Methods of isolating neoantigen-specific T cell receptor sequences
Inventors
Lu, Yong-Chen • Fitzgerald, Peter • Zheng, Zhili • Rosenberg, Steven A.
Assignees
US Department of Health and Human Services
Publication Number
US-11898207-B2
Publication Date
2024-02-13
Expiration Date
2038-03-28
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Abstract
Disclosed are methods of isolating paired T cell receptor (TCR) alpha and beta chain sequences, or an antigen-binding portion thereof. Also disclosed are methods of automatically identifying the TCR alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. Methods of preparing a population of cells that express paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof, are also disclosed. Isolated pairs of TCR alpha and beta chain sequences and isolated populations of cells prepared by the methods are also disclosed.
Core Innovation
The invention provides methods of isolating paired T cell receptor (TCR) alpha and beta chain sequences, or an antigen-binding portion thereof. The methods include isolating T cells from a biological sample that have antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, co-culturing these T cells with antigen presenting cells (APCs) presenting the mutated amino acid sequence to induce expression of T cell activation markers, sorting the co-cultured T cells into single-cell samples, and isolating and sequencing mRNA from each single cell to identify the paired TCR alpha and beta chain sequences.
The methods further involve aligning the sequences of cDNA fragments obtained from sequencing to known sequences of T cell activation markers and a reference TCR sequence database to identify TCR alpha and beta chain variable region sequences and complementarity determining region 3 (CDR3) sequences. The most abundant fragments encoding the same alpha and beta chain CDR3 sequences are collected to assemble one or more nucleotide sequences encoding the paired TCR alpha and beta chains, which have antigenic specificity for the mutated amino acid sequence.
The problem being solved is the difficulty in identifying and isolating functional TCRs that specifically recognize cancer antigens, such as neoantigens, from patients. Due to the large diversity of TCR sequences, the necessity for correct pairing of alpha and beta chains for desired antigen specificity, and the existence of T cells with more than one functional alpha chain, conventional methods such as T cell cloning by limiting dilution are time-consuming, laborious, and have lower success rates. The invention addresses these challenges by providing automated, efficient methods to isolate paired TCR sequences with the desired specificity for personalized TCR therapy targeting cancer-specific mutations.
Claims Coverage
The independent claims cover methods for isolating nucleic acids encoding paired TCR alpha and beta chains with antigen-binding specificity, computerized identification of TCR sequences, and preparation of cell populations expressing such TCRs.
Method of isolating TCR nucleic acids encoding paired alpha and beta chains with antigen specificity
A multi-step method involving: isolating T cells from a biological sample having specificity for a mutated amino acid sequence encoded by a cancer-specific mutation; co-culturing these T cells with antigen presenting cells presenting the mutated sequence to express T cell activation markers; sorting into single-cell samples; isolating and sequencing mRNA with tag labeling; aligning sequences to identify single T cells expressing activation markers; aligning sequences to a TCR database to identify alpha and beta chain V segment sequences; identifying CDR3 sequences in these fragments; counting fragments sharing the same CDR3 amino acid sequences; collecting the most abundant fragments encoding same alpha and beta CDR3 sequences (optionally including second highest alpha sequences); identifying corresponding V segment sequences; and assembling nucleotide sequences encoding TCR alpha and beta chain polypeptides or antigen-binding portions thereof having antigenic specificity for the cancer mutation.
Identification of T cell activation markers for selecting antigen-specific T cells
Use of one or more T cell activation markers selected from interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor alpha (TNF-α), programmed cell death 1 (PD-1), lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1BB, OX40, CD107a, granzyme B, granulocyte/monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22 to identify activated T cells.
Labeling mRNA from single-cell samples with different tags
Labeling the mRNA from each separate single T cell sample with distinct tags (e.g., barcodes) to permit identification of fragments derived from specific cells during sequencing.
Identification of TCR CDR3 sequences by conserved amino acid motifs
Identifying TCR CDR3 sequences by locating cDNA sequences encoding conserved amino acid residues positioned near the C-terminus of the variable (V) segment, specifically motifs that include conserved cysteine and phenylalanine or tryptophan residues.
Identification and assembly of TCR alpha and beta chain constant region sequences
Identifying the TCR alpha and beta chain constant (C) region sequences from the most abundant multiple fragments and assembling nucleotide sequences encoding TCR alpha and beta chain polypeptides with variable, constant, and CDR3 regions.
Use of exogenous constant region sequences in TCR assembly
Assembling nucleotide sequences encoding TCR alpha and beta chains comprising the identified V segment sequence and CDR3 sequence coupled to exogenous (non-native) constant region sequences, allowing for chimeric or hybrid TCRs.
Computerized method for automatic identification of TCR sequences
Automated computational alignment and identification steps where a user computing device receives sequences of cDNA fragments, aligns them to a TCR reference database, identifies CDR3 regions, counts fragment occurrences, collects highest frequency CDR3 sequences, and determines corresponding V segment sequences, thus identifying paired TCR alpha and beta chain sequences.
Preparation of cell populations expressing isolated TCRs
Introducing nucleic acids encoding isolated paired TCR alpha and beta chains, or antigen-binding portions thereof, into host cells to obtain cells expressing the TCR, optionally followed by expansion of these host cells for therapeutic use.
The claims collectively cover an integrated method to isolate, identify, and assemble nucleotide sequences encoding paired TCR alpha and beta chains having antigenic specificity for cancer-specific mutated amino acid sequences, including automated computational identification and subsequent preparation of host cells expressing such TCRs.
Stated Advantages
Significantly reduces time and cost necessary to isolate and identify TCR sequences specific for cancer neoantigens after obtaining a biological sample.
Allows identification of both the cancer antigen and the TCR recognizing that antigen, facilitating highly personalized TCR therapy targeting neoantigens.
Less time-consuming, less laborious, and has a higher success rate compared to conventional T cell cloning by limiting dilution.
Efficiently identifies correct TCR alpha and beta chain pairs in T cells expressing more than one functional alpha chain.
Enables identification and isolation of paired TCR alpha and beta chain sequences from highly diverse T cell populations.
Documented Applications
Adoptive cell therapy using cells genetically engineered to express isolated paired TCR alpha and beta chain sequences or antigen-binding portions thereof for treating or preventing cancer.
Use of pharmaceutical compositions comprising isolated paired TCR alpha and beta chains, antigen-binding portions thereof, or cells expressing these TCRs for cancer immunotherapy.
Personalized TCR therapy targeting mutated amino acid sequences encoded by cancer-specific mutations.
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