Human T cell derived from T cell-derived induced pluripotent stem cell and methods of making and using
Inventors
Blazar, Bruce R. • PATEL, Dharmeshkumar • Webber, Beau R. • Tolar, Jakub
Assignees
University of Minnesota System
Publication Number
US-12270050-B2
Publication Date
2025-04-08
Expiration Date
2036-12-08
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Abstract
Induced pluripotent stem cells (iPSCs) derived from a T cell of a T cell subset. T cells derived from iPSCs derived from a T cell. Methods of deriving iPSCs from a T cell. Methods of deriving T cells from iPSCs including deriving a T cell of a T cell subset from an iPSC. Methods of engineering chimeric antigen receptor (CAR)-expressing or T cell receptor (TCR)-expressing iPSC. Methods of administering T cells derived using the methods disclosed. Induced pluripotent stem cell lines derived from T cells, methods of deriving induced pluripotent stem cell lines, and methods of deriving T cells from induced pluripotent stem cell lines.
Core Innovation
The invention concerns methods and compositions for generating induced pluripotent stem cells (iPSCs) derived from human T cells, particularly from defined T cell subsets such as stem memory T cells (Tsm), naïve T cells, and central memory T cells. The process involves isolating T cells, expanding them as needed, reprogramming them into iPSCs using methods such as Sendai virus-mediated delivery of reprogramming factors, and differentiating these iPSCs back into T cells of desired subsets. These iPSCs may optionally be engineered to express chimeric antigen receptors (CARs) or T cell receptors (TCRs).
The patent addresses the challenge that expansion and ex vivo culture of T stem memory cells from peripheral blood can progressively skew their phenotype away from stemness, and the low frequency of these cells in circulation makes obtaining sufficient quantities difficult. Furthermore, repeated or chronic stimulation of T cells can result in terminal differentiation or exhaustion, restricting their therapeutic effectiveness. The disclosed invention overcomes these hurdles by generating iPSCs from rare T cell subsets, enabling limitless expansion and redifferentiation into functionally relevant T cells while retaining essential phenotypic and functional characteristics.
The methods include reprogramming specific T cell subsets into iPSCs, differentiating the iPSCs into T progenitor cells and mature T cells (including enriching for CD34+ and CD45+ markers), and optionally using defined culture conditions such as the presence of basic fibroblast growth factor (bFGF), bone morphogenetic protein-4 (BMP-4), Wnt pathway activators, TGFβ signaling inhibitors, and Notch ligand-expressing stromal cells. Specific protocols detail the sequential media composition and enrichment steps to optimize T cell lineage differentiation. The invention also allows for genetic manipulation (e.g., BCL6 upregulation, PRDM1 knockdown) to influence and maintain desired T cell states for potential therapeutic use.
Claims Coverage
The claims describe several inventive features related to the derivation, expansion, reprogramming, and differentiation of human T stem memory cells (Tsm) to iPSCs and subsequent T cell development using defined methods and culture components.
Isolation, expansion, reprogramming and differentiation of Tsm into T cells via iPSCs
A method comprising: 1. Isolating at least one stem memory T cell (Tsm). 2. Expanding the Tsm in vitro. 3. Reprogramming at least a portion of the expanded Tsms into induced pluripotent stem cells (iPSCs). 4. Differentiating at least a portion of the iPSCs into T cells. - Differentiation involves forming embryoid bodies (EBs) from the iPSCs and culturing them in the presence of a Wnt pathway activator and a TGFβ signaling inhibitor. - The differentiated T cells comprise a Tsm.
Culturing iPSCs with specific growth factors and cytokines during differentiation
Culturing the iPSCs undergoing differentiation in the presence of one or more of: - Basic fibroblast growth factor (bFGF) - Bone morphogenetic protein-4 (BMP-4) - A hematopoietic cytokine - Stromal cells expressing a Notch ligand - Media comprising a soluble Notch ligand - Stem cell factor (SCF), IL-7, and FMS-like tyrosine kinase 3 ligand (Flt3L)
Sequential culturing steps for iPSC differentiation
Culturing the iPSCs undergoing differentiation sequentially with: 1. Media comprising at least one of bFGF and BMP-4. 2. Media comprising at least one of a Wnt pathway activator and a TGFβ signaling inhibitor. 3. Media comprising at least one hematopoietic cytokine. 4. Media comprising at least one of SCF, IL-7, and Flt3L.
Sequential culturing in animal-product free medium and Notch ligand-expressing stromal media
Culturing the iPSCs undergoing differentiation sequentially in: - Media comprising animal-product free medium (APEL) differentiation medium. - Media comprising a stromal cell expressing a Notch ligand or media comprising a soluble Notch ligand.
Enrichment of differentiating iPSCs for CD34+ or CD45+ cells
The differentiation method includes enriching iPSCs for CD34+ cells. Optionally, subsequent enrichment during or after stimulation via CD3 and/or CD28 can be done for CD45+ cells.
Stimulation of differentiating iPSCs via CD3 and/or CD28
The method includes stimulating the iPSCs undergoing differentiation via at least one of CD3 and CD28, which may further comprise enriching for CD45+ cells and culturing in media supplemented with one or more cytokines, GSK3 inhibitors, or AKT inhibitors.
Generation of chimeric antigen receptor-expressing iPSCs and T cells
The iPSCs may be engineered to express a chimeric antigen receptor (CAR), producing CAR-expressing cells after differentiation into T cells.
Regulation of BCL6 and PRDM1 expression during differentiation
Differentiation of at least a portion of the iPSCs into T cells includes regulating BCL6 and PRDM1 expression, which can be achieved by inducing BCL6 upregulation and PRDM1 down-regulation or by BCL6 knockdown using CRISPRs and inducible PRDM1 expression.
Use of defined Wnt pathway activators and TGFβ signaling inhibitors
The Wnt pathway activator for differentiation includes agonists such as CHIR99021, CHIR-98014, LY2090314, BIO, IM-12, 3F8, A 1070722, CHIR99021 trihydrochloride, L803-mts, SB 216763, SB415286, TC-G 24, TWS119, or combinations thereof. The TGFβ signaling inhibitor includes SB431542 EW-7197, LY2157299, LY2109761, SB525334, SD-208, SB505124, GW788388, RepSox, or combinations thereof.
Specification of Tsm surface marker expression
The at least one isolated Tsm is defined as expressing CD3, CD8, CD45RA, CD62L, CCR7, and CD95.
The inventive features encompass the complete process of isolating and reprogramming human T stem memory cells, differentiating iPSCs into T cells, defining sequential culture and cytokine conditions, optional genetic modifications, and the ability to enrich for specific cell populations and engineer CAR expression. The features are aimed at directing the generation of stem memory T cells from iPSCs using highly defined methods.
Stated Advantages
Provides a virtually limitless source of long-lasting T stem memory cells or CAR T cells for adoptive therapy, especially for patients with low T stem memory cell frequencies.
Improves the safety and efficacy of genetically modified T cells for cancer therapy by enabling reprogramming and expansion without loss of functional phenotype.
Overcomes the skewing and exhaustion that occur with repeated expansion of peripheral blood-derived T stem memory cells.
Permits precise generation and enrichment of defined T cell subsets from iPSCs using specified culture and differentiation methods.
Documented Applications
Adoptive cell therapy for malignant disease, including the use of CAR-expressing T cells generated from iPSCs.
Treatment or prevention of virus infections, cancer, or precancerous conditions using the generated T cells.
Administration of generated T cells for immune surveillance or treatment of tumor cells and pathogens.
Treatment of autoimmune diseases characterized by target cells to which the CAR or TCR can bind.
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