Methods of preparing an isolated or purified population of thymic emigrant cells and methods of treatment using same
Inventors
Vizcardo, Raul E. • Klemen, Nicholas D. • Restifo, Nicholas P.
Assignees
US Department of Health and Human Services
Publication Number
US-12215349-B2
Publication Date
2025-02-04
Expiration Date
2037-12-13
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Abstract
Disclosed are methods of preparing thymic emigrant cells in vitro, isolated or purified thymic emigrant cells prepared by the methods, and pharmaceutical compositions comprising the same. Further disclosed are methods of treating or preventing a condition in a mammal comprising administering the thymic emigrant cells or pharmaceutical compositions comprising the same to the mammal.
Core Innovation
The invention provides methods of preparing an isolated or purified population of thymic emigrant cells in vitro. This is achieved by modifying source cells into pluripotent cells, multipotent cells, or T-lineage cells; culturing the resultant cells in the presence of a Notch receptor agonist to produce CD45+ cells; further culturing these CD45+ cells with thymic tissue to allow their migration into and egress from the tissue; and collecting the egressed thymic emigrant cells which are characterized as CD8α+CD8β+CD4− or CD8α−CD8β−CD4+.
The problem being addressed stems from obstacles in adoptive cell therapy (ACT), particularly compromised efficacy resulting from terminally differentiated T cells. Existing methods, such as differentiation on OP9-DL1 cells, generate aberrant T cells with phenotypic abnormalities like CD8αα+ or double negative phenotypes, reduced MHC-I expression, and impaired positive selection, which limit their therapeutic potential. Thus, there is a need for improved methods for producing minimally differentiated, functional T cells suitable for ACT.
The disclosed methods, including the use of a three-dimensional thymic culture system such as a hanging drop plate incorporating thymic tissue, enable thymic education of T-lineage cells resulting in thymic emigrant cells that more closely resemble natural mature T cells. These cells lack aberrant immature markers (such as Ptcra, Rag1, Rag2, and Rorc), express appropriate surface markers (e.g., CD8α+CD8β+CD4−), retain antigenic specificity, and exhibit in vitro and in vivo functionality including proliferation and anti-tumor activity. The invention further encompasses the isolated thymic emigrant cells and pharmaceutical compositions comprising them, as well as methods of treating or preventing conditions in mammals by administering these cells or compositions.
Claims Coverage
The claims disclose one independent method claim focusing on a method of preparing an isolated or purified population of thymic emigrant cells in vitro with several inventive features.
Modifying antigen-specific T cells into pluripotent, multipotent, or T-lineage cells
The method comprises modifying T cells that have antigenic specificity for a cancer antigen into pluripotent cells, multipotent cells, or T-lineage cells.
Culturing cells with Notch receptor agonist to produce CD45+ cells
The modified cells are cultured in the presence of a Notch receptor agonist to obtain CD45+ cells.
Culturing CD45+ cells with thymic tissue in a hanging drop medium to facilitate migration and egress
The CD45+ cells are cultured in the presence of thymic tissue within a hanging drop of medium, including seeding the thymic tissue with these cells that migrate into the tissue, and egressing occurs between 2 to 5 days after seeding.
Isolating thymic emigrant cells without disrupting thymic tissue characterized by specific surface markers and functional capacity
Thymic emigrant cells are isolated from the tissue without disrupting it. These cells are CD8α+CD8β+CD4− or CD8α−CD8β−CD4+, have capacity to differentiate into T cells with antigenic specificity for the cancer antigen, and do not express Ptcra, Rag1, Rag2 and Rorc.
The claims are centered on a method of preparing thymic emigrant cells in vitro by reprogramming antigen-specific T cells, culturing them with Notch receptor agonist, and inducing their migration into and egress from thymic tissue in a hanging drop culture system. The isolated thymic emigrant cells possess specific phenotypes and functional capabilities relevant to therapeutic applications.
Stated Advantages
Production of minimally differentiated T cells with increased in vivo proliferation, survival, persistence, cytotoxicity, and anti-tumor activity compared to terminally differentiated T cells.
Capability to generate T cells with CD4+ or CD8αβ+ single positive phenotype from various source cells including pluripotent, multipotent, or T-lineage cells.
Reduction or avoidance of generating aberrant T cells with CD8αα+ or double negative phenotypes when using induced pluripotent stem cells.
Three-dimensional thymic culture systems, especially using hanging drop plates, provide improved gas exchange, cellular visualization, and maintenance without mechanical disruption, enhancing thymic education and maturation of T cells.
Documented Applications
Treatment or prevention of cancer, particularly adoptive cell therapy using minimally differentiated T cells derived from induced pluripotent stem cells.
Treatment or prevention of autoimmune conditions by differentiating thymic emigrant cells into regulatory T cells.
Treatment or prevention of infectious conditions, including viral, bacterial, fungal, or protozoan infections.
Use in lymphodepletion contexts, for example, to replace or supplement T cells lost post-transplant or due to aging.
Use in disease modeling and therapeutic validation.
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