Methods for generating populations of t cell lineages from stem / progenitor cells

Abstract

The present disclosure provides a method for generating a population of T cell lineages from T cell progenitor cells. The method comprises culturing the progenitor cell in the presence of a Notch signaling ligand, such as delta-like 4 (DL4), provided on a surface area of at least 7 square centimeter / ml culture volume (7cm2 / mL). Provided are populations of cells produced using the methods and methods of using the populations of cells. Further provided is a method of differentiating a population of T cell progenitor cells enriched for CD4-CD8 + TCR [gamma] [delta] + cells, the method comprising culturing the T cell progenitor cells in the presence of a Notch signaling ligand, such as DL4, provided on a surface area of 0.78 to 4.7 cm2 / mL.

Description

[0001] This application claims priority and benefit to 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 herein by reference as if listed in their entirety. Technical Field

[0002] This invention generally relates to in vitro methods for generating T cell lineage populations from progenitor cells and their uses. Background Technology

[0003] Various feeder cell-based methods can be used for the in vitro generation of T lineage cells. However, in feeder cell-based systems, the expression of cell surface proteins by supporting stromal cells is heterogeneous, resulting in inconsistent outcomes. Furthermore, feeder cell-based systems are not easily scalable to meet the needs of clinical manufacturing.

[0004] Combinations of immobilized Notch signaling ligands (such as DL4) with VCAM-1 have been shown to promote the in vitro generation of T cell progenitors in feed-free and serum-free culture systems (Shukla et al., 2017). Microbeads modified to present DL4 have also been shown to support the in vitro differentiation of T lineage cells, although progress to mature lineages such as CD4-CD8+ cells has been limited (Trotman-Grant et al., 2021). TCR stimulation of T cell progenitors 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). Conjugation of chimeric antigen receptors (CARs) in the absence of Notch signaling has also been shown to induce the generation of CD4-CD8αβ+ cells from CD4+CD8+ cells in TRAC- / - cell lines (Sjoukje et al., 2022). Currently, there are no reported methods for controlling the in vitro emergence of mature T cell lineage populations via Notch signaling. There are also no reports of unique phenotypes of cells that emerge from such processes. Summary of the Invention

[0005] In a first aspect of this disclosure, a method is provided for generating a population of T cell lineage cells from T cell progenitor cells. The method includes providing a population of T cell progenitor cells and, in the presence of these cells, providing them in a culture volume of at least 7 cm² / mL. 2 T cell progenitor cells were cultured with Notch signaling ligands on a surface area of ​​( / mL).

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

[0007] In one embodiment, culturing T cell progenitors in the presence of increased concentrations of Notch signaling ligands increased the absolute and / or relative number of CD4-CD8+ cells in the cell population.

[0008] In one embodiment, at 7 to 56 cm 2 / mL, 7.8 to 55.2cm 2 / mL or 15.7 to 55.2cm 2 Notch signaling ligands are provided on a surface area of ​​ / mL.

[0009] In one embodiment, a 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, 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 beads are made of polystyrene.

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

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

[0015] In one embodiment, the concentration of DL4 is 7.89 x 10⁻⁶. 10 Up to 1.66 x 10 13 molecules / mL.

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

[0017] In one embodiment, the distance is between 7.8 and 55.2 cm. 2 DL4 and VCAM-1 are provided per mL of surface area.

[0018] In one embodiment, the concentration of DL4 is 7.89 x 10⁻⁶. 10 Up to 1.66 x 10 13 The concentration of VCAM-1 was 7.89 x 10⁻⁶ molecules / mL. 10 Up to 1.66 x 10 13 molecules / mL.

[0019] In one embodiment, the cell density is 5 x 10⁻⁶. 5 Up to 2x 10 6 Cells / mL.

[0020] In one embodiment, T cell progenitor 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, T cell progenitor cells are cultured for at least 14 days.

[0022] In one embodiment, the T cell progenitor cells are recultured at least once during or after culturing the T cell progenitor cells 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, CD4-CD8+ cells are CD8αβ+ cells.

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

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

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

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

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

[0029] In one embodiment, pluripotent stem cells contain nucleic acids encoding chimeric antigen receptors (CARs) or exogenous T-cell receptors (TCRs).

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

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

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

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

[0034] In one embodiment, CD4-CD8+ cells express chimeric antigen receptors (CARs).

[0035] In one embodiment, CD4-CD8+ cells express exogenous T cell receptors (TCRs).

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

[0037] In one embodiment, CD4-CD8+ cells are transported in a culture volume of at least 7 cm² / mL (7 cm³ / mL). 2 Notch signaling ligands bound to the surface of cells cultured in vitro from T cell progenitor cells ( / mL) are used.

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

[0039] In a fourth aspect of this disclosure, a method for treating a disease or symptom in a subject is provided. The method includes providing a culture volume of at least 7 square centimeters per milliliter (7 cm² / mL). 2 Culture a cell population containing T cell progenitor cells on a surface area of ​​( / mL) of surface-bound Notch signaling ligand; and administer an effective amount of the T cell lineage cell population to a subject in need of this.

[0040] In one embodiment of the method for treating a subject’s disease or condition provided herein, the T-cell lineage cell population is enriched with CD4-CD8+ cells.

[0041] In one embodiment of the method for treating a subject’s disease or condition provided herein, the CD4-CD8+ cells are CD8αβ+ cells.

[0042] In one embodiment of the method for treating a subject’s disease or condition provided herein, the CD4-CD8+ cells are T-cell receptor (TCR)-cells, CD3-cells, or TCR- / CD3-cells.

[0043] In one embodiment of the method for treating a subject’s disease or condition provided herein, the CD4-CD8αβ+ cells are T-cell receptor (TCR)-cells, CD3-cells, or TCR- / CD3-cells.

[0044] In one embodiment of the method for treating a subject’s disease or condition provided herein, the T-cell lineage cell population contains nucleic acids encoding chimeric antigen receptors (CARs) or exogenous T-cell receptors (TCRs).

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

[0046] In one embodiment of the method for treating a subject’s disease or condition provided herein, the Notch signaling ligand comprises Notch ligand delta-like 4 (DL4) or a variant thereof.

[0047] In one embodiment of the method for treating a subject’s disease or condition provided herein, the Notch signaling ligand is DL4 and the step of culturing T cell progenitor cells further includes culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

[0048] In one embodiment of the method for treating a subject’s disease or condition provided herein, the disease is cancer.

[0049] In a fifth aspect of this disclosure, the use of T-cell lineage cell populations in the manufacture of drugs for treating diseases or symptoms is provided. The T-cell lineage cell populations are generated by a method comprising providing a culture volume of at least 7 cm² / mL. 2 A cell population containing T cell progenitor cells is cultured on a surface area of ​​( / mL) of surface-bound Notch signaling ligand.

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

[0051] In one embodiment of the use of the T-cell lineage populations provided herein in the manufacture of drugs for treating diseases or symptoms, the CD4-CD8+ cells are CD8αβ+ cells.

[0052] In one embodiment of the use of T-cell lineage populations provided herein in the manufacture of medicaments for treating diseases or conditions, 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 the T-cell lineage populations provided herein in the manufacture of medicaments for treating diseases or conditions, 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 T-cell lineage populations provided herein in the manufacture of medicaments for treating diseases or conditions, the T-cell lineage populations contain nucleic acids encoding chimeric antigen receptors (CARs) or exogenous T-cell receptors (TCRs).

[0055] In one embodiment of the use of T-cell lineage populations provided herein in the manufacture of drugs for treating diseases or conditions, Notch signaling ligands are provided on a three-dimensional substrate.

[0056] In one embodiment of the use of the T-cell lineage populations provided herein in the manufacture of drugs for treating diseases or conditions, the Notch signaling ligand comprises Notch ligand delta-like 4 (DL4) or a variant thereof.

[0057] In one embodiment of the use of T-cell lineage populations provided herein in the manufacture of medicaments for treating diseases or conditions, the Notch signaling ligand is DL4, and the step of culturing T-cell progenitor cells further includes culturing in the presence of surface-bound vascular cell adhesion molecule 1 (VCAM-1).

[0058] In one embodiment of the use of T-cell lineage populations provided herein in the manufacture of medicaments for treating a disease or symptom, the disease is cancer.

[0059] In a sixth aspect of this disclosure, a method is provided for differentiating a population of T cell progenitor cells to generate a differentiated cell population enriched with CD4-CD8+TCRγδ+ cells. The method includes providing a population of T cell progenitor cells; and providing cells in a manner that allows for differentiation at a depth of 0.78 to 4.7 cm. 2 T cell progenitor cells are cultured in the presence of surface-bound Notch signaling ligands on a surface area of ​​ / mL.

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

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

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

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

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

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

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

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

[0068] In an eighth aspect of this disclosure, a method is provided for generating a cell population enriched with CD4-CD8+ cells. The method includes providing a population of T cell progenitor cells; and providing cells in a location at least 7 cm in diameter. 2 T cell progenitor cells were cultured with surface-bound Notch signaling ligands on a surface area of ​​ / mL, thereby generating a cell population enriched with CD4-CD8+ cells.

[0069] In a ninth aspect of this disclosure, a method for differentiating a population of T cell progenitor cells is provided. The method includes providing a population of T cell progenitor cells; and in the presence of cells provided at least 7 cm in size... 2T cell progenitor cells were cultured with surface-bound Notch signaling ligands on a surface area of ​​ / mL, in which the differentiated cell population was enriched with CD4-CD8+ cells.

[0070] In a tenth aspect of this disclosure, a method is provided for generating a population of T cell lineage cells from T cell progenitor cells. The method includes providing a population of T cell progenitor cells and culturing the T cell progenitor cells in the presence of Notch signaling ligands 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 population is enriched with CD4-CD8+ cells.

[0072] In one embodiment, culturing T cell progenitor cells in the presence of an increased ratio of substrate surface area to culture container culture surface area increases the absolute and / or relative number 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 container is 1.77:1 to 14:1, 2:1 to 14:1, or 4:1 to 14:1.

[0074] In one embodiment, a 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, one or more beads are made of a material selected from the group consisting of polystyrene, iron oxide, and gold.

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

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

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

[0080] In one embodiment, the concentration of DL4 is 7.89 x 10⁻⁶. 10 Up to 1.66 x 10 13 molecules / mL.

[0081] In one embodiment, the Notch signaling ligand is DL4, and the step of culturing T cell progenitor cells further includes 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 10⁻⁶. 10 Up to 1.66 x 10 13 The concentration of VCAM-1 was 7.89 x 10⁻⁶ molecules / mL. 10 Up to 1.66 x 10 13 molecules / mL.

[0084] In one embodiment, the cell density is 5 x 10⁻⁶. 5 Up to 2x 10 6 Cells / mL.

[0085] In one embodiment, T cell progenitor 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, T cell progenitor cells are cultured for at least 14 days.

[0087] In one embodiment, the T cell progenitor cells are recultured at least once during or after culturing the T cell progenitor cells 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, CD4-CD8+ cells are CD8αβ+ cells.

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

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

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

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

[0093] In one embodiment, pluripotent stem cells contain nucleic acids encoding chimeric antigen receptors (CARs) or exogenous T-cell receptors (TCRs).

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

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

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

[0097] In one embodiment, the T cell progenitor cell contains nucleic acid encoding CAR, and the step of culturing the T cell progenitor cell further includes culturing it in the absence of CAR activator.

[0098] In one embodiment, the Notch signaling ligand is a surface-bound Notch signaling ligand, and this surface-bound Notch signaling ligand is provided in a culture volume of at least 7 cm² / mL. 2 On a surface area of ​​( / mL).

[0099] In one embodiment, the Notch signaling ligand is a surface-bound Notch signaling ligand, and this surface-bound Notch signaling ligand is provided at a culture volume between 7 cm² / mL. 2 / mL) and 56cm 2 The surface area between / mL.

[0100] In one embodiment, 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).

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

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

[0103] In a twelfth aspect of this disclosure, a method is provided for generating a T-cell lineage cell population from hematopoietic stem cells and / or progenitor cells. The method includes providing a population of hematopoietic stem cells and / or progenitor cells and culturing the hematopoietic stem cells / progenitor cells in the presence of Notch signaling ligands and in the absence of T-cell receptor stimulants, wherein the T-cell lineage cell population comprises CD4-CD8+ cells.

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

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

[0106] In one embodiment, hematopoietic stem cells and / or progenitor cells contain nucleic acids encoding CAR, and the step of culturing hematopoietic stem cells and / or progenitor cells further includes culturing in the absence of CAR activators.

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

[0108] In one embodiment, a 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 Notch ligand δ-sample 4 (DL4) or a variant thereof.

[0111] In a thirteenth aspect of this disclosure, a method is provided for generating a population of T cell lineage cells from T cell progenitor cells. The method includes providing a population of T cell progenitor cells and culturing the T cell progenitor cells in the presence of a surface-bound Notch signaling ligand, wherein the Notch signaling ligand is provided at a culture volume of between 7 cm² / mL. 2 / mL) and 56cm 2 The surface area is between / mL, and the T cell lineage cell population contains CD4-CD8+ cells.

[0112] In a fourteenth aspect of this disclosure, a method is provided for generating a population of T cell lineage cells from T cell progenitor cells. The method includes providing a population of T cell progenitor cells and culturing the T cell progenitor 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, and wherein the ratio of the surface area of ​​the substrate to the culture surface area of ​​the culture vessel is between 1.77:1 (1.77:1) and 14:1 (14:1), and wherein the T cell lineage cell population comprises CD4-CD8+ cells. Attached Figure Description

[0113] To facilitate understanding of the subject matter, embodiments are illustrated by non-limiting examples in the accompanying drawings.

[0114] Figure 1 A is a schematic diagram of the process of induced pluripotent stem cells (iPSCs) maturing into T cells, highlighting the progression of T cell progenitor (proT) cells into CD4-CD8+ (CD8 single-positive, CD8SP) cells, which is caused by iPSCs being exposed to a three-dimensional engineered thymic niche (3D ETN) in a concentration-responsive manner.

[0115] Figure 1 B is a description of the thymus niche (2D ETN) using two-dimensional engineered thymus niche, StemSpan. TM Lymphatic differentiation coating material (referred to as SCT or commercial coating; STEMCELL Technologies) and 3D ETN (where the bead concentration gradually increases (0.27x10) 7 Beads / mL – 8.1 x 10⁻⁶ 7 The graph shows the proportions of T cell progenitor cells containing the following four cell populations after culturing T cell progenitor cells for 7 days (0.5x–1.5x bead dose): CD4-CD8+ (CD8SP), CD4+CD8+ (double positive, DP), CD4+CD8- (CD4 immature single positive, CD4 ISP), and CD4-CD8- (double negative, DN).

[0116] Figure 1 C is depicted using 2D ETN, SCT, and 3D ETN (where the bead concentration gradually increases (0.27 x 10⁻⁶)). 7 Beads / mL – 8.1 x 10⁻⁶ 7 A graph showing the percentage of CD4-CD8+ cells that are CD8αα+ or CD8αβ+ cells after culturing T cell progenitor cells for 7 days (beads / mL, 0.5x–1.5x bead dose).

[0117] Figure 2A is a graph depicting cell expansion as a function of 3D ETN bead concentration after 7 days of culture. Two input populations (A and C) were plotted separately. An automatic cell counter determines the cell number.

[0118] Figure 2 B is depicted using gradually increasing 3D ETN bead concentrations (0.27 x 10⁻⁶). 7 beads / mL – 8.1 x 10 7 After culturing T cell progenitor cells at a dose of 0.5x–1.5x beads / mL for 7 days, Figure 1 Figure B shows a graph illustrating the viability of the following four cell populations: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4 ISP), and CD4-CD8- (DN). Cell viability is used... The automated cell counter was determined by acridine orange+ / propidine iodide.

[0119] Figure 3 A is a description of the use of 2D ETN, SCT, and 3D ETN (where the bead concentration gradually increases (0.02x–2x bead dose, or 1.08x 10⁻¹⁰)). 6 Beads / mL – 10.8 x 10 7 Beads / mL (displayed as 0.1–10 x 10⁻⁶) 7 After culturing T cell progenitor cells for 7 days (pills / mL), Figure 1 Figure B shows the proportions of cells comprising the following four cell populations: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4 ISP), and CD4-CD8- (DN), as determined by flow cytometry.

[0120] Figure 3 B is a description of the use of 2D ETN, SCT, and 3D ETN (where the bead concentration gradually increases (0.02x–2x bead dose, or 1.08x 10⁻¹⁰)). 6 Beads / mL – 10.8 x 10 7 Beads / mL (displayed as 0.1–10 x 10⁻⁶) 7 After culturing T cell progenitor cells at a concentration of (pills / mL) for 14 days, Figure 3 Figure A shows the proportions of cells comprising the following four cell populations: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4 ISP), and CD4-CD8- (DN), as determined by flow cytometry.

[0121] Figure 4A is a description of the use of 2D ETN, SCT, and 3D ETN (where the bead concentration gradually increases (0.02x–2x bead dose, or 1.08x 10⁻¹⁰)). 6 Beads / mL - 10.8 x 10 7 Beads / mL (displayed as 0.1–10 x 10⁻⁶) 7 A graph showing the percentage of CD3-TCRαβ-, CD3+TCRαβ-, or CD3+TCRαβ+ cells after 14 days of culture of T cell progenitor cells (pills / mL), as determined by flow cytometry.

[0122] Figure 4 B is a description of the use of 2D ETN, SCT, and 3D ETN (where the bead concentration gradually increases (0.02x–2x bead dose, or 1.08x 10⁻¹⁰)). 6 Beads / mL – 10.8 x 10 7 Beads / mL (displayed as 0.1–10 x 10⁻⁶) 7 A graph showing the expression of TCRαβ and TCRγδ in a cell population after culturing T cell progenitor cells for 14 days (pills / mL), as determined by flow cytometry.

[0123] Figure 4 C is a description of the use of 2D ETN, SCT, and 3D ETN (where the bead concentration gradually increases (0.02x–2x bead dose, or 1.08x 10⁻¹⁰)). 6 Beads / mL - 10.8 x 10 7 Beads / mL (displayed as 0.1–10 x 10⁻⁶) 7 The graph showing the expression of TCRαβ and TCRγδ in CD8+CD4+(DP) cells after 14 days of culture of T cell progenitor cells (pills / mL) is as determined by flow cytometry.

[0124] Figure 4 D is a description of the use of 2D ETN, SCT, and 3D ETN (where the bead concentration gradually increases (0.02x–2x bead dose, or 1.08x 10⁻¹⁰)). 6 Beads / mL - 10.8 x 10 7 Beads / mL (displayed as 0.1–10 x 10⁻⁶) 7 A graph showing the expression of TCRαβ and TCRγδ in CD4-CD8+ (CD8SP) cells after 14 days of culture of T cell progenitor cells (pills / mL), as determined by flow cytometry.

[0125] Figure 5 A is a graph depicting the percentage of viable cells as a function of 3D ETN bead concentration after 7 days of culture. Cell viability is used... The automated cell counter was determined by acridine orange+ / propidine iodide.

[0126] Figure 5 B is a graph depicting the percentage of viable cells as a function of 3D ETN bead concentration after 14 days of culture. Cell viability is used... The automated cell counter was determined by acridine orange+ / propidine iodide.

[0127] Figure 5 C is depicted using gradually increasing 3D ETN bead concentrations (1.08 x 10⁻⁶). 7 Beads / mL – 10.8 x 10 7 After culturing T cell progenitor cells at a dose of 0.02x–2x beads / mL for 7 days, Figure 3 A diagram showing the viability of the following four cell populations: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4 ISP), and CD4-CD8- (DN).

[0128] Figure 5 D is depicted using gradually increasing 3D ETN bead concentrations (1.08 x 10⁻⁶). 7 Beads / mL – 10.8 x 10 7 After culturing T cell progenitor cells at a dose of 0.02x–2x beads / mL for 14 days, Figure 3 A diagram showing the viability of the following four cell populations: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4 ISP), and CD4-CD8- (DN).

[0129] Figure 6 A is a graph depicting cell counts 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 different effector cell:target cell (E:T) ratios.

[0130] Figure 6 B is Figure 6 The magnified view of the data in A omits the target cell control only.

[0131] Figure 7 This graph depicts cell expansion (left axis) and viability (right axis) of cells cultured with 2D ETN and 3D ETN in 12-well cell culture plates (wp) and in a stirred tank reactor (STR) with 3D ETN. A 0.54 x 10⁻⁶ micrometer was used in both the 12-well cell culture plates and the STR. 7 Beads / mL (0.1x bead dose) and 2.7x 10 7 3D ETN bead concentration per bead / mL (0.5x bead dose).

[0132] Figure 8 A is a quantitative graph depicting the percentages of CD4+CD8- (CD4 ISP), CD4-CD8- (DN), CD4-CD8+ (CD8SP), and CD4+CD8+ (DP) cells after culturing cells with 2D ETN and 3D ETN in 12-well cell culture plates (wp) and in STR with 3D ETN. A 0.54 x 10⁻⁶ flow cytometry calibrator was used in both 12-well cell culture plates and STR. 7 Beads / mL (0.1x bead dose) and 2.7x 10 7 3D ETN bead concentration per bead / mL (0.5x bead dose).

[0133] Figure 8 B is a quantitative graph depicting the percentage of cells expressing CD8αα and / or CD8αβ after culturing cells with 2D ETN and 3D ETN in 12-well cell culture plates (wp) and in STRs with 3D ETN. A 0.54 x 10⁻⁶ flow cytometry calibrator was used in both 12-well cell culture plates and STRs. 7 Beads / mL (0.1x bead dose) and 2.7x 10 7 3D ETN bead concentration per bead / mL (0.5x bead dose).

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

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

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

[0137] Figure 10 A is 1.08 x 10 8 Flow cytometry plots of CD5 and CD7 expression in T cell progenitor cell populations cultured at a 3D ETN bead concentration of 3D beads / mL (2x bead dose) for 7 days, in untransduced (top, NTD) and TCR-transduced (bottom) cells.

[0138] Figure 10 B uses 1.08 x 10 8Flow cytometry plots of CD4 and CD8α (CD8A) expression in T cell progenitor cell populations cultured at a 3D ETN bead concentration of 3D beads / mL (2x bead dose) for 7 days. The cells were untransduced (top, NTD) and transduced by TCR (bottom).

[0139] Figure 10 C uses 1.08 x 10 8 Flow cytometry plots of CD3 and TCRαβ (TCRab) expression in T cell progenitor cell populations cultured at a 3D ETN bead concentration of 3D beads / mL (2x bead dose) for 7 days. The cells were untransduced (top, NTD) and transduced by TCR (bottom).

[0140] Figure 10 D is 1.08 x 10 8 Flow cytometry plots of CD8β (CD8B) and CD8α (CD8A) expression in T cell progenitor cell populations cultured at a 3D ETN bead concentration of 3D beads / mL (2x bead dose) for 7 days. The cells were untransduced (top, NTD) and transduced by TCR (bottom).

[0141] Figure 11 A is 1.08 x 10 8 Flow cytometry images of CD5 and CD7 expression in T cell progenitor cell populations cultured at a 3D ETN bead concentration of 3D beads / mL (2x bead dose) for 11 days, in untransduced (top, NTD), TCR-transduced (middle), and CD8β-enriched, TCR-transduced (bottom) cells.

[0142] Figure 11 B uses 1.08 x 10 8 Flow cytometry images of CD4 and CD8α (CD8A) expression in T cell progenitor cell populations cultured at a 3D ETN bead concentration of 3D beads / mL (2x bead dose) for 11 days. The cells were untransduced (top, NTD), transduced by TCR (middle), and enriched by CD8β and transduced by TCR (bottom).

[0143] Figure 11 C uses 1.08 x 10 8 Flow cytometry images of CD3 and TCRαβ (TCRab) expression in T cell progenitor cell populations cultured at a 3D ETN bead concentration of 3D beads / mL (2x bead dose) for 11 days. The cells were untransduced (top, NTD), transduced by TCR (middle), and enriched by CD8β and transduced by TCR (bottom).

[0144] Figure 11 D is 1.08 x 10 8Flow cytometry images of CD8β (CD8B) and CD8α (CD8A) expression in T cell progenitor cell populations cultured at a 3D ETN bead concentration of 3D beads / mL (2x bead dose) for 11 days. These cells were untransduced (top, NTD), transduced by TCR (middle), and enriched by CD8β and transduced by TCR (bottom).

[0145] Figure 12 A is a drawing targeting 1.08 x 10 8 A graph showing the cumulative fold expansion over time of untransduced (NTD, square) and TCR-transduced (TCR, round) cells cultured at a 3D ETN bead concentration of beads / mL (2x bead dose). D17 and D21 correspond to 7 and 11 days of culture with 3D ETN, respectively.

[0146] Figure 12 B is a plotting value for 1.08 x 10. 8 A graph showing cell viability over time for untransduced (NTD, square) and TCR-transduced (TCR, round) cells cultured at a 3D ETN bead concentration of beads / mL (2x bead dose). D17 and D21 correspond to 7 and 11 days of culture with 3D ETN, respectively. TCR-transduced cells were also analyzed after CD8β selection (after CD8B).

[0147] Figure 12 C is used to depict the image with a resolution of 1.08 x 10. 8 A graph showing the cell input / output ratio over time for untransduced (NTD, square) and TCR-transduced (TCR, circle) cells cultured at a 3D ETN bead concentration of beads / mL (2x bead dose). D17 and D21 correspond to 7 and 11 days of culture with 3D ETN, respectively.

[0148] Figure 13A It is depicted using 2D ETN, SCT (commercial coating), and 3D ETN (where the bead concentration gradually increases (0.27x10)). 7 Beads / mL – 8.1 x 10⁻⁶ 7 The graph shows the proportions of T cell progenitor cells containing the following four cell populations after culturing T cell progenitor cells for 7 days at a dose of 0.5x–1.5x beads / mL: CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4 ISP), and CD4-CD8- (DN).

[0149] Figure 13B It is depicted using 2D ETN, SCT (commercial coating), and 3D ETN (where the bead concentration gradually increases (0.27x10)). 7 Beads / mL – 8.1 x 10⁻⁶ 7A graph showing the expression of genes DTX1, TCF7, BCL11B, and GATA3 in T cell progenitor cells cultured for 3 days at a dose of 0.5x–1.5x beads / mL.

[0150] Figure 13C The coatings used were 2D ETN, SCT (commercial coating), and 3D ETN (with bead concentration gradually increasing from 0.27 x 10⁻⁶). 7 Beads / mL – 8.1 x 10⁻⁶ 7 Uniform manifold approximation and projection (UMAP) plot of thirteen-color flow cytometry dataset of T cell progenitor cells cultured for 7 days (beads / mL (0.5x–1.5x bead dose)).

[0151] Figure 13D This is a UMAP map depicting cells in each of six clusters identified using FlowSOM (Van Gassen et al., 2015) via unsupervised machine learning. Each cluster is defined by a different combination of expressed surface proteins identified by flow cytometry.

[0152] Figure 13E It is depicted in Figure 13D The bar chart depicts the relative levels of cell surface proteins expressed in each cluster (e.g., cluster 1 was identified as a cell describing CD4+CD8+(DP)).

[0153] Figure 13F It is depicted as using 2D ETN, SCT (commercial coating), and 3D ETN (where the bead concentration gradually increases (0.27 x 10⁻⁶)). 7 Beads / mL – 8.1 x 10⁻⁶ 7 The function of bead / mL (0.5x–1.5x bead dose) in cultured cells. Figure 13D A bar chart showing the proportion of each cluster identified in the study.

[0154] Figure 13G It uses 5.4 x 10 7 Flow cytometry image of CD5 and CD7 expression, CD4 and CD8α (CD8A) expression, and CD8β (CD8B) and CD8α (CD8A) expression in cells cultured for 14 days at a 3D ETN bead concentration of bead / mL (1x bead dose).

[0155] Figure 14A This 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 source: Park et al., 2020.

[0156] Figure 14BThis is a UMAP map of T cell maturation in the human thymus. Selected single-cell RNAseq data from the Thymus Atlas (Park et al., 2020) are represented as UMAPs and annotated by cell type, including NK-T cell (“NKT”) cell types.

[0157] Figure 15A These are UMAP maps depicting the enrichment of Notch and TCR signaling during T cell maturation in the thymus. Data source: Park et al., 2020.

[0158] Figure 15B These are UMAP maps depicting the enrichment of Notch and TCR signaling during T cell maturation in the thymus. Data source: Park et al., 2020.

[0159] Figure 15C This is a plot depicting the enrichment of Notch and TCR signaling at various T cell developmental stages in the thymus (single-sample gene set enrichment analysis, ssGSEA). Developmental stages were designated as: early DN cells, DNI; proliferating DN cells, DN(p); resting DN cells, DN(q); proliferating DP cells, DP(p); resting DP cells, DP(q); α-βT transition stage cells, αβT; and CD8+ T cells, CD8+T. Data and cell markers are derived from Park et al., 2020.

[0160] Figure 15D This is a UMAP plot of single-cell RNAseq data (Park et al., 2020), generated by the median Notch gene regulatory network (“Notch GRN”). Figure 15D ) and TCR signal transduction ("TCR signal transduction") Figure 15E Transcription signals (ssGEA score) are annotated by population.

[0161] Figure 15E This is a UMAP plot of single-cell RNAseq data (Park et al., 2020), generated by the median Notch gene regulatory network (“Notch GRN”). Figure 15D ) and TCR signal transduction ("TCR signal transduction") Figure 15E Transcription signals (ssGEA score) are annotated by population.

[0162] Figure 16 A is a treatment targeting primary, unstimulated primary T cells with CD8αβ (CD8ab UnStim) and using 5.4 x 10⁻⁶ ppm. 7UMAP plot of iPSC-derived CD4-CD8+ cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of beads / mL (1x bead dose). Cell annotation was performed using the SingleR package, referencing the thymocyte atlas (Park et al., 2020).

[0163] Figure 16 B is a quantification of CD8αβ (CD8ab UnStim) in primary, unstimulated primary T cells and using 5.4 x 10⁻⁶ cells. 7 A graph showing the proportion of iPSC-derived CD4-CD8+ cells (ipsD CD8abUnStim) cultured at a 3D ETN bead concentration of beads / mL (1x bead dose) corresponding to the T cell maturation stage in the thymus (Park et al., 2020).

[0164] Figure 17 A is a depiction of primary, unstimulated CD8αβ+ T cells (CD8ab UnStim) and using 5.4 x 10⁻⁶ cells. 7 UMAP plot of T cell status of iPSC-derived CD4-CD8+ cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of beads / mL (1x bead dose) derived from ProjecTIL (Andreatta et al., 2021).

[0165] Figure 17 B is the quantification of primary, unstimulated CD8αβ+ T cells (CD8ab UnStim) and 5.4 x 10⁻⁶ cells. 7 3D ETN beads at a concentration of 3D beads / mL (1x bead dose) were cultured from unstimulated iPSC-derived CD4-CD8+ T cells (ipsD CD8abUnStim) Figure 17 A graph showing the percentage of T cells in each state of A.

[0166] Figure 18 A is a depiction of primary, unstimulated CD8αβ T cells (CD8ab UnStim) and using 5.4 x 10⁻⁶ cells. 7 A graph showing the expression of selected genes in iPSC-derived CD4-CD8+ cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of 3D beads / mL (1x bead dose), as determined by scRNAseq.

[0167] Figure 18 B is a depiction of primary, unstimulated CD8αβ+ T cells (CD8ab UnStim) and using 5.4 x 10⁻⁶ cells. 7A graph showing the enrichment of gene sets related to Notch signaling, TCR signaling, and downstream TCR signaling in iPSC-derived CD4-CD8+ cells (ipsD CD8ab UnStim) cultured at a 3D ETN bead concentration of 3D beads / mL (1x bead dose), as determined by scRNAseq.

[0168] Figure 19 A is using 2.7 x 10 7 Flow cytometry plot of CD5 and CD7 expression in unmodified iPSC-derived hematopoietic stem cell / progenitor cell populations containing CD34+ cells after culturing at a 3D ETN bead concentration of 3D beads / mL (0.5x bead dose) for 10 days.

[0169] Figure 19 B is using 2.7x 10 7 Flow cytometry plot of CD34 and CD7 expression in unmodified iPSC-derived hematopoietic stem cell / progenitor cell populations containing CD34+ cells after culturing at a 3D ETN bead concentration of 3D beads / mL (0.5x bead dose) for 10 days.

[0170] Figure 19 C is using 2.7x 10 7 Flow cytometry plot of CD4 and CD8α (CD8A) expression in unmodified iPSC-derived hematopoietic stem cell / progenitor cell populations containing CD34+ cells after culturing at a 3D ETN bead concentration of 3D beads / mL (0.5x bead dose) for 10 days.

[0171] Figure 19 D is using 2.7x 10 7 Flow cytometry plot of CAR and CD7 expression in unmodified iPSC-derived hematopoietic stem cell / progenitor cell populations containing CD34+ cells after culturing at a 3D ETN bead concentration of 3D beads / mL (0.5x bead dose) for 10 days.

[0172] Figure 20 A is using 2.7 x 10 7 Flow cytometry plot of CD5 and CD7 expression in CAR-modified iPSC-derived hematopoietic stem cell / progenitor cell populations containing CD34+ cells after culturing at a 3D ETN bead concentration of 3D beads / mL (0.5x bead dose) for 10 days.

[0173] Figure 20 B is using 2.7x 10 7Flow cytometry plot of CD34 and CD7 expression in CAR-modified iPSC-derived hematopoietic stem cell / progenitor cell population containing CD34+ cells after culturing at a 3D ETN bead concentration of 3D beads / mL (0.5x bead dose) for 10 days.

[0174] Figure 20 C is using 2.7x 10 7 Flow cytometry plot of CD4 and CD8α (CD8A) expression in CAR-modified iPSC-derived hematopoietic stem cell / progenitor cell populations containing CD34+ cells after culturing at a 3D ETN bead concentration of 3D beads / mL (0.5x bead dose) for 10 days.

[0175] Figure 20 D is a flow cytometry plot of CAR and CD7 expression in CAR-modified iPSC-derived hematopoietic stem cell / progenitor cell populations containing CD34+ cells after culturing for 10 days at a 3D ETN bead concentration of 2.7 x 107 beads / mL (0.5 x bead dose).

[0176] Figure 21 A is quantized using 1.08 x 10 8 3D ETN bead concentration at 2 x 10⁻⁶ beads / mL (2x bead dose) 6 1 cell / mL or 5 x 10 5 A graph showing the proportion of CD4-CD8α+ (CD8SP) cells relative to unmodified iPSC-derived cells (“4A1”) or CAR-modified iPSC-derived cells (“4B3”) after culturing T cell progenitors at a density of 1 cell / mL for 7 days (17 days of culturing the hematopoietic stem / progenitor cell starting population with 3D ETN).

[0177] Figure 21 B is the quantization value used in 1.08x 10 8 3D ETN bead concentration at 2 x 10⁻⁶ beads / mL (2x bead dose) 6 1 cell / mL or 5 x 10 5 A graph showing the proportion of CD8αβ+ (CD8ab+) cells in unmodified iPSC-derived cells (“4A1”) or CAR-modified iPSC-derived cells (“4B3”) after culturing T cell progenitors at a density of 1 cells / mL for 7 days (17 days of culturing the hematopoietic stem / progenitor cell starting population with 3D ETN).

[0178] Figure 21 C is used for quantization, specifically 1.08 x 10. 8 3D ETN bead concentration at 2 x 10⁻⁶ beads / mL (2x bead dose)6 1 cell / mL or 5 x 10 5 A graph showing cell viability for unmodified iPSC-derived cells (“4A1”) or CAR-modified iPSC-derived cells (“4B3”) after culturing T cell progenitors at a density of 1 cells / mL for 7 days (17 days of culturing the hematopoietic stem / progenitor cell starting population with 3D ETN).

[0179] Figure 21 D is the quantization value used in 1.08x 10. 8 3D ETN bead concentration at 2 x 10⁻⁶ beads / mL (2x bead dose) 6 1 cell / mL or 5 x 10 5 A graph showing the proportion of CAR+ cells relative to unmodified iPSC-derived cells (“4A1”) or CAR-modified iPSC-derived cells (“4B3”) after culturing T cell progenitors at a density of 1 cell / mL for 7 days (17 days of culturing the hematopoietic stem / progenitor cell starting population with 3D ETN).

[0180] Figure 22 A is quantized using 1.08 x 10 8 3D ETN bead concentration at 2 x 10⁻⁶ beads / mL (2x bead dose) 6 1 cell / mL or 5 x 10 5 A graph showing the percentage of CD4-CD8α+ (CD8SP) in unmodified iPSC-derived cells (“4A1”) and CAR-modified iPSC-derived cells (“4B3”) before and after CD8α enrichment, after 11 days of cell culture at a density of cells / mL (21 days of culture with 3D ETN from the hematopoietic stem / progenitor cell starting population).

[0181] Figure 22 B is the quantization value used in 1.08x 10 8 3D ETN bead concentration at 2 x 10⁻⁶ beads / mL (2x bead dose) 6 1 cell / mL or 5 x 10 5 A graph showing the percentage of CD8αβ+ (CD8ab+) cells in unmodified iPSC-derived cells (“4A1”) and CAR-modified iPSC-derived cells (“4B3”) before and after CD8α enrichment, after 11 days of cell culture at a density of cells / mL (21 days of culture with 3D ETN from the hematopoietic stem / progenitor cell starting population).

[0182] Figure 22 C is used for quantization, specifically 1.08 x 10. 8 3D ETN bead concentration at 2 x 10⁻⁶ beads / mL (2x bead dose) 61 cell / mL or 5 x 10 5 A graph showing the percentage of cell viability of unmodified iPSC-derived cells (“4A1”) and CAR-modified iPSC-derived cells (“4B3”) before and after CD8α enrichment, after 11 days of cell culture at a density of 10 cells / mL (21 days of culture with 3D ETN from the hematopoietic stem / progenitor cell starting population).

[0183] Figure 22 D is the quantization value used in 1.08x 10. 8 3D ETN bead concentration at 2 x 10⁻⁶ beads / mL (2x bead dose) 6 1 cell / mL or 5 x 10 5 A graph showing the percentage of CAR expression in unmodified iPSC-derived cells (“4A1”) and CAR-modified iPSC-derived cells (“4B3”) before and after CD8α enrichment, after culturing cells at a density of 1 cells / mL for 11 days (21 days of 3D ETN culture from the hematopoietic stem / progenitor cell starting population).

[0184] Figure 23 A is a graph depicting cell viability over time for unmodified iPSC-derived cells (“4A1”).

[0185] Figure 23 B is a graph depicting the cell viability over time for CAR-modified iPSC-derived cells (“4B3”).

[0186] Figure 24 A is a graph depicting the fold increase over time of unmodified iPSC-derived cells (“4A1”).

[0187] Figure 24 B is a graph depicting the fold increase over time of CAR-modified iPSC-derived cells (“4B3”).

[0188] Figure 25 This is a schematic diagram depicting the typical (gray arrow) and non-physiological (black arrow) differentiation of pluripotent stem cells (e.g., induced pluripotent stem cells, iPSCs) into CD8+CD4- cells. HSPC, hematopoietic stem cells / progenitor cells; DN, double-negative cells; ISP, intermediate single-positive cells; DP, double-positive cells; SP, single-positive cells.

[0189] Figure 26 A is a graph quantifying the percentage of T cell progenitors (CD5+CD7+) and myeloid cells (CD33+CD7-) after different concentrations of ETN were used to differentiate iPSC-derived HSPCs.

[0190] Figure 26B uses 5.4 x 10 6 Beads / mL (0.1x bead dose) or 5.4x 10 7 Representative flow cytometry plots of iPSC-derived HSPCs differentiated at an ETN dose of beads / mL (1x bead dose), outlining the T cell progenitor population (CD5+CD7+) and the bone marrow cell population (CD33+CD7-).

[0191] Figure 27 A depicts two TRAC-deficient cell lines cultured in 3D ETN with bead dosage ranges. - / - (left image) and TRAC defective CD19-CAR-engineered (TRAC) - / - CD19-CAR + (Right Figure) Following this, a quantitative graph of flow cytometry data on the percentages of CD4-CD8- (double negative, DN), CD4+CD8+ (double positive, DP), CD4+CD8- (CD4 ISP), and CD4-CD8+ (CD8 single positive, CD8SP) cells.

[0192] Figure 27 B is a flow cytometry plot showing the expression of CD4 and CD8α (CD8a) in TRAC-deficient CD19-CAR-engineered cells after culturing with three doses of 3D ETN. The annotation boxes on the left side of the plot indicate... Figure 26 The quantified group in A.

[0193] Figure 28A This is a graph depicting changes in gene expression after 2 days of culture in 3D ETN that are positively or negatively correlated with the CD8 SP phenotype, as identified by a machine learning workflow (see...). Figure 28B ).

[0194] Figure 28B It is a graph depicting the accuracy distribution of models used in a machine learning workflow.

[0195] Figure 28C This is a graph depicting the correlation between the expression of DTX1, the gene identified as the most positive predictive trait, and the percentage of CD4-CD8a+ (CD8 SP) cells for both TRAC-deficient (TRAC KO, round) and TRAC-deficient CD19-CAR engineered (TRAC KO+CAR, x's) cell lines.

[0196] Figure 28D It describes the TRAC defect type (TRAC) within a range of bead dosages. - / - ) and TRAC defective CD19-CAR engineering (TRAC - / - CD19-CAR +A graph showing the expression of selected genes in cell lines (showing the average expression normalized to z-scores, n=3).

[0197] Figure 28E This is a graph depicting changes in gene expression after 2 days of culture in 3D ETN that are positively or negatively correlated with the CD8 SP phenotype, as identified by a machine learning workflow (see...). Figure 28F The average weighted regression coefficients of the first 12 predictive features are shown.

[0198] Figure 28F This is a graph depicting the accuracy distribution of the models used in the machine learning workflow. A machine learning workflow trained on gene expression at day -12 was used to predict the CD8αSP phenotype at day -17, with good accuracy (85% cross-validation) and no cell line independence.

[0199] Figure 28G It describes the wild-type (WT, square) and TRAC-deficient type (TRAC). - / - (circular) and TRAC defective CD19-CAR engineering (TRAC - / - CD19-CAR + A graph showing the correlation between the expression of NOTCH1, the gene identified as the most positive predictive trait in the x's cell line, and bead dosage.

[0200] Figure 28H It depicts the wild type (WT, left) and the TRAC defective type (TRAC). - / - (middle) and TRAC defective CD19-CAR engineering (TRAC) - / - CD19-CAR + (Right) A graph showing the expression of selected genes in cell lines within the benzene dose range (showing z-score normalized mean expression, n=3 or 6).

[0201] Figure 28I The graph depicts the variance decomposition analysis, demonstrating the interaction between bead dosage and daily effects and cell line effects, which together account for the majority of the transcriptional variance.

[0202] Figure 28J It is described in the TRAC-deficient CD19-CAR engineered cell line (TRAC - / - CD19-CAR + Plot of differentially expressed pathways (using ssGSEA score) at low and high bead doses and culture times (day 12 and day 17).

[0203] Figure 29AThis is a UMAP diagram of the single-cell transcriptome of iPSC-derived T cells, peripheral blood (PBMC-T) and cord blood (CB-T) CD8+ T cells.

[0204] Figure 29B This is a diagram depicting cell populations of iPSC-derived T cells, peripheral blood (PBMC-T) and cord blood (CB) CD8+ T cells classified using an algorithm derived from ProjecTIL (Haradhvala et al., 2022).

[0205] Figure 29C It is a diagram depicting gene expression of markers for T cells, Notch-related genes, innate lymphocytes (ILCs), T cell activation and depletion transcripts of iPSC-derived T cells, and CD8+ T cells from peripheral blood (PBMC-T) and cord blood (CB-T).

[0206] Figure 29D This is a graph depicting the differential enrichment of selected gene signatures in each cell cluster of iPSC-derived T cells (iPSC-T, numbered 1-4), CD8+ T cells from peripheral blood (PBMC-T), and umbilical cord blood (CB-T) obtained by single-sample gene set enrichment analysis (GSEA).

[0207] Figure 29E This is a diagram depicting the differentially enriched gene signatures of 1473 genes in each cell cluster, obtained through single-sample gene set enrichment analysis (GSEA) targeting iPSC-derived T cells (iPSC-T, numbered 1-4), peripheral blood (PBMC-T), and umbilical cord blood (CB-T) CD8+ T cells.

[0208] Figure 29F The UMAP of the single-cell transcriptome is defined as follows: iPSC-derived T cells (CD34+, ProT, CD8+SP phase and CD19- antigen stimulation) and primary lymphocytes (CD4+ T cells, CD8+ T cells, CD56+ NK cells and T cells activated by Dynabeads (CD3 / CD28- stimulation)).

[0209] Figure 29G It is a bubble chart of selected genes associated with blood progenitor cells, T cell lineage commitment, typical T cell function, innate lymphocytes, cytotoxic T cells, exhausted T cells, and Notch response elements.

[0210] Figure 29H This is a graph depicting the expression of the transcriptional pathway signature (ssGSEA score) as described in the sample.

[0211] Figure 29I This is a UMAP diagram that focuses on the transcriptional similarity annotation of single-cell transcriptomes of iPSC-CD8+ cells, antigen-stimulated iPSC-CD8+ cells, and activated T cells with those of developing thymocytes (DN proliferation, early DN, quiescent DN, CD8+T, NKT, γ-δT (GDT), DP proliferation, and Treg).

[0212] Figure 29J The curves show the emergence of different cell phenotypes in CAR-iPSC-derived cells, determined by single-cell lineage inference and pseudo-time organization.

[0213] Figure 29K This diagram illustrates the emergence of CAR-iPSC-derived cellular transcriptional pathways, determined by single-cell lineage inference and pseudo-time organization.

[0214] Figure 30 A is a graph depicting the target cell (CD19-expressing A549) dynamics in a four-round in vitro killing assay using primary CAR-T and iPSC-CD8 cells. Cell density reads from live-cell imaging, with effector T cells added at a 2:1 E:T ratio (mean ± standard deviation, n = 3).

[0215] Figure 30 B is a graph depicting in vitro cytotoxicity and effector cell expansion against primary CART and iPSC-CART (n = 2 replicates). Cumulative cytotoxicity is the sum of the relative changes in AUC relative to target cells only (not shown) at each stimulation.

[0216] Figure 30 C is a diagram depicting the secretion of inflammatory cytokines IFN-γ, granzyme B, and tumor necrosis factor α (TNFα) by iPSC-derived T cells and primary CAR-T cells after co-culturing with antigen-expressing target cells.

[0217] Figure 31A This is a schematic diagram of a 3D engineered thymus niche (ETN) platform for scalable T cell manufacturing using iPSC-derived CD34+HPC.

[0218] Figure 31BThis is a quantitative graph depicting the percentages of CD4-CD8- (double-negative, DN), CD4+CD8- (CD4 ISP), CD4+CD8+ (double-positive, DP), and CD4-CD8+ (CD8 single-positive, CD8SP) cells after differentiation of iPSC-derived CD34+ HPCs generated in STRs via microplate culture, with differentiation of iPSC-derived HPCs generated in microplate culture as a reference (mean ± SEM, 3 independent STR runs, n = 6 bioreactors).

[0219] Figure 31C This is a quantitative graph (mean ± SEM, 3 independent STR runs, n = 6 bioreactors) depicting the percentage of cells expressing CD8α (CD8a) and CD8β (CD8b) after differentiation from iPSC-derived CD34+ HPCs in microplate culture.

[0220] Figure 32 A is described using ETN doses ranging from 30 times (0.1x to 3x bead dose, or 5.4x 10⁻¹⁰). 6 Beads / mL to 1.62 x 10⁻⁶ 8 A quantitative graph of flow cytometry data showing the percentages of CD4-CD8- (double negative, DN), CD4+CD8- (CD4 ISP), CD4+CD8+ (double positive, DP), and CD4-CD8+ (CD8 single positive, CD8SP) cells after culturing iPSC-derived ProT cells in microplates (beads / mL) for 7 days.

[0221] Figure 32 B is a description of using 2x or 3x bead dosage (1.08 x 10⁻⁶). 8 beads / mL or 1.62 x 10⁻⁶ 8 The graph shows the expression of CD8A, IL7R, CD4 and BCL11A genes within 7 days after ETN culture (beads / mL).

[0222] Figure 33 A is a quantitative graph depicting flow cytometry data showing CD7 and CD5 expression and the percentage of the total ProT cell population after 10 days of culturing iPSC-derived CD34+ cells in microplates and STRs using 3D ETN. In a bioreactor (Eppendorf) and a 24-well plate, 1 x 10 4 and 5x10 4 Two copies of each cell / mL were inoculated into two (2) CD34+ cell banks.

[0223] Figure 33B is a quantitative graph depicting flow cytometry data showing CD7 and CD5 expression and the percentage of the total ProT cell population after 10 days of culturing CD34+ cells with 3D ETN. A CD34+ cell bank was plotted at 5 x 10⁻⁶. 4 10 cells / mL were inoculated in eight (8) bioreactors.

[0224] Figure 34 A is a description of the use of 2.5x bead dosage (1.35x 10⁻⁶) in eight (8) bioreactors. 8 After culturing ProT cells with ETN (beads / mL) for 11 days, a flow cytometry graph showing the percentage of CD4-CD8- (double negative, DN), CD4+CD8- (CD4 ISP), CD4+CD8+ (double positive, DP), and CD4-CD8+ (CD8 single positive, CD8SP) cells and the yield of CD8+ cells was generated.

[0225] Figure 34 B is a description of the use of 2.5x bead dosage (1.35x 10⁻¹⁰) in eight (8) bioreactors. 8 A quantitative graph of flow cytometry data showing the percentages of CD8α+, CD8αβ+ and CD8β+ cells at day 8 of ETN maturation (beads / mL).

[0226] Figure 35 A is a description of the STR system using a 2.5x bead dosage (1.35x 10). 8 A graph showing the expression of CD8A, IL7R, CD4, and BCL11A genes within 7 days of ETN culture (beads / mL).

[0227] Figure 35 B is a graph depicting the correlation between gene expression for 24 genes involved in leukocyte development after culture with 3D ETN in microplate (“microwell”) or stirred tank bioreactor (“STR”) systems (Pearson’s r = 0.71, p < 0.001).

[0228] Figure 36A It is a graph depicting a series of restimulation assays measuring the cytotoxic activity of iPSC-derived CD8+ CAR-T cells (“iPSC CD8CAR”) or primary CD8+ CAR-T cells (“primary CD8 CAR”), with GFP-expressing CD19+ cells as target cells (n = 3 technical replicates).

[0229] Figure 36BThis is a graph depicting the fold increase in iPSC-derived CD8+ CAR-T cells (“iPSC CD8CAR”) or primary CD8+ CAR-T cells (“primary CD8CAR”) at the end of each round of target exposure. The cumulative fold increase in iPSC-derived CD8+ CAR-T cells is indicated by bold letters.

[0230] Figure 36C This diagram depicts the target cell-specific secretion of effector molecules perforin (far left), interferon-γ (IFNγ, middle left), granzyme B (middle right), and tumor necrosis factor α (TNFα, far right) by iPSC-derived CD8+ CAR-T cells (“iPSC CD8CAR”) or primary CD8+ CAR-T cells (“primary CD8 CAR”).

[0231] Figure 36D This is a graph depicting the percentage of T cell subsets targeting iPSC-derived CD8+ CAR-T cells (“iPSC CD8 CAR”) or primary CD8+ CAR-T cells (“primary CD8 CAR”) before consecutive restimulation assays (baseline) and after one round (“Stim1”) or four rounds (“Stim4”) of consecutive restimulation assays. The T cell subsets are categorized based on the expression of CD45RA, CD62L, and CD95 as measured by flow cytometry.

[0232] Figure 36E This is a graph depicting the expression of T cell exhaustion markers PD1, TIM3, LAG3, TIGIT, and CD39 in primary CD8+ CAR-T cells (“primary CD8 CAR”) or iPSC-derived CD8+ CAR-T cells (“iPSC CD8 CAR”). Each section indicates the percentage of cells that do not express (“0”), express one (“1”), two (“2”), three (“3”), or four (“4”).

[0233] Figure 37 A is a flow cytometry image of CAR+CD8+ cells derived from iPSCs after CD8α enrichment.

[0234] Figure 37 B is a flow cytometry image of CAR+CD8+ cells derived from iPSCs after CD8β enrichment.

[0235] Figure 38 A is a graph depicting the viability of iPSC-derived cells during differentiation, both unmodified (“6A1”) and TCR-modified (“172”, “174”).

[0236] Figure 38B is a graph depicting the viability of iPSC-derived cells before and after enrichment following differentiation, targeting unmodified ("6A1") and TCR-modified ("172", "174") cells.

[0237] Figure 38 C is a graph depicting the cumulative expansion fold of unmodified (“6A1”) and TCR-modified (“172”, “174”) iPSC-derived cells during differentiation.

[0238] Figure 39 A is a graph depicting the co-expression of CD5 and CD7 in unmodified ("6A1") and TCR-modified ("172", "174") iPSC-derived cells at the indicated time points.

[0239] Figure 39 B is a graph depicting CD8 expression in unmodified (“6A1”) and TCR-modified (“172”, “174”) iPSC-derived cells at the indicated time points.

[0240] Figure 39 C is a diagram depicting the expression of CD8α and CD8β in iPSC-derived cells after differentiation and enrichment, targeting unmodified ("6A1") and TCR-modified ("172", "174") cells.

[0241] Figure 40 This is a flow cytometry image of cells differentiated from unmodified or TCR-modified iPSCs (“unmodified (6A1)”, “TCR (172)”, or “TCR (174)”).

[0242] Figure 41 A is a graph depicting TCR expression in unmodified (“6A1”) and TCR-modified (“172”, “174”) iPSC-derived cells at the indicated time points.

[0243] Figure 41 B is a flow cytometry plot of TCR (MAGE-A4) expression in TCR-modified ("172", "174") iPSC-derived cells after differentiation and enrichment, as determined by tetramer staining.

[0244] Figure 42 A is a graph depicting the fold change in proliferation of primary T cells and unmodified (“6A1”) and TCR-modified (“172”, “174”) iPSC-derived CD8+ cells during continuous restimulation assays in vitro.

[0245] Figure 42B is a graph depicting the fold change in proliferation of primary T cells and TCR-modified ("172", "174") iPSC-derived CD8+ cells during continuous restimulation assays in vitro.

[0246] Figure 43 This is a graph depicting the cytotoxicity of primary T cells and TCR-modified ("172", "174") iPSC-derived CD8+ cells against A375 target cells at different effector cell to target cell (E:T) ratios as shown.

[0247] Figure 44 Figure A depicts the cytotoxicity of primary T cells and TCR-modified (“174”) iPSC-derived CD8+ cells during an in vitro serial restimulation assay using A375 target cells, compared to target cells and unmodified iPSC-derived cell controls.

[0248] Figure 44 B is from Figure 44 A magnified diagram depicting the cytotoxicity of primary T cells and TCR-modified (“174”) iPSC-derived CD8+ cells.

[0249] Figure 44 C is a graph depicting the cytotoxicity of primary T cells and TCR-modified (“174”) iPSC-derived CD8+ cells during in vitro serial restimulation assays using A375 target cells, compared to target cells and unmodified iPSC-derived cell controls.

[0250] Figure 44 D is by Figure 44 A magnified diagram depicting the cytotoxicity of primary T cells and TCR-modified (“174”) iPSC-derived CD8+ cells.

[0251] Figure 45 This is a diagram depicting the in vitro cytotoxicity of primary T cells and TCR-modified (“174”) iPSC-derived CD8+ cells against different target cells as shown.

[0252] Figure 46A This is a flow cytometry image of iPSC-derived cells during differentiation at the indicated time points. Cellular TCRβ and CD3 were analyzed for intracellular (“ICS”).

[0253] Figure 46B This is a flow cytometry image of iPSC-derived cells on day 24 of differentiation. Cellular analysis focused on intracellular (“ICS”) TCRβ and CD3.

[0254] Figure 46CIt is a diagram depicting the activity and expression of CD4 and / or CD8α in iPSC-derived cells during differentiation.

[0255] Figure 47 This is a flow cytometry image of iPSC-derived cells at the indicated time point during differentiation.

[0256] Figure 48 This is a flow cytometry image of iPSC-derived cells at the indicated time point during differentiation.

[0257] Figure 49A This is a diagram depicting the viability of the following cell lines during differentiation. Figure 50 A): Unmodified iPSC-derived cells (“unmodified”), iPSC-derived cells transduced with TCR on day 7 (“unmodified; transduced with TCR”), CAR-iPSC-derived cells (“CAR SSI”), and TCR-iPSC-derived cells (“TCR SSI”).

[0258] Figure 49B This is a graph depicting the fold increase in the following cell lines during differentiation. Figure 50 B): Unmodified iPSC-derived cells (“unmodified”), iPSC-derived cells transduced with TCR on day 7 (“unmodified; transduced with TCR”), CAR-iPSC-derived cells (“CAR SSI”), and TCR-iPSC-derived cells (“TCR SSI”).

[0259] Figure 49C It is a description of what is described above. Figure 49A and Figure 49B A diagram showing the expression of CAR and / or TCR in the cell lines.

[0260] Figure 50 A is a description of what is described above. Figure 49A and Figure 49B The expression of CD5 and / or CD7 in the cell lines ( Figure 50 Figure A).

[0261] Figure 50 B is a description of what is described above. Figure 49A and Figure 49B The expression of CD56 and / or CD7 in the cell lines ( Figure 50 Figure B). 51A is a depiction of the above-mentioned... Figure 49A and Figure 49B A diagram showing the expression of CD4 and / or CD8α in the cell lines.

[0262] Figure 51B is a flow cytometry image of CD8α and CD8β expression on day 21 of differentiation. Cellular subgating is achieved on CD4-CD8α+.

[0263] Figure 52 A is a description of what is described above. Figure 49A and Figure 49B A diagram showing the expression of CD8α and / or CD8β in the cell lines.

[0264] Figure 52 B is a flow cytometry image of CD4 and CD8β expression on day 21 of differentiation. Cellular subgating is achieved at CD8α+CD8β+.

[0265] Figure 53 This is a flow cytometry image of differentiated, CD8α-enriched, cryopreserved, and CAR-modified cells after thawing (“SSI CAR”).

[0266] Figure 54 A is a graph depicting the fold expansion of CAR-modified cells (“SSI CAR”) during in vitro serial restimulation assays using Raji CD19+ target cells.

[0267] Figure 54 B is a graph depicting the percentage of CD8α+CD8β+, CD8α+CD8β-, CD8α-CD8β-, and CD8α-CD8β+ cells targeting CAR-modified cells (“SSI CAR”) during in vitro serial restimulation assays using Raji CD19+ target cells.

[0268] Figure 54 C is a graph depicting cytotoxicity against CAR-modified cells (“SSI CAR”) during in vitro serial restimulation assays using Raji CD19+ target cells.

[0269] Figure 55 A is a graph depicting the fold expansion of CAR-modified cells (“SSI CAR”) during in vitro serial restimulation assays using A549 CD19+ target cells.

[0270] Figure 55 B is a graph depicting the fold change in proliferation of CAR-modified cells (“SSI CAR”) during in vitro serial restimulation assays using A549 CD19+ target cells.

[0271] Figure 55C is a graph depicting the percentage of CD8α+CD8β+, CD8α+CD8β-, CD8α-CD8β-, and CD8α-CD8β+ cells targeting CAR-modified cells (“SSI CAR”) during in vitro serial restimulation assays using A549 CD19+ target cells.

[0272] Figure 56 This is a diagram depicting cytokine secretion in CAR-modified cells (“SSI CAR”) after in vitro serial restimulation assays using A549 or Raji CD19+ / + target cells or CD19- / - controls.

[0273] Figure 57 A is a description using Raji ( Figure 58 A graph showing the viability of CAR-modified cells (“SSI CAR”) after continuous in vitro restimulation assays of CD19+ target cells.

[0274] Figure 57 B is a description in A549 ( Figure 58 A graph showing the viability of CAR-modified cells (“SSI CAR”) after continuous in vitro restimulation assays of CD19+ target cells.

[0275] Figure 58 This is a flow cytometry image of CAR-iPSC-derived cells after differentiation and CD8 enrichment.

[0276] Figure 59 Figure A depicts the in vivo Raji tumor growth inhibition of CAR-iPSC-derived (“iPSC CD8”) and primary CAR+ T cells (“primary CD8”) in a mouse model. Statistical differences between the tumor group and the CAR-iPSC-derived group alone were assessed by Welch’s two-sample t-test (two-tailed, P < 0.05; ** P < 0.01).

[0277] Figure 59 B is a graph depicting the survival rates of the untreated group (tumor only), the CAR-iPSC-derived CD8+ cell-treated group, and the primary CAR+ T cell-treated group. Statistical differences between the tumor-only group and the CAR-iPSC-derived group were assessed using the Mantel-Cox test (*P<0.05; **P<0.01).

[0278] Figure 59 C is a graph depicting the in vivo toxicity of CAR-iPSC-derived and primary CAR+ T cells, as indicated by percentage changes in body weight.

[0279] Figure 60A is a graph depicting the in vivo cell dynamics of CAR-iPSC-derived and primary CAR+ T cells detected in peripheral blood.

[0280] Figure 60 B is a flow cytometry image of CAR-iPSC-derived CD8+ cells detected in peripheral blood 8 days after infusion.

[0281] Figure 61A These are bioluminescence images over time of untreated mice (tumor only), mice treated with CAR-iPSC-derived CD8+ cells, and mice treated with primary CAR+CD8+ T cells in the A549 CD19 tumor model.

[0282] Figure 61B This is a diagram depicting the in vivo inhibition of A549-CD19 tumor growth by CAR-iPSC-derived and primary CAR+CD8+ T cells.

[0283] Figure 61C This graph depicts the survival rates of the untreated group (tumor only), the CAR-iPSC-derived CD8+ cell-treated group, and the primary CAR+CD8+ T cell-treated group. Statistical differences between the tumor-only group and the CAR-iPSC-derived group were assessed using the Mantel-Cox test (*P<0.05; **P<0.01).

[0284] Figure 61D It is a graph depicting the in vivo toxicity of CAR-iPSC-derived and primary CAR+CD8+ T cells, as indicated by changes in body weight as a percentage.

[0285] Figure 61E This is a diagram depicting the in vivo cell dynamics of CAR-iPSC-derived and primary CAR+ T cells detected in peripheral blood.

[0286] Figure 62 A is a flow cytometry image of CAR-iPSC-derived CD8+ cells detected in peripheral blood 15 days after infusion.

[0287] Figure 62 B is a diagram depicting the expression of CD8α and / or CD8β in CAR-iPSC-derived CD8+ cells before and after infusion.

[0288] Figure 63 This is a graph depicting the secretion of cytokines and cytolytic molecules from CAR-iPSC-derived CD8+ cells and CAR+ primary CD8+ T cells detected in whole blood retrieved 15 days after infusion. Statistical differences between the tumor group and the CAR-iPSC-derived group were assessed by unpaired nonparametric t-tests and Welch correction (p-values ​​as described above).

[0289] Figure 64 A is a graph depicting the survival rates of re-challenge after initial infusion in the untreated group (tumor only), the CAR-iPSC-derived CD8+ cell-treated group, the primary CAR+CD8+ T cell-treated group, and the CAR-iPSC-derived CD8+ cell-treated group.

[0290] Figure 64 B is a graph depicting in vivo toxicity, indicated by percentage change in body weight, after initial infusion in the untreated group (tumor only), the CAR-iPSC-derived CD8+ cell-treated group, the primary CAR+CD8+ T cell-treated group, and the CAR-iPSC-derived CD8+ cell-treated group.

[0291] Figure 65 A is a bioluminescent image of mice treated with CAR-iPSC-derived CD8+ cells after rechallenge.

[0292] Figure 65 B is a graph depicting the in vivo tumor growth inhibition after initial infusion in the CAR-iPSC-derived CD8+ cell treatment group, the primary CAR+T cell treatment group, and the CAR-iPSC-derived CD8+ cell treatment group.

[0293] Figure 65 C is a graph depicting the in vivo cell dynamics of CAR-iPSC-derived and primary CAR+ T cells detected in peripheral blood after initial infusion (iPSC-derived and primary cells) and rechallenge (iPSC-derived cells only).

[0294] Figure 66 A is a graph depicting the detection of iPSC-derived CD8+ cells in the lungs at the endpoint compared to a tumor-only control.

[0295] Figure 66 B is Figure 66 Flow cytometry images of tumor-only controls (left) and iPSC-derived CD8+ cells (center, right) in A.

[0296] Figure 66 C is a graph depicting the expression of CD8α and / or CD8β in CAR-iPSC-derived CD8+ cells before and after infusion (extended to include tumor rechallenge).

[0297] Figure 67 A is a graph depicting the viability of cells targeting CAR-iPSC-derived (“CAR”), TCR-iPSC-derived (“TCR”), and CAR-iPSC-derived TCR transduction (“TCR-CAR”) during differentiation in a stirred tank bioreactor.

[0298] Figure 67 B is a graph depicting the number of cells in a stirred tank bioreactor for CAR-iPSC-derived (“CAR”), TCR-iPSC-derived (“TCR”), and CAR-iPSC-derived TCR transduction (“TCR-CAR”) during differentiation.

[0299] Figure 68 A is a flow cytometry image 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.

[0300] Figure 68 B is a graph depicting the yield of CD8αβ+ cells in a stirred tank bioreactor after differentiation, targeting CAR-iPSC-derived (“CAR”), TCR-iPSC-derived (“TCR”), and CAR-iPSC-derived TCR transduction (“TCR-CAR”) cells.

[0301] Figure 69 A is a graph depicting the in vitro cytotoxicity of TCR-iPSC-derived cells differentiated in a stirred tank bioreactor (“STR TCR+CD8+”) or a well plate (“WP TCR+CD8+”) compared to primary TCR+CD8+ cells (“primary TCR+CD8+”) in a continuous restimulation assay at a 2:1 E:T ratio.

[0302] Figure 69 B is a graph depicting the fold expansion of TCR-iPSC-derived cells differentiated in stirred tank bioreactors ("STR TCR+CD8+") or well plates ("WP TCR+CD8+") compared to primary TCR+CD8+ cells ("primary TCR+CD8+") in a sequential restimulation assay.

[0303] Figure 69 C is a graph depicting the in vitro cytotoxicity of TCR-iPSC-derived cells differentiated in stirred tank bioreactors ("STR TCR+CD8+") or well plates ("WP TCR+CD8+") at different E:T ratios compared to primary TCR+CD8+ cells ("primary TCR+CD8+").

[0304] Figure 69D is a graph depicting the antigen-independent (“antigen-negative”) and antigen-dependent (“antigen-positive”) cytotoxicity of TCR-iPSC-derived cells differentiated in stirred-tank bioreactors (“STR TCR+CD8+”) or well plates (“WP TCR+CD8+”) compared to primary TCR+CD8+ cells (“primary TCR+CD8+”). Detailed Implementation

[0305] 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 pertains.

[0306] Generally, this disclosure provides methods for generating T cell lineage populations from T cell progenitor cells, T cell lineage populations generated by the methods disclosed herein, pharmaceutical compositions comprising T cell lineage populations generated by the methods disclosed herein, use of T cell lineage populations in the manufacture of medicaments for treating diseases or conditions, wherein the T cell lineage populations are generated by the methods disclosed herein, and methods for differentiating T cell progenitor cell populations by the methods disclosed herein.

[0307] definition

[0308] As used herein, the term "stem cell" refers to a cell that can differentiate into more specialized cells and has the capacity for self-renewal. 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 CD34+ stem cells derived from mobilized peripheral blood, umbilical cord blood stem cells, and adult stem cells, which are found in various tissues. Methods for obtaining, deriving, or generating stem cells are known in the art.

[0309] As used herein, the term "progenitor cell" refers to a cell that can differentiate into one or more cell types, but typically has a limited capacity for self-renewal. Progenitor cells are derivatives of stem cells and have a more limited potency relative to their corresponding source stem cells. For example, hematopoietic stem cells (HSCs), found in adult bone marrow, peripheral blood (in smaller numbers), and umbilical cord blood, have the capacity to produce all other blood cells. Hematopoietic progenitor cells are pluripotent or lineage-committed cells derived from HSCs that have the capacity to produce a more limited or specific type of blood cell. Hematopoietic stem cells and progenitor cells (HSPCs) typically exist in vivo as a heterogeneous population and have uses as a heterogeneous population as described herein. Hematopoietic stem cells and progenitor cells can be characterized, for example, by their surface CD34 (CD34+).

[0310] As used herein, the terms “T cell progenitor” and “proT cell” refer to cells derived from pluripotent stem cells or CD34+ hematopoietic stem cells and / or progenitor cells that express at least CD7+ and have the ability to differentiate into one or more types of immature and mature T cells. Examples of T cell progenitor 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.

[0311] As used in this article, “immature T cells” or mature T cells are T lineage cells derived from T cell progenitors. T cell development is characterized by the progressive expression of cell surface receptors, particularly CD4 and CD8. In vivo, T lineage cells progress from T cell progenitors through stages: CD4-CD8- (double-negative, DN), CD4+CD8- (CD4 immature single-positive, CD4ISP), CD4+CD8+ (double-positive, DP), and CD4-CD8+ (CD8 single-positive, CD8SP), as well as CD4 single-positive (CD4SP). CD8 can be expressed as a heterodimer of CD8α and CD8β, resulting in CD8αβ+ cells, or as a homodimer of CD8αα, resulting in CD8αα+ cells. CD4-CD8+ cells can also be characterized by cell surface expression of CD3 and either TCRγδ (γδ T cells) or TCRαβ (αβ T cells).

[0312] As used herein, “serum-free medium” refers to a cell culture medium lacking animal serum. Serum-free medium may include specific, known serum components isolated from animals (including human animals), such as, for example, bovine serum albumin (BSA).

[0313] As used herein, “Notch signaling ligand” refers to any ligand capable of interacting with the Notch protein receptor to regulate T cell lineage commitment and differentiation. Examples of Notch signaling ligands include δ-sample 4 (DL4), δ-sample 1 (DL1), δ-sample 3 (DL3), Jagged1, and Jagged2.

[0314] As used herein, Notch signaling ligands, such as “δ-like 4” and “DL4”, refer to proteins 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 “δ-like ligand 4” and “DLL4”. In this document, references to DL4 are not limited to the entire DL4 protein, but include at least the signaling peptide portion of DL4. For example, a commercially available product (Sino Biologicals) containing an extracellular domain fused to the Fc region of human IgG1 at its C-terminus (Met 1-Pro 524 of human DL4 (full-length DL4 accession number NP 061947.1 (SEQ ID NO:1))) is a suitable DL4 protein for use in the methods provided herein.

[0315] As used herein, Notch signaling ligands also include variants of known Notch signaling ligands, such as DL4. A variant Notch signaling ligand is 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. The definition also includes variants, such as peptides, oligopeptides, peptides, and proteins that have amino acid sequence identity with a given polypeptide, oligopeptide, peptide, or protein. The percentage of 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 specified length, such as over the entire length of the polypeptide.

[0316] 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 sialylate glycoprotein, a type I membrane protein, and a member of the lg superfamily. VCAM-1 is also referred to in the art as “vascular cell adhesion molecule 1 and differentiation cluster 106” (CD106). In this document, references to VCAM-1 are not limited to the entire VCAM-1 protein, but include at least the signal transduction peptide portion of VCAM-1 (QIDSPL (SEQ ID NO:2) or TQIDSPLN (SEQ ID NO:3)). For example, a commercially available mouse VCAM-1-Fc chimeric protein (R&D) containing a region fused to the Fe region of human lgG1 (Phe25-Glu698; a region of mouse VCAM-1 (full-length mouse VCAM-1 accession number CAA47989; (SEQ ID NO:4))) is a suitable VCAM-1 protein for use herein. 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 used in the methods provided herein. References to VCAM-1 herein also include variants that differ from the wild-type VCAM-1 in amino acid sequence by one or more additions, deletions, and / or substitutions, while retaining the desired activity of wild-type VCAM-1. Variants, such as peptides, 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 identity percentage 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 specified length, such as the entire length of the polypeptide. VCAM-1 has been shown to synergistically increase Notch signaling when combined with DL4 (e.g., Shukla et al., 2017).

[0317] As used herein, “two-dimensional engineered thymus 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 may include, for example, tissue culture plates. Methods for immobilizing Notch signaling ligands on 2D substrates are known in the art and described, for example, by Shukla et al., 2017.

[0318] As used herein, “three-dimensional engineered thymus niche (3D ETN)” refers to a three-dimensional substrate immobilized with Notch signaling ligands, such as DL4 and optionally VCAM-1. The three-dimensional (3D) substrate may comprise, for example, micrometer-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. Several methods may be used alone or in combination to produce protein-coated particles, such as: physical adsorption driven by the affinity of the protein for the particulate material, chemical conjugation resulting from reactions with amines, carboxyl groups, thiols, epoxides, azide reactive groups, etc., or by coating with appropriate ligands to capture target proteins by affinity. Examples of affinity tags include, but are not limited to: Fc, biotin, halogenated, aldehydes, Snap, Spy-Catcher, VIPER. The particles or beads may be made of, for example, polystyrene, iron oxide, gold, or other suitable materials known in the art. 3D ETN can be used to culture cells on tissue culture plates, flasks, or other containers used for cell culture.

[0319] As used in this article, “surface-bound” refers to Notch signaling ligands fixed to a 2D ETN or 3D ETN by covalent or non-covalent interactions, affinity-based interactions, or other suitable forms of interaction.

[0320] 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)) that exhibits a higher absolute number or ratio of one of the cell phenotypes (e.g., CD4-CD8+ (CD8SP)) compared to other cell phenotypes (where at least 25% of the cell population consists of a single cell phenotype). For example, compared to T cell progenitor cells cultured with SCT (commercial coating) (where CD4-CD8+ (CD8SP) constitutes less than about 10% of the cell population), when using 10 x 10-1 7 When T cell progenitor cells were cultured at a concentration of 3D ETN beads / mL (1.852x bead dose), the cell population was enriched with CD4-CD8+ (CD8SP) accounting for approximately 50% of the cell population.

[0321] As used herein, an "enriched" cell population also refers to a cell population generated from T cell progenitors cultured with DL4 and optional VCAM-1 when compared to T cell progenitors not cultured with DL4 and optional VCAM-1, provided that the cell population contains one or more cell phenotypes (e.g., CD4-CD8+ (CD8SP), CD4+CD8+ (DP), CD4+CD8- (CD4 ISP), CD4-CD8- (DN)) and exhibits a higher absolute number or ratio of one of the cell phenotypes (e.g., CD4-CD8+ (CD8SP)) compared to other cell phenotypes (where at least 25% of the cell population consists of a single cell phenotype). For example, compared to T cell progenitors cultured with 0.1 x 10⁻⁶ DL4 and optional VCAM-1. 7 Compared to T cell progenitor cells cultured at a 3D ETN bead concentration of 10 x 10 beads / mL (0.018x bead dose) where CD4-CD8+ (CD8SP) comprised less than approximately 10% of the cell population, T cells cultured at a 3D ETN bead concentration of 10 x 10 beads / mL showed significantly better results. 7 When T cell progenitor cells were cultured at a concentration of 3D ETN beads / mL (1.85x bead dose), the cell population was enriched with CD4-CD8+ (CD8SP) accounting for approximately 50% of the cell population.

[0322] As used herein, the term "subject" refers to a vertebrate, preferably a mammal (e.g., a non-human mammal), more preferably a primate, and still more preferably a human. Mammals include, but are not limited to, primates, humans, farm animals, sporting animals, and pets.

[0323] As used herein, the term "treatment" (or "treat" or "treating") refers to a method for obtaining a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: an increased immune response; an increased T-cell response; a reduction in the severity of the disease, symptom, or condition; a shortening of the duration of the disease, symptom, or condition; and / or a reduction in the number, severity, or duration of symptoms associated with the disease, symptom, or condition. This term includes the administration of compounds, reagents, medicines, or pharmaceutical compositions of this disclosure to prevent or delay the onset of one or more symptoms, complications, or biochemical markers of a disease or condition; to alleviate or improve one or more symptoms; to shorten or reduce the duration of symptoms; or to prevent or inhibit the further development of the disease, symptom, or condition. Treatment can be preventative (to prevent or delay the onset of a disease, symptom, or condition, or to prevent the manifestation of its clinical or subclinical symptoms) or therapeutic suppression or relief of symptoms following the manifestation of a disease, symptom, or condition. Beneficial or expected clinical outcomes may be an increase or decrease (where 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% relative to an appropriate control (e.g., a subject not receiving treatment).

[0324] As used herein, the term "administering / administration" means placing the reagents, drugs, compounds, or pharmaceutical compositions disclosed herein into a subject by a method or route that results in at least partial delivery of the composition to the desired location. The compounds and pharmaceutical compositions disclosed herein can be administered via any suitable route that results in effective treatment of the subject. Routes of administration of the compounds and pharmaceutical compositions disclosed herein include, but are not limited to, intravenous or intraperitoneal administration, or combinations thereof.

[0325] The term "effective dose" or "therapeutic effective dose," such as the effective dose or therapeutic effective dose for T-cell lineage populations as used herein, is a dose sufficient to produce any one or more beneficial or desired outcomes. More specifically, an effective dose may alleviate or improve one or more symptoms of a disease; shorten the duration of one or more symptoms of a disease present in a subject; or increase the survival rate of a subject with a disease. For prophylactic use, beneficial or desired outcomes may include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical and / or histological symptoms of infection, its complications, and intermediate pathological phenotypes that occur during the course of disease development. For therapeutic use, beneficial or desired outcomes may include clinical outcomes such as reducing one or more symptoms of a disease; reducing the dose or duration of administration of other drugs required to treat the disease; enhancing the effectiveness of another drug and / or reducing the toxicity of another drug; delaying disease progression in a subject; reducing the duration of one or more symptoms of a disease present in a subject; and / or increasing the overall survival rate of a subject with a disease. An effective dose may be administered in one or more doses, administration cycles, or treatment courses.

[0326] For the purposes of this disclosure, an effective amount of a cell population or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventative or therapeutic treatment. As understood in a clinical context, an effective dose of a compound or pharmaceutical composition may or may not be achieved when combined with another agent, drug, compound, or pharmaceutical composition. Therefore, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and an effective amount of a single agent may be considered if the desired outcome can be obtained or achieved in combination with one or more other agents. The amount may vary from subject to subject and may depend on one or more factors, such as, for example, the subject's sex, age, weight, the subject's health history, and / or the underlying cause of the disease, symptom, or condition to be prevented, suppressed, and / or treated.

[0327] As used herein, the term "pharmaceutical carrier, diluent, or excipient" includes any material that, when combined with an active ingredient, allows that ingredient to remain biologically active and unresponsive to the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, 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 physiological (0.9%) saline. Compositions containing such carriers are formulated using well-known, conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0328] As used in this article, the singular forms “a,” “a,” and “the” are also intended to include the plural forms, unless the context explicitly indicates otherwise.

[0329] The phrase “and / or” should be understood to mean “any one or both” of the elements so connected, that is, elements that exist together in some cases and separately in others. Therefore, as a non-limiting example, when used in conjunction with open-ended terms such as “includes,” a reference to “A and / or B” may in one embodiment refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0330] As used herein, with respect to a list of one or more elements, the phrase "one or more" should be understood as at least one element selected from any one or more elements in the list, but not necessarily including at least one of every element specifically listed in the list, and does not exclude any combination of elements in the list. In addition to the elements specifically identified in the list of elements referred to by the phrase "one or more," this definition also allows for the optional presence of other elements, whether or not they are related to the specifically identified elements. 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") in one embodiment may refer to at least one, optionally including more than one A, with no B (and optionally including elements other than B); in another embodiment, it refers to at least one, optionally including more than one B, with no A (and optionally including elements other than A); in yet another embodiment, it refers to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0331] When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below that value. Generally, the term "about" is used herein to modify values ​​where the difference between the value above and below a specified value is 20%, 10%, 5%, or 1%. Generally, the term "about" is used herein to modify values ​​where the difference between the value above and below a specified value is 10%. Generally, the term "about" is used herein to modify values ​​where the difference between the value above and below a specified value is 5%. Generally, the term "about" is used herein to modify values ​​where the difference between the value above and below a specified value is 1%.

[0332] When this document lists ranges of values, it is intended to cover every value and subrange within that range. For example, “1-5mL” is intended to cover 1mL, 2mL, 3mL, 4mL, 5mL, 1-2mL, 1-3mL, 1-4mL, 1-5mL, 2-3mL, 2-4mL, 2-5mL, 3-4mL, 3-5mL, and 4-5mL.

[0333] It should be further understood that the terms “comprises”, “comprising”, and / or “including”, when used in this specification, specify the presence of the 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.

[0334] As used herein, the term “consisting of” and its derivatives are intended to be closed terms that specify the presence of the stated feature, integer, step, operation, element and / or component, and exclude the presence or addition of one or more other features, integers, steps, operations, elements and / or components.

[0335] General Technology

[0336] Unless otherwise defined herein, scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the terms and techniques used in connection with nuclear tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are well-known and commonly used in the art.

[0337] Unless otherwise indicated, implementation of this disclosure will employ conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. The following references provide a comprehensive explanation of these techniques: Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989), Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E.C. Lis, 1998), Academic Press; Animal Cell Culture (edited by R.R. Freshney, 1987); Introduction to Cell and Tissue Culture (J.M. Pather and P.E. Roberts, 1998), Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J.B. G. R ... Mammalian Cells (edited by J.M. Miller and M.C. Aleus, 1987); Current Protocols in Molecular Biology (edited by F.M. A. Usubel et al., 1987); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); Current Protocols in Immunology (J.E.)Edited by Coligan et al., 1991); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & 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 & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); and lmmunobiology (CA Janeway and P. Travers, 1997). .

[0338] Methods for generating T cell lineage populations in vitro

[0339] Typically, the in vitro method for generating T cell lineage populations provided in this article involves culturing T cell progenitor cells (proT cells) under conditions and time suitable for differentiation into T cell lineage populations in the presence of Notch signaling ligands.

[0340] Methods for generating proT cells from stem cells / progenitor cells (such as hematopoietic progenitor cells (HPC), hematopoietic stem cell / progenitor cells (HSPC), or CD34+ cells) are known in the art, for example, differentiation on immobilized VCAM-1 and Notch signaling ligands (e.g., DL4) under suitable culture conditions (e.g., Shukla et al., 2017) or using DL4-coated microbeads (e.g., Trotman-Grant et al., 2021).

[0341] To confirm the generation of proT cells, one or more characteristics of the cells indicating proT cells can be analyzed, such as, for example, 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 may include, for example, flow cytometry or immunocytochemistry used herein. Cell number and cell viability can be analyzed using techniques well known to those skilled in the art and may include, for example, the use of automated cell counters disclosed herein.

[0342] In one embodiment, CD34+ cells may be obtained from umbilical cord blood, peripheral blood, or bone marrow, or they 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 cells and / or progenitor cells are human cells. In one embodiment, the stem cells are mobilized peripheral blood-derived CD34+ cells. In a preferred embodiment, CD34+ cells are derived in vitro from iPSCs. Methods for generating CD34+ cells from iPSCs are known in the art, for example, differentiation using appropriate culture conditions (e.g., Trotman-Grant et al., 2021).

[0343] Cells can be cultured in cell culture systems known in the art, such as cell culture plates, culture dishes, and bioreactors, including stirred tank reactors (STRs), shake bag bioreactors, and other suitable forms of cell culture. Cell culture can be carried out under static, 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 any type of processing / culture conditions and methods can be used, including, for example, batch processes, fed-batch processes, and perfusion culture methods and conditions.

[0344] This invention, which generates T-cell lineage populations, can be performed using populations of HPC, HSPC, and / or T-cell progenitor cells in the presence of Notch signaling ligands and in the absence of T-cell receptor stimuli. T-cell receptor stimuli can be molecules that bind to and / or activate CD3 signaling, such as anti-CD3 antibodies, antigen-presenting cells, or artificial antigen-presenting cells. This invention can be performed in the absence of exogenous CD3 binding and activation.

[0345] This invention can also be used to generate surface CD3-negative (sCD3-) cells (e.g., cells on which CD3 cannot be detected as a cell surface marker). This invention can also be used to generate TCR- cells (such as cells that do not express endogenous (i.e., natural) TCRs or cells in which undetectable expression is present on the surface of TCR cells). This invention can also be used to generate cells that are both sCD3- and cell surface TCR- (i.e., cells in which neither CD3 nor TCR is expressed on the cell surface).

[0346] The present invention, which generates T cell lineage populations, can also be carried out using populations of HPC, HSPC, and / or T cell progenitors in the presence of Notch signaling ligands and without conjugation and / or stimulation of chimeric antigen receptors (CARs) in CAR+ cells.

[0347] The present invention can also be carried out using serum-free culture conditions and / or feed-free (e.g., matrix cell-free) culture conditions.

[0348] base

[0349] In one embodiment, proT cells are cultured in a two-dimensional culture system using a suitable 2D substrate, which may include, for example, a standard culture plate coated with a Notch signaling ligand (e.g., DL4). The culture plate may also be coated with VCAM-1.

[0350] In one embodiment, proT cells are cultured in a three-dimensional culture system using a suitable 3D substrate, which may include, for example, micrometer-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. Several methods can be used alone or in combination to generate protein-coated particles, such as: physical adsorption driven by the affinity of proteins for particulate material, chemical conjugation resulting from reactions with amines, carboxyl groups, thiols, epoxides, azide reactive groups, etc., or by coating with appropriate ligands to capture target proteins by affinity. Examples of affinity tags include, but are not limited to: Fc, biotin, halogenated, aldehyde, Snap, Spy-Catcher, VIPER.

[0351] The particles or beads may be made of, for example, polystyrene (including carboxylated polystyrene), iron oxide, gold, or other suitable materials known in the art.

[0352] In one instance, as described in Trotman-Grant et al., 2021 and WO2019157597, Notch signaling ligands (e.g., DL4), alone or in combination with VCAM-1, can be conjugated to polystyrene microspheres.

[0353] In another example, 3D ETN beads can be manufactured by affinity capturing DL4 and VCAM-1 carried on beads coated with streptavidin or protein G. The beads are diluted to 0.1% solids in Dulbecco phosphate-buffered saline (DPBS) free of Ca2+ or Mg2+ and supplemented with 0.05% BSA, and incubated at room temperature with a protein solution (0.1x–20x protein molar excess) 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 process is repeated four times, and the 3D ETN beads are then concentrated tenfold for storage.

[0354] Quantification of protein fixation can be performed using methods known in the art, such as colorimetric determination of quinolinic acid (BCA), immunofluorescence assay, or other known detection methods.

[0355] T-cell therapy

[0356] T cells have a wide range of therapeutic applications. T cells can be modified, for example, using conventional gene-editing methods such as nuclease editing or viral vector transduction, to express chimeric antigen receptors (CARs) and / or exogenous T-cell receptors (TCRs), thereby generating 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 stage to contain nucleic acids encoding CARs or TCRs that can be expressed at the pluripotent, progenitor, or derived cell stages. Engineered T-cell therapies are applicable in, for example, oncology and autoimmune diseases. In oncology, engineered T-cell therapies are applicable in, for example, hematologic malignancies such as B-cell lymphoma, B-cell acute lymphoblastic leukemia and other B-cell malignancies, multiple myeloma and other hematologic malignancies, as well as solid tumors such as, for example, mesothelioma, adenocarcinoma, glioma, and sarcoma (Weber et al., 2020). In autoimmune diseases, engineered T-cell therapy is applicable in conditions such as type 1 diabetes, rheumatoid arthritis, multiple sclerosis, and other autoimmune diseases or conditions (Weber et al., 2020).

[0357] Engineered T-cell therapies can target antigens known to be expressed on target cell types, including tumor cells or tumor tissues. Chimeric antigen receptors (CARs) can be engineered to target surface antigens or multivalent soluble antigens. The targeting extracellular domain of a CAR can be a single-chain variable fragment (scFv), a single-domain antibody (a single variable domain on the heavy chain, VHH), a nanobody, or other antigen-binding domains (Qu et al., 2022). CAR-T cell therapies can use different CAR designs or multiple CARs to target a variety of antigens (Qu et al., 2022). Exemplary oncological antigens and corresponding cancer types for CAR-T cell therapies are listed in Table 1 below (Qu et al., 2022; Guha et al., 2022; Drouggkas et al., 2023; Want et al., 2023).

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] 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 oncology 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).

[0368]

[0369]

[0370] The pharmaceutical compositions provided herein can be administered to subjects to relieve or improve one or more symptoms of a disease; shorten the duration of one or more symptoms of a disease present in the subject; and increase the survival rate of subjects with a disease.

[0371] The pharmaceutical compositions described herein can be administered to subjects to treat cancer or autoimmune diseases.

[0372] The pharmaceutical compositions provided herein may be administered to a subject in an effective or therapeutically effective amount. Those skilled in the art will be able to determine such amounts based on factors such as the subject's size (e.g., weight), age and / or sex; the severity of the subject's symptoms; and the specific composition or route of administration chosen. Those skilled in the art will also know how to select an appropriate route of administration and how to administer the compounds and compositions provided herein.

[0373] The dosage of the pharmaceutical compositions disclosed herein depends on many factors, such as the pharmacodynamic properties of the composition, the administration method, the recipient's age, health and weight, the nature and severity of symptoms, the frequency of treatment and the type of concurrent treatment (if any), and the clearance of the compound in the subject being treated. Those skilled in the art can determine an appropriate dosage based on the above factors. In some embodiments, the pharmaceutical composition is initially administered at a suitable dose, which is adjusted as needed based on clinical response.

[0374] Reagent test kit

[0375] The present invention also provides kits comprising the pharmaceutical compositions described herein. The kits of the present invention comprise one or more containers containing the pharmaceutical compositions described herein and instructions for use according to any of the methods of the present invention described herein. Typically, these instructions include a description of the administration of the pharmaceutical compositions for the therapeutic treatment described above. In some embodiments, kits for producing single-dose administration units are provided.

[0376] Instructions for use of a pharmaceutical composition typically include information about the intended therapeutic dose, dosing regimen, and route of administration. Containers may be unit doses, bulk packaging (e.g., multi-dose packs), or subunit doses. Instructions provided in the kits disclosed herein are typically written instructions on a label or packaging insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0377] This disclosure is further described in detail with reference to the following experimental examples. These examples are for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, this disclosure should in no way be construed as limited to the following examples, but should be construed as including any and all variations that become apparent as a result of the teachings provided herein.

[0378] Example 1: Preparation of engineered thymus niche (ETN) and production of DL4 and VCAM-1

[0379] Recombinant DL4-Fc fusion protein was purchased from Sino Biological or manufactured internally using HEK-293T cells and purified using 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 preparing 2D ETN and 3D ETN, as further described below, are shown in Table 3.

[0380]

[0381]

[0382]

[0383] Preparation of 2D ETN

[0384] Tissue culture plates with 6, 12, 24, 48, and 96 wells were coated overnight at 4°C or for 3 hours at 37°C using Notch signaling ligands DL4 and VCAM-1. The 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 phosphate-buffered saline (DPBS) (- / -). Appropriate coating volumes / wells of DL4 and VCAM-1 diluted in DPBS were added to the tissue culture plates as shown in Table 4.

[0385]

[0386] Gently tap the tissue culture plate to ensure the coating solution containing Notch signaling ligand, DL4, and VCAM-1 is evenly distributed across the entire well surface. Place the tissue culture plate with... Seal and store overnight at 4°C or for 3 hours at 37°C. Place the tissue culture plates coated with Notch signaling ligands DL4 and VCAM-1 (prepared overnight at 4°C) in a 37°C incubator for equilibration for 3 hours, then plate the cells. After equilibration or after coating the tissue culture plates with Notch signaling ligands DL4 and VCAM-1 at 37°C for 3 hours, aspirate the coating solution from the wells. Flush the wells with DPBS (- / -) using the volumes shown in Table 5, then immediately add the cell suspension to the tissue culture plates.

[0387]

[0388] Preparation of 3D ETN

[0389] The dosage of 3D ETN can be calculated in proportion to the bead diameter and can be expressed as: a dosage proportional to the surface area of ​​the culture plate or culture flask (e.g., as shown in Table 4), the number of beads per unit culture volume, or the bead surface area per unit culture volume. The bead concentration per mL remains constant for different vessels when calculated per unit volume. A “1x” bead dosage indicates that one layer of beads completely covers the plate surface; since the beads are approximately spherical, the total surface area of ​​the beads is four times the surface area of ​​the plate or culture container.

[0390] Table 6 provides the bead concentration range for 3.05 μm diameter polystyrene beads.

[0391]

[0392]

[0393] The density of Notch signaling ligands on the beads (such as, for example, the density of surface-bound DL4) can range from, for example, 100 molecules per square micrometer (100 molecules / μm²) to 3000 molecules per square micrometer (3000 molecules / μm²). Table 7 provides calculations of Notch signaling ligand concentrations for a range of bead dosages and Notch signaling ligand densities for 3.05 μm polystyrene beads. The concentration of Notch signaling ligands (e.g., DL4) is calculated based on the amount of DL4 capable of binding to soluble Notch1. Therefore, the total concentration of Notch signaling ligands on the beads may be slightly higher.

[0394]

[0395] Table 8 provides a series of notch signaling ligand (such as DL4) surface area / unit volume for both polystyrene beads with diameters of 3.05 μm and 3.29 μm, along with the notch signaling ligand density.

[0396]

[0397] The 3D ETN may also include surface-bound VCAM-1. VCAM-1 can be fixed to the 3D ETN at an input molar ratio ranging from 1:6 to 10:1 DL4:VCAM-1. In one embodiment, the 3D ETN is prepared at an input molar ratio of 2.5:1 DL4:VCAM-1. In one embodiment, the final density of the surface-bound VCAM-1 on the beads is equivalent to the density of the Notch ligand. For example, the surface area of ​​VCAM-1 per unit volume can be equivalent to the surface area of ​​the Notch ligand per unit volume shown in Table 8 above.

[0398] Cells can be cultured at densities suitable for the culture scale and format. In microplate culture, cells can be cultured at, for example, 2.5 x 10⁵–2 x 10⁶ cells / mL. In STR culture, cells can be cultured at, for example, 5 x 10⁴–6 x 10⁶ cells / mL.

[0399] Example 2: Generation of CD4-CD8+ T cells

[0400] To assess the role of 3D ETN bead concentration in T cell differentiation ( Figure 1 A) Two independently derived T cell progenitor cell populations (“proT library A” and “proT library C”) derived from iPSC-derived CD34+ cells, which were cryopreserved 18 days after differentiation with 2D ETN (DL4 and VCAM-1), were thawed and plated at 1 x 10⁶ cells / mL in lymphocyte maturation medium (LMM; STEMCELL Technologies) in 24-well cell culture plates. 3D ETN beads modified with Notch signaling ligand, DL4, and VCAM-1 were added to the cells at six different concentrations: 0.27 x 10⁷, 0.54 x 10⁷, 1.35 x 10⁷, 2.7 x 10⁷, 5.4 x 10⁷, and 8.1 x 10⁷ beads / mL (0.05x, 0.1x, 0.25x, 0.5x, 1x, and 1.5x bead doses). Cells were also plated into 2D ETN (as described in Example 1) and StemSpan in 24-well cell culture plates. TM Cells were cultured on a lymphocyte differentiation coating material (called SCT or a commercial coating; STEMCELL Technologies). Cells were cultured with 2D ETN and 3D ETN or SCT for 3 and 7 days, respectively, and removed from the plate by pipetting and collected for analysis by flow cytometry and by automated cell counters. Cell number and viability were assessed using MX). After 3 days of cell culture, more CD4+CD8- (CD4 immature single-positive, CD4 ISP) cells were generated when cultured with lower 3D ETN bead concentrations (0.27 x 10⁷, 0.54 x 10⁷ beads / mL (0.05x, 0.1x bead doses) and 2D ETN, while more CD4-CD8+ (CD8 single-positive, CD8SP) cells were generated at higher 3D ETN bead concentrations (2.7 x 10⁷, 5.4 x 10⁷, 8.1 x 10⁷ beads / mL (0.5x, 1x, 1.5x bead doses)). This dose-dependent generation of CD4-CD8+ cells was also observed in cells cultured with 3D ETN for 7 days, where the effect was more pronounced. Figure 1(B) The highest concentration of 3D ETN beads (8.1 x 10⁷ beads / mL (1.5x bead dose)) produced the most CD4-CD8+ cells. Among CD4-CD8+ cells, the proportion of CD8αβ cells also responded to the 3D ETN bead dose, with approximately 80% of CD4-CD8+ cells being CD8αβ cells at the highest bead concentration (8.1 x 10⁷ beads / mL (1.5x bead dose)), while only 46% of CD4-CD8+ cells were CD8αβ cells at the lowest bead concentration (0.27 x 10⁷ beads / mL (0.05x bead dose)). In CD4-CD8+ cells cultured with 2D ETN and SCT, less than 40% of the CD4-CD8+ cells were CD8αβ cells. Figure 1 C).

[0401] Live cell expansion varied with 3D ETN bead dosage – after 7 days, cells cultured with 0.54 x 10⁷ and 1.35 x 10⁷ beads / mL (0.1x and 0.25x bead dosages) showed the highest expansion, with most dosages exhibiting expansion between 1 and 1.5-fold. Figure 2 A). When viability was assessed as a phenotypic function, no effect of 3D ETN bead dosage was observed. Instead, high viability (>80%) was observed in CD4+CD8+ (double-positive, CD4+CD8-) and CD4-CD8+ cells, while low viability (<20%) was observed in CD4-CD8- (double-negative, DN) and CD4+CD8- cells, regardless of bead dosage. Figure 2 B).

[0402] As described above, the effect of 3D ETN bead concentration on T cell differentiation was repeatedly evaluated using a wider range of 3D ETN bead concentrations (0.02x–2x bead dose, or 1.08x 10⁶ beads / mL–10.8x 10⁷ beads / mL (shown as 0.1–10x 10⁷ beads / mL)), while T cell progenitor cells were cultured with 2D ETN and SCT. Furthermore, the duration of cell culture with 2D ETN, SCT, and 3D ETN was extended to 7 days and 14 days, respectively. Consistent with the first experiment above, the formation of CD4+CD8- and CD4-CD8- cell populations after 7 days of culture was 3D ETN bead concentration-responsive. More CD4+CD8- cells were observed at lower bead concentrations (0.1 x 10⁷ beads / mL and 0.3 x 10⁷ beads / mL (0.2x and 0.05x bead doses)) and more CD4-CD8+ cells were observed at higher bead concentrations (3 x 10⁷ beads / mL and 10 x 10⁷ beads / mL (0.5x and 2x bead doses)). Figure 3A). These effects were even more pronounced after 7 days of reculturing, with approximately 60% of CD4-CD8+ cells observed at the highest bead concentration (10 x 10⁷ beads / mL (2x bead dose)). Figure 3 B). Interestingly, T cell progenitors cultured at the lowest bead concentration (0.1 x 10⁷ beads / mL (0.02 x bead dose)) produced a cell population similar to that of T cell progenitors cultured with 2D ETN, with a high percentage of CD4+CD8+ cells and a very small percentage of CD4-CD8+ cells.

[0403] The expression of CD3 and TCR was evaluated in T cell progenitor cells cultured with 2D ETN, SCT, and 3D ETN at specific concentrations. By day 14, CD3 and TCR expression increased in cells cultured with 2D ETN and SCT. Specifically, in the 2D ETN case, approximately 60% of cells expressed CD3, and approximately 20% expressed TCRαβ. Figure 4 A). For T cell progenitor cells cultured with 3D ETN, CD3 expression exhibited a negative concentration-responsive pattern, with the lowest bead concentrations (0.1 x 10⁷ beads / mL and 0.3 x 10⁷ beads / mL (0.02x and 0.05x bead doses) producing the highest CD3 expression. At the highest bead concentration (10 x 10⁷ beads / mL (2x bead dose)), <30% of the cells were CD3+ cells, and TCRαβ expression was barely observed (<5%). Figure 4 A and 4B). TCRγδ expression in most populations does not appear to be concentration-responsive, as cells cultured with 3D ETN concentrations ranging from 0.3 x 10⁷ beads / mL to 10 x 10⁷ beads / mL (0.05 x to 2 x bead doses) produced approximately 10% TCRγδ+ cells. Figure 4 B). The expression of TCRαβ and TCRγδ was detected in both CD4+CD8+ and CD4-CD8+ populations, revealing that TCRαβ+ cells were predominantly found in the CD4+CD8+ population. When cultured with 2D ETN, SCT, and 3D ETN (lower bead concentrations (0.1 x 10⁷ beads / mL and 0.3 x 10⁷ beads / mL (0.02x and 0.05x bead doses))), the CD4-CD8+ population contained a large number of TCRγδ+ cells. However, for cells cultured with 3D ETN at higher bead concentrations (3 x 10⁷ and 10 x 10⁷ beads / mL (0.5x and 2x bead doses)), only a small fraction of the cells were TCRγδ+ cells (approximately 10%), and TCRαβ expression was almost entirely absent. Figure 4 D).

[0404] Similar to the first experiment, cell viability showed a moderate concentration response after 7 days of culture with 3D ETN. For proT library A, the highest cell viability was observed at 0.3 x 10⁷ beads / mL (0.02 x bead dose), and for proT library C, the highest cell viability was observed at 3 x 10⁷ beads / mL (0.05 x bead dose). Figure 5 A). However, by day 14, cell viability was observed to increase with increasing bead concentration, although proT library C showed very low viability under all conditions ( Figure 5 B). When activity was assessed as a phenotypic function, 3D ETN bead concentration appeared less phenotypic than that of CD4-CD8+ and CD4+CD8+, as CD4-CD8- and CD4 ISP exhibited consistently high activity, while CD4-CD8- and CD4 ISP showed low activity regardless of bead dosage. Figure 5 (C and 5D). In summary, these data indicate that using 3D ETN beads at a concentration of approximately 3 x 10⁷ beads / mL (0.05x bead dose) or higher produces a population of CD4-CD8+ cells expressing CD8αα and CD8αβ but not TCRαβ, and with only a low percentage of CD3 (<30%) and CD3+TCRγδ (approximately 10%).

[0405] To investigate cytotoxic potential, as described above, T cell progenitors transduced with a lentiviral vector to express CD19 CAR were cultured in 3D ETN (5.4 x 10⁷ beads / mL (1x bead dose)) to generate CAR+CD4-CD8+ cells. These 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 only). Similar to primary CAR-transduced CD8+ T cells (left), CAR+CD4-CD8+ cells generated using ETN beads (right) exhibited concentration-responsive cytotoxicity across multiple rounds of stimulation. Figure 6 A and 6B).

[0406] Example 3: Generation of CD4-CD8+ cells in a stirred tank reactor (STR)

[0407] Evaluation was conducted in a stirred tank reactor (STR) culture system (140 ml). T cell progenitor cells were cultured in Eppendorf with the presence of 3 DETN beads (0.54 x 10⁷ beads / mL and 2.7 x 10⁷ beads / mL, 0.1x and 0.5x bead doses) to show cell expansion and viability, compared with T cell progenitor cells cultured with 2D ETN and 3D ETN in 12-well cell culture plates. T cell progenitor cells were cultured for 17 days in lymphocyte maturation medium (LMM, STEMCELL Technologies) under all conditions. Cell viability decreased during the culture period under all conditions. Figure 7 (Days 18-35). As shown in Example 2 (see...) Figure 3 B) When T cell progenitor cells were cultured in 12-well cell culture plates with 2D ETN or low-concentration 3D ETN beads (0.54 x 10⁷ beads / mL (0.1x bead dose)), a greater number of CD4+CD8+ cells were observed. Figure 8 A). For T cell progenitor cells cultured with 3D ETN in both 12-well cell culture plates and STR conditions, a greater percentage of CD4-CD8+ cells were observed at higher bead concentrations (2.7 x 10⁷ beads / mL (0.5 x bead dose)). Figure 8 A). In both 12-well cell culture plates and STR conditions, CD4-CD8+ cells derived from T cell progenitors were cultured with 3D ETN. At both bead concentrations (0.54 x 10⁷ beads / mL and 2.7 x 10⁷ beads / mL (0.1x and 0.5x bead doses)), CD4-CD8+ cells were predominantly CD8αβ cells. Figure 8 B).

[0408] Example 4: Generating T cells transduced by TCR using 3D ETN

[0409] Based on methods known in the art (e.g., Blassberg, 2022), hematopoietic stem cells / progenitor cells expressing CD34 were self-modified into iPSCs lacking endogenous TCR expression. The cells were then differentiated for 10 days using 2D ETN to generate a population of T-cell progenitor cells (e.g., Shukla et al., 2017), such as those expressing CD7 (…). Figure 9 A) and limited CD4 and CD8 expression ( Figure 9 As demonstrated in B), on day 7, cells were modified for MAGE-A4αβ TCR expression via lentiviral transduction using methods known in the art (e.g., Iriguchi et al., 2021). Approximately 20% of the transduced cells expressed TCRαβ and CD3 by day 10, with no TCR and minimal CD3 expression observed in untransduced cells. Figure 9 C).

[0410] T cell progenitor cells were then cultured for a total of 11 days in lymphocyte maturation medium (LMM, STEMCELL Technologies) and 3D ETN (1.08 x 10⁸ beads / mL (2x bead dose)). During this period, cells were harvested on day 7 and analyzed by flow cytometry, and 3D ETN was removed from the cell population using standard isolation techniques (e.g., Trotman-Grant et al., 2021). Cells were then recultured for another 4 days with fresh 3D ETN (1.08 x 10⁸ beads / mL (2x bead dose)).

[0411] After 7 days of culture with 3D ETN, subsets of cells co-expressing CD5+ and CD7+ were observed in both transduced and untransduced cells. Figure 10 A) and the CD8+ cell population that is CD8αβ+ ( Figure 10 B and 10D). On day 7 of culture with 3D ETN, MAGE-A4αβTCR expression and CD3 expression increased to approximately 50% in transduced cells (B and 10D). Figure 10 C, bottom). Untransduced cells are mainly TCR- / CD3- ( Figure 10 C, top).

[0412] After culturing cells with 3D ETN for 11 days, a larger subset of transduced cells was observed to transform into CD4+CD8+ cells compared to untransduced cells. Figure 11 B). Approximately 25% of cells in both groups retained the CD4-CD8+ phenotype. Figure 11 B), greater CD8αβ+ expression was observed in transduced cells ( Figure 11 D). From day 7 to day 11, TCR and CD3 expression also increased in transduced cells. Figure 11 C, middle). Untransduced cells maintain TCR- / CD3- ( Figure 11 C, top).

[0413] Then, TCR-transduced cells were enriched with CD8β using positive selection (EasySep™ Human PE Positive Selection Kit, STEMCELL Technologies; PE Mouse Anti-Human CD8β, BD Biosciences). After enrichment, the fraction of CD4-CD8+ cells increased, and CD8αβ expression was greater. Figure 11 B, bottom) and TCR expression increased ( Figure 11 C, bottom).

[0414] TCR-transduced and untransduced cells showed similar performance in terms of optimal viability and expansion during the early 2D ETN culture period. Figure 12A-12C, “D0, D10”). However, although improvements in cell output and viability were observed in TCR-transduced cultures 11 days after 3D ETN and CD8β selection, these parameters decreased during the subsequent CD8αβ differentiation phases (“D17, D21”). Figure 12 A-12C). After 11 days of differentiation on 3D ETN (1.08 x 10⁸ beads / mL (2x bead dose)), a 100-fold increase in CD8αβT cell output was observed per CD34+HSPC cell input.

[0415] Example 5: Gene expression and cell phenotype in generated cells

[0416] As described in Example 2, iPSC-derived CD34+ cells were differentiated into proT cells using 2D ETN for 18 days. Subsequently, T cell maturation was induced by culturing cells with 3D ETN beads, where bead concentrations were tested at 30-fold ranges (0.27 to 8.1 x 10⁷ beads / mL (0.05 x to 1.5 x bead dose)). Cells were also cultured with 2D ETN and SCT. Cell phenotype was assessed by flow cytometry after 7 days of cell culture. Figure 13A Higher bead concentrations (5.4 x 10⁷ beads / mL and 8.1 x 10⁷ beads / mL (1x and 1.5x bead doses)) produced more CD4-CD8+ (CD8SP) cells than lower bead concentrations (0.27 x 10⁷ beads / mL and 0.54 x 10⁷ beads / mL (0.05x and 0.1x bead doses)), while lower bead concentrations (0.27 x 10⁷ beads / mL and 0.54 x 10⁷ beads / mL (0.05x and 0.1x bead doses)) produced more CD4+CD8- (CD4 ISP) and CD4-CD8- (DN) cells. Figure 13A , Figure 1 B). After 3 days of cell culture, the expression of Notch signaling-related genes DTX1, TCF7, and BCL11B was concentration-responsive, with saturation occurring around a bead concentration of 5.4 x 10⁷ beads / mL (1x bead dose). Figure 13B The Notch-responsive gene GATA3 showed an inverse concentration response, with the highest expression at lower bead concentrations of 0.27 x 10⁷ beads / mL (0.05 x bead dose) and 0.54 x 10⁷ beads / mL (0.1 x bead dose). Figure 13B ).

[0417] Seven days after cell culture, cell phenotype was assessed using a 13-color flow cytometry group, and dimensionality reduction of the cube was performed using the uniform manifold approximation and projection (UMAP). Figure 13C Cell clustering was observed based on 3D ETN concentration. Figure 13CUnsupervised machine learning (FlowSOM, Van Gassen et al.) was used to identify expression patterns in a multicolor flow cytometry dataset. Six clusters were identified, with the expression of CD8α, CD8β, and CD4 used to describe the clusters as CD4+CD8+(DP), CD4+CD8-(CD4 ISP), CD4-CD8+(CD8SP), and CD4-CD8-(DN). Figure 13E CD4-CD8+ (CD8SP) clusters mapped with high 3D ETN bead concentration, CD4+CD8+ (DP) clusters mapped with 2D ETN, and CD4+CD8- (CD4 ISP) clusters mapped with SCT. Figure 13D The proportion of cells in each cluster depends on the DL4 concentration, with higher ETN bead concentrations (5.4 x 10⁷ beads / mL and 8.1 x 10⁷ beads / mL (1x and 1.5x bead doses)) containing more CD4-CD8+ (CD8SP) clusters, while lower bead concentrations (0.27 x 10⁷ beads / mL and 0.54 x 10⁷ beads / mL (0.05x and 0.1x bead doses) contain a higher proportion of fCD4+CD8- (CD4ISP) clusters. Figure 13F ), similar to Figure 13A The data shown.

[0418] Differentiation at a high ETN bead concentration (5.4 x 10⁷ beads / mL (1x bead dose)) was extended by 7 days. After 14 days of differentiation using a high ETN bead concentration (5.4 x 10⁷ beads / mL (1x bead dose)), over 86% of the cells were CD7+CD5+ cells, over 64% were CD4-CD8α+ cells, and 73% were CD8αβ+ cells. Figure 13G ).

[0419] Example 6: scRNA-Seq analysis of generated cells

[0420] In vivo T cell maturation stages were analyzed using single-cell RNA sequencing data (Park et al., 2020). CellRanger was used to generate trimmed, aligned, demultiplexed, and gene counts from FASTQ files, and the gene count matrix was normalized using Seurat (4.0.1, Satja et al., 2015). Double peaks were identified and removed, and dead cells were removed by filtering for mitochondrial reads greater than 5%. Cell type markers were adapted from Park et al., 2020. As indicated by the arrows, the data showed that T cell maturation progresses from early CD4-CD8- cells (DN(early)) to CD8αα cells, γδ T cells, CD8+ T cells, and CD4+ T cells (…). Figure 14AIn a separate analysis, data from Park et al. were analyzed, including NK-T cell phenotypes. Figure 14B ).

[0421] To examine Notch and TCR signaling during T cell development in vivo, gene set variation analysis (GSVA 1.40.1) was used. et al. (2013) used NOTCH (customized) separated from the dataset of Park et al. The gene sets of Notch (BioCarta) and TCR signaling were used to calculate single-sample GSEA (ssGSEA) scores at the single-cell level. Notch signaling was enriched in the early stages of T cell development in vivo, while TCR signaling was enriched in the later stages of T cell development in vivo. Figures 15A-15C ).

[0422] In further analysis, NOTCH (customized) data separated from the dataset of Park et al. was used. Group) and TCR signal transduction ( The gene set (group) was used to calculate the ssGEA score, and it also showed enrichment of Notch signaling in the early stages of T cell development in vivo and enrichment of TCR signaling in the later stages of T cell development in vivo. Figure 15D , 15E ).

[0423] Single-cell RNA-seq analysis was performed to compare iPSC-derived CD4-CD8+ cells generated with 3D ETN (5.4 x 10⁷ beads / mL (1x bead dose), as described in Example 2) 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). Referring to the Thymic Cell Atlas dataset, primary T cells were annotated as CD8+ memory T cells and CD8, and iPSC-derived CD4-CD8+ cells were annotated as resting double-positive (DP) and double-negative (DN) cells, CD8+ memory T cells, and γδ T cells (γ-δ T cells). Figure 16 A and 16B). Referring to the ProjecTILS dataset, primary T cells were annotated as CD8 effector memory cells and exhausted CD8 cells (CD8_Tex) with a low percentage of Th1 cells and CD8 naive-like cells ( ). Figure 17 A and 17B). A higher percentage of iPSC-derived CD4-CD8+ cells were annotated as CD8 naive cells compared to primary CD8 T cells, with CD8 (CD8_Tex) cells and Th1 cells having roughly the same percentage. Figure 17A and 17B).

[0424] Using single-sample GSEA, it was found that CD8α (CD8A) expression was higher in primary CD8αβT cells than in iPSC-derived cells, and the expression of CD3 subunits CD3γ (CD3G), CD3δ (CD3D), and CD3ε (CD3E) was lower. Figure 18 A). Compared with primary CD8αβT cells, a subset of genes involved in Notch signaling were expressed at higher levels in iPSC-derived CD4-CD8+ cells generated using 3D ETN, and the expression of genes involved in TCR signaling was decreased in iPSC-derived CD4-CD8+ cells. Figure 18 B).

[0425] Example 7: Generation of CD4-CD8+ cells from CAR-modified iPSC-derived HSPCs

[0426] To generate a T-cell progenitor cell population, iPSC-derived hematopoietic stem cell / progenitor cell populations containing CD34+ cells were cultured for 10 days in 24-well cell culture plates using 3D ETN beads (2.7 x 10⁷ beads / mL (0.5x bead dose)). Two cell lines were investigated: unmodified (NTX4A1, (4A1)) and CAR-modified (NTX4B3, (4B3)). On day 10, the cells exhibited a T-cell progenitor cell phenotype, as evidenced by CD5+CD7+ expression, possessing both CD34+ and CD34- populations. Figure 19 Unedited, 4A1; Figure 20 (CAR modified, 4B3).

[0427] T-cell progenitor cells were then cultured for 7 days in lymphocyte maturation medium (LMM, STEMCELL Technologies) and 3D ETN (1.08 x 10⁸ beads / mL (2x bead dose)), and then cultured for another 4 days with fresh 3D ETN beads of the same concentration after harvest. Two cell culture densities were investigated: 2 x 10⁶ cells / mL or 5 x 10⁵ cells / mL. After 11 days of culture at 1.08 x 10⁸ beads / mL (2x bead dose) (21 days for self-differentiated CD34+ cells), a CD8α enrichment step was also performed (EasySep™ Human CD8 Positive Selection Kit II, STEMCELL Technologies).

[0428] After culturing T cell progenitor cells with 3D ETN at a bead concentration of 1.08 x 10⁸ beads / mL (2x bead dose) for 7 days, CD4-CD8α+ (CD8SP) cells (30%-40%) were observed in both the unmodified and CAR cell lines. Figure 21A). The generation of CD8α+CD8β+ cells depends on cell density, as cells cultured at 2 x 10⁶ cells / mL have higher CD8β expression than cells cultured at 5 x 10⁵ cells / mL. Figure 21 B). For both cell lines and cell densities tested, cell viability was similar (40%-50%). Figure 21 C). For two cell densities in the 4B3 cell line, CAR expression was approximately 50% ( Figure 21 D). After culturing for 7 days with 3D ETN at a concentration of 1.08 x 10⁸ beads / mL (2x bead dose), cells were recultured under the same conditions – 5 x 10⁵ cells / mL or 2 x 10⁶ cells / mL – using 3D ETN beads at a concentration of 1.08 x 10⁸ beads / mL (2x bead dose). After reculturing for 4 days with 3D ETN at a concentration of 1.08 x 10⁸ beads / mL (2x bead dose) or a total of 11 days, cells were harvested and CD8α enriched. Under all conditions, the percentage of CD4-CD8α+ cells increased after CD8α enrichment ( Figure 22 A). Prior to enrichment, CD8β expression was higher in cells cultured at 2 x 10⁶ cells / mL than in cells cultured at 5 x 10⁵ cells / mL. CD8α enrichment increased CD8β expression levels in all samples, although higher cell densities (2 x 10⁶ cells / mL) retained higher CD8β expression after enrichment. Figure 22 B). For both unmodified and CAR cell lines, and before and after CD8α enrichment, higher cell viability was observed at lower cell densities (5 x 10⁵ cells / mL). Figure 22 C). CD8α enrichment improved cell viability, although this improvement was more pronounced in CAR cell lines than in unmodified cell lines. For both cell densities tested (5 x 10⁵ cells / mL and 2 x 10⁶ cells / mL), CAR expression was similar, and CD8α enrichment enhanced the percentage of CAR+ cells ( Figure 22 D).

[0429] For unmodified (4A1, Figure 23 A) and CAR(4B3, Figure 23B) Cell viability of both cell lines was monitored throughout the complete 21-day differentiation period (starting from CD34+ cells). At day 0, both cell lines had approximately 90% viable cells, and by day 10, during proT differentiation, viability decreased to approximately 40%. At days 14 and 15 (4 and 5 days of culture with 3D ETN at a concentration of 1.08 x 10⁸ beads / mL, respectively), 4A1 cell viability was density-dependent, with lower cell densities (5 x 10⁵ cells / mL) showing higher viability than higher cell densities (2 x 10⁶ cells / mL). At harvest on day 17 (7 days after culture with 3D ETN at a concentration of 1.08 x 10⁸ beads / mL), cell viability was similar between the two cell lines (4A1 and 4B3) and at cell densities (approximately 40% viability for 5 x 10⁵ cells / mL and 2 x 10⁶ cells / mL). On day 21 (after 11 days of culture with 3D ETN at a concentration of 1.08 x 10⁸ beads / mL), density-dependent activity was observed again, such as... Figure 22 As described by C.

[0430] Compared to a 4A1 rating on day 0 ( Figure 24 A) and 4B3 ( Figure 24 B) Cellular Expansion. In cells cultured at 5 x 10⁵ cells / mL, the relative expansion between day 10 (before culture with 3D ETN at a concentration of 1.08 x 10⁸ beads / mL) and day 17 (after 7 days of culture with 3D ETN at a concentration of 1.08 x 10⁸ beads / mL) was higher than that in cells cultured at 2 x 10⁶ cells / mL (7x vs. 2.5x for 4A1; 5.7x vs. 2.3x for 4B3). At day 21 (after 11 days of culture with 3D ETN at a concentration of 1.08 x 10⁸ beads / mL), cumulative expansion remained high for the lower cell culture density (5 x 10⁵ cells / mL).

[0431] Example 8: Generation and function of CD4-CD8+ cells

[0432] Reducing Notch signaling by decreasing the ETN dose specifies that ProT cells will differentiate into CD4+CD8+ (double-positive: DP) cells via the typical thymic developmental pathway (illustration). Figure 25 In vivo and in previously described methods, these require TCR rearrangement (B-selection) and CD3 / TCR to differentiate into cytotoxic CD4-CD8+ (single-positive: SP) T cells. Figure 25In contrast, sustained supraphysiological Notch signaling mediated by high ETN doses in vitro generated a functional CD8-SP T cell population from ProT cells after only 7 days in the absence of CD3 / TCR stimulation, bypassing the intermediate differentiation stage and the necessity of TCR / CD3 signaling. Figure 25 ).

[0433] iPSC-derived CD34+ HSPCs were cultured in serum-free suspension medium for 10 days with a series of ETN bead doses. T cell lineage induction from iPSC-derived HSPCs showed an ETN-mediated Notch signaling intensity dependence. On day -10 of culture, the generation of T cell progenitors (ProT; CD5+CD7+) and myeloid cells (CD33+CD7-) from iPSC-derived HSPCs was observed to be dose-responsive to ETN bead doses. Figure 26 A). ETN bead dose was normalized to tissue culture plate surface area. Representative flow cytometry plots of CD5, CD7, and CD33 expression are shown for 0.1 and 1 ETN bead doses. Figure 26 B).

[0434] On day -17, CD8-SP cell production was found to be in response to ETN dose in both CD19-CAR-engineered cells and CD19-CAR-deficient cell lines. Figure 27 A). High ETN doses produce CD8-SP cells, while low (physiological) levels produce CD8+CD4+(DP) cells. Representative flow cytometry plots of CD4+ and CD8a expression are shown for ETN bead doses of 0.1, 1, and 3. Figure 27 B, n = 3 technical replicates / cell line).

[0435] The dose-responsive gene expression changes to ETN on day +2 were found to be a quantitative surrogate for Notch signal intensity. (Using custom-designed...) Transcripts were measured. A machine learning workflow was used to predict the CD8-SP phenotype on day -17 based on Notch response gene expression on day -12, and Notch response genes associated with the CD8-SP phenotype were identified. Figure 28AIn the workflow, for the CD4-CD8+ phenotype after 7 days of culture, gene expression data from 2 days of culture and after flow cytometry were randomly divided into 50-50 training-test splits 1000 times. For each training-test split, a regularized linear regression model was fitted with 5-fold cross-validation to predict the CD4-CD8+ cell phenotype from the gene expression data. Model accuracy was measured by the coefficient of determination R² of the experimental data. The weighted linear mean of the regression coefficients, with weights corresponding to the frequencies of genes appearing in the model set, revealed the top 6 positively correlated genes and the top 6 negatively correlated genes with the CD8 SP phenotype (weighted mean as shown in the figure). Figure 28A As shown). All models were highly accurate (mean R² = 0.92 on the test data). Figure 28B DTX1 was identified as the most positive predictive feature and was closely associated with CD8-SP. Figure 28C Similar trends in ETN-responsive gene expression were observed in both cell lines. Figure 28D The average expression of the z-score normalized is shown (n=3).

[0436] Gene expression analysis was extended to other cell lines. As described above, a customized 43-gene assay was performed on T cell progenitor cell populations (ProT) 2 and 7 days after ETN administration. The transcriptional response to ETN was measured. Data were split into 1000 50-50 training-test splits, and a regularized linear model was trained and evaluated using 5-fold cross-validation. The average weighted regression coefficients of the first 12 predicted features are shown below. Figure 28E As shown. Positive predictors (NOTCH1, HES1, DTX1) are known Notch signaling response elements, while negative predictors (RAG2, SPN) are known markers of typical DP stage differentiation. Using a machine learning workflow trained on gene expression at day -12, the prediction of the CD8αSP phenotype at day -17 (7 days after ETN administration to the T cell progenitor cell population) showed good accuracy (85% cross-validation), independent of cell line ( Figure 28F NOTCH1 was identified as the most positive predictive trait, and its value increased with increasing bead dosage. Figure 28G For TRAC-deficient (TRAC- / -) and TRAC-deficient CD19-CAR-engineered (TRAC- / -CD19-CAR+) cell lines, similar trends in ETN-responsive gene expression were observed. Figure 28H Following culture with low (0.1x bead dose) and high (3x bead dose) ETN doses, the more comprehensive 770 gene was used. The transcriptional profiles of WT, TRAC knockout, and CAR-engineered / TRAC-deficient cell lines were assessed. Variance decomposition analysis revealed ETN bead dosage and daily effects: lineage-effect interactions accounted for the majority of the transcriptional variance. Figure 28I In other words, the three cell lines differentiated along different trajectories but responded similarly to Notch exposure. Differential expression pathways were plotted in CAR-engineered TRAC-deficient cell lines using bead dosage and culture time (ssGSEA score). Figure 28J The subset of pathways shown was filtered based on a statistical cutoff of Padj < 0.05 (rank-sum test). High ETN doses are involved in transcriptional programs associated with T cell differentiation and functional activity. It was noted that multiple pathways associated with T cell differentiation and function (interferon signaling, JAK-STAT, cytotoxicity) were induced by enhanced Notch signaling, and this enhancement increased with increasing culture time. Figure 28J ).

[0437] Single-cell transcriptomic characterization of iPSC-derived CD8-SP cells was performed compared to CD8+ T cells derived from peripheral blood and umbilical cord blood. UMAP analysis of single-cell transcriptomes of iPSC-derived T cells, peripheral blood (PBMCs), and umbilical cord blood (CB) CD8+ T cells identified these cell populations as transcriptionally distinct. Figure 29A An algorithm derived from ProjecTIL (Haradhvala et al., 2022) was used to classify cells and identify iPSC-derived cells as primarily naive T cells, similar to umbilical cord blood-derived T cells. Figure 29B ). Selective expression of typical markers of T cell, Notch response genes, innate lymphocytes (ILC), T cell activation and exhaustion transcripts. Figure 29C Single-sample GSEA (ssGSEA) was applied at single-cell resolution to calculate the mean for each cluster, represented by hierarchical clustering z-scores. Differential enrichment of selected gene signatures was found in each cell cluster. Different signal transduction spectra were observed in iPSC-T cell clusters, as labeled and annotated according to UMAP projections. Figure 29D ). 1473 gene signatures were found to be variable across cell clusters (for these signatures, the coefficient of variation (CV), i.e., the ratio of the standard deviation over the cluster to the mean ssGSEA score, was >0.1). Figure 29E Three cells in the iPSC-T cell cluster shared signal transduction similarities with PBMC cells, and one cell shared signal transduction similarities with CB cells. Figure 29E ).

[0438] Furthermore, for iPSC-derived T cells that have undergone differentiation (CD34+, ProT, CD8+SP stages, and after CD19- antigen stimulation), and those derived from... Activated (CD3 / CD28 stimulated) primary lymphocytes (CD4+ T cells, CD8+ T cells, CD56+ NK cells, and T cells) were used to generate a single-cell transcriptome (scRNASeq) UMAP map. Cell* gene expression matrices from single-cell RNASeq sequencing data were calculated using CellRanger (Zheng et al., 2017). Cells smaller than 500 UMI and with 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 applied a regularized negative binomial regression model to adjust for confounding effects of sequencing depth at the cellular level. Further scaling and UMAP (uniform manifold approximation with projection) dimensionality reduction were performed using Seurat (Satja et al., 2015). The iPSC-derived cell population was observed to be transcriptionally unique, but a subset of CD19-stimulated cells was most similar to activated primary cells. Figure 29F Bubble graphs are generated for selected genes associated with blood progenitor cells, T cell lineage commitment, typical T cell function, innate lymphocytes, cytotoxic T cells, exhausted T cells, and Notch response elements. In addition to internally generated samples, autologous CAR-T products are also included. and Single-cell transcriptomes were used as controls (Bai et al., 2022 and Haradhvala et al., 2022). iPSC-derived CD8+ cells acquired typical T cell expression patterns, with enhanced CD3 expression, restricted expression of ILC or depletion markers, and extremely enhanced Notch signaling.

[0439] Single-sample gene set enrichment analysis (ssGSEA) was performed using the Gene Set Variation Analysis (GSVA) ​​package. (Kirouac et al., 2013) to compute enrichment scores of selected gene signatures at single-cell resolution from an internal database of approximately 2500 compilation pathways (Kirouac et al., 2023). Figure 29G Besides superNotch signaling, iPSC-derived CD8+ cells express pathways associated with T cell functional activity (JAK-STAT, IFNG) and TCR signaling, despite being TRAC-deficient cells. CD19 stimulation upregulates transcriptional signatures associated with CAR antigen response and exhaustion (although related to…). and (Compared to limited), this indicates a functional T cell response.

[0440] UMAPs were annotated based on transcriptional similarity to developing thymocytes (DN-proliferating, DN-early, DN-resting, CD8+T, NKT, γ-δT (GDT), and DP-proliferating, as well as Treg; Park et al., 2020) focusing on the single-cell transcriptomes of iPSC-CD8+ cells, antigen-stimulated iPSC-CD8+ cells, and activated T cells. iPSC-derived cells were annotated as a mixture of DN, CD8+T, and NKT, and transformed into NKT-like cells upon antigen stimulation (…). Figure 29I Pseudo-time analysis was performed using Slingshot (Street et al., 2018). CD8+ T cells emerged directly from the resting-DN population and then transformed into NKT-like cells. Figure 29J The expression of transcriptional pathways predicting cell population order (ssGSEA) was achieved using a sequence regression model shown at single-cell resolution. Figure 29K Cell cycle-related pathways gradually give rise to pathways associated with T cell function. Figure 29K ).

[0441] Target-mediated cell killing, proliferation, and cytokine secretion were evaluated in a series of in vitro assays of iPSC-derived CD19-CAR-expressing CD8SP cells compared to primary adult donor-derived CAR-T cells. Target cell (CD19-expressing A549 cells) kinetics were assessed in four rounds of in vitro killing assays, with primary CAR-T and iPSC-CD8 cells demonstrating cell killing activity in each round. Figure 30 A) Use Live-cell imaging was used to measure cell density, with effector T cells (iPSC-derived or primary) added at an effector cell to target cell (E:T) ratio of 2:1. Figure 30 A, mean ± standard deviation, n = 3). The in vitro cytotoxicity of primary CAR-T and iPSC-CART was comparable to that of effector cell expansion. Figure 30 B, n = 2 replicates). Cumulative cytotoxicity was reported as the sum of the relative changes in the area under the curve (AUC) from the target cell-only control at each stimulation (not shown). iPSC-CART secreted inflammatory cytokines at levels comparable to primary CART when co-cultured with antigen-expressing target cells. Figure 30 C). iPSC-derived CD8SP cells expressing CD19-CAR are capable of in vitro continuous target-mediated cell killing, proliferation, and cytokine secretion comparable to primary CAR.

[0442] Example 9: Generation of CD4-CD8+ cells from STR-derived HPC

[0443] Analyze the lymphoid potential of iPSC-derived CD34+ cells generated in the STR system. Figure 31A This paper presents an overview of a 3D engineered thymic niche (ETN) platform for scalable T cell production using iPSC-derived CD34+ HPCs. The ETN is shown to be suitable for use in STRs 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, with HPCs generated in microplate culture serving as a reference. HPCs were cultured with ETN at 2.70 x 10⁷ beads / mL (0.5 x bead dose) for 10 days, followed by 6 days at 1.35 x 10⁸ beads / mL (2.5 x bead dose), and then for the next 5 days at 1.89 x 10⁸ beads / mL (3.5 x bead dose). STR-derived HPCs were equivalent to those derived from microplate culture and generated 58.7 ± 3.7% CD8+ cells, compared to 59.99% CD8+ positive cells in the study platform HPCs. Figure 31B The expression pattern of CD8αβ+ T cells was equivalent across all three STR runs (mean ± SEM, 3 independent STR runs, n = 6 bioreactors). Figure 31C ).

[0444] Example 10: Generation and function of CD4-CD8+ cells in STR culture

[0445] iPSC-derived proT cells were cultured in microplates for 7 days at ETN doses ranging from 0.1x to 3x (5.40 x 10⁶ beads / mL to 1.62 x 10⁸ beads / mL). Cell phenotypes were assessed by flow cytometry. Higher bead doses produced more CD8 SP cells than lower bead doses, while lower bead doses produced more CD4 ISPs and double-positive cells (DPs). Figure 32 A) Through Expression of CD8A, IL7R, CD4, and BCL11A measured over 7 days at ETN doses 2 and 3 showed that CD8A and ILR7 expression increased over time, while CD4 and BCL11A expression decreased over time. Figure 32 B). It was found that T cell phenotype is regulated by ETN bead dosage.

[0446] iPSC-derived CD34+ cells were differentiated into proT cells using ETN at a dose of 2.70 x 10⁷ beads / mL (0.5x bead) in microplates and STRs for 10 days. Two (2) CD34+ cell banks were seeded in duplicate in a bioreactor (Eppendorf) and 24-well plates at 1 x 10⁴ and 5 x 10⁴ cells / mL. For both cell banks, the percentage of CD5+CD7+ ProT cells at D10 was equivalent in both the STR and microplate, with a higher percentage of proT cells at the 5 x 10⁴ cells / mL seeding condition. Figure 33 A). For STR culture, each input CD34+ cell produces a higher number of ProT cells (A). Figure 33 A). STR culture using ETN generates T cell progenitor cells at an equivalent ratio to those cultured in 24-well plates, with high reproducibility.

[0447] Eight (8) bioreactors were seeded with a CD34+ cell bank at 5 x 10⁴ cells / mL. Differentiation and expansion were reproducible, with 28% ± 4% CD5+CD7+ cells at D10 and a yield of 23 ± 6 proT cells per input CD34+ cell bank (mean ± 1 SD). Figure 33 B).

[0448] Based on optimal conditions in microplate culture ( Figure 32 ), to generate proT cells from STR ( Figure 33 Reseeded at 1 x 10⁶ cells / mL and cultured with ETN at 1.35 x 10⁸ beads / mL (2.5x relative bead dose) for 11 days. In all eight bioreactors, the maximum CD8⁺ cell yield occurred 8 days after maturation, with a mean percentage of CD8⁺ of 41% ± 1.5%. Figure 34 A). The number of CD8+ cells generated by each input CD34+ cell was 35±5 (A). Figure 33 A). For microplate culture, this expression pattern is similar to... Figure 32 The expression patterns observed were consistent across all eight STRs. The proportion of CD8αβ+ cells on day 8 of maturity was uniform, at 28% ± 1%. Figure 34 B). In microplate culture, the proportion of CD8αβ+ cells has been observed to gradually increase until day 14 of maturity.

[0449] Gene expression dynamics (by) The assessment revealed similar trends in CD8A, IL7R, CD4, and BCL11A expression between STR and microplate culture. Figure 35 A, Figure 32Gene expression dynamics were quantified by the sign of regression coefficients over time and the logarithm of the Benjamini / Hochberg adjusted p-values ​​for linear models fitted to each gene expression. For 24 genomes associated with leukocyte development, a significant correlation was found between micropore and STR gene expression dynamics (Pearson's r = 0.71, p < 0.001). Figure 35 B).

[0450] Functional comparisons were performed between iPSC-derived CD8+ CAR-T cells generated from STR culture and donor peripheral blood CD8+ CAR-T cells (primary CD8+ CAR-T cells). (Sartorius) live-cell imaging assays were developed using GFP-expressing CD19+ cells as target cells (n = 3 technical replicates) to measure cytotoxic activity via a sequential restimulation assay. 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 reduction of GFP surface area. iPSC-derived CD8+ CAR-T cells showed comparable activity to primary CD8+ CAR-T cells after 4 rounds of antigen exposure. Figure 36A Cell expansion was calculated by counting at the end of each round of target exposure. iPSC-derived CAR-T cells proliferated 57,000-fold over 4 rounds of antigen exposure at levels comparable to primary CD8+ CAR-T cells. Figure 36B ).

[0451] The secretion of 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 ).

[0452] 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 performance, were found to be enriched in iPSC-derived CD8+ CAR-T cells at baseline and upon chronic antigen exposure. Figure 36D The expression of exhaustion markers was also assessed in both cell populations. Compared to primary CD8+ CAR-T cells, iPSC-derived CD8+ CAR-T cells showed lower co-expression of several exhaustion markers (PD1, TIM3, LAG3, TIGIT, and CD39), which have been reported as hallmarks of T cell exhaustion. Figure 36E ).

[0453] iPSC-derived CD8+ CAR-T cells generated from STR culture were further enriched by CD8α or CD8β selection and analyzed by flow cytometry. After CD8α enrichment, both CD8αα+ and CD8αβ+ cells were present. Figure 37 A). Lower CD8α levels were detected because the enrichment process blocked the CD8α epitope. Most CD8β-enriched cells were CD8αβ+ cells. Figure 37 B), and both CD8α-enriched and CD8β-enriched cells lacked surface CD3 expression. Figure 37 A, 37B).

[0454] Example 11: Generation and function of exogenous TCR+ cells

[0455] Two independent iPSC lines (“172” and “174”) were generated at the TRAC locus using site-specific integration (SSI) of the MAGE-A4 TCR 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. The CD34+ cells were then seeded at 5 x 10⁴ cells / mL in wells and differentiated into T cell progenitors for 10 days, with the addition of 2.70 x 10⁷ beads / mL (0.5X bead dose) of 3D ETN on day 1, and half-culture medium replacement on days 4 and 7. Cells were then reseeded at 1.5 x 10⁶ cells / mL and cultured for another 11 days (21 days in total from CD34+ phase) with 3D ETN at 1.62 x 10⁸ beads / mL (3X bead dose). The medium was replaced at days 12, 14, 16, and 19, and on day 17, cells were reseeded at 1.5 x 10⁶ cells / mL with 3D ETN at 1.62 x 10⁸ beads / mL (3X bead dose). On day 21 (from CD34+ phase), cells were harvested and CD8 was enriched using the EasySep™ Human CD8 Positive Enrichment II Kit (STEMCELL Technologies).

[0456] For all cell lines, decreased viability was observed at the end of differentiation, and this was improved upon CD8α enrichment. Figure 38 A, 38B). At the end of differentiation, one of the TCR-modified cell lines ("174") had the highest cumulative fold increase. Figure 38 C).

[0457] As determined by flow cytometry, CD5+CD7+ cells were observed on day 10 of differentiation in all cell lines, and the expression of CD5 and CD7 further increased from day 17 onwards. Figure 39A). CD8 expression was observed on day 17, and the CD8+ population increased after CD8 enrichment on day 21. Figure 39 B). After enrichment, both CD8αα+ and CD8αβ+ cells were present in all cell lines, with a larger proportion of CD8αβ+ cells observed in the TCR SSI lines (“172”, “174”). Figure 39 C). In all cell lines, a large proportion of CD8αβ+ cells are CD4-CD8+ cells ( Figure 40 ).

[0458] For the TCR SSI lineage, cells maintain high expression of MAGE-A4 TCR during differentiation. Figure 41 A, 41B).

[0459] Cell characteristics after differentiation using 3D ETN and CD8 enrichment are shown in Table 9 below.

[0460]

[0461] Next, the in vitro function of the TCR-iPSC-derived CD4-CD8+ cells was assessed using a serial restimulation assay targeting A375 human malignant melanoma cells. Both the TCR-modified iPSC-derived CD8+ cells and the TCR-modified primary CD8+ cells proliferated throughout the assay, with the "174" line showing increased proliferation compared to the "172" line. Figure 42 A, 42B). Following the first round of stimulation, the two TCR-modified iPSC-derived CD8+ cell lines exhibited similar cytotoxicity across multiple effector cell:target cell ratios. Figure 43 In two independent experiments, TCR-modified iPSC-derived CD8+ cells functioned through four rounds of continuous killing, similar to the TCR-modified primary CD8+ cell control. Figure 44 A, 44B and Figure 44 C, 44D).

[0462] Cell specificity was analyzed by comparing cytotoxicity against wild-type A375 target cells (A375 WT, presenting the target antigen), β2-microglobulin (B2M) knockout A375 cells (A375 B2M KO, lacking antigen presentation), MC-38 mouse colon adenocarcinoma cells, and human umbilical vein endothelial cells (HUVEC). MC-38 and HUVEC cells were treated with Cytolight Orange. The fluorescent label was lost over time due to cell division, whereas A375WT and A375 B2M KO cells were labeled with Nuclight Green. The marker is used for stable expression. Upon initial activation, TCR-modified iPSC-derived CD8+ cells exhibit specificity for the A375 target. Figure 45 ).

[0463] Example 12: Intracellular TCRβ Analysis

[0464] As described in Example 11 above, 3D ETN was used to differentiate CAR-modified TRAC- / -iPSC-derived cells. Cell analysis was performed on days 0 (CD34+ stage), 10 (T cell progenitor stage), 17, 21, and 24 targeting intracellular CD3 and intracellular TCRβ (TCR VβF1). By day 10, a high proportion of cells were positive for intracellular CD3 (…). Figure 46A ), and intracellular CD3 ( ) continued to be detected until day 24. Figure 46A , 46B In contrast, intracellular TCRβ was not detected at any time point. Figure 46A , 46B Cell viability decreased over time, and the proportion of CD4-CD8α+ cells increased. Figure 46C , Figure 47 The CD8α+β+ population also increased over time. Figure 47 A small number of CD3+TCRγδ- and CD3+TCRγδ+ cells were observed on days 21 and 24. Figure 48 ).

[0465] Example 13: Generation and function of exogenous TCR+ and CAR+ cells

[0466] Four cell lines were generated and compared: iPSC-derived CD34+ cells (“unmodified”), iPSC-derived CD34+ cells transduced with TCR on day 7 (“unmodified TCR-transduced”), TRAC- / -CD19 CAR SSI iPSC-derived CD34+ cells (“CAR SSI”), and TCR SSI iPSC-derived CD34+ cells (“TCR SSI”). Cells were differentiated with 3D ETN as described in Example 11 above. Over time, the viability of the different lineages was comparable. Figure 49A Furthermore, the expansion factor of the TCR SSI series is higher. Figure 49B ).

[0467] CAR or TCR expression was analyzed during differentiation. For different cell lines, TCRαβ expression levels on day 10 ranged from 20% to 30%. Figure 49C ).

[0468] Cells were further characterized by flow cytometry to target the expression of CD5, CD7, CD56, CD4, CD8α, and CD8β. Compared with their untransduced counterparts, TCR-transduced cells showed a higher percentage of CD5+CD7+ and a lower percentage of CD56+CD7+ populations during late differentiation. Figure 50 A, 50B). TCR-transduced cells showed higher CD4 expression on day 21, resulting in a lower percentage of CD4-CD8α+ cells compared to the untransduced counterpart. Figure 51 A). However, compared to CD8αα+ cells, the proportion of CD8α+CD8β+ cells was higher in this fraction ( Figure 51 B, subgated on CD4-CD8α+. Furthermore, compared to untransduced controls, higher expression of CD8α+CD8β+ was observed in the total cell population at day 21 in TCR-transduced cells. Figure 52 A). However, the composition of this subpopulation is attributed to both CD4-CD8+ cells (CD8SP) and CD4+CD8+ cells (DP). Figure 52 B, subgating on CD8α+CD8β+).

[0469] Next, in an in vitro serial restimulation assay, cells enriched with CAR-modified CD8α (“CAR SSI”) were cryopreserved before exposure to target cells. Upon thawing, the cells retained the expression of key markers (…). Figure 53 In both the first and second rounds of stimulation, CAR SSI cells expanded in the presence of Raji target cells. Figure 54 A). However, a lack of CD8β+ expression was observed ( Figure 54 B). Cells maintained cytotoxicity through multiple rounds of stimulation, among which activity against CD19- / - target cells was observed. Figure 55 C).

[0470] In subsequent in vitro serial restimulation assays, CAR SSI cells expanded in three rounds of target cell co-culture in the presence of A549 target cells. Figure 55 A, 55B). As reported above, loss of CD8β+ expression was observed during assays using A549 target cells in co-culture with Raji target cells. Figure 55 C). Cytokine secretion in iPSC-derived CAR SSI cells is antigen-dependent, with greater cytokine secretion observed in co-culture with A549 target cells compared to Raji target cells. Figure 56Following the first round of stimulation with Raji or A549 target cells, the viability of iPSC-derived CARSSI cells decreased and continued to decline in co-culture with Raji cells, remaining stable in subsequent rounds of stimulation with A549 target cells. Figure 57 ).

[0471] Example 14: In vivo delivery of CAR+ cells

[0472] As described above, cells expressing CD19 CAR were generated from iPSCs using 3D ETN. Cells were characterized at harvest and after CD8 enrichment (Table 10). Figure 58 After enrichment).

[0473]

[0474] In vivo function of CAR+iPSC-derived CD8 cells was evaluated in a Raji tumor implantation model. Raji cells were delivered intravenously (iv) to immunocompromised NOD-scid IL2Rgnull (NSG) mice and exogenous cytokine support was administered intraperitoneally (ip) three times weekly for four weeks. Two days after tumor implantation, CAR+iPSC-derived CD8+ cells (3 doses of 1x107 cells, 3 days apart (Q3Dx3), iv) or primary CAR+CD8+ T cells (single dose of 3x106 cells, iv) were administered. Repeated administration of CAR+iPSC-derived CD8+ cells significantly delayed Raji tumor growth and thus prolonged median survival compared to untreated controls. Figure 59 A, 59B). Monitoring changes in animal body weight as a surrogate indicator of toxicity ( Figure 59 C). In peripheral blood, a lower number of CAR+iPSC-derived CD8+ cells were detected compared to primary CAR+CD8+ T cells. Figure 60 A). On day 8 post-infusion, CAR+iPSC-derived CD8+ cells were highly CD3-CAR+ fused ( Figure 60 B).

[0475] In another study, the in vivo function of CAR+iPSC-derived CD8 cells was evaluated in a metastatic A549-CD19 tumor model. A459-CD19 tumor cells were intravenously delivered to NSG mice, and exogenous cytokine support was provided via intraperitoneal (ip) injection three times weekly for four weeks. Two days after tumor implantation, CAR+iPSC-derived CD8+ cells (3 doses of 1x10⁷ cells, 3 days apart (Q3Dx3), iv) or primary CAR+CD8+ T cells (a single dose of 3x10⁶ cells, iv) were administered. Repeated administration of CAR+iPSC-derived CD8+ cells maintained complete remission against A549-CD19+ metastatic tumors comparable to that achieved with primary CAR+CD8+ T cells. Figures 61A-61C Monitoring changes in animal body weight was used as a surrogate indicator of toxicity, where comparable weight gain was observed between groups. Figure 61D In peripheral blood, a lower number of CAR+iPSC-derived CD8+ cells were detected compared to primary CAR+CD8+ T cells. Figure 61D On day 15 post-infusion, compared to pre-infusion analysis, retrieved CAR+iPSC-derived CD8+ cells were highly CD3+ oxidized. Figure 62 A). In the CAR+ population, iPSC-derived CD8+ cells were highly CD8αβ+-mediated from day 15 onwards. Figure 62 B). Furthermore, CAR+iPSC-derived CD8+ cells retrieved on day 15 post-infusion secreted inflammatory cytokines and cytolytic molecules (B). Figure 63 ).

[0476] The A549-CD19 study was extended in due course to investigate the ability of CAR+iPSC-derived CD8+ cells to control tumor rechallenge. On day 36, 2.5 x 10⁵ A549-CD19 tumor cells were IV-delivered to animals previously treated with CAR+iPSC-derived CD8+ cells. Cytokine support was restarted until the end of the study on day 50. The study investigated the animals' tolerance to rechallenge of A549-CD19+ tumors, with extended complete survival and sustained weight gain. Figure 64 A, 64B). Sustained CAR+iPSC-derived CD8+ cells maintain tumor growth inhibition ( Figure 65 A-65C) and was detected at low levels in peripheral blood after re-challenge. Figure 65 D). On day 50, CAR+iPSC-derived CD8+ cells were also detected in the lungs, and these cells were highly CD3+ oxidized. Figure 66 A, 66B). During the rechallenge, CAR+iPSC-derived CD8+ cells retrieved from peripheral blood remained highly CD8αβ+ mediated. Figure 66C).

[0477] Example 15: Generation and function of exogenous TCR+ and CAR+ cells in STRs

[0478] CD34+ cells derived from CAR-modified (CD19 CAR) and TCR-modified (MAGE-A4 TCR) iPSCs were differentiated using 3D ETN in a stirred tank reactor. Cells were initially seeded on day 0 and cultured from day 1 with 2.7 x 10⁷ beads / mL (0.5 x bead dose) of 3D ETN to generate T cell progenitors. Cells were reseeded on day 10 with 1.35 x 10⁸ beads / mL (2.5 x relative bead dose) of 3D ETN, and on day 16 with 1.89 x 10⁸ beads / mL (3.5 x bead dose), and then cultured for another 5 days (total 21 days). Compared to TCR-modified cells, CAR-modified cells showed lower viability on day 21. Figure 67 A), and for TCR-modified cells, a greater cell density was observed ( Figure 67 B). On day 21, the TCR-modified cell line also had higher purity of CD8αβ+ cells compared to the CAR-modified cells. Figure 68 A, 68B).

[0479] In vitro serial restimulation assays were used to study TCR-modified cells generated in STR and well plate (WP) culture systems using 3D ETN. Similar cytotoxicity and fold expansion were observed in both groups. Figure 69 A, 69B). Furthermore, for both STR-generated and WP-generated cells, different E:T ratios were observed ( Figure 69 C) and antigen specificity ( Figure 69 Comparable features under D).

[0480] It is anticipated that T cell lineage populations derived from T cell progenitor cells cultured using the methods provided herein with 3D ETN can be included in the pharmaceutical composition.

[0481] It is also anticipated that T cell lineage populations derived from T cell progenitor cells cultured using the methods provided herein with 3D ETN can be used to treat a subject's disease or condition. "Treatment" refers to the administration of an effective amount of cells, as provided herein, to a subject under conditions suitable for increasing the subject's T cell count, which can result in preventative, suppressive, and / or therapeutic treatment of a medical condition. "Effective amount" refers to a therapeutically effective amount, such as the amount of cells sufficient, for example, to achieve the intended purpose (e.g., treatment), upon administration to the subject. The amount may vary from subject to subject and may depend on one or more factors, such as, for example, the subject's sex, age, weight, the subject's health history, and / or the underlying cause of the condition to be prevented, suppressed, and / or treated.

[0482] For example, subjects with oncology or autoimmune diseases, symptoms or conditions may benefit from administration of T-cell lineage populations as described in this article.

[0483] Although this disclosure has been described with reference to certain specific embodiments, various modifications thereto will be apparent to those skilled in the art. Any examples provided herein are included for illustrative purposes only and are not intended to limit the disclosure in any way. Any drawings provided herein are for illustrating various aspects of the disclosure only and are not intended to be drawn to scale or to limit the disclosure in any way. The scope of the appended claims should not be limited to the preferred embodiments set forth in the foregoing description, but should be given the broadest interpretation consistent with the entirety of this specification. All disclosures of the technology described herein are incorporated herein by reference in their entirety.

[0484] Referenced files

[0485] 1. Andreatta et al. Interpretation of T cell states from single-cell transcriptomics data using reference atlases. Nat. Commun. 12, 2965 (2021). Doi:10.1038 / s41467-021-23324-4

[0486] 2. Bai, Z. et al. Single-cell antigen-specific landscape of CAR T infusionproduct identifies determinants of CD19-positive relapse in patients with ALL. Sci Adv 8, (2022). Doi: 10.1126 / sciadv.abj2820

[0487] 3. Baulu et al. TCR-engineered T cell therapy in solid tumors: State of the art and perspectives. Science Advances eadf3700 (2023). Doi: 10.1126 / sciadv.adf3700

[0488] 4. Blassberg. Genome Editing of Pluripotent Stem Cells for Adoptive and Regenerative Cell Therapies. GEN Biotechnology. 2022. 77 - 90. Doi: 10.1089 / genbio.2021.0010

[0489] 5. Drougkas et al. Comprehensive clinical evaluation of CAR-T cell immunotherapy for solid tumors: a path moving forward or a dead end?J Cancer Res Clin Oncol 149:2709 - 2734 (2023). Doi: 10.1007 / s00432-022-04547-4

[0490] 6. Guha et al. Assessing the Future of Solid Tumor Immunotherapy. Biomedicines 10:655 (2022). Doi: 10.3390 / biomedicines10030655

[0491] 7. Hafemeister, C., Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol 20, 296 (2019). Doi: 10.1186 / s13059-019-1874-1

[0492] 8. S., Castelo, R. & Guinney, J. GSVA: gene set variation analysis for microarray and RNA-Seq data. BMC Bioinformatics 14, 7 (2013). Doi: 10.1186 / 1471-2105-14-7

[0493] 9. Haradhvala, N. J. et al. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nat Med 1 - 12 (2022). Doi: 10.1038 / s41591-022-01959-0

[0494] 10. Iriguchi et al. A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nat. Commun. 12, 430 (2021). Doi: 10.1038 / s41467-020-20658-3

[0495] 11. Kirouac, D. C. et al. Deconvolution of clinical variance in CAR-T cell pharmacology and response. Nat Biotechnol 1 – 12 (2023). Doi: 10.1038 / s41587-023-01687-x.

[0496] 12. Michaels et al. DLL4 and VCAM1 enhance the emergence of T cell–competent hematopoietic progenitors from human pluripotent stem cells. Science Advances 2022 8(34) doi:10.1126 / sciadv.abn5522

[0497] 13. Montel-Hagen et al., Generation of artificial thymic organoids from human and murine hematopoietic stem and progenitor cells. Curr. Protoc. 2022, 2(4): e403 doi:10.1002 / cpz1.403

[0498] 14. Park et al., A cell atlas of human thymic development defines T cell repertoire formation. Science 2020, 367(6480): 1 - 11. Doi:10.1126 / science.aay3224

[0499] 15. Qu et al., Tumor buster - where will the CAR-T cell therapy ‘missile’ go? Molecular Cancer 21:201(2022). Doi:10.1186 / s12943-022-01669-8

[0500] 16. Satija, R. et al., Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015, 33, 495–502. Doi:10.1038 / nbt.3192

[0501] 17. Shukla et al., Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1. Nat. Methods. 2017, 14(5): 531 - 538. Doi:10.1038 / nmeth.4258

[0502] 18. Sjoukje et al. Generation of T-cell-receptor-negative CD8αβ-positive CAR T cells from T-cell-derived induced pluripotent stem cells. Nature Biomedical Engineering 1-14 (2022). Doi: 10.1038 / s41551-022-00915-0

[0503] 19. Street, K. et al. Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics 19, 477 (2018). Doi: 10.1186 / s12864-018-4772-0

[0504] 20. Sun et al. Evolution of CD8+ T Cell Receptor (TCR) Engineered Therapies for the Treatment of Cancer. Cells 10:2379 (2021) Doi: 10.3390 / cells10092379

[0505] 21. Trotman-Grant et al. DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell free system. Nat. Commun. 2021 12(5023)1-11. Doi: 10.1038 / s41467

[0506] 22. Van Gassen et al. FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data. Cytometry Part A 87.7 2015 87(7):636-645. Doi: 10.1002 / cyto.a.22625

[0507] 23. Want et al., T Cell Based Immunotherapy for Cancer: Approaches and Strategies. Vaccines 11:835 (2023). Doi: 10.3390 / vaccines11040835

[0508] 24. Weber et al., The Emerging Landscape of Immune Cell Therapies. Cell. 2020 181(1):46 - 62. Doi: 10.1016 / j.cell.2020.03.001

[0509] 25. Zheng et al., Massively parallel digital transcriptional profiling of single cells. Nat Commun. 2017 8(14049). Doi: 10.1038 / ncomms14049

[0510] 26. Zuniga - Pflucker et al., WO 2019 / 157597

Claims

1. A method for generating a population of T cell lineage cells, the method comprising: a) Provide a population of T cell progenitor cells; as well as b) Culture the T cell progenitor cells in the presence of surface-bound Notch signaling ligands. The Notch signaling ligand is provided at a culture volume of at least 7 cm² / mL (7 cm³). 2 On a surface area of ​​( / mL).

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

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

4. The method according to any one of claims 1 to 3, wherein the Notch signaling ligand is provided at a distance of 7 to 56 cm. 2 / mL, 7.8 to 55.2cm 2 / mL or 15.7 to 55.2cm 2 / mL of surface area.

5. The method according to any one of claims 1 to 4, wherein the Notch signaling 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. The method of claim 6, wherein the one or more beads are made 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 according to any one of claims 6 to 8, wherein the Notch signaling ligand is covalently conjugated to the one or more beads.

10. The method according to any one of claims 1 to 9, wherein the Notch signaling ligand comprises Notch ligand δ-sample 4 (DL4) or a variant thereof.

11. The method of claim 10, wherein the concentration of DL4 is 7.89 x 10⁻⁶. 10 Up to 1.66 x 10 13 molecule / mL culture volume.

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

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

14. The method of claim 12, wherein the concentration of DL4 is 7.89 x 10⁻⁶. 10 Up to 1.66 x 10 13 The concentration of VCAM-1 was 7.89 x 10⁻⁶ molecules / mL culture volume. 10 Up to 1.66 x 10 13 molecule / mL culture volume.

15. The method according to claim 11 or claim 14, wherein the cell density is 5 x 10⁻⁶. 5 Up to 2x 10 6 Cells / mL.

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

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

18. The method of claim 16, wherein the T cell progenitor cells are recultured at least once during or after culturing the T cell progenitor cells for at least 3 days, at least 7 days, at least 11 days, or at least 14 days, wherein the reculture 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+ cell is a T cell receptor (TCR)-cell, a surface CD3-negative (sCD3-) cell, or a TCR- / sCD3-cell.

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

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

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

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

25. A population of CD4-CD8+ cells, said population being prepared by the method according to any one of claims 1 to 24.

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

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

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

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

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

31. The pharmaceutical composition of claim 30, wherein the CD4-CD8+ cells are generated by T cell progenitor cells in a culture volume of at least 7 cm² / mL (7 cm³ / mL). 2 Notch signaling ligands are derived in vitro by being cultured on a surface area of ​​( / mL) of surface-bound ligands.

32. The pharmaceutical composition of claim 31, wherein the T cell progenitor 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 exogenous T cell receptors (TCRs).

35. A method for treating a disease or symptom in a subject, the method comprising: a) Culture a cell population containing T cell progenitor cells in the presence of a surface-bound Notch signaling ligand, wherein the Notch signaling ligand is provided in a culture volume of at least 7 cm² / mL. 2 On a surface area of ​​( / mL), T cell lineage populations are generated; and b) Administer an effective amount of the T cell lineage population to the subject in need.

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

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

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

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

41. The method according to any one of claims 35 to 40, wherein the Notch signaling ligand comprises Notch ligand δ-sample 4 (DL4) or a variant thereof.

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

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

44. Use of T-cell lineage populations in the manufacture of medicaments for treating diseases or symptoms, wherein the T-cell lineage populations are generated by a method comprising: a) Culture a cell population containing T cell progenitor cells in the presence of surface-bound Notch signaling ligands. The surface-bound Notch signaling ligand provided at a culture volume of at least 7 cm² / mL (7 cm³) 2 On a surface area of ​​( / mL).

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

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

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

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

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

52. The use according to claim 51, wherein the Notch signaling ligand is DL4, and wherein the step of culturing the T cell progenitor 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. A method for differentiating a population of T cell progenitor cells, the method comprising: a) Provide a population of T cell progenitor cells; as well as b) The T cell progenitor cells were cultured in the presence of a surface-bound Notch signaling ligand, wherein the Notch signaling ligand provided at a culture volume of 0.78 to 4.7 cm² / mL. 2 The surface area is 1 / mL, and the differentiated cell population therein is enriched with CD4-CD8+TCRγδ+ cells.

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

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

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

59. The method according to any one of claims 56 to 58, wherein the Notch signaling ligand is covalently conjugated to the one or more beads.

60. The method according to any one of claims 54 to 59, wherein the Notch signaling ligand comprises Notch ligand δ-sample 4 (DL4) or a variant thereof.

61. The method of claim 60, wherein the Notch signaling ligand is DL4, and wherein the step of culturing the T cell progenitor 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, said population being generated in vitro by the method of any one of claims 52 to 59.

63. A method for generating a population of T cell lineage cells, the method comprising: a) Provide a population of T cell progenitor cells; as well as b) Culture the T cell progenitor cells in a culture vessel in the presence of a surface-bound Notch signaling ligand. The Notch signaling ligand is provided on a 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).

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

65. The method according to 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 and / or the relative number of CD4-CD8+ cells in the cell population.

66. The method according to any one of claims 63 to 65, wherein 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.

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

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

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

71. The method according to any one of claims 68 to 70, wherein the Notch signaling ligand is covalently conjugated to the one or more beads.

72. The method according to any one of claims 63 to 71, wherein the Notch signaling ligand comprises Notch ligand δ-sample 4 (DL4) or a variant thereof.

73. The method of claim 72, wherein the concentration of DL4 is 7.89 x 10⁻⁶. 10 Up to 1.66 x 10 13 molecule / mL culture volume.

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

75. The method of claim 74, wherein the DL4 and the VCAM-1 are provided on a substrate, wherein 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.

76. The method of claim 75, wherein the density of DL4 is 7.89 x 10⁻⁶. 10 Up to 1.66 x 10 13 The concentration of VCAM-1 was 7.89 x 10⁻⁶ molecules per mL of culture volume. 10 Up to 1.66 x 10 13 molecule / mL culture volume.

77. The method according to any one of claims 63 to 76, wherein the cell density is 5 x 10⁻⁶. 5 Up to 2x 10 6 Cells / mL.

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

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

80. The method of claim 78, wherein the T cell progenitor cells are recultured at least once during or after culturing the T cell progenitor cells for at least 3 days, at least 7 days, at least 11 days, or at least 14 days, wherein the reculture 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. The method according to 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. The method according to any one of claims 63 to 82, wherein the T cell progenitor cells are derived from pluripotent stem cells.

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

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

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

87. A population of CD4-CD8+ cells, said population being prepared by the method according to any one of claims 63 to 86.

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

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

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

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

92. A method for generating a population of T cell lineage cells, the method comprising: a) Provide a population of T cell progenitor cells; as well as b) The T cell progenitor cells were cultured in the presence of Notch signaling ligand and in the absence of T cell receptor stimulants; The T cell lineage population mentioned therein comprises CD4-CD8+ cells.

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

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

95. The method of claim 92, wherein the T cell progenitor cell comprises nucleic acid encoding CAR, and wherein the step of culturing the T cell progenitor cell further comprises culturing in the absence of CAR activator.

96. The method of claim 92, wherein the Notch signaling ligand is surface-bound, and wherein the surface-bound Notch signaling ligand is provided in a culture volume of at least 7 cm² / mL (7 cm³). 2 On a surface area of ​​( / mL).

97. The method of claim 92, wherein the Notch signaling ligand is surface-bound, and wherein the surface-bound Notch signaling ligand is provided in a culture volume of between 7 cm² / mL. 2 / mL) and 56cm 2 The surface area between / mL.

98. The method of claim 92, wherein the Notch signaling ligand is provided on a three-dimensional 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).

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 1.77:1 (1.77:1) and 14:1 (14:1).

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

101. A method for generating a population of T cell lineage cells, the method comprising: a) Providing a population of hematopoietic stem cells and / or progenitor cells; as well as b) Culture the hematopoietic stem cells / progenitor cells in the presence of Notch signaling ligand and in the absence of T cell receptor stimulants. The T cell lineage population mentioned therein comprises CD4-CD8+ cells.

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

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

104. The method of claim 101, wherein the hematopoietic stem cells and / or progenitor cells comprise nucleic acids encoding CAR, and wherein the step of culturing the hematopoietic stem cells and / or progenitor cells further comprises culturing in the absence of CAR activators.

105. The method according to any one of claims 101 to 104, wherein the Notch signaling ligand is surface-bound.

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

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

108. The method according to any one of claims 92 to 107, wherein the Notch signaling ligand comprises Notch ligand δ-sample 4 (DL4) or a variant thereof.

109. A method for generating a population of T cell lineage cells, the method comprising: a) Provide a population of T cell progenitor cells; as well as b) Culture the T cell progenitor cells in the presence of surface-bound Notch signaling ligands. The Notch signaling ligand provided is available in a culture volume of between 7 cm² / mL. 2 / mL) and 56cm 2 On the surface area between / mL; Furthermore, the T cell lineage population mentioned above includes CD4-CD8+ cells.

110. A method for generating a population of T cell lineage cells, the method comprising: a) Provide a population of T cell progenitor cells; as well as b) Culture the T cell progenitor cells in a culture vessel in the presence of Notch signaling ligand. 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 1.77:1 (1.77:1) and 14:1 (14:1). Furthermore, the T cell lineage population mentioned above includes CD4-CD8+ cells.

Images