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US 12,178,831 B2Granted
Techniques for generating cell-based therapeutics using recombinant T cell receptor genes
AssigneeUniversity of Kansas
InventorsBrandon Dekosky, Cheng-Yu Chung
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Abstract
The present technology relates generally to compositions and methods for creating recombinant T cell receptor (TCR) libraries and methods of their therapeutic use. The compositions and methods of the present technology are useful for rapid isolation of antigen-specific TCR repertoires as personalized, targeted therapies for cancer and viral infection.
Claims5
A method for preparing a recombinant TCR library, the method comprising
- (a) lysing a single T cell in a compartment having a volume of 5 nL or less,
- (b) isolating mRNA of the lysed single T cell in the compartment using an mRNA capture reagent;
- (c) reverse transcribing the mRNA into cDNA in the compartment by overlap extension reverse transcription PCR (OE-RT-PCR) to generate linked cDNA products, wherein the linked cDNA products comprise
- (i) a first polynucleotide encoding a T cell receptor (TCR) a polypeptide and a second polynucleotide encoding a TCRβ polypeptide;
- or
- (ii) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide,
- wherein the first and second polynucleotides are a cognate pair and are operably linked via a linker polynucleotide;
- (d) amplifying the linked cDNA products using nested PCR to generate TCR amplicons;
- (e) generating a recombinant TCR library by introducing the TCR amplicons into an expression vector or a population of cells.
6 more elements
Dependencies are inferred from the claim text.