Modular polypeptide libraries and methods of making and using same
The present disclosure provides synthetic modular polypeptide libraries and nucleic acids encoding such synthetic modular polypeptide libraries. Also provided are methods of making synthetic modular polypeptide libraries and nucleic acids encoding synthetic modular polypeptide libraries. Methods of screening a synthetic modular polypeptide library to identify a selected phenotype associated with a member of a synthetic modular polypeptide library are also provided where such methods find use in both in vitro and in vivo assays.
1. A method of screening, comprising:
(a) introducing a library of nucleic acids into a population of cells, wherein:
(i) the members of the library each comprise a barcode region and a coding sequence that is operably linked to a promoter;
(ii) within each member, the barcode region is separate from the coding sequence;
(iii) the coding sequence encodes a modular protein having at least a first domain, a second domain, and a third domain;
(iv) the sequences of the first domain, the second domain and the third domain independently vary in the library;
(v) the barcode region comprises at least a first barcode, a second barcode and a third barcode, and
(vi) within each member, the first barcode, the second barcode and the third barcode correspond to and identify the sequence of the first domain, the sequence of the second domain, and the sequence of the third domain of the modular protein, respectively;
(b) exposing the cells to a stimulus;
(c) sequencing the barcode region from a cell that exhibits a phenotype in response the stimulus, thereby determining the identity of the first, second, third domains of the modular protein in the cell.
2. The method of claim 1 , wherein the modular protein is a receptor.
3. The method of claim 1 , wherein the receptor comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
4. The method of claim 2 , wherein the receptor is a chimeric antigen receptor (CAR).
5. The method of claim 4 , wherein the stimulus is an antigen-presenting cell that displays on its surface an antigen that is bound by the CAR.
6. The method of claim 1 , wherein the cells are eukaryotic cells.
7. The method of claim 6 , wherein the eukaryotic cells are human cells.
8. The method of claim 7 , wherein the human cells are human T cells.
9. The method of claim 1 , wherein, within each member, the barcode region is 3′ of the coding sequence.
10. The method of claim 1 , wherein the members of the library contain, from 5′ to 3′,
a sequence that encodes the first domain of the modular protein;
a sequence that encodes the second domain of the modular protein;
a sequence that encodes the third domain of the modular protein;
the third barcode;
the second barcode; and
the first barcode.
11. The method of claim 1 , wherein the library comprises at least 100 members.