Antigen-specific immune effector cells
Inventors
Vodyanyk, Maksym A. • Zhang, Xin • BRANDL, Andrew J. • Rajesh, Deepika • Swanson, Bradley • MUNN, Christie • BURTON, Sarah • Wang, Wen Bo • McLEOD, Ethan
Assignees
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Publication Number
US-12163155-B2
Publication Date
2024-12-10
Expiration Date
Abstract
Provided herein are methods for the production of antigen-specific effector T cells and NK cells from pluripotent stem cells which express a chimeric antigen receptor (CAR). Further provided herein are methods for the adoptive cell therapy by administering the effector T cells and/or NK cells provided herein.
Core Innovation
The invention relates to producing antigen-specific effector T cells and/or NK cells by engineering induced pluripotent stem cells (iPSCs) to express a chimeric antigen receptor (CAR), thereby producing CAR-iPSCs. The CAR-iPSCs are directed through mesoderm induction toward CD34+ hematopoietic progenitor cells (HPCs) and then further differentiated into T cells and/or NK cells.
CAR-iPSC priming for mesoderm induction is performed by directed differentiation in which embryoid bodies (EBs) are generated in the presence of a ROCK inhibitor, a GSK-3 inhibitor, FGF2, and VEGF, and the EBs are contacted with BMP-4, VEGF, and FGF2 to induce mesoderm induction. The resulting differentiation produces a population of HPCs comprising at least 40% CD34+CD43+ cells.
The HPCs are further differentiated to T cells and/or NK cells by culturing the HPC population on a fibronectin or fragment thereof and a Notch DLL-4 coated surface in the presence of ascorbic acid and nicotinamide under hypoxic conditions. Antigen-specific effector T cells and/or NK cells are then produced by expanding the differentiated T cells and/or NK cells through co-culturing with antigen-specific target cells.
Claims Coverage
The document provides one independent claim defining a multistage production method for antigen-specific effector T cells and/or NK cells, with dependent claims that add measurable composition or functional thresholds and specify particular culture components and starting materials.
CAR engineering of iPSCs to produce CAR-iPSCs
Engineering induced pluripotent stem cells (iPSCs) to express a chimeric antigen receptor (CAR), thereby producing CAR-iPSCs.
Directed differentiation of CAR-iPSCs to CD34+ HPCs using EB mesoderm induction
Priming CAR-iPSCs for mesoderm induction by directed differentiation of the CAR-iPSCs to CD34+ hematopoietic progenitor cells (HPCs) by generating embryoid bodies (EBs) in the presence of a ROCK inhibitor, a GSK-3 inhibitor, FGF2, and VEGF, contacting the EBs with BMP-4, VEGF, and FGF2 to induce mesoderm induction.
HPC generation with CD34+CD43+ composition threshold
Differentiating the EBs in the presence of Flt-3 ligand, IL3, SCF, and TPO, thereby producing a population of HPCs comprising at least 40% CD34+CD43+ cells.
Differentiation of HPCs to T cells and/or NK cells on fibronectin fragment and Notch DLL-4 under hypoxia
Further differentiating the HPCs in the population to T cells and/or NK cells comprising culturing the HPC population on a fibronectin or fragment thereof and Notch DLL-4 coated surface in the presence of ascorbic acid and nicotinamide under hypoxic conditions.
Expansion by co-culture with antigen-specific target cells
Expanding the T cells and/or NK cells by co-culturing with antigen-specific target cells, thereby producing antigen-specific effector T cells and/or NK cells.
Overall, the claim set centers on CAR-engineered iPSCs differentiated through EB-based mesoderm induction to CD34+CD43+ HPCs, followed by hypoxia-associated differentiation on fibronectin or fragment thereof and Notch DLL-4 with ascorbic acid and nicotinamide, and final antigen-specific expansion via co-culture with antigen-specific target cells.
Stated Advantages
Not explicitly described in patent.
Documented Applications
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