Methods of Sequencing, Determining, Pairing, and Validating Therapeutic Agents and Disease Specific Antigens

Abstract

Provided herein are methods and composition for immune repertoire sequencing and single cell barcoding. The methods and compositions can be used to pair any two sequences originating from a single cell, such as heavy and light chain antibody sequences, for antibody discovery, disease and immune diagnostics, and low error sequencing.

1 - 255 . (canceled) 
     
     
         256 . An immunoglobulin (Ig) or T-cell receptor (TCR) polypeptide produced by a process comprising:
 (a) forming a plurality of vessels, at least one vessel comprising:
 a single tumor infiltrating lymphocyte (TIL) cell or a single non-TIL cell, wherein the single cell is isolated from a first biological sample from a first subject, 
 a molecular barcoded polynucleotide, 
 a vessel barcoded polynucleotide, 
 a forward primer and a reverse primer for amplifying the vessel barcoded polynucleotide, and 
 a reverse transcriptase, 
   wherein the molecular barcoded polynucleotide comprises a 5′ region complementary to a region of the vessel barcoded polynucleotide;   (b) generating a cDNA polynucleotide by reverse transcription of an RNA from the single cell, wherein the reverse transcriptase adds three or more non-template nucleotides to the 3′ end of the cDNA polynucleotide;   (c) annealing the molecular barcoded polynucleotide to the three or more non-template nucleotides of the cDNA polynucleotide, and extending the cDNA polynucleotide to generate a single-barcoded cDNA polynucleotide;   (d) amplifying the vessel barcoded polynucleotide using the forward primer and the reverse primer, thereby generating an amplified product, annealing the amplified product to the single-barcoded cDNA polynucleotide, and extending the single-barcoded cDNA polynucleotide to generate a dual-barcoded cDNA polynucleotide;   (e) sequencing the dual-barcoded cDNA polynucleotide thereby obtaining sequence information;   (f) selecting an Ig or a TCR polynucleotide sequence from a TIL based on the sequence information;   (g) producing an Ig or TCR polypeptide encoded by the selected polynucleotide; and   (h) engineering the produced polynucleotide to have additional nonproteinaceous moieties, modifying glycosylation patterns of the produced polynucleotide, and/or conjugating a therapeutic moiety or a detectable moiety to the produced polynucleotide.   
     
     
         257 . The Ig or TCR polypeptide of  claim 256 , wherein the RNA from the single cell encodes a variable region of an immunoglobulin (Ig) or a T-cell receptor (TCR) polypeptide. 
     
     
         258 . The Ig or TCR polypeptide of  claim 256 , wherein the single cell is not sorted or selected based on an extracellular cell marker prior to the sequencing. 
     
     
         259 . The Ig or TCR polypeptide of  claim 256 , wherein the selected polynucleotide sequence comprises 1-500 unique Ig or TCR polynucleotide sequences. 
     
     
         260 . The Ig or TCR polypeptide of  claim 256 , wherein the selecting is based on comparing the sequence information to sequence information obtained from a second biological sample, wherein the second biological sample comprises:
 (i) a normal tissue sample from the first subject,   (ii) a normal biological sample from a healthy subject, and/or   (iii) a biological sample from a second subject, wherein the first and second subject have a same disease.   
     
     
         261 . The Ig or TCR polypeptide of  claim 256 , wherein the selecting is based on:
 (i) determining that the Ig or TCR polynucleotide sequence is present in the sequence information but absent in sequence information obtained from a second biological sample,   (ii) determining that the Ig or TCR polynucleotide sequence is enriched in the sequence information compared to sequence information obtained from a second biological sample,   (iii) a pre-determined isotype profile of the Ig or TCR polynucleotide sequence in the sequence information,   (iv) a size or a frequency of a phylogenetic cluster of the Ig or TCR polynucleotide sequence in the sequence information, or   (v) a similarity between the Ig or TCR polynucleotide sequence in the sequence information and the sequence information obtained from a second biological sample.   
     
     
         262 . The Ig or TCR polypeptide of  claim 256 , wherein the selected polynucleotide sequence comprises an Ig polynucleotide encoding at least one of an Ig heavy chain (IgH), an Ig light chain (IgL), an Ig constant domain region, an Ig heavy chain variable region (VH), or an Ig light chain variable region (VL); or wherein the selected polynucleotide comprises a TCR polynucleotide encoding at least one of a TCRα chain, a TCRβ chain, a TCRγ chain, a TCRδ chain, a TCRα chain variable region (Vα), a TCRβ chain variable region (νβ), a TCRγ chain variable region (Vγ), a TCRδ chain variable region (Vδ), a TCRα constant domain, a TCRβ constant domain, a TCRγ constant domain, or a TCRδ constant domain. 
     
     
         263 . The Ig or TCR polypeptide of  claim 256 , wherein the process further comprises, prior to producing the Ig or TCR polypeptide, at least one of pairing an IgH with an IgL from a same B-cell, pairing a TCRα chain with a TCRβ chain from a same T-cell, or pairing a TCRγ chain with a TCRδ chain from a same T-cell. 
     
     
         264 . The Ig or TCR polypeptide of  claim 256 , wherein the Ig or TCR polypeptide comprises a framework region sequence comprising one or more mutated framework residues. 
     
     
         265 . The Ig or TCR polypeptide of  claim 256 , wherein the Ig or TCR polypeptide does not substantially interact with or bind to a cell of normal adjacent tissue or a cell from a corresponding tissue in a healthy subject. 
     
     
         266 . The Ig or TCR polypeptide of  claim 256 , wherein the Ig or TCR polypeptide has a K D  of about 1×10 −7  M or less for a disease-associated protein or a disease-specific protein. 
     
     
         267 . The Ig or TCR polypeptide of  claim 256 , wherein the process further comprises identifying a target antigen of the produced Ig or TCR polypeptide. 
     
     
         268 . The Ig or TCR polypeptide of  claim 267 , wherein the identifying comprises performing a whole genome siRNA screen, a protein display screen, a yeast-two-hybrid screen, a 2D gel electrophoresis, a protein array, a proteome screen, an immunoprecipitation, a mass spectrometry, a cell-mediated cytotoxicity assay, or a binding assay. 
     
     
         269 . The Ig or TCR polypeptide of  claim 267 , wherein the target antigen is a disease-associated or disease-specific target antigen. 
     
     
         270 . The Ig or TCR polypeptide of  claim 269 , wherein the disease is an autoimmune disease, a cancer, or a precancerous disease. 
     
     
         271 . The Ig or TCR polypeptide of  claim 256 , wherein the Ig or TCR polypeptide is capable of killing a diseased cell. 
     
     
         272 . The Ig or TCR polypeptide of  claim 256 , wherein the first biological sample comprises at least one TIL cell and at least one non-TIL cell present in the first biological sample at a ratio of 1:10,000 or less. 
     
     
         273 . The Ig or TCR polypeptide of  claim 256 , wherein the first biological sample is a solid tissue sample. 
     
     
         274 . The Ig or TCR polypeptide of  claim 256 , wherein the TIL cell comprises a T cell or a B cell, or wherein the non-TIL cell comprises an epithelial cell, a lymphocyte, or a cancer cell. 
     
     
         275 . The Ig or TCR polypeptide of  claim 256 , wherein the Ig or TCR polypeptide comprises a variable domain of an Ig or TCR polypeptide, or a fragment thereof.