Generation of type 1 regulatory T cells through transcription factor targeting
Inventors
Uyeda, Molly Kathryn Javier • Roncarolo, Maria Grazia
Assignees
Leland Stanford Junior University
Publication Number
US-12319935-B2
Publication Date
2025-06-03
Expiration Date
2040-02-10
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Abstract
Methods, compositions and kits for producing functional antigen-specific regulatory T cells (Tr1 cells) by reprogramming non-Tr1 target cells with suitable transcription factors.
Core Innovation
The invention provides methods, compositions, and kits for inducing Type 1 regulatory T cells (Tr1 cells) by reprogramming non-Tr1 target cells with specific transcription factors. A Tr1 reprogramming system comprising one or more of BHLHE40, ID2, HLF, and AHR transcription factors is used to convert target cells, such as T cells, hematopoietic stem cells, lymphoid progenitors, or pluripotent stem cells, into induced Tr1 (iTr1) cells. This induction leads to functional changes in the target cells, such as upregulation of IL-10, reduced proliferation upon stimulation, and expression of inhibitory molecules including LAG3, PD-1, CTLA-4, TIM-3, TIGIT, OX40, and ICOS.
The primary problem addressed by this invention is the difficulty in producing functional, antigen-specific Tr1 cells due to limited understanding of the transcriptional machinery governing human Tr1 cell differentiation. While some transcription factors have been implicated in Tr1 cell differentiation in mice, equivalent systems in humans were lacking, and master regulators of Tr1 cells had not been identified. This has hampered the ability to generate, study, and utilize Tr1 cells therapeutically or in research.
By identifying and utilizing specific transcription factors, particularly BHLHE40 alone, or combinations such as ID2 and AHR, or BHLHE40 with ID2 and AHR, this invention enables the direct reprogramming of non-Tr1 cells into iTr1 cells. The methods include delivering genetic sequences encoding these transcription factors into target cells, including autologous or allogeneic CD4+ T cells, via various means such as genetic constructs, protein, or mRNA. The produced iTr1 cells can be antigen-specific and are characterized by Tr1 markers such as CD49b and LAG3, as well as a functional cytokine profile distinctive for Tr1 cells.
Claims Coverage
The patent comprises three independent claims, each covering a key inventive feature related to methods for converting non-Tr1 cells into induced Tr1 cells using a Tr1 reprogramming system that includes specific transcription factors.
Conversion of non-Tr1 cells to iTr1 cells using BHLHE40 protein
A method of converting human non-Type 1 regulatory (Tr1) cells into induced Tr1 cells (iTr1) by contacting a population of non-Tr1 cells with a Tr1 reprogramming system that comprises a genetic sequence encoding BHLHE40 protein. - The contact is performed for a period sufficient to reprogram the non-Tr1 cells into LAG3+/CD49b+T cells. - The resulting product is a population of iTr1 cells.
Conversion of non-Tr1 cells to iTr1 cells using a Tr1 reprogramming system comprising a sequence encoding protein
A method of converting human non-Type 1 regulatory (Tr1) cells into induced Tr1 cells (iTr1) by contacting a population of non-Tr1 cells with a Tr1 reprogramming system that includes a sequence encoding a protein for a period sufficient to reprogram the non-Tr1 cells. - This feature enables generation of iTr1 cells by using protein-encoding sequences within the reprogramming system.
Ex vivo conversion of human CD4+ T cells using BHLHE40 protein
A method of converting human CD4+ T cells ex vivo into induced LAG3+/CD49b+ Tr1 cells (iTr1) by contacting the population with a Tr1 reprogramming system comprising a genetic sequence encoding BHLHE40 protein. - The process is performed ex vivo for a time sufficient to reprogram the CD4+ T cells to LAG3+/CD49b+ iTr1 cells. - The resulting population consists of LAG3+/CD49b+ iTr1 cells.
The inventive features collectively cover methods for generating induced Tr1 cells from various human cell types through reprogramming with specific transcription factors, particularly emphasizing the use of BHLHE40, with the methods applicable to general non-Tr1 cells as well as specifically to CD4+ T cells.
Stated Advantages
The methods and compositions enable more efficient production of human in vitro induced Tr1 cells for clinical cell therapy.
The protocols allow for the specific identification, quantification, and tracking of Tr1 cells in vivo using intracellular markers.
The invention enables screening of small molecules, biologics, and drugs that interact with Tr1-relevant transcription factors for use as immunomodulators in immune-mediated diseases and cancer.
The invention provides an approach for generating antigen-specific Tr1 cells, which may be more efficacious for cell-based therapies tailored to disease settings.
The described methods require only a single blood collection for CD4+ T cell isolation, streamlining production compared to certain clinical trial protocols.
Overexpressing upstream transcription factors allows intrinsic regulation of IL-10 expression in Tr1 cells, as opposed to constitutive expression systems.
Documented Applications
Producing Tr1 cells for transplantation therapies to generate human in vitro induced Tr1 cells for clinical cell therapy.
Quantifying and tracking Tr1 cells in vivo in healthy and patient individuals using intracellular markers.
Screening small molecules, biologics, and other drugs that interact with Tr1-specific transcription factors to be used as immunomodulators in immune-mediated diseases and cancer.
Treating patients with inflammatory disorders, including autoimmune diseases such as type 1 diabetes, multiple sclerosis, psoriasis, and rheumatoid arthritis.
Treating allergies, graft versus host disease, and graft rejection by providing compositions of iTr1 cells generated by the described methods.
Providing cell-based therapy for chronic inflammatory diseases, including protocols involving the production and transfer of Tr1 cells.
Developing protocols and kits for generating antigen-specific IL-10 producing human T cells for potential use in disease-specific therapy.
Establishing GMP-compatible, feeder-free systems to differentiate and expand IL-10 producing human CD4+ T cells.
Basic research and drug discovery applications, including evaluation of disease phenotypes, identification of target cells, and screening of candidate agents for modulating T cell survival or function.
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