Methods of enriching cell populations for cancer-specific T cells using in vitro stimulation of memory T cells
Inventors
Cafri, Gal • Rosenberg, Steven A.
Assignees
US Department of Health and Human Services
Publication Number
US-12221627-B2
Publication Date
2025-02-11
Expiration Date
2038-12-03
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Abstract
Disclosed are methods of obtaining a cell population enriched for T cells having antigenic specificity for a cancer-specific mutation using in vitro stimulation of memory T cells. Also disclosed are related methods of isolating a T cell receptor (TCR), populations of cells, TCRs or antigen-binding portions thereof, pharmaceutical compositions, and methods of treating or preventing cancer.
Core Innovation
The invention provides methods of obtaining a cell population enriched for T cells having antigenic specificity for a cancer-specific mutation by using in vitro stimulation (IVS) of memory T cells or TEMRA phenotype T cells. The method includes obtaining monocytes from a mammal, differentiating them into dendritic cells (DCs), inducing the DCs to present mutated amino acid sequences encoded by genes with cancer-specific mutations, and stimulating T cells with a memory or TEMRA phenotype from the peripheral blood mononuclear cells (PBMCs) of the mammal with these DCs in vitro. The cells are re-stimulated, selected based on expression of markers of T cell stimulation, and screened for specificity to the mutated amino acid sequences to provide an enriched population of cancer-specific T cells.
The inventive methods address the problem of identifying and isolating cancer-specific T cells and T cell receptors (TCRs), which is challenging because these cells are often rare in peripheral blood compared to tumor tissues. Although adoptive cell therapy can produce positive clinical outcomes, the isolation of cancer-reactive T cells remains a hurdle to its widespread use. This invention improves the identification of antigen-experienced T cells by focusing on memory and TEMRA phenotypes, believed to have been stimulated by cognate antigen at tumor sites or draining lymph nodes, thus providing better candidates for T cell or TCR isolation.
By using autologous DCs to present mutated amino acid sequences in the context of the mammal's MHC molecules, the inventive methods enable the isolation of T cells or TCRs specific for cancer mutations from the more accessible peripheral blood rather than tumor tissues. This approach enriches for cancer-specific mutation-stimulated memory T cells, overcoming the low frequency of such cells in peripheral blood and avoiding interference by naïve T cells that have not encountered antigen, thereby isolating clinically relevant T cells or TCRs for adoptive transfer or therapy.
Claims Coverage
The patent discloses eleven claims including eleven inventive features, focusing on methods for enriching, isolating, and utilizing T cells and T cell receptors (TCRs) specific for cancer-specific mutations, particularly via in vitro stimulation of central memory T cells.
Method of enriching T cells with central memory phenotype specific for cancer mutations
An in vitro method of obtaining a cell population enriched for T cells having antigenic specificity for a cancer-specific mutation by providing monocytes and PBMCs from an epithelial cancer patient, differentiating monocytes into dendritic cells (DCs), inducing DCs to present mutated amino acid sequences encoded by genes with cancer-specific mutations, specifically selecting central memory T cells (CCR7+, CD62L+, CD45RO+, CD45RA−) from PBMCs, stimulating and re-stimulating those T cells with DCs in vitro, selecting cells expressing T cell stimulation markers, screening for recognition of mutated sequences, and selecting antigen-specific cells.
Selection of re-stimulated T cells using specific T cell activation markers
Specifically selecting re-stimulated T cells expressing one or more markers of T cell stimulation including programmed cell death 1 (PD-1), lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1BB, OX40, and CD107a.
Expansion of T cells expressing stimulation markers
Expanding the number of selected T cells that express one or more markers of T cell stimulation to increase the cell population for further use.
Inclusion of additional T cell markers in phenotype selection
The T cell phenotype selected may further comprise one or more of CD3+, CD4+, and CD8+ markers.
Timing of screening for mutated amino acid recognition
Screening of cells expressing stimulation markers for antigenic specificity occurs about 11 to about 16 days after separation.
Induction of DCs to present mutated amino acid sequences by peptide pulsing
Inducing dendritic cells to present mutated amino acid sequences by pulsing them with peptides or peptide pools each containing different mutated amino acid sequences.
Induction of DCs to present mutated sequences by nucleotide introduction
Inducing dendritic cells to present mutated amino acid sequences by introducing nucleotide sequences encoding the mutated amino acids into the DCs.
Use of tandem minigene constructs in dendritic cells
Introducing nucleotide sequences as tandem minigene (TMG) constructs into DCs, where each minigene encodes a different gene containing a cancer-specific mutation encoding a mutated amino acid sequence.
Isolation of TCR having antigen specificity from enriched T cells
Isolating a nucleotide sequence encoding a T cell receptor (TCR), or an antigen-binding portion thereof, having antigenic specificity for a mutated amino acid sequence by obtaining a cell population enriched for such T cells and isolating the nucleotide sequence encoding the specific TCR or antigen binding portion.
Preparation of cells expressing isolated TCR
Preparing a population of cells expressing a TCR or antigen-binding portion thereof, having antigenic specificity for a mutated amino acid sequence by isolating the TCR and introducing its nucleotide sequence into peripheral blood mononuclear cells (PBMC) to obtain the expressing cells.
Method of treating epithelial cancer by administering enriched T cell population
Treating epithelial cancer in a patient by obtaining a cell population enriched for T cells having antigenic specificity for a cancer-specific mutation and administering this population in an amount effective to treat the cancer.
The claims cover inventive features including enriching T cells with antigen specificity by selecting central memory phenotypes and stimulating with dendritic cells presenting mutated sequences, identifying specific T cell activation markers for selection, expanding enriched T cells, methods of inducing DC presentation by peptide pulsing or nucleotide introduction including use of tandem minigenes, isolating TCR nucleotide sequences from enriched T cells, engineering cells expressing isolated TCRs, and methods of treating epithelial cancer using the enriched cell populations.
Stated Advantages
The inventive methods enable isolation of cancer mutation-specific T cells from peripheral blood, which is more accessible and abundant than tumor tissue.
Focusing on memory and TEMRA T cells enriches for antigen-experienced T cells likely stimulated by tumor antigens, potentially increasing clinical relevance.
By using autologous dendritic cells to present mutated amino acid sequences, the methods enable identification of T cells or TCRs specific for patient-expressed MHC molecules, improving personalized therapy.
Isolating T cells from antigen-experienced populations rather than bulk PBMC reduces interference by naïve T cells and enriches for clinically relevant cancer-specific T cells or TCRs.
The methods can isolate very low-frequency cancer mutation-specific T cells from peripheral blood, facilitating adoptive transfer or TCR therapy.
Documented Applications
Obtaining cell populations enriched for T cells having antigenic specificity for cancer-specific mutations for use in adoptive cell therapy.
Isolating T cell receptors (TCRs) or antigen-binding portions thereof that have specificity for mutated amino acid sequences encoded by cancer-specific mutations.
Preparing a population of cells expressing isolated TCRs for use in patient treatment.
Formulating TCRs, antigen-binding portions thereof, or cell populations into pharmaceutical compositions for treating or preventing cancer.
Treating or preventing various cancers including epithelial cancers, cholangiocarcinoma, melanoma, colon cancer, rectal cancer, and others by administering enriched T cells or engineered T cells expressing isolated TCRs.
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