Methods of isolating T cells having antigenic specificity for a cancer-specific mutation
Inventors
Tran, Eric • Lu, Yong-Chen • Robbins, Paul F. • Rosenberg, Steven A.
Assignees
US Department of Health and Human Services
Publication Number
US-12171818-B2
Publication Date
2024-12-24
Expiration Date
2034-10-02
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Abstract
Disclosed are methods of isolating T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in the nucleic acid of a cancer cell of a patient, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; inducing autologous APCs of the patient to present the mutated amino acid sequence; co-culturing autologous T cells of the patient with the autologous APCs that present the mutated amino acid sequence; and selecting the autologous T cells. Also disclosed are related methods of preparing a population of cells, populations of cells, pharmaceutical compositions, and methods of treating or preventing cancer.
Core Innovation
The invention provides methods of isolating T cells that have antigenic specificity for mutated amino acid sequences encoded by cancer-specific mutations. The methods involve identifying one or more genes in the nucleic acid of cancer cells from a patient that contain cancer-specific mutations, inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequences, co-culturing autologous T cells of the patient with the autologous APCs presenting the mutated amino acid sequences, and selecting the autologous T cells that specifically recognize the mutated amino acid sequences in the context of major histocompatibility complex (MHC) molecules expressed by the patient.
The methods enable rapid assessment of a large number of mutations restricted by all MHC molecules of the patient simultaneously, thereby identifying the full repertoire of mutation-reactive T cells. By distinguishing immunogenic cancer mutations from silent or non-immunogenic mutations, the methods identify one or more cancer-specific mutated amino acid sequences that may be targeted by T cells. The invention provides isolated, personalized populations of T cells unique to the patient, which may be useful for adoptive cell therapies to treat or prevent the patient's cancer.
The methods avoid technical biases and limitations of traditional cancer antigen identification methods, are less time-consuming and laborious, and may select mutation-reactive T cells without co-culturing with tumor cell lines. The invention may identify and isolate T cells targeting cancer cells while minimizing destruction of normal cells, potentially reducing toxicity. The isolated T cells may treat or prevent cancers that do not respond to other treatments such as chemotherapy, surgery, or radiation.
Claims Coverage
The patent includes one independent claim describing a method for preparing a pharmaceutical composition comprising T cells with antigenic specificity for mutated amino acid sequences encoded by cancer-specific mutations.
Method of preparing a pharmaceutical composition with mutation-specific T cells
A method comprising identifying genes with cancer-specific mutations encoding mutated amino acid sequences in a patient's tumor cells; inducing autologous APCs to present these mutated sequences; co-culturing autologous T cells with these APCs; assessing T cells for antigenic specificity to mutated sequences presented by MHC class I and II molecules; selecting those specific T cells; and combining them with a pharmaceutically acceptable carrier to provide the pharmaceutical composition.
Inducing APCs to present mutated amino acid sequences by peptide pulsing
Inducing autologous APCs to present mutated amino acid sequences by pulsing with peptides comprising the mutated sequences or pools of such peptides.
Inducing APCs to present mutated amino acid sequences by nucleotide sequence introduction
Inducing autologous APCs to present mutated amino acid sequences by introducing nucleotide sequences encoding the mutated amino acid sequences into the APCs.
Use of tandem gene sequence constructs for APC induction
Introducing tandem minigene constructs comprising multiple different mutated gene sequences encoding mutated amino acid sequences into autologous APCs.
Assessing antigenic specificity of T cells from multiple tumor fragments
Obtaining multiple tumor fragments, separately co-culturing autologous T cells from each fragment with APCs presenting mutated amino acid sequences, and separately assessing antigenic specificity of T cells from each fragment.
Selective growth of antigen-specific T cells
Selecting antigen-specific autologous T cells by culturing the T cells to selectively grow those with antigenic specificity for mutated amino acid sequences.
Selecting T cells based on expression of activation markers
Selecting antigen-specific T cells expressing activation markers including PD-1, LAG-3, TIM-3, 4-1BB, OX40, and CD107a.
Selecting T cells based on cytokine secretion
Selecting antigen-specific T cells based on secretion of cytokines upon co-culture with APCs presenting mutated amino acid sequences, including higher cytokine secretion compared to controls or at least twice the number of cytokine-secreting cells compared to negative controls.
Cytokines indicative of T cell activation
Using cytokines such as IFN-γ, IL-2, TNF-α, GM-CSF, IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22 as indicators for selecting antigen-specific T cells.
Sequencing of tumor nucleic acids to identify mutated genes
Identifying mutated genes by sequencing the whole exome, whole genome, or whole transcriptome of tumor cells.
Expanding selected antigen-specific T cells
Expanding the number of selected autologous T cells after selection.
Expansion method using feeder PBMCs, IL-2, and OKT3 antibody
Expanding selected T cells by culturing with feeder peripheral blood mononuclear cells, interleukin-2, and OKT3 antibody.
Formulating pharmaceutical compositions for injection
Formulating the pharmaceutical composition comprising selected T cells for injection, including intravenous administration.
Formulation components of pharmaceutically acceptable carriers
Using pharmaceutically acceptable carriers such as normal saline, electrolyte solutions, 5% dextrose in water, or Ringer's lactate, optionally supplemented with human serum albumin.
Separating selected T cells from non-specific T cells
Separating selected antigen-specific T cells from autologous T cells lacking antigenic specificity for the mutated amino acid sequences.
Method of treating tumors by administering selected T cells
Treating tumors by administering to a patient the pharmaceutical composition comprising autologous T cells having antigenic specificity for mutated amino acid sequences expressed by the tumor.
Treatment of epithelial cancers including specific cancer types
The method of treatment applies to epithelial cancers including cholangiocarcinoma, melanoma, colon cancer, and rectal cancer.
The claims cover methods for identifying mutated genes in tumors, inducing autologous APCs to present mutated amino acid sequences, co-culturing and selecting autologous T cells specific to these mutations, expanding these T cells, formulating pharmaceutical compositions for administration, and methods of treating cancer using these compositions.
Stated Advantages
Methods rapidly assess a large number of mutations restricted by all patient's MHC molecules simultaneously, identifying the full repertoire of mutation-reactive T cells.
Distinguishes immunogenic cancer mutations from non-immunogenic or silent ones, enabling targeted isolation of effective T cells.
Provides personalized populations of T cells unique to each patient for adoptive cell therapies.
Avoids technical biases and limitations of traditional antigen identification methods, reducing time and labor.
Selects mutation-reactive T cells without co-culture with tumor cell lines, which are difficult to generate.
Potentially reduces toxicity by minimizing destruction of normal cells while targeting cancer cells.
Enables treatment or prevention of cancers non-responsive to other treatments such as chemotherapy, surgery, or radiation.
Documented Applications
Preparation of populations of T cells having antigenic specificity for mutated amino acid sequences encoded by cancer-specific mutations for adoptive cell therapy.
Obtaining T cells with specificity for cancer-specific mutations from various bodily samples including blood and tumor tissues.
Co-culturing autologous T cells with autologous APCs presenting mutated amino acid sequences for selection and expansion.
Formulation of pharmaceutical compositions comprising selected T cells and pharmaceutically acceptable carriers for injection, preferably intravenous administration.
Treatment and prevention of various cancers including epithelial cancers such as cholangiocarcinoma, melanoma, colon cancer, and rectal cancer by administering the pharmaceutical compositions containing mutation-specific T cells.
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