283] [Figure 61E] Figure 61E is a graph showing the in vivo cell kinetics of CAR-iPSC-derived T cells and primary CAR+ T cells as detected in peripheral blood.

[0284] [Figure 62A] Figure 62A is a flow cytometry plot of CAR-iPSC-derived CD8+ cells detected in peripheral blood 15 days after infusion.

[0285] [Figure 62B] Figure 62B is a graph showing the expression of CD8α and / or CD8β in CAR-iPSC-derived CD8+ cells before and after infusion.

[0286] [Figure 63]Figure 63 is a graph showing the secretion of cytokines and cytolytic molecules of CAR-iPSC-derived CD8+ cells and CAR+ primary CD8+ T cells detected in whole blood collected 15 days after infusion. Statistical differences between tumor-only and CAR-iPSC-derived groups were assessed by unpaired nonparametric t-test with Welch's correction (p-values ​​as indicated).

[0287] [Figure 64A] Figure 64A is a graph showing survival after first infusion for re-administration in untreated (tumor only), CAR-iPSC-derived CD8+ cell-treated, primary CAR+CD8+ T cell-treated, and CAR-iPSC-derived CD8+ cell-treated groups.

[0288] [Figure 64B] Figure 64B is a graph showing in vivo toxicity by percent body weight change after initial infusion for untreated (tumor only), CAR-iPSC-derived CD8+ cell-treated, and primary CAR+CD8+ T cell-treated groups, and re-administration in the CAR-iPSC-derived CD8+ cell-treated group.

[0289] [Figure 65A] Figure 65A is a bioluminescence image of a CAR-iPSC-derived CD8+ cell-treated mouse after re-administration.

[0290] [Figure 65B] Figure 65B is a graph showing in vivo tumor growth inhibition after initial infusion for the CAR-iPSC-derived CD8+ cell treatment group, the primary CAR+ T cell treatment group, and re-administration in the CAR-iPSC-derived CD8+ cell treatment group.

[0291] [Figure 65C] Figure 65C is a graph showing the in vivo cell kinetics of CAR-iPSC-derived T cells and primary CAR+ T cells as detected in peripheral blood after initial infusion (iPSC-derived cells and primary cells) and re-administration (iPSC-derived cells only).

[0292] [Figure 66A] Figure 66A is a graph showing detection of iPSC-derived CD8+ cells in the lung at endpoint compared to tumor-only controls.

[0293] [Figure 66B] Figure 66B is a flow cytometry plot of the tumor-only control (left) and iPSC-derived CD8+ cells (center, right) from Figure 66A.

[0294] [Figure 66C] Figure 66C is a graph showing CD8α and / or CD8β expression in CAR-iPSC-derived CD8+ cells before and after infusion, expanded to include tumor re-challenge.

[0295] [Figure 67A] Figure 67A is a graph showing the viability of CAR-iPSC-derived ("CAR"), TCR-iPSC-derived ("TCR"), and CAR-iPSC-derived TCR-transduced ("TCR-CAR") cells during differentiation in a stirred tank bioreactor.

[0296] [Figure 67B] Figure 67B is a graph showing cell numbers of CAR-iPSC-derived ("CAR"), TCR-iPSC-derived ("TCR"), and CAR-iPSC-derived TCR-transduced ("TCR-CAR") cells during differentiation in a stirred tank bioreactor.

[0297] [Figure 68A] Figure 68A is a flow cytometry plot of CAR-iPSC-derived ("CAR"), TCR-iPSC-derived ("TCR"), and CAR-iPSC-derived TCR-transduced ("TCR-CAR") cells after differentiation in a stirred tank bioreactor.

[0298] [Figure 68B]Graph showing CD8αβ+ cell yield for CAR-iPSC-derived (“CAR”), TCR-iPSC-derived (“TCR”), and CAR-iPSC-derived TCR-transduced (“TCR-CAR”) cells after differentiation in a stirred tank bioreactor.

[0299] [Figure 69A] Figure 69A is a graph showing the in vitro cytotoxicity of TCR-iPSC-derived cells differentiated in stirred tank bioreactors ("STRTCR+CD8+") or well plates ("WPTCR+CD8+") compared to primary TCR+CD8+ cells ("Primary TCR+CD8+") in a serial restimulation assay at an E:T ratio of 2:1.

[0300] [Figure 69B] Figure 69B is a graph showing the fold expansion of TCR-iPSC-derived cells differentiated in stirred tank bioreactors ("STRTCR+CD8+") or well plates ("WPTCR+CD8+") compared to primary TCR+CD8+ cells ("Primary TCR+CD8+") in a serial restimulation assay.

[0301] [Figure 69C] Figure 69C is a graph showing the in vitro cytotoxicity of TCR-iPSC-derived cells differentiated in stirred tank bioreactors ("STRTCR+CD8+") or well plates ("WPTCR+CD8+") at various E:T ratios compared to primary TCR+CD8+ cells ("Primary TCR+CD8+").

[0302] [Figure 69D] Figure 69D is a graph showing the antigen-independent ("antigen negative") and antigen-dependent ("antigen positive") cytotoxicity of TCR-iPSC-derived cells differentiated in stirred tank bioreactors ("STRTCR+CD8+") or well plates ("WPTCR+CD8+") compared to primary TCR+CD8+ cells ("Primary TCR+CD8+") in a serial restimulation assay. DETAILED DESCRIPTION OF THE INVENTION

[0303] Detailed Description of Disclosure Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0304] Generally, the disclosure provides methods for generating T cell lineage cell populations from progenitor cells, T cell lineage populations generated by the methods disclosed herein; pharmaceutical compositions comprising the T cell lineage populations generated by the methods disclosed herein; the use of a T cell lineage population in the manufacture of a medicament for treating a disease or condition, wherein the T cell lineage population is generated by the methods disclosed herein; and methods for differentiating progenitor T cell populations by the methods disclosed herein.

[0305] definition As used herein, the term "stem cell" refers to a cell that can differentiate into more specialized cells and has the ability to self-renew.Stem cells include pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), as well as pluripotent stem cells, such as mobilized peripheral blood-derived CD34+ stem cells, umbilical cord blood stem cells, and adult stem cells, found in various tissues.Methods for obtaining, inducing, or producing stem cells are known in the art.

[0306] As used herein, the term "progenitor cells" refers to cells that can differentiate into one or more types of cells but typically have limited self-renewal capacity. Progenitor cells are derivatives of stem cells and have more limited potency compared to their corresponding source stem cells. For example, hematopoietic stem cells (HSCs) present in adult bone marrow, peripheral blood (in lower numbers), and umbilical cord blood have the ability to give rise to all other blood cells. Hematopoietic progenitor cells are pluripotent or lineage-committed cells derived from HSCs that have the ability to give rise to more limited or specific types of blood cells. Hematopoietic stem and progenitor cells (HSPCs) typically exist as heterogeneous populations in vivo and are used as such as described herein. Hematopoietic stem and progenitor cells can be characterized, for example, by surface CD34 (CD34+).

[0307] As used herein, the terms "precursor T cells" and "pro T cells" refer to cells derived from pluripotent stem cells or CD34+ hematopoietic stem and / or progenitor cells, expressing at least CD7+, and capable of differentiating into one or more types of immature and mature T cells. Examples of progenitor T cells include, but are not limited to, CD7+ cells, CD7+CD5+ cells, CD7+CD5+CD34+ cells, CD7+CD5+CD45RA+ cells, and / or CD7+CD5+CD1a+ cells.

[0308] As used herein, "immature T cells" or mature T cells are T lineage cells derived from precursor T cells. T cell differentiation can be characterized by the progressive expression of cell surface receptors, particularly CD4 and CD8. In vivo, T lineage cells differentiate from precursor T cells through CD4-CD8- (double negative, DN), CD4+CD8- (CD4 immature single positive, CD4ISP), CD4+CD8+ (double positive, DP), and CD4-CD8+ (CD8 single positive, CD8SP) and CD4 single positive (CD4SP) stages. CD8 can be expressed as a heterodimer of CD8α and CD8β, resulting in CD8αβ+ cells, or as a CD8αα homodimer, resulting in CD8αα+ cells. CD4-CD8+ cells can also be characterized by the cell surface expression of CD3 and either TCRγδ (γδ T cells) or TCRαβ (αβ T cells).

[0309] As used herein, "serum-free medium" refers to a cell culture medium that lacks animal serum. Serum-free medium may contain certain known serum components isolated from animals (including human animals), such as bovine serum albumin (BSA).

[0310] As used herein, "Notch signaling ligand" refers to any ligand that can interact with Notch protein receptor to regulate T cell lineage commitment and differentiation. Examples of Notch signaling ligands include Delta-like 4 (DL4), Delta-like 1 (DL1), Delta-like 3 (DL3), Jagged1 and Jagged2.

[0311] As used herein, Notch signaling ligands, such as "Delta-like 4" and "DL4," refer to the protein encoded by the DLL4 gene in humans. DL4 is a member of the Notch signaling pathway and is also referred to in the art as "Delta-like ligand 4" and "DLL4." Reference to DL4 herein is not limited to the entire DL4 protein, but includes at least the signaling peptide portion of DL4. For example, a commercially available product (Sino Biologicals) containing the extracellular domain (Met1-Pro524) of human DL4 (full-length DL4 accession number NP061947.1 (SEQ ID NO: 1)) fused at the C-terminus to the Fc region of human IgG1 is a DL4 protein suitable for use in the methods provided herein.

[0312] As used herein, the term "Notch signaling ligand" also includes variants of known Notch signaling ligands, such as DL4. A variant Notch signaling ligand refers to a protein molecule whose amino acid sequence differs from the wild-type amino acid sequence by one or more additions, deletions, and / or substitutions, and which retains the desired Notch signaling activity of wild-type DL4. Variants, such as polypeptides, oligopeptides, peptides, and proteins, that have amino acid sequence identity with a given polypeptide, oligopeptide, peptide, or protein are also included in the definition. The percent identity can be, for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with a given polypeptide, oligopeptide, peptide, or protein over a particular length, for example, the entire length of the polypeptide.

[0313] As used herein, "vascular cell adhesion molecule 1" and "VCAM-1" refer to the protein encoded by the VCAM1 gene in humans. VCAM-1 is a cell surface sialoglycoprotein, a type I membrane protein that is a member of the Ig superfamily. VCAM-1 is also referred to in the art as "vascular cell adhesion protein 1 and cluster of differentiation 106" (CD106). References herein to VCAM-1 are not limited to the entire VCAM-1 protein, but include at least the signaling peptide portion of VCAM-1 (QIDSPL (SEQ ID NO: 2) or TQIDSPLN (SEQ ID NO: 3)). For example, the (Phe25-Glu698; Fe region of commercially available mouse VCAM-1 (full length mouse VCAM-1 accession number CAA47989; (SEQ ID NO: 4)) fused to the human IgG1 Fe region is a VCAM-1 chimeric protein suitable for use herein. Use of at least a portion of human VCAM-1 (full length human VCAM-1 accession numbers P19320, NP001069, EAW72950; (SEQ ID NO: 5)) may also be suitable for use in the methods provided herein. As used herein, reference to VCAM-1 also includes variants that differ in amino acid sequence from the wild-type amino acid sequence of VCAM-1 by one or more additions, deletions, and / or substitutions, and that retain the desired activity of wild-type VCAM-1. The definition also includes variants such as polypeptides, oligopeptides, peptides, and proteins that have amino acid sequence identity with a peptide, oligopeptide, peptide, or protein. The percent identity can be, for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with a given polypeptide, oligopeptide, peptide, or protein over a specific length, for example, the entire length of the polypeptide. VCAM-1 has been shown to synergistically increase Notch signaling in combination with DL4 (e.g., Shukla et al., 2017).

[0314] As used herein, "two-dimensional engineered thymic niche (2D ETN)" refers to a two-dimensional substrate immobilized with Notch signaling ligands, such as DL4, and optionally VCAM-1. Two-dimensional (2D) substrates can include, for example, tissue culture plates. Methods for immobilizing Notch signaling ligands on 2D substrates are known in the art and are described, for example, in Shukla et al., 2017.

[0315] As used herein, "three-dimensional engineered thymic niche (3D ETN)" refers to a three-dimensional substrate immobilized with Notch signaling ligands, such as DL4 and optionally VCAM-1. Three-dimensional (3D) substrates can include, for example, micron-sized particles (or beads) with or without magnetic cores coated with one or more complete proteins, protein domains (e.g., extracellular, intracellular, or other domains), peptides, or protein fragments to activate Notch signaling. To produce protein-coated particles, several approaches can be used, individually or in combination, including physical adsorption driven by protein affinity to the particle material; chemical conjugation, particularly by reaction with amine, carboxyl, thiol, epoxy, or azide reactive groups; or chemical conjugation by coating with an appropriate ligand to capture the protein of interest by affinity. Examples of affinity tags include, but are not limited to, Fc, biotin, Halo, aldehyde, Snap, Spy-Catcher, and VIPER. The particles or beads may be composed of, for example, polystyrene, iron oxide, gold, or other suitable materials known in the art. 3D ETNs can be used to culture cells on tissue culture plates, flasks, or other containers utilized for culturing cells.

[0316] As used herein, "surface-bound" refers to a Notch signaling ligand immobilized on a 2D or 3D ETN via covalent or non-covalent interactions, affinity-based interactions, or other suitable forms of interactions.

[0317] As used herein, an "enriched" cell population refers to a cell population containing one or more cell phenotypes (e.g., CD4-CD8+(CD8SP), CD4+CD8+(DP), CD4+CD8-(CD4 ISP), CD4-CD8-(DN)), which exhibits a higher absolute number or ratio of a cell phenotype (e.g., CD4-CD8+(CD8SP)) compared to other cell phenotypes, and at least 25% of the cell population consists of a single cell phenotype. For example, 10 x 10 progenitor T cells 7 When cultured at a 3D ETN bead concentration of 1.852 beads / mL (1.852 x bead dose), the cell population is enriched in CD4-CD8+ (CD8SP) cells, which comprise approximately 50% of the cell population, when compared to precursor T cells cultured with SCT (commercially coated), where CD4-CD8+ (CD8SP) cells comprise approximately less than 10% of the cell population.

[0318] As used herein, an "enriched" cell population also refers to a cell population generated from culturing precursor T cells with DL4 and optionally VCAM-1, wherein the cell population comprises one or more cell phenotypes (e.g., CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4ISP), CD4-CD8- (DN)), exhibits a higher absolute number or ratio of one cell phenotype (e.g., CD4-CD8+ (CD8SP)) compared to other cell phenotypes, and at least 25% of the cell population consists of a single cell phenotype when compared to precursor T cells not cultured with DL4 and optionally VCAM-1. For example, 10 x 10 precursor T cells 7 When cultured at a 3D ETN bead concentration of 1.85x beads / mL (1.85x bead dose), the CD4-CD8+ (CD8SP) cell population was 0.1x10 7 Compared to precursor T cells cultured at a 3D ETN bead concentration of 0.018 × beads / mL (0.018 × bead dose), the CD4-CD8+ (CD8SP) cells comprise approximately 50% of the cell population, whereas CD4-CD8+ (CD8SP) cells comprise only approximately less than 10% of the cell population.

[0319] As used herein, the term "subject" refers to a vertebrate, preferably a mammal (e.g., a non-human mammal), more preferably a primate, and even more preferably a human. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, pets, etc.

[0320] As used herein, the terms "treatment," "treat," or "treating" refer to an approach to obtaining beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of: an increased immune response; an increased T-cell response; a decrease in the extent of damage from a disease, condition, or disorder; a decrease in the duration of a disease, condition, or disorder; and / or a decrease in the number, extent, or duration of symptoms associated with a disease, condition, or disorder. This term includes administering a compound, agent, drug, or pharmaceutical composition of the present disclosure to prevent or delay the onset of one or more symptoms, complications, or biochemical manifestations of a disease or condition; alleviating or ameliorating one or more symptoms; shortening or reducing the duration of symptoms; or preventing or inhibiting further development of a disease, condition, or disorder. Treatment can be prophylactic (to prevent or delay the onset of a disease, condition, or disorder, or to prevent the onset of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the onset of a disease, condition, or disorder. A beneficial or desired clinical result can be an increase or decrease (as appropriate) of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% compared to a suitable control, e.g., a subject not receiving the treatment.

[0321] As used herein, the term "administer" or "administration" refers to the placement of a drug, agent, compound, or pharmaceutical composition disclosed herein into a subject by a method or route that results in at least partial delivery of the composition to a desired site. The compounds and pharmaceutical compositions disclosed herein can be administered by any suitable route that results in effective treatment in the subject. The administration routes of the compounds and pharmaceutical compositions disclosed herein include, but are not limited to, intravenous or intraperitoneal administration routes, or combinations thereof.

[0322] The term "effective amount" or "therapeutically effective amount," for example, an effective amount or therapeutically effective amount of a T cell lineage population as used herein, is an amount sufficient to produce any one or more beneficial or desired results. In more specific embodiments, an effective amount can alleviate or ameliorate one or more symptoms of a disease; can shorten the period during which one or more symptoms of a disease exist in a subject; or can increase the survival rate of a subject with a disease. For prophylactic use, beneficial or desired results can include eliminating or reducing the risk of, reducing the severity of, or delaying the onset of a disease, including biochemical and / or histological symptoms of infection, its complications, and intermediate pathological phenotypes that appear during the development of the disease. For therapeutic use, beneficial or desired results can include clinical results such as alleviating one or more symptoms of a disease, reducing the dosage or length of administration of other drugs required to treat the disease, enhancing the effectiveness and / or reducing the toxicity of another drug, delaying the progression of a disease in a subject, shortening the period during which one or more symptoms of a disease exist in a subject, and / or increasing the overall survival rate of a subject with a disease. An effective amount can be administered in one or more doses, rounds of administration, or series of treatments.

[0323] For purposes of this disclosure, an effective dosage of a cell population or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective dosage of a compound or pharmaceutical composition may or may not be achieved in combination with another agent, drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in relation to the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be achieved or is achieved. Amounts may vary from subject to subject and may depend on one or more factors, such as the subject's gender, age, weight, health history, and / or the underlying cause of the disease, condition, or disorder being prevented, inhibited, and / or treated.

[0324] As used herein, the term "pharmaceutically acceptable carrier, diluent, or excipient" includes any substance that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (e.g., Remington's Pharmaceutical Sciences, 18th ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th ed., Mack Publishing, 2000).

[0325] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0326] The phrase "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0327] As used herein, the phrase "one or more" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "one or more" refers, whether related to the specifically identified elements or not, to be optionally present. Thus, as a non-limiting example, "one or more of A and B" (or, equivalently, "one or more of A or B," or, equivalently, "one or more of A and / or B") may, in one embodiment, refer to at least one, possibly more than one, A, in which B is absent (and may include elements other than B). In another embodiment, it may refer to at least one, possibly more than one, B, in which A is absent (and may include elements other than A). In yet another embodiment, there is at least one, and may include more than one, A, and at least one, and may include more than one, B (and optionally other elements); etc.

[0328] When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. Generally, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 20%, 10%, 5%, or 1%. In certain embodiments, the term "about" is used to modify numerical values ​​above and below the stated value by a variance of 10%. In certain embodiments, the term "about" is used to modify numerical values ​​above and below the stated value by a variance of 5%. In certain embodiments, the term "about" is used to modify numerical values ​​above and below the stated value by a variance of 1%.

[0329] When a range of values ​​is recited herein, it is intended to encompass each value and subrange within that range. For example, "1 to 5 mL" is intended to encompass 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 1 to 2 mL, 1 to 3 mL, 1 to 4 mL, 1 to 5 mL, 2 to 3 mL, 2 to 4 mL, 2 to 5 mL, 3 to 4 mL, 3 to 5 mL, and 4 to 5 mL.

[0330] It will be further understood that the terms "comprises," "comprising," "includes," and / or "including," as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0331] As used herein, the term "consisting of" and its derivatives are intended to be closed terms specifying the presence of stated features, integers, steps, operations, elements, and / or components, and excluding the presence or addition of one or more other features, integers, steps, operations, elements, and / or components.

[0332] General Technology Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0333] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of those in the art. Such techniques are described in the literature, e.g., in Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCales, eds., 1987); Current Protocols in Molecular Biology (FMAusubel eta / ., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis eta / ., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991; Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, NY (2002); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley and Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); and Immunobiology (C.A. Janeway and P. Travers, 1997).

[0334] Methods for generating T cell lineage populations in vitro Generally, the in vitro methods for generating T cell lineage populations provided herein involve culturing precursor T cells (proT cells) in the presence of a Notch signaling ligand under conditions and for a time suitable for differentiation into a T cell lineage population.

[0335] Methods for generating proT cells from stem / progenitor cells, such as hematopoietic progenitor cells (HPCs), hematopoietic stem / progenitor cells (HSPCs), or CD34+ cells, are known in the art and include, for example, differentiation on immobilized VCAM-1 and Notch signaling ligands, such as DL4, under appropriate media conditions (e.g., Shukla et al., 2017), or using DL4-coated microbeads (e.g., Trotman-Grant et al., 2021).

[0336] To confirm the generation of proT cells, cells can be analyzed for one or more characteristics indicative of proT cells (e.g., one or more cell surface markers, such as CD5 and CD7). Suitable techniques for analyzing cell surface markers are known to those skilled in the art and can include, for example, flow cytometry or immunocytochemistry, as used herein. Cell number and cell viability can be analyzed by techniques well known to those skilled in the art and can include, for example, the use of an automated cell counter as disclosed herein.

[0337] In one embodiment, CD34+ cells may be obtained from umbilical cord blood, peripheral blood, or bone marrow, or may be derived in vitro from pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) or other intermediate stem cells. In a preferred embodiment, the stem and / or progenitor cells are human cells. In one embodiment, the stem cells are mobilized peripheral blood-derived CD34+ cells. In a preferred embodiment, the CD34+ cells are derived in vitro from iPSCs. Methods for generating CD34+ cells from iPSCs are known in the art, such as differentiation in appropriate culture conditions (e.g., Trotman-Grant et al., 2021).

[0338] Cells can be cultured in cell culture systems of the type known in the art, such as cell culture plates, culture dishes, and bioreactors, including stirred tank reactors (STRs), swing-bag bioreactors, and other suitable cell culture formats. Cell culture can be carried out under static conditions, dynamic or stirred conditions, or a combination of static and dynamic conditions. The bioreactor can be any type of bioreactor known in the art and can use any type of processing / culture conditions and methods, including, for example, batch processes, fed-batch processes, and perfusion culture methods and conditions.

[0339] The present invention for generating a T cell lineage population can be carried out using a population of HPCs, HSPCs, and / or progenitor T cells in the presence of a Notch signaling ligand and in the absence of a T cell receptor stimulator. The T cell receptor stimulator can be a molecule that mediates and / or activates CD3 signaling, such as an anti-CD3 antibody, an antigen-presenting cell, or an artificial antigen-presenting cell. The present invention can be carried out without the involvement and activation of exogenous CD3.

[0340] The present invention can also be used to generate cells that are surface CD3 negative (sCD3-) (e.g., cells in which CD3 is not detectable as a cell surface marker). The present invention can also be used to generate cells that are TCR- (e.g., cells that do not express an endogenous (i.e., native) TCR, or cells in which TCR cell surface expression is not detectable). The present invention can also be used to generate cells that are both sCD3- and cell surface TCR- (i.e., cells in which both CD3 and TCR are not expressed on the cell surface).

[0341] The present invention for generating T cell lineage populations may also be practiced using populations of HPCs, HSPCs, and / or progenitor T cells in the presence of a Notch signaling ligand, without chimeric antigen receptor (CAR) engagement and / or stimulation in cells that are CAR+.

[0342] The present invention may also be practiced using serum-free and / or feeder-free (eg, stromal cell-free) culture conditions.

[0343] Base material In one embodiment, pro-T cells are cultured in a two-dimensional culture system utilizing a suitable 2D substrate, which may include, for example, standard culture plates coated with a Notch signaling ligand, e.g., DL4. The culture plates may also be coated with VCAM-1.

[0344] In one embodiment, proT cells are cultured in a three-dimensional culture system utilizing a suitable three-dimensional substrate, which may comprise, for example, micron-sized particles (or beads) with or without a magnetic core, coated with one or more intact proteins, protein domains (e.g., extracellular, intracellular, or other domains), peptides, or protein fragments to activate Notch signaling. To produce protein-coated particles, several approaches may be used, individually or in combination, including physical adsorption driven by protein affinity to the particle material; chemical conjugation by reaction with, among others, amine, carboxyl, thiol, epoxy, or azide reactive groups; or chemical conjugation by coating with an appropriate ligand to affinity capture the protein of interest. Examples of affinity tags include, but are not limited to, Fc, biotin, Halo, aldehyde, Snap, Spy-Catcher, and VIPER.

[0345] The particles or beads may be composed of, for example, polystyrene, including carboxylated polystyrene, iron oxide, gold, or other suitable materials known in the art.

[0346] In one example, a Notch signaling ligand, such as DL4, alone or in combination with VCAM-1, can be bound to polystyrene microbeads as described in Trotman-Grant et al., 2021 and WO 2019157597.

[0347] In another example, 3D ETN beads can be produced by affinity capturing DL4 and VCAM-1 carrying appropriate affinity tags onto streptavidin- or protein G-coated beads. The beads are diluted to 0.1% solids in Ca2+- or Mg2+-free Dulbecco's phosphate-buffered saline (DPBS) supplemented with 0.05% BSA and incubated with the protein solution (0.1x to 20x protein molar excess) at room temperature for 60 minutes with continuous stirring. At the end of the incubation period, excess free protein is removed by magnetic separation, followed by buffer exchange. This procedure is repeated four more times, after which the 3D ETN beads are concentrated 10x for storage.

[0348] Quantitation of protein immobilization can be performed according to methods known in the art, such as colorimetric bicinchoninic acid (BCA) assay, immunofluorescence assay, or other known detection methods.

[0349] T cell therapy T cells have a wide range of therapeutic applications. For example, T cells can be modified by traditional gene editing approaches, such as nuclease editing or viral vector transduction, to express chimeric antigen receptors (CARs) and / or exogenous T cell receptors (TCRs) to generate engineered T cell therapies (Weber et al., 2020). T cells derived from progenitor cells, including pluripotent stem cells, can be genetically engineered at the pluripotent or progenitor cell stage to contain nucleic acids encoding CARs or TCRs that can be expressed at the pluripotent, progenitor, or derivative cell stage. Engineered T cell therapies are applicable, for example, in oncology and autoimmune disorders. In oncology, engineered T cell therapies are applicable, for example, to hematological cancers, such as B-cell lymphoma, B-cell acute lymphoblastic leukemia, and other B-cell malignancies, multiple myeloma, and other hematological cancers, as well as solid tumors, such as mesothelioma, adenocarcinoma, glioma, and sarcoma (Weber et al., 2020). In autoimmune disorders, engineered T cell therapy is applicable, for example, in type 1 diabetes, rheumatoid arthritis, multiple sclerosis, and other autoimmune disorders or conditions (Weber et al., 2020).

[0350] Engineered T cell therapy can target antigens known to be expressed on target cell types, including tumor cells or tumor tissue. Chimeric antigen receptors (CARs) can be designed to target surface antigens or multivalent soluble antigens. The targeting ectodomain of the CAR can be a single-chain variable fragment (scFv), a single-domain antibody (single variable domain on the heavy chain, VHH), a nanobody, or other antigen-binding domain (Qu et al., 2022). CAR-T cell therapy can be directed against multiple antigens using various CAR designs or multiple CARs (Qu et al., 2022). Exemplary tumor antigens and corresponding cancer types for CAR-T cell therapy are listed in Table 1 below (Qu et al., 2022; Guha et al., 2022; Drougkas et al., 2023; Want et al., 2023). [Table 1] TIFF2026504155000002.tif255170TIFF2026504155000003.tif254170TIFF2026504155000004.tif255170TIFF2026504155000005.tif7170

[0351] TCR-T cell therapy targets antigens expressed as peptide-human leukocyte antigen (HLA) complexes on the surface of target cells. These targets may include tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) (Baulu et al., 2023). Exemplary tumor antigens and corresponding cancer types for TCR-T cell therapy are listed in Table 2 below (Baulu et al., 2023; Sun et al., 2021; Want et al., 2023). [Table 2]

[0352] The pharmaceutical compositions provided herein can be administered to a subject to alleviate or ameliorate one or more symptoms of a disease; to reduce the duration that one or more symptoms of a disease are present in a subject; or to increase the survival rate of a subject with a disease.

[0353] The pharmaceutical compositions provided herein can be administered to a subject to treat cancer or an autoimmune disorder in the subject.

[0354] The pharmaceutical compositions provided herein can be administered to subjects in effective or therapeutically effective amounts.Those skilled in the art can determine such amounts based on factors such as the size (e.g., body weight), age and / or sex of the subject.The severity of the subject's symptoms; a specific composition or administration route will be selected.Those skilled in the art will also know how to select suitable administration routes and administer the compounds and compositions provided herein.

[0355] The dosage of the pharmaceutical composition of the present disclosure varies depending on many factors, such as the pharmacodynamic properties of the composition, the mode of administration, the recipient's age, health condition and weight, the nature and severity of symptoms, the frequency of treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the treated subject.Those skilled in the art can determine the appropriate dosage based on the above factors.In some embodiments, the pharmaceutical composition is initially administered at an appropriate dosage, which depends on clinical response and is adjusted as necessary.

[0356] kit The present invention also provides a kit comprising the pharmaceutical composition described herein.The kit of the present invention comprises one or more containers containing the pharmaceutical composition described herein and instructions for use according to any of the methods of the present invention described herein.Generally, these instructions include instructions for administering the pharmaceutical composition for the above-mentioned therapeutic treatment.In some embodiments, a kit for producing a single-dose unit is provided.

[0357] The instructions for use of the pharmaceutical composition generally include information on the dosage, administration schedule, and administration route for the intended treatment. The container can be a unit dose, bulk package (e.g., a multi-dose package), or sub-unit dose. The instructions provided in the kit of the present invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions distributed on a magnetic or optical storage disk) are also acceptable.

[0358] The present disclosure will be further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present disclosure should not be interpreted as being limited to the following examples in any way, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0359] Example 1: Preparation of engineered thymic niche (ETN) Production of DL4 and VCAM-1 Recombinant DL4-Fc fusion protein was purchased from Sino Biological or produced in-house using HEK-293T cells and purified on a HiTrap Protein G affinity column (GE Healthcare) as previously described (e.g., Trotman-Grant et al., 2017). Recombinant VCAM-1-FC fusion protein was purchased from R&D Systems. DL4 and VCAM-1 suitable for preparation of 2D and 3D ETNs, as further described below, are listed in Table 3. [Table 3] TIFF2026504155000008.tif27170

[0360] Preparation of 2D ETNs Six-, 12-, 24-, 48-, and 96-well tissue culture plates were coated with Notch signaling ligands, DL4 and VCAM-1, overnight at 4°C or for 3 hours at 37°C. Tissue culture plates could be stored at 4°C for up to 2 weeks after coating. For coating, a solution of 20 μg / mL DL4 and 10 μg / mL VCAM-1 was prepared in Dulbecco's phosphate-buffered saline (DPBS) (- / -). The appropriate coating volume per well of DL4 and VCAM-1 diluted in DPBS was added to the tissue culture plates, as shown in Table 4. [Table 4]

[0361] The tissue culture plate was gently tapped to ensure that the coating solution containing the Notch signaling ligands, DL4 and VCAM-1, was evenly spread across the entire well surface. The tissue culture plate was sealed with Parafilm® and then stored at 4°C overnight or at 37°C for 3 hours. The tissue culture plate coated with the Notch signaling ligands, DL4 and VCAM-1, overnight at 4°C was placed in a 37°C incubator for 3 hours for equilibration before cells were seeded. After equilibration or after coating the tissue culture plate with the Notch signaling ligands, DL4 and VCAM-1, at 37°C for 3 hours, the coating solution was aspirated from the wells. The wells were washed with DPBS(- / -) using the volumes shown in Table 5, and the cell suspension was immediately added to the tissue culture plate. [Table 5]

[0362] Preparation of 3D ETNs The dose of 3D ETN can be calculated to fit the bead diameter and expressed as a dose proportional to the surface area of ​​the culture plate or flask (e.g., as in Table 4), the number of beads per unit volume of culture, or the bead surface area per unit volume of culture. As calculated on a per-unit-volume basis, the beads per mL concentration does not vary across different vessels. A "1x" bead dose indicates complete coverage of the plate surface with one layer of beads. Because the beads are approximately spherical, the total surface area of ​​the beads is four times the surface area of ​​the plate or culture vessel surface. Table 6 shows a range of bead concentrations for 3.05 μm diameter polystyrene beads. [Table 6]

[0363] The Notch signaling ligand density on the beads (e.g., the density of surface-bound DL4) can be, for example, 100 molecules / square micrometer (100 molecules / µm 2 ) ~ 3000 molecules / square micrometer (3000 molecules / μm2 ) can vary. Table 7 provides the calculation of Notch signaling ligand concentration for a range of bead doses and Notch signaling ligand density for 3.05 μm polystyrene beads. The concentration of Notch signaling ligand, for example, DL4, was calculated based on the amount of DL4 that can bind to soluble Notch1. Therefore, the total concentration of Notch signaling ligand on beads may be slightly higher. [Table 7]

[0364] Table 8 provides the Notch signaling ligand (eg, DL4, etc.) surface area per unit volume for a range of bead doses and the Notch signaling ligand density for both 3.05 μm and 3.29 μm diameter polystyrene beads. [Table 8]

[0365] The 3D ETNs may also contain surface-bound VCAM-1. VCAM-1 may be immobilized on the 3D ETNs at an input molar ratio of DL4:VCAM-1 ranging from 1:6 to 10:1. In one embodiment, the 3D ETNs are prepared at an input molar ratio of DL4:VCAM-1 of 2.5:1. In one embodiment, the final density of surface-bound VCAM-1 on the beads is equivalent to the density of the Notch ligand. For example, the VCAM-1 surface area per unit volume may be equivalent to the Notch ligand surface area per unit volume shown in Table 8 above.

[0366] Cells can be cultured at a density appropriate for the culture scale and format. For microplate culture, cells can be cultured at a density of, for example, 2.5 x 10 5 ~2×10 6 For STR cultures, the cells can be cultured at, for example, 5 x 10 cells / mL. 4 ~6×10 6 The cells can be cultured at 1000 cells / mL.

[0367] Example 2: Generation of CD4-CD8+ T cells To assess the effect of 3D ETN bead concentration on T cell differentiation (Figure 1A), two independently derived populations of progenitor T cells ("proT Bank A" and "proT Bank C"), both cryopreserved after 18 days of differentiation with 2D ETNs (DL4 and VCAM-1) from iPSC-derived CD34+ cells, were thawed and collected at 1 × 10 6 Cells / mL were seeded in lymphocyte maturation medium in 24-well cell culture plates. 3D ETN beads modified with Notch signaling ligands, DL4, and VCAM-1 were added at six different concentrations: 0.27, 0.54, 1.35, 2.7, 5.4, and 8.1 × 10 cells / mL. 7 Beads / mL (0.05x, 0.1x, 0.25x, 0.5x, 1x, and 1.5x bead doses) were added to the cells. Cells were also plated on 2D ETNs (described in Example 1) and StemSpan™ lymphocyte differentiation coating material (referred to as SCT or commercial coating; STEMCELL Technologies) in 24-well cell culture plates. Cells were cultured with 2D ETNs and 3D ETNs or SCT for 3 and 7 days, and then removed from the plate by pipetting and harvested for analysis by flow cytometry and assessment of cell number and viability by an automated cell counter (Cellaca® MX). After culturing the cells for 3 days, lower 3D ETN bead concentrations (0.27, 0.54 x 10 7 beads / mL (0.05, 0.1x bead volume)) and 2D ETN, more CD4+CD8- (CD4 immature single positive, CD4ISP) cells were generated when cultured with 3D ETN beads at higher concentrations (2.7, 5.4, 8.1x10 7 beads / mL (0.5, 1, and 1.5x bead doses) generated more CD4-CD8+ (CD8 single positive, CD8SP) cells. This 3D ETN dose-dependent generation of CD4-CD8+ cells was also observed in cells cultured with 3D ETNs for 7 days, at which point the effect was more pronounced (Figure 1B), with the highest number of CD4-CD8+ cells generated at the highest 3D ETN bead concentration (8.1 x 10 7beads / mL (1.5× bead dose). Among CD4-CD8+ cells, the proportion of CD8αβ cells was also observed to be responsive to 3D ETN bead dose, with approximately 80% of CD4-CD8+ cells at the highest bead concentration (8.1×10 7 beads / mL (1.5 × bead dose)) of CD8αβ cells, whereas only 46% of CD4− CD8+ cells were detected at the lowest bead concentration (0.27 × 10 7 The CD8αβ cells were 100% of the CD4-CD8+ cells cultured in 2D ETN and SCT (Figure 1C).

[0368] Viable cell proliferation varied with 3D ETN bead dose—after 7 days, 0.54 × 10 7 and 1.35 × 10 7 Cells cultured at 0.1x and 0.25x bead dose had the highest proliferation, with most doses having 1-1.5x proliferation (Figure 2A). When viability was assessed as a function of phenotype, 3D ETN bead dose was not observed to have an effect. Instead, regardless of bead dose, CD4+CD8+ (double positive, CD4+CD8-) and CD4-CD8+ cells were observed to have high viability (>80%), whereas CD4-CD8- (double negative, DN) and CD4+CD8- cells were observed to have low viability (<20%) (Figure 2B).

[0369] Assessment of the effect of 3D ETN bead concentration on T cell differentiation as described above was performed by culturing precursor T cells with 2D ETN and SCT, as well as a wider range of 3D ETN bead concentrations (0.02×–2× bead dose, or 1.08×10). 6 beads / mL ~ 10.8 x 10 7 beads / mL (0.1–10 × 10 7The experiment was repeated at different bead concentrations (shown as beads / mL). Furthermore, the duration of cell culture with 2D ETN, SCT, and 3D ETN was extended to 7 and 14 days. Consistent with the first experiment above, the generation of CD4+CD8-CD4+CD8- and CD4-CD8+ cell populations was 3D ETN bead concentration-responsive after 7 days of culture, with a higher bead concentration (0.1 × 10 7 beads / mL and 0.3 x 10 7 More CD4+CD8CD4+CD8− cells were observed at higher bead concentrations (3 × 10 7 beads / mL and 10 x 10 7 More CD4-CD8+ cells were observed at 0.5x and 2x beads / mL (Figure 3A). After an additional 7 days of culture, these effects were even more pronounced, with approximately 60% of CD4-CD8+ cells at the highest bead concentration (10x10 7 beads / mL (2x bead dose)) (Figure 3B). Interestingly, at the lowest bead concentration (0.1 x 10 7 Precursor T cells cultured in 2D ETNs (0.02 × beads / mL) yielded a similar cell population to those cultured in 2D ETNs, with a high proportion of CD4+CD8+ cells and a very low proportion of CD4-CD8+ cells.

[0370] Precursor T cells cultured with 2D ETNs, SCTs, and 3D ETNs at various bead concentrations were assessed for CD3 and TCR expression. CD3 and TCR expression increased in cells cultured with 2D ETNs and SCTs by day 14, with approximately 60% of cells expressing CD3 and approximately 20% expressing TCRαβ with 2D ETNs (Figure 4A). In precursor T cells cultured with 3D ETNs, CD3 expression was inversely concentration-responsive, increasing at the lowest bead concentration (0.1 x 10 7 beads / mL and 0.3 x 10 7 The highest CD3 expression was obtained at the highest bead concentration (10 × 10 beads / mL (0.02 × and 0.05 × bead dose)). 7At 0.3 × 10 beads / mL (2 × bead dose), less than 30% of cells were CD3+, and TCRαβ expression was barely observed (less than 5%) (Figures 4A and 4B). 7 beads / mL ~ 10 x 10 7 Expression of TCRγδ in the bulk population did not appear to be concentration-responsive, as cells cultured at 3D ETN concentrations ranging from 0.05x to 2x bead dose yielded approximately 10% TCRγδ cells (Figure 4B). Examination of TCRαβ and TCRγδ expression in the CD4+CD8+ and CD4-CD8+ populations revealed that TCRαβ cells were found primarily in the CD4+CD8+ population. The CD4-CD8+ population was significantly higher at lower bead concentrations (0.1x10). 7 beads / mL and 0.3 x 10 7 Culturing with 2D ETNs, SCTs, and 3D ETNs at 0.02x and 0.05x beads / mL (0.02x and 0.05x bead dose) contained significant TCRγδ cells. However, at higher bead concentrations (3x10 7 and 10×10 7 In cells cultured with 3D ETN at 0.5x and 2x beads / mL (0.5x and 2x bead dose), only a small fraction of cells were TCRγδ+ (approximately 10%), and almost no TCRαβ expression was observed (Figure 4D).

[0371] Similar to the first experiment, cell viability showed a moderate concentration response after 7 days of culture in 3D ETNs, with the highest cell viability observed for proT bank A at 0.3 x 10 7 beads / mL (0.02 x bead dose) and 3 x 10 for proT Bank C 7beads / mL (0.05x bead dose) (Figure 5A). However, by day 14, cell viability was observed to increase with bead concentration, although proT bank C showed very low viability in all conditions (Figure 5B). When viability was assessed as a function of phenotype, 3D ETN bead concentration appeared less significant than phenotype, as CD4-CD8+ and CD4+CD8+ phenotypes consistently had high viability, while CD4-CD8- and CD4ISP phenotypes had low viability regardless of bead dose (Figure 5C and Figure 5D). Taken together, these data suggest that 3D ETN beads at approximately 3x10 7 When used at concentrations of 1000 beads / mL (0.05 × bead dose) or higher, it generates a population of CD4-CD8+ cells that express CD8αα and CD8αβ but not TCRαβ, suggesting a low proportion of CD3 (less than 30%) and CD3+TCRγδ (approximately 10%).

[0372] To examine cytotoxicity, precursor T cells transduced with a lentiviral vector to express a CD19 CAR were cultured in 3D ETNs (5.4 × 10 cells) as described above. 7 CAR+CD4-CD8+ cells were generated by culturing them with 1× beads / mL (1× bead dose). The cells were then co-cultured with target CD19 A549 cells (effector cell to target cell ratios of 4:1, 2:1, 1:1, or target cells alone). Similar to primary CAR-transduced CD8+ T cells (left), CAR+CD4-CD8+ cells generated using ETN beads (right) demonstrated concentration-responsive cytotoxicity across multiple stimulations (Figures 6A and 6B).

[0373] Example 3: Generation of CD4-CD8+ cells in a stirred tank reactor (STR) 3D ETN beads (0.54 × 10) were cultured in a stirred tank reactor (STR) culture system (140 ml DASbox®, Eppendorf). 7 beads / mL and 2.7 x 10 7Cell proliferation and viability of precursor T cells cultured in the presence of beads / mL (0.1x and 0.5x bead dose) were evaluated and compared with precursor T cells cultured with 2D ETN and 3D ETN using 12-well cell culture plates. Precursor T cells were cultured for 17 days under all conditions using lymphocyte maturation medium (LMM, STEMCELL Technologies). Cell viability decreased over the culture period in all conditions (Figure 7; days 18-35). As demonstrated in Example 2 (see Figure 3B), precursor T cells were cultured in 12-well cell culture plates with 2D ETN or low-concentration 3D ETN beads (0.54 x 10 7 Higher numbers of CD4+CD8+ cells were observed when cultured at 1000 beads / mL (0.1x bead dose) (Figure 8A). Precursor T cells cultured with 3D ETN in both 12-well cell culture plates and STR conditions showed a higher bead concentration (2.7x10 7 A higher percentage of CD4-CD8+ cells was observed at 0.54 × 10 beads / mL (0.5 × bead dose) (Figure 8A). Among the CD4-CD8+ cells generated by culturing precursor T cells with 3D ETNs in both 12-well cell culture plates and STR conditions, CD4-CD8+ cells were significantly higher at both bead concentrations (0.54 × 10 7 beads / mL and 2.7 x 10 7 beads / mL (0.1× and 0.5× bead dose) were predominantly CD8αβ cells (FIG. 8B).

[0374] Example 4: Generation of TCR-transduced T cells with 3D ETN Hematopoietic stem / progenitor cells expressing CD34 were derived from iPSCs modified to lack endogenous TCR expression, based on methods known in the art (e.g., Blassberg, 2022). The cells were then differentiated for 10 days in 2D ETN to generate a progenitor T cell population, as demonstrated by CD7 expression (Figure 9A) and restricted CD4 and CD8 expression (Figure 9B) (e.g., Shukla et al., 2017). On day 7, the cells were modified for MAGE-A4αβ TCR expression by lentiviral transduction, based on methods known in the art (e.g., Iriguchi et al., 2021). Approximately 20% of the transduced cells expressed TCRαβ and CD3 at day 10, whereas no TCR was expressed, with minimal CD3 expression observed in untransduced cells (Figure 9C).

[0375] The precursor T cells were then cultured in lymphocyte maturation medium (LMM, STEMCELL Technologies) and 3D ETN (1.08 × 10 8 The cells were cultured with 1.08 × 10 beads / mL (2 × bead dose) for a total of 11 days. During this period, cells were harvested and analyzed by flow cytometry on day 7. 3D ETNs were removed from the cell population using conventional separation techniques (e.g., Trotman-Grant et al., 2021). The cells were then resuspended in fresh 3D ETNs (1.08 × 10 8 beads / mL (2x bead dose)) for another 4 days.

[0376] After 7 days of culture with 3D ETNs, a subset of cells co-expressed CD5+ and CD7+ (Figure 10A), and a population of CD8+ cells that were CD8αβ+ was also observed in both transduced and non-transduced cells (Figures 10B and 10D). MAGE-A4αβ TCR expression and CD3 expression increased to approximately 50% in transduced cells by day 7 of culture with 3D ETNs (Figure 10C, bottom). Non-transduced cells were primarily TCR- / CD3- (Figure 10C, top).

[0377] After 11 days of culturing cells in 3D ETNs, a larger subset of transduced cells was observed to transition to CD4+CD8+ cells compared to untransduced cells (Figure 11B). Approximately 25% of cells in both groups retained the CD4-CD8+ phenotype (Figure 11B), and greater expression of CD8αβ+ was observed in transduced cells (Figure 11D). TCR and CD3 expression also increased in transduced cells from day 7 to day 11 (Figure 11C, center). Untransduced cells remained TCR- / CD3- (Figure 11C, top).

[0378] TCR-transduced cells were then enriched for CD8β by positive selection (EasySep™ Human Pe Positive Selection Kit, STEMCELL Technologies; PE Mouse Anti-Human CD8β, BD Biosciences). After enrichment, the percentage of CD4-CD8+ cells increased, with greater expression of CD8αβ (FIG. 11B, bottom) and increased TCR expression (FIG. 11C, bottom).

[0379] TCR-transduced and non-transduced cells behaved similarly with respect to optimal viability and proliferation over the initial 2D ETN culture period (Figures 12A-12C, "D0, D10"). However, these metrics decreased during the late CD8αβ differentiation phase ("D17, D21") despite improved cell output and viability observed in TCR-transduced cultures after 11 days in 3D ETN and CD8β selection, respectively (Figures 12A-12C). A 100-fold increase in CD8αβ T cell output per CD34+ HSPC cell input was observed after 11 days of differentiation in 3D ETN (1.08 x 10 8 beads / mL (2x bead dose).

[0380] Example 5: Gene expression and cell phenotype of generated cells iPSC-derived CD34+ cells were differentiated into proT cells for 18 days using 2D ETN as described in Example 2. Then, a 30-fold range of bead concentrations tested (0.27 to 8.1 × 10) was used. 7T cell maturation was induced by culturing cells with 3D ETN beads at 0.05x to 1.5x beads / mL (0.05x to 1.5x bead dose). Cells were cultured with 2D ETN and SCT. After culturing the cells for 7 days, cell phenotype was assessed by flow cytometry (Figure 13A). At higher bead concentrations (5.4x10 7 beads / mL and 8.1 x 10 7 beads / mL (1× and 1.5× bead dose) at lower bead concentrations (0.27×10 7 beads / mL and 0.54 x 10 7 beads / mL (0.05× and 0.1× bead dose)), while lower bead concentrations (0.27×10 7 beads / mL and 0.54 x 10 7 Beads / mL (0.05x and 0.1x bead dose) produced more CD4+CD8- (CD4ISP) and CD4-CD8- (DN) cells (Figure 13A, Figure 1B). After culturing the cells for 3 days, the expression of Notch signaling-related genes DTX1, TCF7, and BCL11B was concentration-responsive, reaching 5.4x10 7 Saturation occurred at a bead concentration of approximately 10 beads / mL (1× bead dose) (FIG. 13B). The Notch-responsive gene GATA3 showed an inverse concentration response, with a 0.27×10 7 beads / mL (0.05 x bead dose) and 0.54 x 10 7 A lower bead concentration of 100 beads / mL (0.1× bead dose) showed the highest expression (FIG. 13B).

[0381] After culturing the cells for 7 days, we assessed cell phenotype using a 13-color flow cytometry panel and performed dimensionality reduction of this multidimensional dataset using uniform manifold approximation and projection (UMAP) (Figure 13C). Cells were observed to form clusters based on the concentration of 3D ETNs (Figure 13C). Unsupervised machine learning (FlowSOM, Van Gassen et al.) was used to identify expression patterns within the multicolor flow cytometry dataset. Six clusters were identified, and the clusters were designated CD4+CD8+ (DP), CD4+CD8- (CD4ISP), CD4-CD8+ (CD8SP), and CD4-CD8- (DN) clusters based on the expression of CD8α, CD8β, and CD4 (Figure 13E). The CD4-CD8+ (CD8SP) cluster was mapped with high 3D ETN bead concentration, the CD4+CD8+ (DP) cluster was mapped with 2D ETNs, and the CD4+CD8- (CD4ISP) cluster was mapped with SCT (Figure 13D). Similar to the data shown in Figure 13A, the proportion of cells in each cluster depended on the concentration of DL4, with higher ETN bead concentrations (5.4 × 10 7 beads / mL and 8.1 x 10 7 beads / mL (1× and 1.5× bead dose) contained more CD4-CD8+ (CD8SP) clusters, while lower bead concentrations (0.27×10 7 beads / mL and 0.54 x 10 7 beads / mL (0.05x and 0.1x bead dose)) contained a higher proportion of CD4+CD8- (CD4ISP) clusters (Figure 13F).

[0382] High ETN bead concentration (5.4 × 10 7 Differentiation was extended for an additional 7 days at a high ETN bead concentration (5.4 × 10 beads / mL (1 × bead dose)). 7 After 14 days of differentiation using 10 ...

[0383] Example 6: scRNA-Seq analysis of generated cells Analysis of in vivo T cell maturation stages was performed using single-cell RNA sequencing data (Park et al., 2020). Trimming, alignment, demultiplexing, and gene counts were generated from FASTQ files using CellRanger, and a gene count matrix normalized using Seurat (4.0.1, Satja et al., 2015) was used. Duplicate samples were identified and removed, and dead cells were removed by filtering those with more than 5% mitochondrial reads. Cell type labels were adapted from Parket et al., 2020. As indicated by arrows, the data demonstrate the progression of T cell maturation from early CD4-CD8- cells (DN(early)) to CD8αα cells, γδ T cells, CD8+ T cells, and CD4+ T cells (Figure 14A). In a separate analysis, the data from Park et al. was analyzed, including the NK-T cell (NK-T) cell phenotype (Figure 14B).

[0384] To examine Notch and TCR signaling during in vivo T cell development, we calculated single-sample GSEA (ssGSEA) scores at the single-cell level with Gene Set Variation Analysis (GSVA1.40.1, Hanzelmann et al., 2013) using NOTCH (custom NanoString® panel) and TCR signaling (BioCarta) gene sets isolated from the Park et al. dataset. Notch signaling was enriched in early stages of in vivo T cell development, whereas TCR signaling was enriched in later stages of in vivo T cell development (Figures 15A-C).

[0385] In further analysis, we calculated ssGEA scores using NOTCH (custom NanoString® panel) and TCR signaling (NanoString®) gene sets isolated from the Park et al. dataset and also demonstrated enriched Notch signaling at early stages of in vivo T cell development and enriched TCR signaling at later stages of in vivo T cell development (Figure 15D, E).

[0386] 3D ETNs (5.4 x 10 as described in Example 2) 7 Single-cell RNA-seq analysis was performed to compare iPSC-derived CD4-CD8+ cells generated using 1x beads / mL (1x bead dose) with primary CD8αβ T cells. Cells were annotated using two reference datasets: the Human Thymic Cell Atlas (Park et al., 2020) and ProjecTILS (Andreatta et al., 2021). Using the Thymic Cell Atlas dataset, primary T cells were annotated as CD8+ memory T cells and CD8+, while iPSC-derived CD4-CD8+ cells were annotated as resting double-positive (DP) and double-negative (DN) cells, CD8+ memory T cells, and gamma-delta T cells (Figures 16A and 16B). Using the ProjecTILS dataset, primary T cells were annotated as CD8 effector memory cells, exhausted CD8 (CD8_Tex) cells, and low proportions of Th1 and CD8 naive-like cells (Figures 17A and 17B). Compared to primary CD8 T cells, a higher proportion of iPSC-derived CD4-CD8+ cells were annotated as CD8 naive-like cells, accompanied by similar proportions of CD8 (CD8_Tex) and Th1 cells (Figures 17A and 17B).

[0387] Using single-sample GSEA, we found that primary CD8αβ T cells expressed higher levels of CD8α (CD8A) and lower levels of the CD3 subunits CD3γ (CD3G), CD3δ (CD3D), and CD3ε (CD3E) compared to iPSC-derived cells (Figure 18A). A subset of genes associated with Notch signaling was more highly expressed in iPSC-derived CD4 − CD8 + cells generated with 3D ETNs compared to primary CD8αβ T cells, and expression of genes associated with TCR signaling was reduced in iPSC-derived CD4 − CD8 + cells (Figure 18B).

[0388] Example 7: Generation of CD4-CD8+ cells from CAR-modified iPSC-derived HSPCs To generate progenitor T cell populations, iPSC-derived hematopoietic stem / progenitor cell populations containing CD34+ cells were incubated with 3D ETN beads (2.7 × 10) for 10 days in 24-well cell culture plates. 7 The cells were cultured with 1000 CAR-modified NTX4B3 cells (4B3, 4A1, 4B3, 4B4) and 1000 CAR-modified NTX4B3 cells (4B3, 4B4). At day 10, the cells displayed a precursor T cell phenotype as evidenced by CD5+CD7+ expression, and both CD34+ and CD34- populations were observed (Figure 19, unmodified, 4A1; Figure 20, CAR-modified, 4B3).

[0389] The precursor T cells were then cultured in lymphocyte maturation medium (LMM, STEMCELL Technologies) and 3D ETN (1.08 × 10 8 The cells were cultured at 2 × 10 beads / mL (2 × bead dose) for 7 days, and then harvested and re-cultured with fresh 3D ETN beads at the same bead concentration for another 4 days. 6 cells / mL or 5 x 10 5 Cells / mL was determined: 1.08 x 10 8 After 11 days of culture at 2x beads / mL (2x bead dose) (21 days from differentiation of CD34+ cells), a CD8α enrichment step was also performed (EasySep™ Human CD8 Positive Selection Kit II, STEMCELL Technologies).

[0390] 1.08 × 10 precursor T cells 8 After 7 days of culture in 3D ETNs at a bead concentration of 2 × 10 beads / mL (2 × bead dose), CD4-CD8α+ (CD8SP) cells were observed in both unmodified and CAR cell lines (30–40%, Figure 21A). The generation of CD8α+CD8β+ cells was cell density dependent, exceeding 2 × 10 6 Cells cultured at 5 x 10 cells / mL 5Cells cultured at 1.08 × 10 cells / mL had higher CD8β expression than cells cultured at 1.08 × 10 cells / mL (Figure 21B). Cell viability was similar for both cell lines and cell densities tested (40-50%; Figure 21C). CAR expression was approximately 50% for both cell densities in the 4B3 cell line (Figure 21D). Cells were cultured at 1.08 × 10 cells / mL. 8 After 7 days of culture with 3D ETN at a concentration of 5 x 10 beads / mL (2 x bead dose), the same conditions were 5 cells / mL or 2 x 10 6 Using cells / mL, 1.08 x 10 8 The cells were re-cultured at a 3D ETN bead concentration of 1.08 × 10 beads / mL (2 × bead dose) for an additional 4 days, or 1.08 × 10 8 After a total of 11 days of culture with 3D ETN at a concentration of 100 beads / mL (2x bead dose), cells were harvested and enriched for CD8α. The percentage of CD4-CD8α+ cells increased in all conditions after CD8α enrichment (Figure 22A). Before enrichment, CD8β expression was 5x10 5 cells / mL than 2 x 10 6 CD8α enrichment increased the level of CD8β expression in all samples, but not in cells cultured at high cell densities (2 × 10 6 Higher CD8β expression was retained after enrichment at lower cell densities (5 × 10 cells / mL) (Figure 22B). Cell viability was significantly higher at lower cell densities (5 × 10 cells / mL) for both unmodified and CAR cell lines, both before and after CD8α enrichment. 5 cells / mL) was higher (Figure 22C). CD8α enrichment improved cell viability, but the improvement was more pronounced in the CAR cell lines than in the unmodified cell lines. CAR expression was significantly higher at the two cell densities tested (5 × 10 5 cells / mL and 2 x 10 6 cells / mL), and CD8α enrichment increased the percentage of CAR+ cells (Figure 22D).

[0391] Cell viability (from CD34+ cells) was monitored over the full 21-day differentiation time course for both unmodified (4A1, Figure 23A) and CAR (4B3, Figure 23B) cell lines. At day 0, both cell lines were approximately 90% viable, and viability decreased to approximately 40% by day 10 during pro-T differentiation. At days 14 and 15 (1.08 x 10 respectively), viability was approximately 90%. 8 After 4 and 5 days of culture with 3D ETN at a concentration of 5000 beads / mL, 4A1 cell viability was cell density dependent, with cell densities as low as 5 × 10 5 cells / mL) is obtained at a higher cell density (2 x 10 6 The survival rate was higher than that of the control group (1.08 × 10 cells / mL). 8 After 7 days of culture with 3D ETN at a concentration of 5 × 10 beads / mL, cell viability was significantly higher for two cell lines (4A1 and 4B3) and at a cell density of 5 × 10 5 cells / mL and 2 x 10 6 The results were similar between day 21 (1.08 × 10 cells / mL) and day 22 (1.08 × 10 cells / mL). 8 After 11 days of culture with 3D ETNs at a concentration of 1000 beads / mL, density-dependent survival was again observed, as shown in Figure 22C.

[0392] Cell proliferation was assessed for 4A1 (Figure 24A) and 4B3 (Figure 24B) cells compared to day 0. On day 10 (1.08 x 10 8 Before culturing in 3D ETNs at a concentration of beads / mL (before 17 days) and on day 17 (1.08 × 10 8 The relative proliferation of 2 × 10 6 cells / mL than 5 x 10 5 The 4A1-induced cytotoxicity was higher in cells cultured at 1000kJ / mL (7-fold vs. 2.5-fold for 4A1; 5.7-fold vs. 2.3-fold for 4B3). Day 21 (1.08 × 10 8 After 11 days of culture with 3D ETNs at a concentration of beads / mL, a lower cell culture density (5 × 10 5 cells / mL), cumulative proliferation remained higher.

[0393] Example 8: Generation and function of CD4-CD8+ cells Diminished Notch signaling via reduced ETN dose induces ProTs to differentiate via the canonical thymic developmental pathway into CD4+CD8+ cells (double positive: DP) (schematic, Figure 25). In vivo and previously described methods, these require TCR rearrangement (B-selection) and CD3 / TCR for differentiation into cytotoxic CD4-CD8+ (single positive: SP) T cells (Figure 25). In contrast, sustained supraphysiological Notch signaling in vitro mediated by high ETN dose culture in the absence of CD3 / TCR stimulation bypasses the intermediate differentiation step and the need for TCR / CD3 signaling, resulting in a population of functional CD8-SP T cells from ProTs after 7 days (Figure 25).

[0394] iPSC-derived CD34+ HSPCs were cultured in serum-free suspension medium for 10 days with various ETN bead doses. T cell lineage induction from iPSC-derived HSPCs was shown to depend on the strength of ETN-mediated Notch signaling. Generation of precursor T (ProT; CD5+CD7+) cells versus myeloid (CD33+CD7-) cells from iPSC-derived HSPCs on day 10 of culture was observed to be dose-responsive to ETN bead dose (Figure 26A). ETN bead dose was normalized to the surface area of ​​the tissue culture plate. Representative flow cytometry plots of CD5, CD7, and CD33 expression are shown for 0.1 and 1 ETN bead doses (Figure 26B).

[0395] CD8-SP cell yield at day 17 was found to be responsive to ETN dose in TRAC-deficient cell lines, both in CD19-CAR-engineered cells and in lines lacking CD19-CAR (Figure 27A). High ETN doses resulted in CD8-SP cells, while low (physiological) levels resulted in CD8+CD4+ (DP) cells. Representative flow cytometry plots of CD4 versus CD8a expression are shown for 0.1, 1, and 3 ETN bead doses (Figure 27B, n=3 technical triplicates / cell line).

[0396] Dose-responsive gene expression changes to ETN on day 2 were found to be a quantitative surrogate for Notch signal strength. Transcripts were measured using a custom NanoString® panel. A machine learning workflow was used to predict CD8-SP phenotype on day 17 based on Notch-responsive gene expression on day 12 and to identify Notch-responsive genes associated with the CD8-SP phenotype (Figure 28A). In the workflow, gene expression data after 2 days of culture and flow cytometry CD4-CD8+ phenotype after 7 days of culture were randomly split into 50 / 50 test splits 1000 times. For each train-test split, a regularized linear regression model was fitted with 5-fold cross-validation to predict CD4-CD8+ cell phenotype from the gene expression data. Model accuracy was measured using the coefficient of determination (R) for the test data. 2 A weighted linear average of the regression coefficients, with weights corresponding to the frequency of genes appearing in the ensemble of models, revealed the top six positively and top six negatively associated genes with the CD8 SP phenotype (weighted averages shown in Figure 28A). All models were highly accurate (average R on the test data). 2 = 0.92, Figure 28B). DTX1 was identified as the most positive predictive feature and was strongly correlated with CD8-SP (Figure 28C). Trends in ETN-responsive gene expression were observed to be similar in both cell lines (Figure 28D, z-score normalized mean expression is shown, n = 3).

[0397] Gene expression analysis was extended to additional cell lines. As described above, the transcriptional response to ETN was measured with a custom 43-gene NanoString® panel 2 and 7 days after ETN administration to progenitor T cell populations (ProTs). Data were separated into 1,000 50-50 train-test splits, and regularized linear models were trained and evaluated using 5-fold cross-validation. The average weighted regression coefficients of the top 12 predictive features are shown in Figure 28E. Positive predictors (NOTCH1, HES1, DTX1) are known Notch signaling response elements, while negative predictors (RAG2, SPN) are known markers of canonical DP stage differentiation. Day 17 CD8α SP phenotype (7 days after ETN administration to progenitor T cell populations) was predicted with good accuracy (85% cross-validation) independent of cell line using a machine learning workflow trained on day 12 gene expression (Figure 28F). NOTCH1 was identified as the most positive predictive feature and increased with increasing bead dose (Figure 28G). Similar trends in ETN-responsive gene expression were observed for TRAC-deficient (TRAC- / -) and TRAC-deficient CD19-CAR-operated (TRAC- / -CD19-CAR+) cell lines (Figure 28H). After culturing with low (0.1x bead dose) versus high (3x bead dose) doses of ETN, the transcriptional profiles of WT, TRAC knockout, and CAR-operated / TRAC-deficient cell lines were assessed using a more comprehensive 770-gene NanoString® panel. Variance partitioning analysis revealed that ETN bead dose and day:line interaction effects accounted for the majority of transcriptional variance (Figure 28I). Thus, the three cell lines differentiated along different trajectories but responded similarly to Notch exposure. Differentiation potential expression pathways (ssGSEA scores) were plotted by bead dose and culture time in the CAR-operated TRAC-deficient cell lines (Figure 28J). The pathway subset shown was filtered based on a statistical cutoff (rank sum test) of Padj<0.05. High ETN doses engaged transcriptional programs associated with T cell differentiation and functional activity.Note that multiple pathways associated with T cell differentiation and function (interferon signaling, JAK-STAT, cytotoxicity) are all induced by elevated Notch signaling and increase with time in culture (Figure 28J).

[0398] We performed single-cell transcriptome analysis of iPSC-derived CD8- SP cells compared with CD8+ T cells derived from peripheral blood and umbilical cord blood. UMAP of the single-cell transcriptomes of iPSC-derived T cells, peripheral blood (PBMC), and umbilical cord blood (CB)-derived CD8+ T cells identified these cell populations as transcriptionally distinct (Figure 29A). Using an algorithm derived from ProjecTILs (Haradhvala et al., 2022), we classified the cells and identified iPSC-derived cells as primarily T-naive, similar to cord blood-derived T cells (Figure 29B). We assessed canonical selective expression for T cells, Notch-responsive genes, innate lymphoid cells (ILCs), and T cell activation and exhaustion transcripts (Figure 29C). Single-sample GSEA (ssGSEA) was applied at single-cell resolution, and means were calculated for each cluster and expressed via hierarchical clustering z-scores. Selected gene signatures were found to be differentially enriched in each cell cluster. iPSC-T cell clusters were observed to have distinct signaling profiles, as labeled and annotated from the UMAP projection (Figure 29D). 1,473 gene signatures were found to be variable across cell clusters (coefficient of variation (CV), the ratio of the standard deviation to the mean ssGSEA score for the cluster, >0.1 for these signatures) (Figure 29E). Three of the iPSC-T cell clusters were observed to share signaling similarities with PBMC cells, and one with CB cells (Figure 29E).

[0399] Additionally, UMAP plots were generated for single-cell transcriptomes (scRNASeq) of iPSC-derived T cells during differentiation (CD34+, ProT, CD8+ SP stages, and after CD19 antigen stimulation) and primary lymphocytes (CD4+ T cells, CD8+ T cells, CD56+ NK cells, and T cells activated with Dynabeads® (CD3 / CD28 stimulation)). Cell* gene expression matrices from single-cell RNASeq sequencing data were calculated using CellRanger (Zheng et al., 2017). Cells with a UMI less than 500 and a mitochondrial ratio less than 0.2 were removed. Expression values ​​for each transcript were normalized at single-cell resolution using SCTransfor (Hafemeister and Satija, 2019), which applies a regularized negative binomial regression model to adjust for the confounding effects of sequencing depth at the cellular level. Further scaling and UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction were performed using Seurat (Satja et al., 2015). While iPSC-derived cell populations were transcriptionally unique, the CD19 stimulatory subset was identified as most similar to activated primary cells (Figure 29F). Bubble plots were generated for selected genes associated with blood progenitors, T cell lineage commitment, canonical T cell function, innate lymphoid cells, cytotoxic T cells, exhausted T cells, and Notch response elements. In addition to internally generated samples, single-cell transcriptomes of autologous CAR-T products Yescarta® and Kymriah® were included for reference (Bai et al., 2022 and Haradhvala et al., 2022). iPSC-derived CD8+ cells acquired canonical T cell expression patterns, with increased CD3 expression, limited expression of ILC or exhaustion markers, and highly increased Notch signaling.

[0400] Single-sample gene set enrichment analysis (ssGSEA) was performed with the Gene Set Variation Analysis (GSVA) ​​package (Hanzelmann et al., 2013) to calculate enrichment scores for select gene signatures at single-cell resolution from an internal database of approximately 2500 compiled pathways (Kirouac et al., 2023) (Figure 29G). In addition to supra-Notch signaling, iPSC-derived CD8+ cells expressed pathways related to T cell functional activity (JAK-STAT, IFNG) and TCR signaling, despite being TRAC-deficient. After CD19 stimulation, iPSC-derived CD8+ cells upregulated transcriptional signatures associated with CAR antigen response and exhaustion (although to a limited extent compared to Yescarta® and Kymriah®), indicating functional T cell responses.

[0401] UMAP of single-cell transcriptomes focused on iPSC-CD8+ cells, iPSC-CD8+ cells after antigen stimulation, and activated T cells were annotated by their transcriptional similarity to developing thymocytes (DN proliferating, DN early, DN resting, CD8+ T, NKT, gamma delta T (GDT), and DP proliferating, as well as Tregs; Park et al., 2020). iPSC-derived cells were annotated as a mixed composition of DN, CD8+ T, and NKT cells, which transitioned to NKT-like cells after antigen stimulation (Figure 29I). Pseudotime analysis was performed using Slingshot (Street et al., 2018). CD8+ T cells emerged directly from the resting DN population and then transitioned to NKT-like cells (Figure 29J). Expression of transcriptional pathways predicting cell population order via an ordinal regression model (ssGSEA, Figure 29K) shown at single-cell resolution. Cell cycle-related pathways gradually gave rise to pathways related to T cell function (Figure 29K).

[0402] The target-mediated cell killing, proliferation, and cytokine secretion of iPSC-derived CD19-CAR-expressing CD8SP cells were evaluated in an in vitro serial assay compared with primary adult donor-derived CAR-T cells. Target cell (CD19-expressing A549 cells) kinetics was assessed over four rounds of in vitro cytotoxicity assays, with primary CAR-T and iPSC-CD8 cells demonstrating cytotoxicity over each round (Figure 30A). Effector T cells (iPSC-derived or primary) were added at a 2:1 effector-to-target (E:T) ratio (Figure 30A, mean ± standard deviation, n = 3). Cell density was measured using Incucyte® live-cell imaging. In vitro cytotoxicity versus effector cell expansion of primary CAR-Ts and iPSC-CAR-Ts was comparable (Figure 30B, n = 2). Cumulative cytotoxicity was reported as the sum of the relative change in area under the curve (AUC) from the target cell-only control for each stimulus (not shown). iPSC-CARTs secreted proinflammatory cytokines when cocultured with antigen-expressing target cells at levels comparable to primary CARTs (Figure 30C). iPSC-derived CD19-CAR-expressing CD8SP cells were capable of continuous target-mediated cell killing, proliferation, and cytokine secretion in vitro comparable to primary CARTs.

[0403] Example 9: Generation of CD4-CD8+ cells from STR-derived HPCs The lymphocyte potential of iPSC-derived CD34+ cells generated by the STR system was analyzed. An overview of the 3D engineered thymic niche (ETN) platform for scalable T cell manufacturing using iPSC-derived CD34+ HPCs is shown in Figure 31A. ETN was shown to be suitable for use with STR to differentiate CD34+ HPCs into CD8 single-positive T cells. iPSC-derived CD34+ HPCs from three different STR runs were differentiated into CD8 cells using microplate culture, using HPCs generated in microplate culture as a reference. HPCs were cultured with ETN at 2.70 x 10 7 beads / mL (0.5× bead dose) for 10 days, followed by 1.35 × 10 8 beads / mL (2.5× bead dose) for 6 days, 1.89 × 108 beads / mL (3.5x bead dose) for subsequent 5 days of culture. STR-derived HPCs were comparable to microplate-culture-derived cells, generating 58.7 ± 3.7% CD8+ cells compared to 59.99% CD8-positive cells from research platform HPCs (Figure 31B). The CD8αβ+ T cell expression pattern was comparable across all three STR runs (mean ± SEM, three independent STR runs, n = 6 bioreactors) (Figure 31C).

[0404] Example 10: Generation and function of CD4-CD8+ cells in STR cultures iPSC-derived ProT cells were cultured in a 30x range of ETN doses (0.1x to 3x bead dose (5.40 x 10) 6 beads / mL ~ 1.62 x 10 8 T cells were cultured in microplates at 100 μg / mL (beads / mL) for 7 days. The resulting cell phenotypes were evaluated by flow cytometry. Higher bead doses produced more CD8 SP cells than lower bead doses, whereas lower bead doses produced more CD4 SP cells and double-positive cells (DP) (Figure 32A). Expression of CD8A, IL7R, CD4, and BCL11A measured by Nanostring® at ETN doses 2 and 3 over 7 days shows that CD8A and ILR7 expression increased over time, while CD4 and BCL11A expression decreased over time (Figure 32B). T cell phenotype was found to be modulated by ETN bead dose.

[0405] iPSC-derived CD34+ cells were cultured at 2.70 × 10 7 The two CD34+ cell banks were differentiated into ProT cells for 10 days in microplates and STRs using ETN at 1 × 10 beads / mL (0.5 × bead dose). The two CD34+ cell banks were cultured in DASbox® bioreactors (Eppendorf) and 24-well plates at 1 × 10 4 and 5 × 10 4 The percentage of CD5+CD7+ProT cells at D10 was comparable in STR and microplate for both cell banks, with 5 × 10 4The percentage of ProT cells was higher in the condition seeded at 1000 cells / mL (Figure 33A). The number of ProT cells produced per input CD34+ cell was higher for STR cultures (Figure 33A). STR cultures with ETN generated a similar percentage of precursor T cells as 24-well plate cultures and were highly reproducible.

[0406] Eight bioreactors were filled with 5 × 10 CD34+ cell banks of one type. 4 Differentiation and expansion was reproducible at D10 with 28±4% CD5+CD7+ cells, generating 23±6 ProT cells per input CD34+ cell (mean±1SD) (Figure 33B).

[0407] STR-generating ProT cells (Figure 33) at 1 × 10 6 Reseeded at 1.35 x 10 cells / mL, based on optimal conditions for microplate culture (Figure 32). 8 The cells were cultured for 11 days in 1000 sputum / mL ETN (2.5x relative bead dose). CD8+ cell yield was highest after 8 days of maturation, with an average CD8+ percentage of 41±1.5% across all eight bioreactors (Figure 34A). The number of CD8+ cells produced per input CD34+ cell was 35±5 (Figure 33A). This expression pattern was comparable to that seen in Figure 32 for the microplate cultures. The percentage of CD8αβ+ cells at day 8 of maturation was 28±1%, consistent across all eight STRs (Figure 34B). In microplate cultures, an increase in the percentage of CD8αβ+ cells was observed by day 14 of maturation.

[0408] Gene expression kinetics (assessed by Nanostring®) revealed similar trends in CD8A, IL7R, CD4, and BCL11A expression between STR and microplate cultures (Figure 35A, Figure 32). Gene expression kinetics was quantified by the sign of the regression coefficient and the logarithm of the Benjamini / Hochberg adjusted p-value of the linear model fitted to the expression of each gene over time. Microwell and STR gene expression kinetics were found to be significantly correlated (Pearson's r=0.71, p<0.001) for a panel of 24 genes related to leukocyte development (Figure 35B).

[0409] We performed a functional comparison of iPSC-derived CD8+ CAR-T cells generated by STR culture with donor peripheral blood CD8+ CAR-T (primary CD8+ CAR-T cells). Using GFP-expressing CD19+ cells as target cells, we developed a serial restimulation assay to measure cytotoxic activity using an Incucyte® (Sartorius) live cell imaging assay (n = 3 technical replicates). T cells were co-cultured with target cells and exogenous cytokine support at a 2:1 effector cell to target cell (E:T) ratio every 5 days. Target clearance was measured by the decrease in GFP surface area. iPSC-derived CD8+ CAR-T cells exhibited comparable activity to primary CD8+ CAR-T cells over four antigen exposures (Figure 36A). Cell expansion folds were calculated by counting cells at the end of each target exposure. iPSC-derived CAR-T cells expanded 57,000-fold over four antigen exposures, at levels comparable to primary CD8+ CAR-T cells (Figure 36B).

[0410] Secretion of the effector molecules perforin, interferon-γ (IFNγ), granzyme B, and tumor necrosis factor α (TNFα) was also assessed. iPSC-derived CD8+ CAR-T cells secreted effector molecules in a target-specific manner at levels comparable to primary CD8+ CAR-T cells (Figure 36C).

[0411] T cell subsets in iPSC-derived CD8+ CAR-T cells and primary CAR-T cells were classified based on the expression of CD45RA, CD62L, and CD95. Stem cell memory (TSCM) and central memory (TCM) subsets, previously shown to be associated with better in vivo outcomes, were found to be enriched in iPSC-derived CD8+ CAR-T cells at baseline and upon chronic antigen exposure (Figure 36D). Expression of exhaustion markers was also assessed in both cell populations. Co-expression of multiple exhaustion markers (PD1, TIM3, LAG3, TIGIT, and CD39), which have been reported as hallmarks of T cell exhaustion, was observed to be lower in iPSC-derived CD8+ CAR-T cells compared to primary CD8+ CAR-T cells (Figure 36E).

[0412] iPSC-derived CD8+ CAR-T cells generated by STR culture were also further enriched by CD8α or CD8β selection and analyzed by flow cytometry. After CD8α enrichment, both CD8αα+ and CD8αβ+ cells were present (Figure 37A). Lower levels of CD8α were detected due to blocking of the CD8α epitope by the enrichment process. The majority of CD8β-enriched cells were CD8αβ+ (Figure 37B), and both CD8α- and CD8β-enriched cells lacked surface CD3 expression (Figures 37A, B).

[0413] Example 11: Generation and function of exogenous TCR+ cells Two independent iPSC lines ("172" and "174") were generated using site-specific integration (SSI) of the MAGE-A4 TCR at the TRAC locus under the ubiquitin C (UBC) promoter. These cell lines, along with an unmodified cell line control ("6A1"), were differentiated into CD34+ cells as described above. CD34+ cells were then cultured at 5 x 10 4 cells / mL in well plates and differentiated into precursor T cells for 10 days, with 2.70 × 10 7 3D ETN was added at 1.5 × 10 beads / mL (0.5 × bead dose) and half the medium was replaced on days 4 and 7. The cells were then cultured at 1.5 × 10 6Re-seeded at 1.62 x 10 cells / mL 8 The cells were cultured for an additional 11 days (21 days total from CD34+ cells) in 3D ETN at 3x beads / mL (3x bead dose). Half-medium changes were performed on days 12, 14, 16, and 19, and on day 17, 1.62 x 10 8 1.5 x 10 beads / mL (3 x bead dose) with 3D ETN 6 Cells were replated at 1000 cells / mL. Cells were harvested on day 21 (from the CD34+ stage) and CD8 enriched using EasySep™ Human CD8 Positive Enrichment II Kit (STEMCELL Technologies).

[0414] For all cell lines, a decrease in viability was observed at the end of differentiation, which improved after CD8α enrichment (Figure 38A, B). The cumulative fold expansion was highest in one of the TCR-modified cell lines ("174") at the end of differentiation (Figure 38C).

[0415] As determined by flow cytometry, CD5+CD7+ cells were observed in all cell lines by day 10 of differentiation, and CD5 and CD7 expression further increased from day 17 onwards (Figure 39A). CD8 expression was observed by day 17, and the CD8+ population increased after CD8 enrichment on day 21 (Figure 39B). After enrichment, both CD8αα+ and CD8αβ+ cells were present in all cell lines, with a greater proportion of CD8αβ+ cells ("172" and "174") observed in the TCRSSI line (Figure 39C). In all cell lines, the majority of CD8αβ+ cells were CD4-CD8+ cells (Figure 40).

[0416] For the TCRSSI line, cells maintained high expression of MAGE-A4 TCR throughout the course of differentiation (FIGS. 41A, 41B).

[0417] Cell characteristics following differentiation by 3D ETN and CD8 enrichment are shown in Table 9 below. [Table 9]

[0418] The in vitro functionality of the TCR-iPSC-derived CD4-CD8+ cells described above was then evaluated using a serial restimulation assay against A375 human melanoma target cells. Both the TCR-modified iPSC-derived CD8+ cells and the TCR-modified primary CD8+ cells proliferated over the course of the assay, with the "174" line showing increased proliferation compared to the "172" line (Figure 42A, Figure 42B). After the first stimulation, cytotoxicity was similar at multiple effector:target ratios for the two TCR-modified iPSC-derived CD8+ cell lines (Figure 43). In two independent experiments, the TCR-modified iPSC-derived CD8+ cells were functional throughout four serial killings, similar to the TCR-modified primary CD8+ cell control (Figure 44A, B and Figure 44C, D).

[0419] The specificity of the cells was analyzed by comparing cytotoxicity against wild-type A375 target cells (A375WT, presenting the antigen of interest), β2-microglobulin (B2M) knockout A375 cells (A375B2MKO, no antigen presentation), MC-38 mouse colon adenocarcinoma cells, and human umbilical vein endothelial cells (HUVEC). MC-38 and HUVEC cells were labeled with Cytolight Orange (Incucyte®) and therefore showed loss of fluorescent label over time due to cell division, whereas A375WT and A375B2MKO cells were labeled with Nuclight Green (Incucyte®) for stable expression. Upon initial activation, TCR-modified iPSC-derived CD8+ cells were specific for the A375 target (Figure 45).

[0420] Example 12: Intracellular TCRβ analysis CAR-modified TRAC- / - iPSC-derived cells were differentiated using 3D ETN as described in Example 11 above. Cells were analyzed for intracellular CD3 and intracellular TCRβ (TCRVβF1) on days 0 (CD34+ stage), 10 (precursor T cell stage), 17, 21, and 24. By day 10, a high percentage of cells were positive for intracellular CD3 (Figure 46A), and intracellular CD3 continued to be detected up to day 24 (Figure 46A, Figure 46B). In contrast, intracellular TCRβ was not detected at any time point (Figure 46A,B). Cell viability decreased over time, and the percentage of CD4-CD8α+ cells increased (Figure 46C, Figure 47). The CD8α+β+ population also increased over time (Figure 47). Small numbers of CD3+TCRγδ- and CD3+TCRγδ+ cells were observed on days 21 and 24 (Figure 48).

[0421] Example 13: Generation and function of exogenous TCR+ and CAR+ cells Four cell lines were generated and compared: iPSC-derived CD34+ cells ("unmodified"), iPSC-derived CD34+ cells transduced with a TCR on day 7 ("unmodified TCR transduced"), TRAC- / -CD19 CARSSI iPSC-derived CD34+ cells ("CARSSI"), and TCRSSI iPSC-derived CD34+ cells ("TCRSSI"). Cells were differentiated in 3D ETNs as described in Example 11 above. Viability was comparable between lines over time (Figure 49A), and fold expansion was higher for the TCRSSI line (Figure 49B).

[0422] CAR or TCR expression was analyzed over the course of differentiation. TCRαβ expression levels at day 10 ranged from 20-30% for the various cell lines (Figure 49C).

[0423] Cells were further characterized for expression of CD5, CD7, CD56, CD4, CD8α, and CD8β by flow cytometry. TCR-transduced cells had a higher proportion of CD5+CD7+ and a lower proportion of CD56+CD7+ populations during late differentiation compared to their untransduced counterparts (Figure 50A, B). TCR-transduced cells had higher expression of CD4 and a lower proportion of CD4-CD8α+ cells compared to their untransduced counterparts at day 21 (Figure 51A). However, the proportion of CD8α+CD8β+ cells within this fraction was higher compared to CD8αα+ cells (Figure 51B, subgated on CD4-CD8α+). Furthermore, higher expression of CD8α+CD8β+ in the overall cell population was observed at day 21 in TCR-transduced cells compared to untransduced controls (Figure 52A). However, the composition of this subpopulation was attributable to both CD4-CD8+ cells (CD8SP) and CD4+CD8+ cells (DP) (subgated on CD8α+CD8β+ Figure 52B).

[0424] The CAR ("CARSSI")-modified CD8α-enriched cells were then cryopreserved and then exposed to target cells in an in vitro serial restimulation assay. After thawing, the cells retained expression of key markers (Figure 53). In both the first and second stimulations, CAR SSI cells proliferated in the presence of Raji target cells (Figure 54A). However, a loss of CD8β+ expression was observed (Figure 54B). The cells retained cytotoxicity across multiple stimulations, and activity was observed against CD19- / - target cells (Figure 55C).

[0425] In subsequent in vitro serial restimulation assays, CAR SSI cells were expanded in three cycles of coculture with target cells in the presence of A549 target cells (Figure 55A, B). As reported above for coculture with Raji target cells, loss of CD8β+ expression was observed over the course of the assay using A549 target cells (Figure 55C). Cytokine secretion was antigen-dependent in iPSC-derived CAR SSI cells, with greater cytokine secretion observed in coculture with A549 target cells compared to Raji target cells (Figure 56). The viability of iPSC-derived CAR SSI cells decreased after the first stimulation with both Raji or A549 target cells, continued to decrease in coculture with Raji cells, and remained stable in subsequent stimulations with A549 target cells (Figure 57).

[0426] Example 14: In vivo delivery of CAR+ cells Cells expressing CD19 CAR were generated from iPSCs using 3D ETNs as described above. Cells were characterized at harvest and after CD8 enrichment (Table 10, Figure 58 after enrichment). [Table 10]

[0427] The in vivo function of CAR+ iPSC-derived CD8 cells was evaluated in a Raji tumor implant model. Raji cells were transfected with immunocompromised NOD-scidIL2Rg null (NSG) mice were intravenously (i.v.) delivered and supplemented with exogenous cytokines by intraperitoneal (i.p.) injection three times per week for 4 weeks. Two days after tumor implantation, CAR iPSC-derived CD8 cells (3 doses of 1 x 10 7 cells, 3 days apart (Q3Dx3), iv) or primary CAR CD8 T cells (3 x 10 6A single dose of cells (iv) was administered. Repeated administration of CAR+ iPSC-derived CD8+ cells significantly delayed Raji tumor growth and prolonged median survival compared with untreated controls (Figure 59A, Figure 59B). Changes in animal weight were monitored as a surrogate indicator of toxicity (Figure 59C). Fewer CAR+ iPSC-derived CD8+ cells were detected in peripheral blood compared with primary CAR+ CD8+ T cells (Figure 60A). Eight days after injection, CAR+ iPSC-derived CD8+ cells were highly CD3-CAR+ (Figure 60B).

[0428] In another study, the in vivo function of CAR+ iPSC-derived CD8+ cells was evaluated in a disseminated A549-CD19 tumor model. NSG mice were administered A459-CD19 tumor cells intravenously and supplemented with exogenous cytokines via intraperitoneal (ip) injection three times per week for four weeks. Two days after tumor implantation, CAR+ iPSC-derived CD8+ cells (three doses of 1 x 10 7 cells, 3 days apart (Q3Dx3), iv) or primary CAR CD8 T cells (3 x 10 6 A single dose of cells (iv) was administered. Repeated administration of CAR+ iPSC-derived CD8+ cells maintained complete remission comparable to primary CAR+ CD8+ T cells against A549-CD19+ disseminated tumors (Figures 61A-61C). Animal weight change was monitored as a surrogate for toxicity, and comparable weight gain was observed between groups (Figure 61D). Fewer CAR+ iPSC-derived CD8+ cells were detected in peripheral blood compared with primary CAR+ CD8+ T cells (Figure 61D). At day 15 post-infusion, recovered CAR+ iPSC-derived CD8+ cells had a higher proportion of CD3+ compared with pre-infusion analysis (Figure 62A). Within the CAR+ population, iPSC-derived CD8+ cells had a higher proportion of CD8αβ+ from day 15 onwards (Figure 62B). Furthermore, CAR+ iPSC-derived CD8+ cells harvested 15 days post-infusion secreted proinflammatory cytokines and cytolytic molecules (Figure 63).

[0429] The A549-CD19 study was advantageously expanded to examine the ability of CAR+ iPSC-derived CD8+ cells to exert control over tumor rechallenge. At day 36, 2.5 x 10 5 A549-CD19 tumor cells were administered intravenously to animals previously treated with CAR+ iPSC-derived CD8+ cells. Cytokine support was resumed until the end of the study on day 50. Test animals tolerated the rechallenge with A549-CD19+ tumors, with extended full survival and continued weight gain (Figure 64A, B). Persistent CAR+ iPSC-derived CD8+ cells maintained tumor growth inhibition (Figure 65A-C) and were detected at low levels in the peripheral blood after rechallenge (Figure 65D). At day 50, CAR+ iPSC-derived CD8+ cells were also detected in the lungs, and these cells were enriched in CD3+ (Figure 66A, B). CAR+ iPSC-derived CD8+ cells recovered from the peripheral blood during rechallenge maintained a high proportion of CD8αβ+ (Figure 66C).

[0430] Example 15: Generation and function of exogenous TCR+ and CAR+ cells in STR CAR-modified (CD19 CAR) and TCR-modified (MAGE-A4 TCR) iPSC-derived CD34+ cells were differentiated in a stirred tank reactor containing 3D ETN. Cells were initially seeded on day 0 and 2.7 x 10 cells were seeded on day 1. 7 Precursor T cells were generated by culturing with 3D ETNs at 1.35 × 10 beads / mL (0.5 × bead dose). Cells reached 1.35 × 10 8 beads / mL (2.5× relative bead dose) of 3D ETN and 1.89×10 8 The cells were then replated with 3D ETN at 3.5x bead dose per mL and cultured for an additional 5 days (21 days total). At day 21, viability was lower for CAR-modified cells compared to TCR-modified cells (Figure 67A), and a higher cell density was observed for TCR-modified cells (Figure 67B). At day 21, the TCR-modified cell line also had a higher purity of CD8αβ+ cells compared to CAR-modified cells (Figure 68A, B).

[0431] Using an in vitro serial restimulation assay, we investigated TCR-modified cells generated in both STR and well-plate (WP) culture systems with 3D ETN. Similar cytotoxicity and fold expansion were observed in both groups (Figures 69A, B). Furthermore, comparable function at various E:T ratios (Figure 69C) and antigen specificities (Figure 69D) was observed for both STR- and WP-producing cells.

[0432] It is contemplated that T cell lineage populations derived from culturing progenitor T cells in 3D ETNs using the methods provided herein can be included in pharmaceutical compositions.

[0433] It is further contemplated that a T cell lineage population derived from culturing precursor T cells in 3D ETNs using the methods provided herein can be used to treat a disease or condition in a subject. "Treating" refers to administering an effective amount of the cells provided herein to a subject under conditions suitable for increasing the number of the subject's T cells, which can result in the prevention, inhibition, and / or therapeutic treatment of a medical condition. An "effective amount" refers to a therapeutically effective amount, e.g., an amount of cells sufficient to achieve the intended purpose (e.g., treatment) when administered to a subject. The amount may vary from subject to subject and may depend on one or more factors, such as the subject's gender, age, weight, health history, and / or the underlying cause of the condition being prevented, inhibited, and / or treated.

[0434] For example, a subject suffering from an oncological or autoimmune disease, condition or disorder may benefit from administration of a T cell lineage population as described herein.

[0435] Although the present disclosure has been described with reference to specific embodiments, various modifications thereof will be apparent to those skilled in the art. Any examples provided herein are included solely for the purpose of illustrating the present disclosure and are not intended to limit the disclosure in any way. Any drawings provided herein are intended solely for the purpose of illustrating various aspects of the present disclosure and are in no way intended to be drawn to scale or to limit the disclosure. The claims appended hereto should not be limited by the preferred embodiments set forth in the above description, but should be accorded the broadest interpretation consistent with the present specification as a whole. The disclosures of all technologies listed herein are incorporated herein by reference in their entirety. 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Claims

1. 1. A method of generating a T cell lineage cell population, comprising: a) providing a population of precursor T cells; and b) culturing said precursor T cells in the presence of a surface-bound Notch signaling ligand; The Notch signaling ligand is present in an amount of at least 7 square centimeters (7 cm) per milliliter of culture volume. ２ / mL) of surface area.

2. The method of claim 1, wherein the T cell lineage cell population is enriched for CD4-CD8+ cells.

3. 3. The method of claim 1 or 2, wherein increasing the Notch signalling ligand surface area increases the absolute number of CD4-CD8+ cells and / or the relative number of CD4-CD8+ cells in the cell population.

4. The Notch signaling ligand is 7 to 56 cm ２ / mL, 7.8-55.2 cm ２ / mL, or 15.7 to 55.2 cm ２ The method according to any one of claims 1 to 3, wherein the surface area of ​​the spheroid is provided to the spheroid in an amount of 1 / mL.

5. The method of any one of claims 1 to 4, wherein the Notch signalling ligand is provided on a three-dimensional substrate.

6. The method of claim 5 , wherein the three-dimensional substrate is one or more beads.

7. 7. The method of claim 6, wherein the one or more beads are comprised of a material selected from the group consisting of polystyrene, iron oxide, and gold.

8. The method of claim 7, wherein the one or more beads are made of polystyrene.

9. The method of any one of claims 6 to 8, wherein the Notch signalling ligand is covalently bound to the one or more beads.

10. 10. The method of any one of claims 1 to 9, wherein the Notch signalling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

11. The concentration of DL4 is 7.89 x 10 １０ ~1.66 x 10 １３ The method of claim 10, wherein the concentration is in molecules / mL of culture volume.

12. 11. The method of claim 10, wherein the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

13. The DL4 and the VCAM-1 are 7.8 to 55.2 cm ２ 13. The method of claim 12, wherein the surface area of ​​the solution is provided to a surface area of ​​1 mL / mL.

14. The concentration of DL4 is 7.89 x 10 １０ ~1.66 x 10 １３ molecules / mL culture volume, and the concentration of VCAM-1 was 7.89 × 10 １０ ~1.66 x 10 １３ The method of claim 12, wherein the concentration is in molecules / mL of culture volume.

15. The cell density is 5 × 10 ５ ~2 x 10 ６ The method of claim 11 or claim 14, wherein the concentration is cells / mL.

16. 10. The method of claim 1, wherein the progenitor T cells are cultured for at least 3 days, at least 7 days, at least 11 days, or at least 14 days.

17. 17. The method of claim 16, wherein the progenitor T cells are cultured for at least 14 days.

18. 17. The method of claim 16, wherein the progenitor T cells are re-cultured at least once during or after culturing the progenitor T cells for at least 3 days, at least 7 days, at least 11 days, or at least 14 days, and wherein the re-culture is performed in the presence of the surface-bound Notch signaling ligand.

19. The method according to any one of claims 2 to 18, wherein the CD4-CD8+ cells are CD8αβ+ cells.

20. The method of claim 2, wherein the CD4-8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

21. The method of any one of claims 1 to 20, wherein the progenitor T cells are derived from pluripotent stem cells.

22. 21. The method of any one of claims 1 to 20, wherein the progenitor T cells are derived from induced pluripotent stem cells (iPSCs).

23. 23. The method of any one of claims 1 to 22, wherein the progenitor T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

24. 23. The method of claim 21 or 22, wherein the pluripotent stem cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

25. A population of CD4-CD8+ cells produced according to the method of any one of claims 1 to 24.

26. The population of claim 25, wherein the CD4-CD8+ cells are CD8αβ+ cells.

27. 26. The population of claim 25, wherein the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

28. 28. The population of any one of claims 25 to 27, wherein the CD4-CD8+ cells express a chimeric antigen receptor (CAR).

29. 28. The population of any one of claims 25 to 27, wherein the CD4-CD8+ cells express an exogenous T cell receptor (TCR).

30. 1. A pharmaceutical composition comprising CD4-CD8+ cells and a pharmaceutically acceptable carrier, wherein the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

31. The CD4-CD8+ cells are distributed over at least 7 square centimeters (7 cm) per milliliter of culture volume. ２ 31. The pharmaceutical composition of claim 30, wherein the T cell is derived in vitro from a progenitor T cell by culturing the T cell at a surface area of ​​1000 μg / mL in the presence of a surface-bound Notch signaling ligand.

32. 32. The pharmaceutical composition of claim 31, wherein the progenitor T cells are derived in vitro from pluripotent stem cells.

33. The pharmaceutical composition of claim 30, wherein the CD4-CD8+ cells express a chimeric antigen receptor (CAR).

34. The pharmaceutical composition of claim 30, wherein the CD4-CD8+ cells express an exogenous T cell receptor (TCR).

35. 1. A method of treating a disease or condition in a subject, comprising: a) culturing a cell population comprising progenitor T cells in the presence of a surface-bound Notch signaling ligand, wherein the Notch signaling ligand is present in an area of ​​at least 7 square centimeters (7 cm) per milliliter of culture volume; ２ generating a T cell lineage cell population by culturing the cells so as to provide a surface area of ​​100 μg / mL; and b) administering an effective amount of said population of T cell lineage cells to a subject in need of treatment for a disease or condition. A method comprising:

36. 36. The method of claim 35, wherein the T cell lineage cell population is enriched for CD4-CD8+ cells.

37. The method of claim 36, wherein the CD4-CD8+ cells are CD8αβ+ cells.

38. 36. The method of claim 35, wherein the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

39. 36. The method of claim 35, wherein the T cell lineage cell population comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

40. 40. The method of any one of claims 35 to 39, wherein the Notch signalling ligand is provided on a three-dimensional substrate.

41. 41. The method of any one of claims 35 to 40, wherein the Notch signalling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

42. 42. The method of claim 41, wherein the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

43. The method of any one of claims 35 to 42, wherein the disease is cancer.

44. 1. Use of a T cell lineage cell population in the manufacture of a medicament for treating a disease or condition, said T cell lineage cell population comprising: a) culturing a population of cells comprising progenitor T cells in the presence of a surface-bound Notch signalling ligand; The surface-bound Notch signaling ligand is at least 7 square centimeters (7 cm) per milliliter of culture volume. ２ / mL) surface area, produced by the method, use.

45. 45. The use of claim 44, wherein the T cell lineage cell population is enriched for CD4-CD8+ cells.

46. The use according to claim 45, wherein the CD4-CD8+ cells are CD8αβ+ cells.

47. 45. The use of claim 44, wherein the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3- (sCD3-) cells, or TCR- / sCD3- cells.

48. 48. The use of any one of claims 44 to 47, wherein the T cell lineage cell population comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

49. 49. The use of any one of claims 44 to 48, wherein the T cell lineage cell population expresses a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

50. 50. The use according to any one of claims 44 to 49, wherein the Notch signalling ligand is provided on a three-dimensional substrate.

51. 51. The use according to any one of claims 44 to 50, wherein the Notch signalling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

52. 52. The use of claim 51, wherein the Notch signaling ligand is DL4 and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

53. The use according to any one of claims 44 to 52, wherein the disease is cancer.

54. 1. A method for differentiating a population of precursor T cells, comprising: a) providing a population of precursor T cells; and b) culturing said precursor T cells in the presence of a surface-bound Notch signaling ligand. and wherein the Notch signaling ligand is present in an amount of 0.78 to 4.7 square centimeters (cm) per milliliter of culture volume. ２ / mL) and the differentiated cell population is enriched for CD4-CD8+TCRγδ+ cells.

55. 55. The method of claim 54, wherein the Notch signaling ligand is provided on a three-dimensional substrate.

56. 56. The method of claim 55, wherein the three-dimensional substrate is one or more beads.

57. 57. The method of claim 56, wherein the one or more beads are made of a material selected from the group consisting of polystyrene, iron oxide, and gold.

58. 58. The method of claim 57, wherein the one or more beads are made of polystyrene.

59. 59. A method according to any one of claims 56 to 58, wherein the Notch signalling ligand is covalently attached to the one or more beads.

60. 60. The method of any one of claims 54 to 59, wherein the Notch signalling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

61. 61. The method of claim 60, wherein the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

62. A population of CD4-CD8+TCRγδ+ cells generated in vitro according to the method of any one of claims 52 to 59.

63. 1. A method of generating a T cell lineage cell population, comprising: a) providing a population of precursor T cells; and b) culturing said precursor T cells in a culture vessel in the presence of a surface-bound Notch signaling ligand. Including, A method wherein the Notch signalling ligand is provided on a substrate, and wherein the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is at least 1.77 to 1 (1.77:1).

64. 64. The method of claim 63, wherein the T cell lineage cell population is enriched for CD4-CD8+ cells.

65. 65. The method of claim 63 or 64, wherein increasing the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel increases the absolute number of CD4-CD8+ cells and / or the relative number of CD4-CD8+ cells in the cell population.

66. 66. The method of any one of claims 63-65, wherein the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is between 1.77:1 and 14:1, between 2:1 and 14:1, or between 4:1 and 14:

1.

67. 67. The method of any one of claims 63 to 66, wherein the Notch signalling ligand is provided on a three-dimensional substrate.

68. 68. The method of claim 67, wherein the three-dimensional substrate is one or more beads.

69. 69. The method of claim 68, wherein the one or more beads are made of a material selected from the group consisting of polystyrene, iron oxide, and gold.

70. 70. The method of claim 69, wherein the one or more beads are made of polystyrene.

71. 71. A method according to any one of claims 68 to 70, wherein the Notch signalling ligand is covalently attached to the one or more beads.

72. 72. The method of any one of claims 63 to 71, wherein the Notch signalling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

73. The concentration of DL4 is 7.89 x 10 １０ ~1.66 x 10 １３ 73. The method of claim 72, wherein the nuclease activity is in molecules / mL of culture volume.

74. 73. The method of claim 72, wherein the Notch signaling ligand is DL4, and the step of culturing the progenitor T cells further comprises culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

75. 75. The method of claim 74, wherein the DL4 and VCAM-1 are provided on a substrate, and the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is between 1.77:1 and 14:1, between 2:1 and 14:1, or between 4:1 and 14:

1.

76. The density of DL4 is 7.89 x 10 １０ ~1.66 x 10 １３ molecules / mL culture volume, and the density of VCAM-1 was 7.89 × 10 １０ ~1.66 x 10 １３ 76. The method of claim 75, wherein the concentration is in molecules / mL culture volume.

77. The cell density is 5 × 10 ５ ~2 x 10 ６ The method of any one of claims 63 to 76, wherein the concentration is cells / mL.

78. 78. The method of any one of claims 63 to 77, wherein the precursor T cells are cultured for at least 3 days, at least 7 days, at least 11 days or at least 14 days.

79. 79. The method of claim 78, wherein the progenitor T cells are cultured for at least 14 days.

80. 79. The method of claim 78, wherein the progenitor T cells are re-cultured at least once during or after culturing the progenitor T cells for at least 3 days, at least 7 days, at least 11 days, or at least 14 days, and said re-culture is performed in the presence of the surface-bound Notch signaling ligand.

81. The method of claim 64, wherein the CD4-CD8+ cells are CD8αβ+ cells.

82. 65. The method of claim 64, wherein the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or T cell receptor (TCR)- / sCD3- cells.

83. 83. The method of any one of claims 63 to 82, wherein the progenitor T cells are derived from pluripotent stem cells.

84. 83. The method of any one of claims 63 to 82, wherein the progenitor T cells are derived from induced pluripotent stem cells (iPSCs).

85. 85. The method of any one of claims 63-84, wherein the progenitor T cells comprise nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

86. 84. The method of claim 83, wherein the pluripotent stem cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

87. A population of CD4-CD8+ cells produced according to the method of any one of claims 63 to 86.

88. 88. The population of claim 87, wherein the CD4-CD8+ cells are CD8αβ+ cells.

89. 88. The population of claim 87, wherein the CD4-CD8+ cells are T cell receptor (TCR)- cells, surface CD3 negative (sCD3-) cells, or TCR- / sCD3- cells.

90. 90. The population of any one of claims 87 to 89, wherein the CD4-CD8+ cells express a chimeric antigen receptor (CAR).

91. 90. The population of any one of claims 87 to 89, wherein the CD4-CD8+ cells express an exogenous T cell receptor (TCR).

92. 1. A method of generating a T cell lineage cell population, comprising: a) providing a population of precursor T cells; and b) culturing said precursor T cells in the presence of a Notch signaling ligand and in the absence of a T cell receptor stimulator. Including; The method, wherein the T cell lineage cell population comprises CD4-CD8+ cells.

93. 93. The method of claim 92, wherein the CD4-CD8+ cells are surface CD3 negative (sCD3-).

94. The method of claim 92 or 93, wherein the CD4-CD8+ cells are T cell receptor negative (TCR-) cells.

95. 93. The method of claim 92, wherein the progenitor T cells comprise a nucleic acid encoding a CAR, and wherein culturing the progenitor T cells further comprises culturing in the absence of a CAR activator.

96. the Notch signaling ligand is surface-bound, and the surface-bound Notch signaling ligand is present in an amount of at least 7 square centimeters (7 cm) per milliliter of culture volume. ２ 93. The method of claim 92, wherein the surface area of ​​the solution is provided to a surface area of ​​1 / mL.

97. The Notch signaling ligand is surface-bound, and the surface-bound Notch signaling ligand is present in an amount of 7 square centimeters (7 cm) per milliliter of culture volume. ２ / mL) ~ 56 cm ２ 93. The method of claim 92, wherein the surface area of ​​the solution is provided to a surface area of ​​1 mL.

98. 93. The method of claim 92, wherein the Notch signaling ligand is provided on a three-dimensional substrate, and the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is at least 1.77 to 1 (1.77:1).

99. 99. The method of claim 98, wherein the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is between 1.77: to 1 (1.77:1) and 14: to 1 (14:1).

100. 100. The method of claim 98 or 99, wherein the three-dimensional substrate is one or more beads.

101. 1. A method of generating a T cell lineage cell population, comprising: a) providing a population of hematopoietic stem and / or progenitor cells; and b) culturing said hematopoietic stem cells / hematopoietic progenitor cells in the presence of a Notch signaling ligand and in the absence of a T cell receptor stimulator. Including; The method, wherein the T cell lineage cell population comprises CD4-CD8+ cells.

102. The method of claim 101, wherein the CD4-CD8+ cells are surface CD3 negative (sCD3-).

103. The method of claim 101 or 102, wherein the CD4-CD8+ cells are T cell receptor negative (TCR-) cells.

104. 102. The method of claim 101, wherein the hematopoietic stem cells and / or hematopoietic progenitor cells comprise a nucleic acid encoding a CAR, and wherein culturing the hematopoietic stem cells and / or hematopoietic progenitor cells further comprises culturing in the absence of a CAR activating agent.

105. A method according to any one of claims 101 to 104, wherein the Notch signalling ligand is surface bound.

106. 106. The method of claim 105, wherein the Notch signaling ligand is provided on a three-dimensional substrate.

107. 107. The method of claim 106, wherein the three-dimensional substrate is one or more types of beads.

108. 108. The method of any one of claims 92 to 107, wherein the Notch signalling ligand comprises the Notch ligand Delta-like-4 (DL4) or a variant thereof.

109. 1. A method of generating a T cell lineage cell population, comprising: a) providing a population of precursor T cells; and b) culturing said precursor T cells in the presence of a surface-bound Notch signaling ligand. Including, The Notch signaling ligand is present in an amount of 7 cm per milliliter of culture volume. ２ / mL) ~ 56 cm ２ / mL of surface area provided; The method, wherein the T cell lineage cell population comprises CD4-CD8+ cells.

110. 1. A method of generating a T cell lineage cell population, comprising: a) providing a population of precursor T cells; and b) culturing the precursor T cells in a culture vessel in the presence of a Notch signaling ligand. Including, the Notch signaling ligand is provided on a three-dimensional substrate, and the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is between 1.77 to 1 (1.77:1) and 14 to 1 (14:1); The method, wherein the T cell lineage cell population comprises CD4-CD8+ cells.

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