Methods of isolating T cells and T cell receptors having antigenic specificity for a cancer-specific mutation from peripheral blood
Inventors
Gros, Alena • Rosenberg, Steven A.
Assignees
US Department of Health and Human Services
Publication Number
US-11629334-B2
Publication Date
2023-04-18
Expiration Date
2036-04-29
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Abstract
Disclosed are methods of isolating T cells and TCRs having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. Also disclosed are related methods of preparing a population of cells, populations of cells, TCRs, pharmaceutical compositions, and methods of treating or preventing cancer.
Core Innovation
The invention provides methods of isolating T cells and T cell receptors (TCRs) that have antigenic specificity for mutated amino acid sequences encoded by cancer-specific mutations. The methods comprise obtaining peripheral blood mononuclear cells (PBMCs) from a patient, selecting and enriching for T cells expressing programmed cell death 1 (PD-1) from the bulk population, identifying cancer-specific mutations in the patient's cancer cell nucleic acid, inducing autologous antigen presenting cells (APCs) to present mutated amino acid sequences, co-culturing the PD-1-expressing T cells with the APCs presenting the mutated sequences, and selecting T cells that have antigenic specificity for the mutated amino acid sequence in the context of a major histocompatibility complex (MHC) molecule expressed by the patient.
The invention addresses the challenge that T cells and TCRs specifically recognizing cancer antigens can be difficult to identify and isolate from a patient, hindering effective adoptive cell therapy (ACT). Existing methods may require invasive tissue sampling such as biopsies or surgery to obtain tumor-infiltrating lymphocytes (TIL). The inventive methods enable isolation of cancer-reactive T cells from peripheral blood, a more accessible and abundant source, without the need for invasive procedures. This approach facilitates identification of antigen-specific T cells and TCRs even when tumor tissue is unavailable, potentially enabling personalized immunotherapy.
The methods also provide advantages by enabling rapid assessment of a large number of cancer mutations restricted by all of the patient’s MHC molecules simultaneously, allowing identification of the full repertoire of mutation-reactive T cells. By distinguishing immunogenic cancer-specific mutated amino acid sequences from silent or non-immunogenic mutations, the inventive methods identify targets for T cell or TCR-based therapies, vaccines, or pharmaceutical compositions. The identified TCRs or antigen-binding portions thereof can be used to engineer host cells for adoptive cell therapies, improving specificity against cancer cells while minimizing toxicity to normal cells.
Claims Coverage
The claims encompass methods for isolating T cells and TCRs with specificity for mutated amino acid sequences encoded by cancer-specific mutations, involving steps of enrichment, identification, co-culture, and selection. Several inventive features related to selection markers, antigen presentation, and TCR isolation are specified.
Selection of PD-1-expressing T cells
The method involves selecting T cells that express programmed cell death 1 (PD-1) from a bulk population of peripheral blood mononuclear cells to enrich for cancer antigen-reactive T cells.
Identification of cancer-specific mutations
Identifying one or more genes in the nucleic acid of a cancer cell of the patient, each containing a cancer-specific mutation encoding a mutated amino acid sequence.
Inducing autologous APCs to present mutated amino acid sequences
Inducing autologous antigen presenting cells of the patient to present mutated amino acid sequences either by pulsing them with peptides comprising the mutated sequences or introducing nucleotide sequences encoding these mutations, including use of tandem minigene (TMG) constructs.
Co-culture and selection of antigen-specific T cells
Co-culturing enriched PD-1-expressing T cells or TCR-expressing T cells with autologous APCs presenting mutated amino acid sequences and selecting those T cells that show antigenic specificity for the mutated amino acid sequences presented in the context of the patient's MHC molecules.
Use of additional markers for selecting T cells
Selecting T cells that co-express PD-1 and markers such as CD3, CD4, CD8, TIM-3, and CD27, including specific combinations like CD8+PD-1+, PD-1+TIM-3+, CD8+PD-1hi, and others.
Selection criteria based on activation markers or cytokine secretion
Selecting antigen-specific T cells based on expression of activation markers (e.g., PD-1, LAG-3, TIM-3, 4-1BB, OX40, CD107a) or secretion of cytokines such as interferon-gamma (IFN-γ), interleukins, and tumor necrosis factor alpha (TNF-α) upon co-culture with APCs presenting the mutated amino acid sequence.
Isolation and use of nucleotide sequences encoding TCRs
Isolating nucleotide sequences encoding TCRs or antigen-binding portions thereof from selected antigen-specific T cells, cloning into recombinant expression vectors, and introducing them into host cells to prepare populations expressing these TCRs.
The claims collectively disclose a comprehensive approach for isolating, characterizing, and using T cells and TCRs with specificity for cancer-specific mutated amino acid sequences from peripheral blood, utilizing PD-1-based enrichment, mutation identification, antigen presentation by autologous APCs, co-culture assays, and recombinant expression to generate therapeutic cell populations.
Stated Advantages
Obtaining cancer antigen-reactive T cells from peripheral blood, a more accessible and abundant source than tumor tissue, eliminating need for invasive procedures.
Rapidly assessing a large number of mutations restricted by all the patient's MHC molecules simultaneously to identify the full repertoire of mutation-reactive T cells.
Distinguishing immunogenic mutated amino acid sequences from silent or non-immunogenic mutations, enabling targeted immunotherapies including personalized TCRs and vaccines.
Avoiding technical biases and laborious processes inherent in traditional methods such as cDNA library screening or tumor cell line generation.
Enabling administration of adoptive cell therapy to patients lacking accessible tumors for TIL harvest.
Potentially reducing the cost and expanding the availability of adoptive cell therapy to a larger number of patients.
Producing T cells and TCRs that target cancer cells while minimizing destruction of normal cells, reducing toxicity in treatments.
Documented Applications
Isolating T cells and TCRs with antigenic specificity for mutated amino acid sequences encoded by cancer-specific mutations from peripheral blood to prepare populations of cells for adoptive cell therapy.
Using isolated TCRs or antigen-binding portions thereof to engineer host cells for personalized immunotherapy targeting patient-specific cancer mutations.
Preparing pharmaceutical compositions comprising isolated TCRs, polypeptides, host cells, or populations thereof, for treating or preventing cancer.
Treating a wide range of cancers including melanoma, colorectal cancer, cholangiocarcinoma, and other epithelial cancers using the isolated T cells, TCRs, or engineered cells.
Screening peripheral blood populations from cancer patients to identify mutation-specific tumor-reactive T cells without requiring tumor biopsy or generation of tumor cell lines.
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