US2021115594A1PendingUtilityA1

Variant libraries of the immunological synapse and synthesis thereof

67
Assignee: TWIST BIOSCIENCE CORPPriority: Dec 16, 2016Filed: Dec 23, 2020Published: Apr 22, 2021
Est. expiryDec 16, 2036(~10.4 yrs left)· nominal 20-yr term from priority
Inventors:Anthony James CoxSiyuan Chen
G01N 33/575A61K 40/4269A61K 40/4268A61K 40/11A61K 40/32A61K 39/001188A61K 39/001186A61K 39/001182C40B 30/04C07K 2317/70C12N 15/1068G01N 33/505C07K 2317/94C40B 40/08C07K 14/7051C07K 2317/565G01N 33/6812G01N 2800/7028C12N 15/102G01N 33/6893C07K 2317/92G01N 33/574A61K 39/0011
67
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Claims

Abstract

Disclosed herein are methods for the generation of highly accurate nucleic acid libraries encoding for predetermined variants of a nucleic acid sequence. The nucleic acid sequence may encode for all or part of a TCR or a TCR-binding antigen. The degree of variation may be complete, resulting in a saturated variant library, or less than complete, resulting in a non-saturating library of variants. The variant nucleic acid libraries described herein may designed for further processing by transcription or translation. The variant nucleic acid libraries described herein may be designed to generate variant RNA, DNA and/or protein populations. Further provided herein are method for identifying variant species with increased or decreased activities, with applications in regulating biological functions and the design of therapeutics for treatment or reduction of a disease, such as cancer.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A nucleic acid library, wherein the nucleic acid library comprises at least 3000 variant nucleic acids, wherein each variant nucleic acid encodes for a variant gene or gene fragment for a T cell receptor (TCR) protein, and wherein a region selected for variation spans up to 1000 bases in length. 
     
     
         2 . The nucleic acid library of  claim 1 , wherein the variant gene or gene fragment is from a variable domain. 
     
     
         3 . The nucleic acid library of  claim 2 , wherein the variable domain is a variable domain of TCR alpha, TCR beta, TCR gamma, or TCR delta. 
     
     
         4 . The nucleic acid library of  claim 2 , wherein the variable domain is specific to a cancer antigen. 
     
     
         5 . The nucleic acid library of  claim 4 , wherein the cancer antigen is MAGE A3, MAGE A12, MAGE A2, MAGE A6, NY-ESO-1, or CEA. 
     
     
         6 . The nucleic acid library of  claim 1 , wherein the variant gene or gene fragment is from a constant domain. 
     
     
         7 . The nucleic acid library of  claim 1 , wherein the variant gene or gene fragment allows for generation of a TCR protein having increased specificity, avidity, affinity, stability, or expression. 
     
     
         8 . A polynucleotide library, wherein the polynucleotide library comprises at least 3000 variant nucleic acids, wherein each polynucleotide is at least 15 bases in length, wherein each polynucleotide encodes for a variant within a variable domain of a T cell receptor (TCR) protein or fragment thereof, wherein the variable domain comprises up to 1000 bases, and wherein each variant nucleic acid comprises at least one variation at a preselected codon for an amino acid residue in an antigen contacting interface. 
     
     
         9 . The polynucleotide library of  claim 8 , wherein the variable domain is a variable domain of TCR alpha, TCR beta, TCR gamma, or TCR delta. 
     
     
         10 . The polynucleotide library of  claim 8 , wherein the antigen is a cancer antigen. 
     
     
         11 . The polynucleotide library of  claim 10 , wherein the cancer antigen is MAGE A3, MAGE A12, MAGE A2, MAGE A6, NY-ESO-1, or CEA. 
     
     
         12 . The polynucleotide library of  claim 8 , wherein each variant nucleic acid comprises a plurality of variations at preselected codons for amino acid residues in the antigen contacting interface. 
     
     
         13 . The polynucleotide library of  claim 12 , wherein the plurality of variations at preselected codons for amino acid residues comprises up to 100 residues. 
     
     
         14 . The polynucleotide library of  claim 12 , wherein the plurality of variations at preselected codons for amino acid residues comprises up to 30 residues. 
     
     
         15 . The polynucleotide library of  claim 12 , wherein the plurality of variations at preselected codons for amino acid residues comprises up to 5 residues. 
     
     
         16 . The polynucleotide library of  claim 8 , wherein the variant allows for generation of a TCR protein having increased specificity, avidity, affinity, stability, or expression. 
     
     
         17 . The polynucleotide library of  claim 8 , wherein each variant nucleic acid further comprises at least one variation at a preselected codon for an amino acid residue within a constant domain of the T cell receptor (TCR) protein or fragment thereof. 
     
     
         18 . A polynucleotide library, wherein the polynucleotide library comprises at least 3000 variant polynucleotides, wherein each polynucleotide is at least 15 bases in length, wherein each polynucleotide encodes for a variant within a constant domain of a T cell receptor (TCR) protein or fragment thereof, wherein the constant domain comprises up to 1000 bases, and wherein the variant comprises at least one variation at a preselected codon for an amino acid residue in an antigen contacting interface. 
     
     
         19 . The polynucleotide library of  claim 18 , wherein the constant domain is a constant domain of TCR alpha, TCR beta, TCR gamma, or TCR delta. 
     
     
         20 . The polynucleotide library of  claim 18 , wherein the antigen is a cancer antigen. 
     
     
         21 . The polynucleotide library of  claim 20 , wherein the cancer antigen is MAGE A3, MAGE A12, MAGE A2, MAGE A6, NY-ESO-1, or CEA. 
     
     
         22 . The polynucleotide library of  claim 18 , wherein each polynucleotide comprises a plurality of variations at preselected codons for amino acid residues. 
     
     
         23 . The polynucleotide library of  claim 22 , wherein the plurality of variations at preselected codons for amino acid residues comprises up to 100 residues. 
     
     
         24 . The polynucleotide library of  claim 22 , wherein the plurality of variations at preselected codons for amino acid residues comprises up to 30 residues. 
     
     
         25 . The polynucleotide library of  claim 22 , wherein the plurality of variations at preselected codons for amino acid residues comprises up to 5 residues. 
     
     
         26 . A nucleic acid library, the nucleic acid library comprising at least 10,000 variant nucleic acids, wherein each variant nucleic acid is about 500 to about 1000 bases in length, wherein each variant nucleic acid encodes for a variant of a reference sequence that encodes an exon sequence for a T cell receptor protein or fragment thereof, and wherein the reference sequence is from a subject that has a cancer. 
     
     
         27 . A nucleic acid library, the library comprising at least about 1,000,000 variant nucleic acids, wherein each variant nucleic acid is about 500 to about 1000 bases in length, wherein each variant nucleic acid encodes for a variant of a reference sequence that encodes an exon sequence for a T cell receptor protein or fragment thereof, wherein the reference sequence of the T cell receptor protein or fragment thereof is selected based on comparing T cell receptor gene sequence from a plurality of subjects for a nucleic acid sequence that is common amongst the subjects, and wherein at least a portion of the subjects are diagnosed with a cancer. 
     
     
         28 . The nucleic acid library of any one of  claim 26  or  27 , wherein the cancer is a solid cancer or a hematopoietic cancer. 
     
     
         29 . The nucleic acid library of any one of  claim 26  or  27 , wherein the library comprises about 10,000,000 variant nucleic acids. 
     
     
         30 . The nucleic acid library of any one of  claim 26  or  27 , wherein each variant nucleic acid is about 600 to about 900 bases in length. 
     
     
         31 . The nucleic acid library of any one of  claim 26  or  27 , wherein each variant nucleic acid is in a vector sequence. 
     
     
         32 . The nucleic acid library of  claim 31 , wherein the vector sequence is a viral vector sequence. 
     
     
         33 . A protein library comprising proteins encoded by the nucleic acid library of any one of  claims 1  to  7  or  26  to  32 . 
     
     
         34 . A cell library comprising the nucleic acid library of any one of  claims 1  to  7  or  26  to  32 .

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