Methods of producing T memory stem cell populations
Inventors
Gattinoni, Luca • Lugli, Enrico • Roederer, Mario • Restifo, Nicholas P.
Assignees
US Department of Health and Human Services
Publication Number
US-10316289-B2
Publication Date
2019-06-11
Expiration Date
2032-09-06
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Abstract
Provided are methods of producing an isolated T memory stem cell population, the method comprising a) isolating nave T cells from a mammal, wherein the mammal is not a mouse; b) activating the nave T cells and expanding the numbers of nave T cells in the presence of one or more non-specific T cell stimuli, one or more cytokines, and a GSK-3beta inhibitor. Also provided are methods of producing an isolated T memory stem cell population, the method comprising a) isolating lymphocytes from a mammal; b) sorting the lymphocytes using flow cytometry into a population comprising a phenotype comprising i) CD95+, CD45RO−, and CCR7+; and ii) CD62L+ or one or more of CD27+, CD28+, CD45RA+, and CD127+ to produce an isolated T memory stem cell population. Further embodiments of the invention provide related cells, populations of cells, pharmaceutical compositions, and methods of treating or preventing cancer.
Core Innovation
The invention provides methods of producing an isolated T memory stem cell population (TSCM), which are T cells with enhanced stem cell-like qualities compared with central memory T cells. These memory stem T cells possess an enhanced capacity for self-renewal and multipotency, capable of repopulating differentiated effector lymphocytes upon antigenic stimulation. The methods include isolating naïve T cells or lymphocytes from a mammal, activating and expanding naïve T cells in the presence of non-specific T cell stimuli, cytokines, and a GSK-3beta inhibitor, and sorting lymphocytes by flow cytometry into populations expressing specific T memory stem cell phenotypes.
The methods further include producing TSCM cells by isolating lymphocytes and sorting them into phenotypes comprising markers such as CD95+, CD45RO−, CCR7+, CD62L+, and others like CD27+, CD28+, CD45RA+, and CD127+. The invention also covers populations of these TSCM cells, pharmaceutical compositions containing them, and methods of treating or preventing cancer using these cells.
The problem being addressed is that adoptive cell therapy (ACT) using tumor reactive T cells, while effective, faces challenges such as insufficient tumor-specific reactivity and poor persistence of T cells isolated from a host's peripheral blood upon reinfusion. Therefore, there is a need for improved methods to obtain antigen-specific T cells exhibiting sufficient tumor reactivity and persistence in patients. The invention solves this by isolating and producing T memory stem cells with improved self-renewal, multipotency, and antitumor efficacy.
Claims Coverage
The claims focus on a method comprising isolating lymphocytes and sorting them into a T memory stem cell population with a characteristic phenotype, including additional steps such as in vitro expansion and genetic modification.
Method of isolating T memory stem cells by flow cytometry based on specific phenotype
The method comprises isolating lymphocytes from a mammal and sorting them by flow cytometry into a population comprising a phenotype characterized by (i) CD95+, CD45RO−, and CCR7+; and (ii) CD62L+ or one or more of CD27+, CD28+, CD45RA+, and CD127+ to produce an isolated T memory stem cell population.
Phenotypic refinement of sorted T memory stem cells
The sorting may further include cells expressing one or more of CD58+, CD122+, CD3+, CD4+, and CD8+ to refine the isolated T memory stem cell population.
In vitro expansion of isolated T memory stem cells
The isolated T memory stem cells can be expanded in vitro to increase their number.
Genetic modification of isolated T memory stem cells with antigen-specific receptors
The method includes transducing isolated T memory stem cells with a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) with specificity for a cancer antigen or a viral antigen.
Exclusion of certain phenotypes and cell types
The isolated T memory stem cells produced are characterized by the absence of CD161+ phenotype, IL-18Rα+ phenotype, are not mucosal-associated invariant T cells (MAITs), and do not express RORC or IL17A.
The claims cover methods of isolating, characterizing, expanding, and genetically modifying T memory stem cells with defined phenotypes, intended for therapeutic applications such as cancer treatment.
Stated Advantages
The invention provides TSCM cells with enhanced capacity for self-renewal and multipotency compared to central memory T cells.
TSCM cells have increased proliferative capacity, survival, and antitumor activity compared to other T cell subsets.
The methods allow efficient generation and identification of human and non-human primate TSCM cells using specific markers, facilitating therapeutic application.
TSCM cells enable effective treatment or prevention of cancer, potentially requiring significantly lower numbers of cells than other adoptive immunotherapy protocols.
Documented Applications
Producing isolated T memory stem cell populations for adoptive cell therapy.
Use of isolated TSCM cells in pharmaceutical compositions for treating or preventing cancer in mammals.
Genetic modification of TSCM cells with chimeric antigen receptors or T cell receptors targeting cancer or viral antigens.
Isolation and expansion of TSCM cells from humans and non-human primates for research and therapeutic purposes.
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