Variant libraries of the immunological synapse and synapse and synthesis thereof

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 reference domain of a CAR. 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 be 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.

What is claimed is: 
     
         1 . A nucleic acid library, the nucleic acid library comprising at least 10,000 nucleic acids, wherein each nucleic acid encodes for a preselected variant of a reference sequence that encodes for a chimeric antigen receptor or a functional domain thereof. 
     
     
         2 . The nucleic acid library of  claim 1 , wherein the functional domain of the chimeric antigen receptor comprises an antigen recognition domain, a hinge domain, a transmembrane domain, or an intracellular domain. 
     
     
         3 . The nucleic acid library of  claim 1 , wherein the library comprises at least 1,000,000 nucleic acids. 
     
     
         4 . The nucleic acid library of  claim 1 , wherein each nucleic acid is 100 bases to 2000 bases in length. 
     
     
         5 . The nucleic acid library of  claim 1 , wherein each nucleic acid is attached to a vector sequence. 
     
     
         6 . The nucleic acid library of  claim 5 , wherein the vector sequence is a viral vector sequence. 
     
     
         7 . A nucleic acid library, the nucleic acid library comprising a plurality of nucleic acids, wherein each nucleic acid encodes for a preselected variant of a reference sequence that encodes for a chimeric antigen receptor or a functional domain thereof, wherein the plurality of nucleic acids comprises all variations for at least two positions in the reference sequence, and wherein the at least two positions encode sequences from 5 to 20 different codons. 
     
     
         8 . The nucleic acid library of  claim 7 , wherein the functional domain of the chimeric antigen receptor comprises an antigen recognition domain, a hinge domain, a transmembrane domain, or an intracellular domain. 
     
     
         9 . The nucleic acid library of  claim 7 , wherein the plurality of nucleic acids comprises all variations for 2, 3, 4, or 5 positions in the reference sequence. 
     
     
         10 . The nucleic acid library of  claim 7 , wherein the at least two positions encode sequences from 10 to 20 different codons. 
     
     
         11 . The nucleic acid library of  claim 7 , wherein the at least two positions encode sequences for about 10 different codons. 
     
     
         12 . The nucleic acid library of  claim 7 , wherein each nucleic acid is 100 bases to 2000 bases in length. 
     
     
         13 . An oligonucleotide library, the oligonucleotide library comprising at least 10,000 oligonucleotides, wherein each oligonucleotide encodes for a preselected variant of a reference sequence that encodes for a region of a chimeric antigen receptor, wherein the region comprises a portion of an antigen recognition domain, a hinge domain, a transmembrane domain, or an intracellular domain. 
     
     
         14 . The oligonucleotide library of  claim 13 , wherein the library comprises at least 1,000,000 oligonucleotides. 
     
     
         15 . The oligonucleotide library of  claim 13 , wherein each oligonucleotide is 12 to 500 bases in length. 
     
     
         16 . The oligonucleotide library of  claim 13 , wherein each oligonucleotide is attached to a vector sequence. 
     
     
         17 . An oligonucleotide library, the oligonucleotide library comprising a plurality of oligonucleotides, wherein each oligonucleotide encodes for a preselected variant of a reference sequence that encodes for a region of a chimeric antigen receptor, wherein the region comprises a portion of an antigen recognition domain, a hinge domain, a transmembrane domain, or an intracellular domain, wherein each oligonucleotide is at least 12 bases in length, wherein the plurality of oligonucleotides comprises all variations for at least two positions in the reference sequence, and wherein the at least two positions encode sequences from 5 to 20 different codons. 
     
     
         18 . The oligonucleotide library of  claim 17 , wherein the plurality of oligonucleotides comprises all variations for 2, 3, 4, or 5 positions. 
     
     
         19 . The oligonucleotide library of  claim 17 , wherein the at least two positions encode sequences from 10 to 20 different codons. 
     
     
         20 . The oligonucleotide library of  claim 17 , wherein the at least two positions encode sequences for about 10 different codons. 
     
     
         21 . The oligonucleotide library of  claim 17 , wherein each oligonucleotide is 12 to 500 bases in length. 
     
     
         22 . A nucleic acid library comprising at least 400 nucleic acids, wherein a first plurality of the at least 400 nucleic acids encodes for a variant of a reference sequence that encodes for an antigen recognition domain, and wherein a second plurality of the at least 400 nucleic acids encodes for a variant of a reference sequence that encodes for a hinge domain, a transmembrane domain, or an intracellular domain of a chimeric antigen receptor (CAR). 
     
     
         23 . The nucleic acid library of  claim 22 , wherein each nucleic acid is at least 100 bases in length. 
     
     
         24 . The nucleic acid library of  claim 22 , wherein each nucleic acid is 100 to 2000 bases in length. 
     
     
         25 . The nucleic acid library of  claim 22 , wherein the first plurality of the at least 400 nucleic acids comprises all variations for at least two positions in the reference sequence. 
     
     
         26 . The nucleic acid library of  claim 22 , wherein the first plurality of the at least 400 nucleic acids comprises all variations for 2, 3, 4, or 5 positions in the reference sequence. 
     
     
         27 . The nucleic acid library of  claim 22 , wherein the second plurality of the at least 400 nucleic acids comprises all variations for at least two positions in the reference sequence. 
     
     
         28 . The nucleic acid library of  claim 22 , wherein the second plurality of the at least 400 nucleic acids comprises all variations for 2, 3, 4, or 5 positions in the reference sequence. 
     
     
         29 . The nucleic acid library of  claim 25 , wherein the at least two positions encode sequences from 5 to 20 different codons. 
     
     
         30 . The nucleic acid library of  claim 25 , wherein the at least two positions encode sequences from 10 to 20 different codons. 
     
     
         31 . The nucleic acid library of  claim 25 , wherein the at least two positions encode sequences for about 10 different codons.