Methods of producing enriched populations of tumor reactive T cells from peripheral blood
Inventors
Gros, Alena • Rosenberg, Steven A.
Assignees
US Department of Health and Human Services
Publication Number
US-11679128-B2
Publication Date
2023-06-20
Expiration Date
2033-04-30
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Abstract
Methods of obtaining a cell population enriched for tumor-reactive T cells, the method comprising: (a) obtaining a bulk population of peripheral blood mononuclear cells (PBMCs) from a sample of peripheral blood; (b) specifically selecting CD8+ T cells that also express PD-1 and/or TIM-3 from the bulk population; and (c) separating the cells selected in (b) from unselected cells to obtain a cell population enriched for tumor-reactive T cells are disclosed. Related methods of administering a cell population enriched for tumor-reactive T cells to a mammal, methods of obtaining a pharmaceutical composition comprising a cell population enriched for tumor-reactive T cells, and isolated or purified cell populations are also disclosed.
Core Innovation
The invention provides methods of obtaining a cell population enriched for tumor-reactive T cells by specifically selecting CD8+ T cells that express programmed cell death protein 1 (PD-1) and/or T-cell immunoglobulin and mucin domain 3 (TIM-3) from a bulk population of peripheral blood mononuclear cells (PBMCs) obtained from peripheral blood. The selected cells are then separated from unselected cells to yield a population enriched for tumor-reactive T cells. This approach allows for the enrichment of tumor-reactive T cells without the necessity to screen for autologous tumor recognition or non-specifically stimulate the bulk T cell population.
The method encompasses the use of flow cytometry, preferably fluorescence-activated cell sorting (FACS), employing antibodies specific to CD3, CD8, PD-1, and TIM-3 to isolate these T cells. The cell populations obtained may include single positive PD-1+ or TIM-3+ cells, or double positive PD-1+/TIM-3+ cells, and can be further expanded in vitro with cytokines or other agents to increase their numbers. The invention also provides related methods of administering these enriched cell populations to mammals and methods of producing pharmaceutical compositions containing them.
The problem addressed is that T cells isolated from peripheral blood often lack adequate tumor-specific reactivity, presenting a barrier to the successful use of adoptive cell therapy (ACT) in cancer treatment. Therefore, there is a need for improved methods for isolating tumor-reactive T cells from peripheral blood, which this invention addresses by identifying PD-1 and TIM-3 expression as markers for tumor-reactive CD8+ T cells and providing a selective isolation strategy.
Claims Coverage
The claims describe a method for treating cancer by obtaining and administering a cell population enriched for tumor-reactive T cells through specific selection of CD8+ T cells expressing PD-1 and/or TIM-3 from peripheral blood, with various optional steps enhancing the method.
Method of treating cancer by enrichment and administration of tumor-reactive T cells
A method comprising obtaining PBMCs from peripheral blood, specifically selecting CD8+ T cells expressing PD-1 and/or TIM-3, separating these cells from unselected cells to enrich for tumor-reactive T cells, and administering the enriched cell population to a mammal in an effective amount to treat cancer.
Formulation of pharmaceutical composition including enriched tumor-reactive T cells
Combining the enriched tumor-reactive cell population with a pharmaceutically acceptable carrier to produce a pharmaceutical composition suitable for administration.
Specific selection of CD8+ T cells expressing TIM-3
Selecting CD8+ T cells from the bulk PBMC population that express TIM-3.
Specific selection of CD8+ T cells expressing PD-1
Selecting CD8+ T cells from the bulk PBMC population that express PD-1.
Selection of CD8+ T cells with specific PD-1 and TIM-3 expression profiles
Selecting CD8+ T cells that are either TIM-3 positive and PD-1 negative, TIM-3 positive and PD-1 positive, or TIM-3 negative and PD-1 positive.
Enrichment for tumor-reactive T cells without autologous tumor recognition screening
Obtaining the enriched tumor-reactive T cell population without conducting a screening process for autologous tumor recognition.
No non-specific stimulation of bulk T cells prior to selection
Performing the method without applying non-specific stimulation to the bulk T cell population before specifically selecting PD-1 and/or TIM-3 positive CD8+ T cells.
In vitro expansion of enriched tumor-reactive T cells
Expanding the numbers of the tumor-reactive T cells after isolation in vitro.
Culturing enriched T cells with immune-modulating agents
Culturing the enriched cell population in the presence of one or more of TWS119, interleukin (IL-21), IL-12, IL-15, IL-7, transforming growth factor beta, and AKT inhibitor.
Stimulation of enriched cells with tumor antigen and/or autologous tumor T cells
Stimulating the enriched tumor-reactive T cell population with a tumor antigen and/or autologous tumor T cells to potentially enhance anti-tumor activity.
Genetic modification of enriched cells to express immune-related factors
Transducing or transfecting the enriched tumor-reactive T cells with nucleotides encoding IL-12, IL-7, IL-15, IL-2, IL-21, mir155, or anti-PD-1 siRNA.
Treatment of a range of cancers with enriched tumor-reactive T cells
Applying the method to treat various cancers including acute myeloid leukemia, breast cancer, cervical cancer, chronic lymphocytic leukemia, colon cancer, esophageal cancer, hepatocellular/liver cancer, lung cancer, melanoma, ovarian cancer, sarcoma, and thyroid cancer.
The claims comprehensively cover methods of isolation, enrichment, expansion, modification, formulation, and administration of CD8+ T cells expressing PD-1 and/or TIM-3 to treat various cancers, emphasizing selective isolation without non-specific stimulation and without tumor recognition screening.
Stated Advantages
The method enriches for greater numbers of tumor-reactive T cells compared to CD8+ cells lacking PD-1 and/or TIM-3 expression.
Allows shortening of in vitro culture time during T cell preparation.
Enables selection of tumor-reactive T cells without the need to screen for autologous tumor recognition.
Avoids the need for non-specific stimulation of bulk T cells prior to selection.
Provides enriched tumor-reactive T cells that can specifically recognize, lyse, and kill tumor cells.
The enriched cell population can be expanded extensively in vitro to obtain large numbers of tumor-reactive T cells.
Culturing with certain immune modulators may enhance the anti-tumor reactivity of the enriched T cells.
Documented Applications
Treatment or prevention of cancer in mammals by administering enriched tumor-reactive T cells or pharmaceutical compositions comprising these cells.
Use of the enriched T cells for adoptive cell therapy in various cancers including melanoma, lung, breast, colon, esophageal, cervical, ovarian, liver/hepatocellular, thyroid cancer, sarcoma, acute myeloid leukemia, and chronic lymphocytic leukemia.
Production of pharmaceutical compositions containing tumor-reactive T cells for injection, including intravenous administration.
In vitro expansion and culturing of T cells from peripheral blood for therapeutic uses.
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