EA041085B1 - LIBRARIES OF MODULAR POLYPEPTIDES AND METHODS FOR THEIR PRODUCTION AND APPLICATION - Google Patents

Description

cross reference

This application claims priority under U.S. Provisional Application No. 62/212,999, filed September 1, 2015, the contents of which are hereby incorporated by reference in their entirety.

Federally funded research statement.

The present invention was made with government support under grants EY016546, P50 GM081879, R01 CA196277, F32 GM006499 and R01 GM055040 awarded by the National Institutes of Health (USA). The US Government has certain rights to the present invention.

Inclusion of a sequence listing provided as a text file via a link.

The sequence listing is provided in this application as a text file UCSF518WO SeqList_ST25.txt created on August 29, 2016 and having a size of 145 kilobytes. The contents of the text file are incorporated herein by reference in their entirety.

Introduction

Many eukaryotic proteins carry out their functions through modular domains or motifs that control or facilitate the input and output functions of the protein as a whole. Currently, there are ways to rearrange and recombine the modular domains of eukaryotic proteins, allowing you to create new proteins with modified ratios of input/output functions and completely new overall functions. The successful construction of individual modular proteins by simple recombination of domains that mediate input and output functions served as proof of the principle of the mentioned modular approach to the development of new proteins.

The construction of new synthetic proteins, for example for use in therapeutic cells, has hitherto been based on a sequential construction approach. Similarly, transfection and screening of such synthetic proteins was also performed using a low throughput approach, using single constructs that were screened on an individual basis, or, in some cases, a small number of single constructs were screened simultaneously. Simultaneous screening, while more efficient than screening individual constructs on an individual basis, has limited scalability due to the requirement that individual constructs must remain physically separate to facilitate eventual identification of effective constructs. Individual screening of a large number of new proteins is cumbersome in in vitro quantitation, but becomes even more unacceptably difficult and costly when testing moves to the quantification stage in in vivo models. This individual production and individual screening significantly limits the rate of development of new synthetic proteins.

Brief description of the invention

The present invention provides synthetic modular polypeptide libraries and nucleic acids encoding said synthetic modular polypeptide libraries. The present invention also provides methods for obtaining libraries of synthetic modular polypeptides and nucleic acids encoding libraries of synthetic modular polypeptides. The present invention also provides methods for screening a library of synthetic modular polypeptides to identify a selected phenotype associated with an element of the library of synthetic modular polypeptides, which methods can be used in quantitative studies in vitro and in vivo conditions.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said method comprising: the barcode nucleic acid library contains a plurality of elements, each of the plurality of elements containing a nucleotide sequence encoding a different synthetic modular polypeptide; and b) identifying the genetically modified host cell in the heterogeneous population that exhibits the selected phenotype in response to the stimulus.

The present invention also provides a method wherein the synthetic modular polypeptide is a chimeric antigen receptor (CAR) polypeptide and wherein the stimulus is an antigen presenting cell that expresses on its surface an antigen that is associated with the CAR.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said stimulus being contacted with a co-stimulatory molecule.

The present invention also provides a method for identifying a selected phenotype in a cell,

- 1 041085 associated with a synthetic modular polypeptide, and the specified synthetic modular polypeptide is a modular receptor polypeptide.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said modular receptor polypeptide being a chimeric Notch receptor polypeptide.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said stimulus being a chimeric Notch receptor polypeptide ligand.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said synthetic modular polypeptide being a modular scaffold protein.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said synthetic modular polypeptide being a protein kinase or protein phosphatase modular protein.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said synthetic modular polypeptide being a modular transcriptional regulatory protein.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said synthetic modular polypeptide being a modular epigenetic regulatory protein.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said synthetic modular polypeptide being a recombinase or nuclease modular protein.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said selected phenotype having a phenotypic feature comprising two or more phenotypes.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, comprising sequencing a nucleic barcode sequence from an identified genetically modified host cell to identify a synthetic modular polypeptide associated with the phenotype.

The present invention also provides a method for identifying a selected phenotype associated with a synthetic modular polypeptide in a cell, comprising quantifying the synthetic modular polypeptide associated with the phenotype.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, comprising quantifying a single module of synthetic modular polypeptides from a nucleic acid library containing barcode nucleic acid sequences that is associated with the phenotype.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, wherein each nucleotide sequence encoding a different synthetic modular polypeptide contains a sequence encoding a detectable reporter that is operably linked to a nucleotide sequence encoding a synthetic modular polypeptide, wherein said the method further comprises separating a heterogeneous population of genetically modified host cells based on an expressed detectable reporter.

The present invention also provides a method for identifying a selected phenotype associated with a synthetic modular polypeptide in a cell, the identification being performed in vitro or ex vivo.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said phenotype being one or more of: a) proliferation; b) production of cytokines; c) expression of a cell surface marker; d) reporter protein expression.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said method comprising a nucleic acid library containing nucleic barcode sequences that contains 100 or more unique elements, and the identification includes screening 100 or more unique elements of the library for the chosen phenotype.

The present invention also provides a method for identifying in a cell a selected phenotype associated with a synthetic modular polypeptide, said method comprising a nucleic acid library containing nucleic barcode sequences containing a plurality of elements, each of the plurality of elements containing a nucleotide sequence encoding a synthetic modular polypeptide containing a modular domain selected from the group consisting of: an antigen-binding domain, a specific binding domain, or a protein

- 2 041085 specific binding partner, co-stimulatory domain, co-inhibitory domain, intracellular signaling domain, transmembrane domain, scaffold protein domain, protein kinase protein domain, protein phosphatase protein domain, receptor tyrosine kinase protein domain, lipid kinase protein domain, lipid phosphatase protein domain, ubiquitinylase protein domain, a deubiquitinylase protein domain, a SUMOylase protein domain, an acetylase protein domain, a deacetylase protein domain, a methylase protein domain, a demethylase protein domain, a nuclease protein domain, a recombinase protein domain, a transcription factor protein domain, and combinations thereof.

The present invention provides a library of nucleic acids containing nucleic barcode sequences, said library containing: a plurality of unique polynucleotides, each containing a nucleotide sequence encoding a unique synthetic modular polypeptide, each unique polynucleotide containing: i) a coding region encoding a unique a synthetic modular polypeptide comprising a first coding sequence encoding a first module connected in open reading frame to a second coding sequence encoding a second module, ii) a barcode nucleic sequence region containing a first barcode nucleic sequence specific for the first coding sequence, connected with a second nucleic sequence-barcode specific for the second coding sequence, wherein said first and second nucleic sequences-barcodes arranged in reverse order from the 5' end to the 3' end with respect to the first and second coding sequences; and while sequencing each region of the nucleic sequence-barcode allows you to identify each unique synthetic modular polypeptide.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences in which said first and second coding sequences are directly linked without any intermediate non-coding nucleotides.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, in which a plurality of unique polynucleotides contains at least 1000 unique polynucleotides.

The present invention also provides a library of nucleic acids containing barcode nucleic acid sequences, in which the barcode nucleic acid sequence region is located in the 5' direction relative to the coding region.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, in which the coding region is located in the direction of the 5'end relative to the region of the nucleic barcode sequence.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and said unique synthetic modular polypeptide contains a costimulatory domain.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, which contains the first and second modules, and the specified first and second modules contain different costimulatory domains.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences that includes a plurality of unique polynucleotides, each containing a nucleotide sequence encoding a unique synthetic modular polypeptide, each unique polynucleotide further comprising a promoter sequence operably linked to a coding region and a reporter a sequence encoding a detectable polypeptide, wherein said detectable polypeptide is an optically detectable polypeptide containing, for example, an optically detectable fluorescent polypeptide.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, which contains a coding region, and the specified coding region additionally contains a third coding sequence encoding the third module connected with the preservation of the open reading frame with the second coding sequence, and the region of the nucleic sequence-barcode contains the third nucleic sequence-barcode, specific for the third coding sequence, connected to the second nucleic sequence-barcode, and the specified first, second and third nucleic sequences -barcodes are arranged in reverse order from the 5' end to the 3' end with respect to the first, second and third coding sequences.

- 3 041085

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and these unique synthetic modular polypeptides encoded by each unique polynucleotide of the set are chimeric antigen receptor (CAR) polypeptides.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and these unique synthetic modular polypeptides encoded by each unique polynucleotide of the set are modular receptor polypeptides.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences encoding modular receptor polypeptides, said modular receptor polypeptides being chimeric Notch receptor polypeptides.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and these unique synthetic modular polypeptides encoded by each unique polynucleotide of the set are modular scaffold proteins.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and these unique synthetic modular polypeptides encoded by each unique polynucleotide of the set are modular proteins of protein kinases or protein phosphatases.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and these unique synthetic modular polypeptides encoded by each unique polynucleotide of the set are modular transcriptional regulatory proteins.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and these unique synthetic modular polypeptides encoded by each unique polynucleotide of the set are modular epigenetic regulatory proteins.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and these unique synthetic modular polypeptides encoded by each unique polynucleotide of the set are modular proteins of recombinases or nucleases.

The present invention also provides a library of nucleic acids containing nucleic barcode sequences, which contains a plurality of unique polynucleotides, each of which contains a nucleotide sequence encoding a unique synthetic modular polypeptide, and these unique synthetic modular polypeptides, encoded by each unique polynucleotide of the set, contain a modular a domain selected from the group consisting of: antigen-binding domain, specific binding domain or specific binding partner protein, co-stimulatory domain, co-inhibitory domain, intracellular signaling domain, transmembrane domain, scaffold protein domain, protein kinase protein domain, protein phosphatase protein domain, receptor tyrosine kinase protein domain , lipid kinase protein domain, lipid phosphatase protein domain, ubiquitinylase protein domain, deubiquitinylase protein domain, SUMOylase protein domain, acetylase protein domain, protein domain deacetylase, methylase protein domain, demethylase protein domain, nuclease protein domain, recombinase protein domain, transcription factor protein domain, and combinations thereof.

The present invention provides a cell library comprising: a plurality of cells each containing a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, each unique polynucleotide

- 4 041085 leotide contains: i) a coding region encoding a unique synthetic modular polypeptide containing the first coding sequence encoding the first module, connected with maintaining an open reading frame with the second coding sequence encoding the second module, ii) the region of the nucleic sequence-barcode containing a first nucleic barcode sequence specific to a first coding sequence, connected to a second nucleic barcode sequence specific to a second coding sequence, wherein said first and second barcode sequences are arranged in reverse order from the 5' end to the 3' end with respect to to the first and second coding sequences; and while sequencing each region of the nucleic sequence of the barcode allows you to identify each unique synthetic modular polypeptide of each cell of the library.

The present invention also provides a cell library in which the cells are prokaryotic cells or eukaryotic cells. The present invention also provides a cell library in which the eukaryotic cells are human cells. The present invention also provides a cell library in which the human cells are human T cells.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, which contains a coding region encoding a unique synthetic modular polypeptide, containing a first coding sequence encoding a first module, linked in open reading frame to a second coding sequence encoding a second module, said first and second coding sequences being directly linked without any intermediate non-coding nucleotides.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, which contains a coding region encoding a unique synthetic modular polypeptide, containing a first coding sequence encoding a first module, coupled in open reading frame to a second coding sequence encoding a second module, said coding region further comprising a sequence encoding a reporter coupled in open reading frame to the second coding sequence.

The present invention also provides a cellular library that contains a reporter-coding sequence fused in an open reading frame to a second coding sequence, said reporter being an epitope tag.

The present invention also provides a cellular library that contains a reporter-coding sequence fused in open reading frame to a second coding sequence, said reporter being a fluorescent protein.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, and said plurality of cells contains at least 1000 unique polynucleotides.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence that contains a coding region encoding a unique synthetic modular polypeptide, and said coding region further comprises a third coding sequence encoding a third module, connected while maintaining an open reading frame with the second coding sequence, and the region of the barcode nucleic sequence contains a third barcode sequence specific for the third coding sequence, connected to the second barcode nucleic sequence, and the specified first, second and third barcode nucleic sequences are located in the reverse order from 5'-end to 3'-end relative to the first, second and third coding sequences.

The present invention also provides a cell library that contains a plurality of cells, each containing a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, wherein the unique synthetic modular polypeptides encoded by each unique polynucleotide from the plurality of cells are chimeric antigen receptor polypeptides. (CAR).

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, wherein said unique synthetic modular polypeptides encoded by each unique polynucleotide from the plurality of cells are are modular receptor polypeptides.

The present invention also provides a cell library that contains a plurality of cells each containing a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, said modular receptor polypeptides being chimeric Notch receptor polypeptides.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, wherein said unique synthetic modular polypeptides encoded by each unique polynucleotide from the plurality of cells are modular scaffold proteins. .

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, wherein said unique synthetic modular polypeptides encoded by each unique polynucleotide from the plurality of cells are protein kinase modular proteins. or protein phosphatase.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, and said unique synthetic modular polypeptides encoded by each unique polynucleotide from a plurality of cells are modular transcriptional regulatory proteins.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, and said unique synthetic modular polypeptides encoded by each unique polynucleotide from a plurality of cells are modular epigenetic regulatory proteins.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, wherein said unique synthetic modular polypeptides encoded by each unique polynucleotide from the plurality of cells are recombinase modular proteins. or nuclease.

The present invention also provides a cell library that contains a plurality of cells, each of which contains a unique polynucleotide containing a nucleotide sequence encoding a unique synthetic modular polypeptide, and said unique synthetic modular polypeptides encoded by each unique polynucleotide from a plurality of cells contain a modular domain selected from the group consisting of: antigen-binding domain, specific binding domain or specific binding partner protein, co-stimulatory domain, co-inhibitory domain, intracellular signaling domain, transmembrane domain, scaffold protein domain, protein kinase protein domain, protein phosphatase protein domain, receptor tyrosine kinase protein domain, lipid kinase protein domain, lipid phosphatase protein domain, ubiquitinylase protein domain, deubiquitinylase protein domain, SUMOylase protein domain, acetylase protein domain, deacetylase protein domain, methylase protein domain, a demethylase protein domain, a nuclease protein domain, a recombinase protein domain, a transcription factor protein domain, and combinations thereof.

The present invention provides a method for obtaining a nucleic acid library containing barcode nucleic acid sequences, each of which encodes a unique synthetic modular polypeptide, and this method includes: bringing the first polynucleotide containing the sequence encoding the first module, connected to the first nucleic barcode sequence, into contact with a second polynucleotide containing a sequence encoding a second module connected to a second nucleic sequence by a barcode, under conditions sufficient to introduce the first polynucleotide into the second polynucleotide at the junction of the second coding sequence with the second nucleic sequence-barcode to obtain a bimodular polynucleotide containing nucleic sequences - barcodes, wherein said bimodular polynucleotide containing nucleic barcode sequences contains a second modular coding sequence b, coupled in an open reading frame to a first modular coding sequence coupled to a first barcode nucleoside sequence coupled to a second barcode nucleoside sequence.

The present invention also provides a method for obtaining a library of nucleic acids containing nucleic barcode sequences, each of which encodes a unique synthetic modular polypeptide, and this method further includes bringing the first

- 6 041085 and a second polynucleotide in contact with a third polynucleotide containing a third modular coding sequence connected to a third nucleic barcode sequence, wherein said third polynucleotide is inserted into the first polynucleotide at the junction of the first coding sequence and the first nucleic barcode sequence to obtain a three-module polynucleotide containing nucleic barcode sequences, while the three-modular polynucleotide containing nucleic barcode sequences contains a second modular coding sequence connected in an open reading frame to the first modular coding sequence connected in an open reading frame to a third modular coding sequence connected to a third barcode nucleic acid sequence linked to a first barcode nucleic acid sequence linked to a second barcode nucleic acid sequence barcode research.

The present invention also provides a method for preparing a nucleic acid library containing barcode nucleic acid sequences, each of which encodes a unique synthetic modular polypeptide, wherein said first and second modular coding sequences are linked in an open reading frame without any intervening non-coding nucleotides.

The present invention also provides a method for obtaining a nucleic acid library containing barcode nucleic acid sequences, each of which encodes a unique synthetic modular polypeptide, and said nucleic acid library containing barcode nucleic sequences contains 1000 or more unique nucleic acids, each of which encodes unique synthetic modular polypeptide.

The present invention also provides a method for obtaining a nucleic acid library containing barcode nucleic acid sequences, each of which encodes a unique synthetic modular polypeptide, and said nucleic barcode sequences are located in the 5'-terminus relative to the sequences encoding the modules, or in the 3' direction -end relative to sequences encoding modules.

The present invention also provides a method for obtaining a nucleic acid library containing barcode nucleic acid sequences, each of which encodes a unique synthetic modular polypeptide, wherein the introduction of the first polynucleotide into the second polynucleotide at the junction of the second coding sequence and the second nucleic barcode sequence is mediated by the activity of a restriction enzyme that recognizes a restriction enzyme recognition site on the second polynucleotide and cleaves the second polynucleotide between the second coding sequence and the second barcode nucleic sequence, including cases where the restriction enzyme used is a type II restriction enzyme, including cases where the restriction enzyme used is a type IIS restriction enzyme.

The present invention also provides a method for obtaining a library of nucleic acids containing nucleic barcode sequences, each of which encodes a unique synthetic modular polypeptide, and the first polynucleotide also contains a restriction enzyme recognition site that is present on the second polynucleotide.

The present invention also provides a method for preparing a library of nucleic acids containing barcode nucleic acid sequences each encoding a unique synthetic modular polypeptide, said method further comprising contacting the bimodular polynucleotides containing the barcode nucleic acid sequences with a reporter-encoding polynucleotide in conditions sufficient to introduce a reporter-encoding polynucleotide into a bimodular polynucleotide containing barcode nucleic sequences at the junction of the first modular coding sequence and the first barcode nucleic sequence to obtain a reporter-bound bimodular polynucleotide containing barcode nucleic acid sequences, said reporter-bound bimodular polynucleotide containing nucleic barcode sequences, contains a second modular coding sequence connected with preservation an open reading frame with a first modular coding sequence connected in an open reading frame with a sequence encoding a reporter connected to a first nucleic barcode sequence connected to a second nucleic sequence with a barcode. The present invention also provides a method for obtaining a nucleic acid library containing nucleic barcode sequences, in which the sequence encoding the reporter encodes an epitope tag or a fluorescent reporter.

The present invention provides a chimeric antigen receptor (CAR) identified by screening a library of nucleic acids encoding synthetic modular CAR polypeptides, said CAR containing at least one comodulatory domain listed in Table 1. 3 or 4.

The present invention also provides a CAR identified by screening a library of nucleic acids encoding synthetic modular CAR polypeptides, said

- 7 041085

CAR contains a CD19 specific antigen binding domain.

The present invention also provides a CAR identified by screening a library of nucleic acids encoding synthetic modular CAR polypeptides, said

CAR contains the primary signaling domain of CD3-zeta.

The present invention also provides a CAR identified by screening a library of nucleic acids encoding synthetic modular CAR polypeptides, said CAR stimulating T cell activity and containing at least one co-stimulatory domain listed in Table 1. 3.

The present invention also provides a CAR identified by screening a library of nucleic acids encoding synthetic modular CAR polypeptides, said CAR inhibiting T cell activity and containing at least one co-inhibitory domain listed in Table 1. 4.

The present invention also provides a nucleic acid encoding a CAR identified by screening a library of nucleic acids encoding synthetic modular CAR polypeptides, including, for example, any of the CARs described herein.

Brief description of the drawings

In FIG. 1 shows a modular sequence containing a barcode nucleic sequence subcloned to assemble a library according to one embodiment of the present invention.

In FIG. 2 is a modular sequence containing the nucleic barcode sequence shown in FIG. 1 after IIS digestion in preparation for library assembly.

In FIG. 3 shows an exemplary fragment of the sequence according to FIG. 1 (before digestion with restriction enzyme type IIS (RE), SEQ ID NO: 16) and FIG. 2 (after cleavage with restriction enzyme IIS, SEQ ID NO: 17) containing a sequence encoding the costimulatory domain of CD28 (translation of the fragment after cleavage with restriction enzyme IIS, SEQ ID NO: 18).

In FIG. 4 is presented in Table. 1. Identification numbers of sequences for the sequences presented in table. 1 were as follows: index 1 - SEQ ID NO: 19; index 2 - SEQ ID NO: 20; index 3 - SEQ ID NO: 21; index 4 - SEQ ID NO: 22; index 5 - SEQ ID NO: 23; index 6 - SEQ ID NO: 24; index 7 - SEQ ID NO: 25; index 8 - SEQ ID NO: 26; index 9 - SEQ ID NO: 27; index 10 - SEQ ID NO: 28; index 11 - SEQ ID NO: 29; index 12 - SEQ ID NO: 30; index 13 -SEQ ID NO: 31; index 14 - SEQ ID NO: 32; index 15 - SEQ ID NO: 33; index 16 - SEQ ID NO: 34; index 17 - SEQ ID NO: 35; index 18 - SEQ ID NO: 36; index 19 - SEQ ID NO: 37; index 20 - SEQ ID NO: 38; index 21 - SEQ ID NO: 39; index 22 - SEQ ID NO: 40; index 23 - SEQ ID NO: 41; index 24 - SEQ ID NO: 42; index 25 - SEQ ID NO: 43; index 26 - SEQ ID NO: 44; index 27 - SEQ ID NO: 45; index 28 - SEQ ID NO: 46; index 29 - SEQ ID NO: 47; index 30 - SEQ ID NO: 48; index 31 - SEQ ID NO: 49; index 32 - SEQ ID NO: 50; index 33 - SEQ ID NO: 51; index 34 - SEQ ID NO: 52; index 35 - SEQ ID NO: 53; index 36 - SEQ ID NO: 54; index 37 - SEQ ID NO: 55; index 38 - SEQ ID NO: 56; index 39 - SEQ ID NO: 57; index 40 - SEQ ID NO: 58; index 41 - SEQ ID NO: 59; index 42 - SEQ ID NO: 60; index 43 - SEQ ID NO: 61; index 44 - SEQ ID NO: 62; index 45 - SEQ ID NO: 63; index 46 - SEQ ID NO: 64; index 47 - SEQ ID NO: 65; index 48 - SEQ ID NO: 66; index 49 - SEQ ID NO: 67; index 50 - SEQ ID NO: 68; index 51 - SEQ ID NO: 69; index 52 - SEQ ID NO: 70; index 53 - SEQ ID NO: 71; index 54 - SEQ ID NO: 72; index 55 - SEQ ID NO: 73; index 56 - SEQ ID NO: 74; index 57 - SEQ ID NO: 75; index 58 - SEQ ID NO: 76; index 59 - SEQ ID NO: 77; index 60 - SEQ ID NO: 26; index 61 - SEQ ID NO: 26; index 62 - SEQ ID NO: 26.

In FIG. 5 is a general diagram of a staggered assembly of a two-dimensional library of comodulating modules containing nucleic barcode sequences, in accordance with one embodiment of the present invention.

In FIG. 6 shows a general scheme for obtaining a two-dimensional library of 62x62 comodulating modules according to one embodiment of the present invention.

In FIG. 7 shows the general configuration of the elements of a one-dimensional chimeric antigen receptor (CAR) library in accordance with one embodiment of the present invention.

In FIG. 8 shows the results of functional sorting (ie, binning) of stimulated CAR-expressing T cells based on the end level of activation.

In FIG. 9 shows the results of a quantitative assessment of the relative effect of each comodulatory domain of the library on T cell activity, which was performed using quantitative sequencing of module-specific barcode sequences.

In FIG. 10 shows the dose-response profiles for six comodulatory domains at three increasing antigen loading levels obtained using a 62-element one-dimensional library.

In FIG. 11 is a non-limiting example of a CAR library showing that different CAR domains can vary within a library according to one variant.

- 8 041085 implementation of the present invention.

In FIG. 12 is a diagram of an assembly strategy for the coding modules of the sequences containing the barcode nucleic acid sequences described herein.

In FIG. 13 is a diagram of an assembly strategy for the coding modules of the sequences containing the barcode nucleic acid sequences described herein.

In FIG. 14 is a diagram of an assembly strategy for the coding modules of the sequences containing the barcode nucleic acid sequences described herein.

In FIG. 15 is a diagram of an assembly strategy for the coding modules of the sequences containing the barcode nucleic acid sequences described herein.

In FIG. 16 is a diagram of an assembly strategy for the coding modules of the sequences containing the barcode nucleic acid sequences described herein.

In FIG. 17 is a diagram of an assembly strategy for the coding modules of the sequences containing the barcode nucleic acid sequences described herein.

In FIG. 18 is a diagram of an assembly strategy for the coding modules of the sequences containing the barcode nucleic acid sequences described herein.

In FIG. 19 is a diagram of an assembly strategy for the coding modules of the sequences containing the barcode nucleic acid sequences described herein.

In FIG. 20 is a diagram of an assembly strategy for the coding modules of the sequences containing the nucleic barcode sequences described herein.

In FIG. 21 is a diagram of an assembly strategy for the coding modules of the sequences containing the nucleic barcode sequences described herein.

In FIG. 22 is a schematic diagram of a combinatorial nested assembly strategy for a modular chimeric antigen receptor (CAR) library in accordance with an embodiment of the present invention.

In FIG. 23 is a diagram for the production of a combined CAR cellular module library in accordance with an embodiment of the present invention.

In FIG. 24 is a schematic example of an integrated method for phenotype detection and modular polypeptide identification for in vitro and/or in vivo use in accordance with an embodiment of the present invention.

In FIG. 25 is presented in Table. 2.

In FIG. 26 is an exhaustive representation of each element of a two-dimensional library of 61x61 elements, as determined by deep sequencing of the assembled library.

In FIG. 27 shows the results of element quantification of the pre-normalized combinatorial nucleic acid library described herein.

In FIG. 28 is a linear equation used in calculating normalization adjustments related to library items quantified as shown in FIG. 27.

In FIG. 29 presents results confirming the predictability of normalization adjustments made in accordance with the method described herein.

Definitions

In this application, the terms polynucleotide and nucleic acid are used interchangeably and refer to the polymeric form of nucleotides of any length, both ribonucleotides and deoxyribonucleotides. Therefore, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

In this application, the terms polypeptide, peptide and protein are used interchangeably and refer to the polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically labeled proteins; and so on.

In this application, the terms domain and motif are used interchangeably and refer to structured domains having one or more specific functions and unstructured segments of a polypeptide that, despite not being structured, retain one or more specific functions. For example, a structured domain may include, but is not limited to, a continuous or discrete set of amino acids, or parts thereof, in a folded polypeptide that has a three-dimensional structure that contributes to a particular function of the polypeptide. In other cases, the domain may include an unstructured polypeptide segment containing a plurality of two or more amino acids, or portions thereof, that supports a particular function of the polypeptide, whether unfolded or disordered. This definition also includes domains that may be random or unstructured, but become structured or ordered when bound to a target or binding partner. Non-limiting examples of natively unstructured domains and domains of natively unstructured proteins are described, for example, in Dyson & Wright. Nature Reviews Molecular Cell Biology 6:197-208.

In the present application, the term module refers to a continuous polypeptide sequence or fragment thereof, which is associated with a particular function, in particular with a biological function.

In this application, the terms chimeric antigen receptor and CAR are used interchangeably and refer to artificial multi-module molecules capable of initiating or inhibiting immune cell activation, which usually, but not exclusively, contain an extracellular domain (for example, a ligand/antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. The term CAR is not limited to specific CAR molecules, but also includes CAR variants. CAR variants include separated CARs in which an extracellular portion (eg, ligand binding site) and an intracellular portion (eg, intracellular signaling portion) of the CAR are present on two separate molecules. CAR variants also include CAR switches and CAR switches, which are conditionally activated/suppressed CARs, for example, these variants include a cleaved CAR in which the conditional heterodimerization of the two parts of the cleaved CAR is controlled pharmacologically. CAR variants also include bispecific CARs that contain an additional CAR binding domain that can enhance or inhibit the activity of the main CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs), which can be used, for example, as a component of a bispecific CAR system where binding of a binding domain to an additional CAR results in inhibition of activation of the primary CAR. CAR molecules and their derivatives (ie, CAR variants) are described, for example, in PCT application US2014/016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2): 151-5; Riddell et al. Cancer J (2014) 20(2): 141-4; Pegram et al. Cancer J (2014) 20(2): 127-33; Headle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304, the contents of which are hereby incorporated by reference in their entirety.

The term gene refers to a specific unit of heredity present at a specific locus in the genetic component of an organism. A gene may be a nucleic acid sequence, such as a DNA or RNA sequence, present in the nucleic acid genome, DNA or RNA genome, of an organism, and, in some cases, may be present on a chromosome. A gene may be a DNA sequence encoding an mRNA that codes for a protein. A gene may consist of a single exon and may have no introns, or may contain multiple exons and one or more introns. One of two or more identical or alternative forms of a gene present at a particular locus is called an allele and, for example, a diploid organism will typically have two alleles for a particular gene. New alleles for a particular gene may arise naturally or artificially through natural or induced mutation, and may be propagated by reproduction or cloning. The gene or allele may be isolated from the genome of the organism and replicated and/or manipulated, or the gene or allele may be modified in situ by gene therapy techniques. A gene locus or allele may have associated regulatory elements, and gene therapy, in some cases, may involve modification of the regulatory elements of a gene or allele, leaving the coding sequences of the gene or allele unmodified.

The term operably linked refers to an alignment in which the described components are interconnected, allowing them to function in the intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.

An expression vector or vector is a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment can be attached, i.e. insertion to cause replication of the attached segment in the cell.

In this application, the term heterologous means a nucleotide or polypeptide sequence that does not occur in native (eg, natural) nucleic acid or protein, respectively.

The terms antibodies and immunoglobulin include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins containing the antigen-binding portion of an antibody and a non-antibody protein.

Antibody fragments contain a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab fragments,

- 10 041085

Fab', F(ab')2 and Fv; diabody; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single chain antibody molecules; and polyspecific antibodies formed from antibody fragments. Cleavage of antibodies with papain produces two identical antigen-binding fragments, called Fab fragments, each containing one antigen-binding site, and a residual Fc fragment, a designation that reflects the ability of the fragment to easily crystallize. Pepsin treatment produces an F(ab') ₂ fragment that contains two antigen binding sites and is still capable of antigen crosslinking.

Single chain Fv or sFv fragments of an antibody contain the VH and V _L domains of the antibody, these domains being present in the same polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

In this application, the term affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd).

Affinity can be at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold , at least 7 times more, at least 8 times more, at least 9 times more, at least 10 times more, at least 20 times more, at least 30 times more, according to at least 40 times more, at least 50 times more, at least 60 times more, at least 70 times more, at least 80 times more, at least 90 times more, at least 100 times greater, or at least 1000 times greater, or greater than the affinity of the antibody for non-target amino acid sequences. The affinity of an antibody for a target protein can be, for example, from about 100 to about 0.1 nanomoles nM, from about 100 nM to about 1 picomole (pM), or from about 100 nM to about 1 femtomol (fM) or more.

The term bonding refers to the direct bonding of two molecules through, for example, covalent, electrostatic, hydrophobic and ionic and/or hydrogen bonds, including such interactions as salt bridges and water bridges. Non-specific binding refers to binding with an affinity value of less than about 10 ^-7 M, such as binding with an affinity of 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, and so on.

In this application, the term immune cells generally includes white blood cells (leukocytes) that are derived from hematopoietic stem cells (HSC) produced in the bone marrow. Immune cells include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and cells of myeloid origin (neutrophils, eosinophils, basophils, monocytes, macrophages and dendritic cells).

The term T cell includes all types of CD3-expressing immune cells, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), regulatory T cells (Treg), and gamma delta T cells.

The term cytotoxic cell includes CD8+ T cells, natural killer (NK) cells, and neutrophils that are capable of mediating cytotoxic responses.

In this application, the term stem cell usually includes pluripotent or multipotent stem cells. Stem cells include, for example, embryonic stem cells (ES); mesenchymal stem cells (MSC); induced pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC), cells derived from bone marrow, etc.).

In the present application, the term treatment, treating, etc. relate to obtaining the desired pharmacological and/or physiological action. The action may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse event associated with the disease. As used herein, the term treatment encompasses any method of treating a disease in a mammal, such as a human, and includes: (a) preventing the onset of a disease in a subject who may be predisposed to the disease but in which the disease has not yet been diagnosed; (b) inhibition of the disease, ie. cessation of its development; and (c) alleviating the disease, ie. induction of disease regression.

In this application, the terms individual, subject, host, and patient are used interchangeably and refer to a mammal, including, but not limited to, murine rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canine, feline, ungulate (e.g. horse, cattle, sheep, pig, goat), etc.

A therapeutically effective amount or effective amount refers to the amount of an agent, or combined amounts of the two agents, which, when administered to a mammal or

- 11 041085 to another subject for the treatment of the disease is sufficient to carry out the specified treatment of the disease. A therapeutically effective amount will vary depending on the agent(s), the disease and its severity, as well as the age, weight, etc., of the subject being treated.

Terms control, control reaction, control quantitative study, etc. refer to a reaction, test or other part of an experimental or diagnostic procedure or experimental design for which the expected outcome is known with a high degree of certainty, e.g. experimental results indicate the true result and/or to provide calibration of the experimental results. For example, in some cases, the control may be a negative control such that an essential component of the assay is excluded from the negative control reaction so that the experimenter can have a high degree of confidence that the negative control reaction will not lead to a positive result. In some cases, the control may be a positive control such that all components of a particular assay are characterized and known, when combined, to produce a specific result in a assay performed by an experimenter who can have a high degree of confidence that the positive control's response is not lead to no positive outcome.

As used herein, the term primer or oligonucleotide primer refers to an oligonucleotide that initiates the synthesis of a complementary nucleic acid strand under conditions where synthesis of a primer extension product is induced, for example, in the presence of nucleotides and a polymerization-inducing agent such as DNA or RNA polymerase, and at suitable temperature, pH, metal concentration and salt concentration. Primers are generally of a length compatible with their use in the synthesis of primer extension products and may be in the range of 8 to 100 nucleotides, e.g. 10 to 75, 15 to 60, 15 to 40, 18 to 30 , 20 to 40, 21 to 50, 22 to 45, 25 to 40, etc., including the range of 18-40, 20-35, 21-30 nucleotides, and any length within these ranges. In some cases, the length of the primers may be within the range of 10 to 50 nucleotides, such as 15-45, 18-40, 20-30, 21-25, etc., and the primer may be any length within these ranges. In some embodiments of the present invention, primers typically contain no more than 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60 , 65 or 70 nucleotides.

The terms hybridize and hybridize refer to the formation of complexes between nucleotide sequences that are complementary enough to form complexes via Watson-Crick base pairing. For example, when hybridizing a primer with a target (template), such complexes (or hybrids) are stable enough to provide the priming necessary, for example, to initiate DNA synthesis by DNA polymerase.

The term biological sample includes many types of samples obtained from an individual or a population of individuals and can be used in a diagnostic, control, or screening quantitative study. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy samples or tissue cultures or cells derived from them, and their progeny. The definition also includes samples that have been processed in some way after they were received, for example, by mixing or combining individual samples, treatment with reagents, solubilization or enrichment with certain components, such as cells, polynucleotides, polypeptides, etc. The term biological sample includes a clinical sample and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. The term biological sample includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions such as blood plasma and blood serum, and the like. The term biological sample also includes solid tissue samples, tissue culture samples, and cell samples.

The term evaluation includes any form of measurement and includes determining the presence or absence of an element. The terms definition, measurement, research, evaluation and quantitative research are used interchangeably and include quantitative and qualitative definitions. The assessment can be relative or absolute. Presence estimation includes determining the amount of an object present and/or determining its presence or absence. In this application, the terms definition, measurement and evaluation and quantitative research are used interchangeably and include quantitative and qualitative definitions.

The present invention will be further described below, however, it should be understood that the present invention is not limited to the specific embodiments described, as it is obvious that they may vary. It should also be understood that the terminology used herein is for the sole purpose of describing particular embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended

- 12 041085 claims.

Where a range of values is provided, it should be understood that each intermediate value, up to a tenth of a unit of the lower limit, unless the context clearly requires otherwise, between the upper and lower limits of the specified range and any other declared or intermediate value in the specified range, included in the scope of the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included within the scope of the present invention, subject to any specifically excluded limit within the specified range. In cases where the specified range includes one or both of the limits, ranges excluding one or both of the specified included limits are also included in the scope of the present invention.

Unless otherwise indicated, in this application, all technical and scientific terms have the same meaning as is usually understood by a person skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the implementation of the present invention or when testing the present invention, preferred methods and materials are described herein. All publications mentioned in this document are incorporated into this application by reference for the disclosure and description of the methods and/or materials in connection with which the publications are cited.

It should be noted that in this application and in the appended claims, singular forms include plural forms of referents, unless the context clearly indicates otherwise. For example, a reference to a polypeptide includes a plurality of such polypeptides, and a reference to an antigen includes a reference to one or more antigens and their equivalents known to those skilled in the art, and so on. It should also be noted that the claims of the present invention may be drafted to exclude any optional element. Therefore, this statement is intended to be used as a foregoing basis for the use of such exclusionary terminology as exclusively, only, and the like. regarding the description of the elements of the claims of the present invention or the use of a negative limitation.

It should be understood that some of the features of the present invention, which for clarity are described in relation to individual implementation options, can also be presented in combination in one embodiment. Conversely, various features of the invention, which are described for brevity in relation to one implementation variant, can also be presented individually or in any suitable additional combination. All combinations of embodiments relating to the present invention are specifically covered by the present invention and are disclosed herein in the same way as if each combination were individually and expressly disclosed. In addition, all additional combinations of various embodiments and their elements are also specifically included in the scope of the present invention and are disclosed herein in the same way as if each such additional combination were individually and expressly disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing contained herein should be construed as an admission that the invention has no right to precede such disclosure by virtue of prior invention. In addition, the dates of the submitted publications may differ from the actual dates of publications, which may need to be confirmed independently.

Detailed description of the invention

The present invention provides synthetic modular polypeptide libraries and nucleic acids encoding said synthetic modular polypeptide libraries. The present invention also provides methods for obtaining libraries of synthetic modular polypeptides and nucleic acids encoding libraries of synthetic modular polypeptides. The present invention also provides methods for screening a library of synthetic modular polypeptides to identify a selected phenotype associated with an element of the library of synthetic modular polypeptides, which methods can be used in quantitative studies in vitro and in vivo conditions.

Libraries.

Aspects of the present invention relate to libraries of nucleic acids containing nucleic barcode sequences that encode a plurality of synthetic modular polypeptides. Aspects of the present invention also include cell libraries in which each cell expresses a single nucleic acid containing a barcode nucleic acid sequence encoding a unique synthetic modular polypeptide, or a plurality of said individual nucleic acids containing barcode nucleic acid sequences. As described in more detail below, aspects of the present invention also include methods for obtaining said libraries and methods for screening said libraries for detection.

- 13 041085 specific phenotypes.

Nucleic acid libraries of the present invention are typically assembled combinatorially from modular components and include a ladder barcode sequence that can be sequenced to determine the identity and configuration of the modular components of each element of the library. Therefore, each individual element of the nucleic acid library will contain at least a coding region and a barcode nucleic sequence region. In this application, the term coding region, as it refers to individual elements of a nucleic acid library, refers to the region of each nucleic acid that encodes a synthetic polypeptide of interest, for example, a synthetic polypeptide containing one or more polypeptide modules that can be screened to determine their influence on a particular desirable or undesirable phenotype. In some cases, the coding region may further comprise sequences encoding a reporter molecule, such as a molecule that is used to detect and/or measure the expression of a synthetic polypeptide of interest.

The coding region typically encodes a single modular polypeptide, described in more detail below; however, the present description does not exclude libraries in which the coding region of each element of the library encodes two or more separate modular polypeptides. Such library elements may include a single coding region that is polycistronic (e.g., bicistronic, polycistronic, etc.), e.g., by including a separable linker (e.g., internal ribosome entry site (IRES), ribosomal shunt sequence, nucleic acid encoding self-cleaving peptide, etc.) between sequences encoding shared modular polypeptides. A coding region containing a sequence encoding two or more separate modular polypeptides will be contiguous and will be assembled and screened according to the methods described herein for single-module polypeptide constructs.

The coding region will contain variable modules and non-variable modules, and the intended use of the library will determine whether a particular portion of the synthetic modular polypeptide is variable or non-variable. Coding region variable modules are typically those modules that are screened, individually or combinatorially, to determine a functional property and/or effect on a phenotype of interest. In general, variable modules will be associated with a barcode sequence identifying the module, while non-variable modules will not be associated with a barcode sequence. Any module may serve as a variable or non-variable module depending on the design of the particular library and/or the screening method with which the library is associated.

As a non-limiting example, in some embodiments in which the coding region encodes a chimeric antigen receptor (CAR), any CAR module may serve as a variable module depending on the activity of the CAR being screened, including but not limited to extracellular domain, coregulatory domain or primary signaling domain. For example, in FIG. 11 shows one element of the CAR library, in which each member of the library contains an extracellular domain, a coregulatory domain, and a primary signaling domain, and provides non-limiting examples of cases where each domain can serve as a variable module.

In some cases, the extracellular domain may be a variable module, for example, in cases where the targeting of the antigen is of interest, in cases where the strength of the interaction between the antigen and the extracellular domain is of interest, etc. In some cases, the coregulatory domain may be a variable module, such as when an individual coregulatory domain or combinations of coregulatory domains are to be screened for co-modulation of functional activity. In some cases, the primary signaling domain may be a variable module, such as when different intracellular signaling pathways are to be screened.

As a non-limiting example, in some cases, the encoded synthetic modular polypeptide may be a chimeric Notch receptor polypeptide comprising an extracellular binding domain, a cleavage domain of the Notch receptor polypeptide (including a binding-inducible cleavage site), and an intracellular domain. Suitable chimeric Notch receptor polypeptides and their domains include, but are not limited to, for example, those described in US patent application PCT US0201/019188; the contents of which are incorporated herein by reference in their entirety. In some cases, one or more domains of a chimeric Notch receptor polypeptide serve as a variable domain, including, but not limited to, for example, an extracellular binding domain, a Notch receptor polypeptide domain, an intracellular domain, an extracellular binding domain, and a Notch receptor polypeptide domain, an extracellular binding domain. and an intracellular domain or a Notch receptor polypeptide domain and an intracellular domain.

Non-variable modules included in the coding region will be selected if needed.

- 14 041085 mo, so that all elements of the library have the function of a non-variable module, i.e. when a function that is provided by a non-variable module is kept constant across all members of the library. As non-limiting examples, in some cases, the extracellular domain, coregulatory domain, or primary signaling domain described above can serve as non-variable modules, if necessary, or if it is necessary for quantitative research that all elements of the library have the function of an extracellular domain, coregulatory domain or primary signaling domain. As non-limiting examples, in some cases, the extracellular binding domain, Notch receptor polypeptide cleavage domain, or intracellular domain described above may be non-variable modules, if necessary, or if it is necessary for quantitative analysis that all elements of the library have the function of extracellular a binding domain, a cleavable domain of a Notch receptor polypeptide, or an intracellular domain.

Depending on the specific modules used and the screening assay for which the library of the present invention is used, the coding region of each library element can encode any combination of variable and non-variable modules, with the simplest coding region encoding only the variable module. In some cases, each individual element of the library, for example, each vector into which a variable module is inserted, may contain, prior to insertion of the variable module, one or more coding sequences such that when the variable module is inserted, the coding region will contain the variable module and pre-existing coding sequences, which may or may not contain a non-variable module and/or a non-modular coding sequence. In some cases, such a pre-existing coding sequence that is present in all elements of the library may be referred to as a constant domain.

In some cases, a coding region may encode two or more variable modules, including but not limited to, for example, three or more variable modules, four or more variable modules, five or more variable modules, six or more variable modules, seven or more variable modules. modules, eight or more variable modules, etc. In some cases, a coding region may encode two or more non-variable modules, including but not limited to, for example, three or more non-variable modules, four or more non-variable modules, five or more non-variable modules, six or more non-variable modules, seven or more non-variable modules. modules, eight or more non-variable modules, etc. The number of variable and non-variable modules in a coding region may be limited by the practical limitations of the method for cloning a coding sequence and constructing a library.

The number of unique elements in a library encoding unique synthetic modular polypeptides will depend on the number of variable modules used in constructing the library and the overall perceived complexity of the library. In some embodiments of the present invention, the complexity of a library can be described in terms of library dimension, with library dimension being correlated with the number of variable module encoding sequences that are present in the coding region of library elements. Therefore, the one-dimensional library contains one variable module encoding sequence in each coding region of each library element, the two-dimensional library contains two variable module encoding sequences in each coding region of each library element, the three-dimensional library contains three variable module encoding sequences in each coding region of each element of the library, the four-dimensional library contains four sequences encoding variable modules in each coding region of each element of the library, and so on.

The dimensions of a library need not be uniform throughout the library, and therefore, in some cases, a library may have mixed dimensions. By mixed dimension is meant that the library contains elements of the library that are different from the one-dimensional ones described above. For example, the library may be partially one-dimensional, partially two-dimensional, partially three-dimensional, and so on. These mixed dimensional libraries may be mixed 1D and 2D libraries, 2D and 3D libraries, 1D, 2D and 3D libraries, and so on. The descriptions of mixed dimensional libraries provided are not limiting, and one of ordinary skill in the art will readily appreciate that the variety of mixed dimensional libraries included within the scope of the present invention goes well beyond what is explicitly described.

Therefore, the number of unique library elements encoding unique synthetic modular polypeptides will vary and will be a result of the size of the library and the number of modules used in the construction of the library. For example, a one-dimensional library constructed from 20 unique variable modules would contain 20 unique library elements. A two-dimensional library constructed from 20 unique variable modules will contain 20x20 (ie 400) unique library elements. 3D Library,

- 15 041085 constructed from 20 unique variable modules will contain 20x20x20 (ie 8000) unique library elements. In some cases, a unique library element from a multidimensional library may contain two or more sequences encoding the same variable module.

In some cases, where each element of the library contains two or more variable modules, these modules may have a specific position, which means that a subset of the variable modules can only be placed in a specific location within the synthetic modular polypeptide in relation to other variable modules. For example, a synthetic modular polypeptide containing two variable modules may contain a first set and a second set of variable modules, with the modules of the first set always in a certain position relative to the modules of the second set. Therefore, a two-dimensional library designed with a specific module position, consisting of 30 unique variable modules, including a first set of 10 unique variable modules and a second set of 20 unique variable modules, may contain 10x20 (i.e., 200) unique library elements.

One of skill in the art will readily appreciate the significant variability in library configurations given the variable dimension described above and the variable numbers of modules that can be combined, with or without specific positions, when constructing libraries according to the present invention. Therefore, the total number of unique library items will vary greatly and may range from less than 20 to 50,000 or more, including but not limited to, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more , 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, 1000 or more, 1500 or more, 2000 or more, 2500 3000 or more 3500 or more 4000 or more 4500 or more 5000 or more 5500 or more 6000 or more 6500 or more 7000 or more 7500 or more 8000 or more 8500 or more , 9000 or more, 9500 or more, 10000 or more, 20000 or more, 30000 or more, 40000 or more, 50000 or more, etc.

Where multidimensional libraries are constructed by pooling, the total number of unique library items in the pool may vary considerably and may range from less than 20 to 50,000 or more as described above, and may have a higher degree of diversity, including, but not limited to, for example, 105 or more, 106 or more, 10 ⁷ or more, 108 or more, or 109 or more.

The unique elements of the libraries described herein need not be physically ordered for library construction or screening purposes. As described in more detail below, the combinatorial assembly of the nucleic acid components encoding each synthetic modular polypeptide and the co-assembly of the multi-unit barcode sequences in a manner that characterizes the identity and orientation of the assembled modules allows for a one-pot synthesis and overall screening of the libraries described herein. Therefore, a library, and a plurality of unique library elements, may be present in one suitable solution and/or present in one suitable container.

Synthetic modular polypeptides.

The present invention provides synthetic modular polypeptide libraries and nucleic acids encoding synthetic modular polypeptides. By modular polypeptide is meant a functional protein containing two or more operably linked modular components such that the modules are physically connected and co-function in a single polypeptide molecule. Modular polypeptide modules may have related or unrelated functions or activities. In many embodiments, at least two of the modular components of synthetic modular polypeptides are derived from separate proteins. Modules derived from individual proteins can be derived from different proteins that are operably unrelated or operably linked, and can be derived from different body molecules, from the same body molecule, different orthologous proteins, different paralogous proteins, etc. .

In some embodiments of the present invention, individual elements of the synthetic modular polypeptide library described herein will include, at a minimum, a module that is a member of a specific binding pair (e.g., antigen-antibody binding pair, ligand-receptor binding pair, etc.). .), and a functional or signaling module that is involved in the induction of a cellular response. In some cases, an element of a particular binding pair may be referred to herein as an extracellular domain or extracellular recognition domain. In some cases, the element of a particular binding pair may refer to a protein involved in protein-protein signaling interaction, or to a protein involved in protein-lipid interaction.

- 16 041085

In many embodiments, the element of a specific binding pair that can be used in individual library elements may include an antigen binding domain. An antigen-binding domain suitable for use in the library elements of the present invention may be any antigen-binding polypeptide, a wide range of which is known in the art. In some cases, the antigen binding domain is a single chain Fv fragment (scFv). Other antibody-based recognition domains are suitable for use (cAb VHH (variable domains of camelid antibodies) and humanized variants, IgNAR VH (variable domains of shark antibodies) and humanized variants, sdAb VH (variable domains of single-domain antibodies) and variable domains of antibodies containing antibody fragments T cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single chain dual domain TCR containing VaVe) are also suitable for use in some cases.

An antigen binding domain suitable for use in the library elements of the present invention may have many types of antigen binding specificity. In some instances, the antigen-binding domain is specific for an epitope present on an antigen that is expressed (synthesized) by the cancer cell, ie. in the antigen associated with the cancer cell. The cancer cell-associated antigen may be an antigen associated with, for example, breast cancer cell, B-cell lymphoma, Hodgkin's lymphoma cell, ovarian cancer cell, prostate cancer cell, mesothelioma, lung cancer cell (e.g. small cell lung cancer cell), non-Hodgkin B-cell lymphoma (B-NHL) cell, ovarian cancer cell, prostate cancer cell, mesothelioma cell, lung cancer cell (eg, small cell lung cancer cell), melanoma cell, chronic lymphocytic leukemia cell, acute lymphocytic leukemia, neuroblastoma cell, glioma, glioblastoma, medulloblastoma, colorectal cancer cell, etc. An antigen associated with a cancer cell can also be expressed by a non-cancerous cell.

Non-limiting examples of antigens to which the antigen-binding domain of the library of the present invention can bind include, for example, CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1, prostate specific membrane antigen (PSMA), molecule CD44, mesothelin, oncoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor 2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R -a2, GD2, etc.

In some cases, the element of a particular binding pair suitable for use in the elements of the libraries according to the present invention is a ligand for the receptor. Ligands include, but are not limited to, cytokines (eg, IL-13, etc.); growth factors (eg, heregulin, vascular endothelial growth factor (VEGF), etc.); an integrin-binding peptide (eg, a peptide containing the sequence Arg-Gly-Asp); Notch ligands (eg, Delta, Serrate, Delta-like, X-Delta, Jagged, etc. and their homologues and orthologues), and the like.

In the event that an element of a particular binding pair in the elements of the libraries of the present invention is a ligand, then the particular element of the library can be activated in the presence of a second member of the particular binding pair, the second element of the particular binding pair being the receptor for the ligand. For example, if the ligand is VEGF, the second member of the specific binding pair may be a VEGF receptor, including a soluble VEGF receptor. In another example, if the ligand is a Notch ligand, then the second member of the specific binding pair may be a Notch receptor or a ligand-binding site thereof. As another example, if the ligand is heregulin, then the second member of the specific binding pair may be Her2.

In some cases, the member of a specific binding pair that is included in the library elements of the present invention is a receptor, such as a receptor for a ligand, a co-receptor, etc. The receptor may be a ligand-binding fragment of the receptor. Suitable receptors include, but are not limited to, growth factor receptor (eg, VEGF receptor); lectin-like killer cell receptor subfamily member 1 (NKG2D) member polypeptide (receptor for MICA, MICB and ULB6); a cytokine receptor (eg, IL-13 receptor, IL-2 receptor, etc.); Her2; CD27; a natural cytotoxicity receptor (NCR) polypeptide (eg, NKP30 (NCR3/CD337) (receptor for HLA-B-associated transcript 3 (BAT3) and B7-H6), etc.); a Notch receptor (eg, human NOTCH1, human NOTCH2, human NOTCH3, human NOTCH4, etc.); and the like.

Specific binding partners also include dimer pairs. Individual members of dimer pairs are suitable for use as members of the libraries of the present invention and include, but are not limited to, dimerizer-binding pairs. Dimerizer-binding pairs bind to different regions of the same molecule (referred to herein as a dimerizer). In the presence of a dimerizer, both members of the dimerizer-binding pair bind to different regions of the dimerizer and hence approach each other.

- 17 041085

According to some embodiments of the present invention, the binding to the dimerizer is reversible. According to some embodiments of the present invention, binding to the dimerizer is irreversible. In some embodiments of the present invention, the binding to the dimerizer is non-covalent. In some embodiments of the present invention, the binding to the dimerizer is covalent.

Other suitable dimer pairs include dimer-binding pairs that dimerize when the first member of the dimer pair is bound to the dimerizing agent, wherein the dimerizing agent induces a conformational change in the first member of the dimer pair, wherein the conformational change allows the first member of the dimer pair to bind ( covalently or non-covalently) with the second member of the dimeric pair. Other dimer pairs suitable for use include dimer pairs in which exposure to light (eg, blue light) induces dimerization of the dimer pair.

In some cases, a member of a specific binding pair may include a protein involved in protein-protein signaling interaction or protein-lipid signaling interaction, and in this regard, the synthetic modular polypeptides described herein may include one or more protein-protein interaction domains or domains protein-lipid interactions. Suitable protein-protein interaction domains or protein-lipid interaction domains include, but are not limited to, for example, the 14-3-3 domain (for example, the one present in the 2B05 PDB structure (RCSB Protein Structure Database available on the website www.rcsb.org)), actin depolymerization factor (ADF) domain (e.g. present in the 1CFY PDB structure), ANK domain (e.g. present in the 1SW6 PDB structure), ANTH domain (N-terminal homology of AP180) (e.g. present in structure 5AHV PDB), Armadillo domain (ARM) (e.g. present in structure 1BK6 PDB), BAR domain (Bin/Ampphiphysin/Rvs) (e.g. present in structure 1I4D PDB), BEACH domain (beige and CHS) (e.g., present in the 1MI1 PDB structure), BH domains (Bcl-2 homologies) (BH1, BH2, BH3 and BH4) (e.g. present in the 1BXL PDB structure), baculovirus IAP repeat (BIR) domain (e.g. present in the 1G73 PDB structure) , a BRCT domain (BRCA1 C-terminal) (e.g., present in the 1T29 P DB), bromo domain (e.g. present in the 1E6I PDB structure), BTB domain (BR-C, ttk and bab) (e.g. present in the 1R2B PDB structure), C1 domain (e.g. present in the 1PTQ PDB structure), C2 domain ( e.g., present in the 1A25 PDB structure), caspase attraction domains (CARD) (e.g., present in the 1CWW PDB structure), supercoiled domain (CC) (e.g., present in the 1QEY PDB structure), CALM domain (Clathrin Assembly Lymphoid Myeloid) (e.g. , present in the 1HFA PDB structure), caloponin (CH) homology domain (e.g., present in the 1BKR PDB structure), chromatin organization modifying (Chromo) domain (e.g., present in the 1KNA PDB structure), CUE domain (e.g., present in the 1KNA PDB structure), 1OTR PDB), death domains (DD) (e.g., present in the 1FAD PDB structure), death effector domain (DED) (e.g., present in the 1A1W PDB structure), Discheveled domain, EGL-10, and plextrin (DEP) (e.g., present in the 1FSH PDB structure), d Dbl homology domain (DH) (e.g., present in the 1FOE PDB structure), EF-arm (EFh) domain (e.g., present in the 2PMY PDB structure), Eps15 homology (EH) domain (e.g., present in the 1EH2 PDB structure), epsin NH2-terminal homology (ENTH) domain (eg, present in the 1EDU PDB structure), Ena/Vasp 1 (EVH1) homology domain (eg, present in the 1QC6 PDB structure), F-box domain (eg, present in the 1FS1 PDB structure ), FERM domain (lane 4.1, ezrin, radixin, moesin) (e.g., present in the 1GC6 PDB structure), FF domain (e.g., present in the 1UZC PDB structure), formin-2 (FH2) homology domain (e.g., present in the 1UX4 PDB), Forkhead Associated (FHA) domain (e.g., present in the 1G6G PDB structure), FYVE domain (Fab-1, YGL023, Vps27, and EEA1) (e.g., present in the 1VFY PDB structure), GAT domain (GGA and Tom1) (e.g. present in the 1O3X PDB structure), gelsolin homology domain (GEL) (e.g. present in structure 1H1V PDB), GLUE domain (GRAM-like ubiquitin-binding domain in EAP45) (e.g. present in structure 2CAY PDB), GRAM domain (from glucosyltransferases, Rab-like GTPases activators and myotubularins) (e.g. present in structure 1LW3 PDB), GRIP domain (e.g. present in the 1UPT PDB structure), glycine tyrosine phenylalanine (GYF) domain (e.g. present in the 1GYF PDB structure), HEAT domain (Huntington, extension factor 3, PR65/A, TOR) ( e.g., present in the 1IBR PDB structure), a domain homologous to the C-terminal E6-AP domain (HEST) (e.g., present in the 1C4Z PDB structure), IQ domain (e.g., present in the 1N2D PDB structure), LIM domain (Lin-1 , Isl-1 and Mec-3) (e.g., present in the 1QLI PDB structure), leucine rich repeat (LRR) domain (e.g., present in the 1YRG PDB structure), brain tumor (MBT) domain (e.g., present in the 1OYX PDB), MH1 domain (Mad 1 homologies) (for example, the presence present in the 1OZJ PDB structure), MH2 domain (Mad 2 homology) (e.g., present in the 1DEV PDB structure), MIU domain (ubiquitin-interacting motif) (e.g., present in the 2C7M PDB structure), NZF domain (Npl4 zinc finger) ( e.g. present in the 1Q5W PDB structure), PAS domain (Per-ARNT-Sim)

- 18 041085 (eg, present in the 1P97 PDB structure), Phox and Bem1 (PB1) domain (eg, present in the 1IPG PDB structure), PDZ domain (post-synaptic density 95, PSD-85, discs large, Dlg, zonula occludens-1 , ZO-1) (e.g., present in the 1BE9 PDB structure), plextrin homology domain (PH) (e.g., present in the 1MAI PDB structure), Polo-Box domain (e.g., present in the 1Q4K PDB structure), phosphotyrosine binding domain (PTB ) (e.g. present in the 1SHC PDB structure), Pumilio/Puf (PUF) domain (e.g. present in the 1M8W PDB structure), PWWP domain (e.g. present in the 1KHC PDB structure), Phox (PX) homology domain (e.g. present in the 1H6H PDB structure), RGS domain (regulator of G protein-mediated signaling) (e.g., present in the 1AGR PDB structure), RING domain (e.g., present in the 1FBV PDB structure), SAM domain (sterile alpha motif) (e.g., present in 1B0X PDB structure), Shadow Chromo domain ( SC) (e.g. present in structure 1E0B PDB), Src homology domain 2 (SH2) (e.g. present in structure 1SHB PDB), Src homology domain 3 (SH3) (e.g. present in structure 3SEM PDB), SOCS domain (suppressors of signaling involving cytokines) (for example, present in the 1VCB PDB structure), SPRY domain (for example, present in the 2AFJ PDB structure), steroidogenic acute regulatory protein (StAR) related lipid transfer domain (START) (for example, present in the structure 1EM2 PDB), SWIRM domain (e.g. present in structure 2AQF PDB), Toll/Il-1 receptor (TIR) domain (e.g. present in structure 1FYV PDB), tetratricopeptide repeat (TPR) domain (e.g. present in structure 1ELW PDB ), a TRAF domain (tumor necrosis factor (TNF) receptor-associated factors) (e.g. present in the 1F3V PDB structure), a tSNARE domain (SNARE domain (soluble N-ethylmaleimide sensitive protein attachment receptor (SNA P)) (e.g. present in structure 1SFC PDB), Tubby domain (e.g. present in structure 1I7E PDB), TUDOR domain (e.g. present in structure 2GFA PDB), ubiquitin-associated (UBA) domain (e.g. present in structure 1IFY PDB), UEV domain (E2 ubiquitin variant) (e.g., present in the 1S1Q PDB structure), Ubiquitin Interacting Motif (UIM) domain (e.g., present in the 1Q0W PDB structure), VHL domain (e.g., present in the 1LM8 PDB structure ), VHS domain (Vps27p, Hrs, and STAM) (eg, present in the 1ELK PDB structure), WD40 domain (eg, present in the 1NEX PDB structure), WW domain (eg, present in the 1I6C PDB structure), and the like.

Individual elements of the library according to the present invention described herein may contain a modulating domain. Modulating domains include domains with stimulatory and inhibitory functions and domains that modulate the activating and/or inhibitory functions of other signaling domains, the reactions involving which proceed before the reactions involving the modulating domains. In some cases, modulating domains include costimulatory domains. In some cases, modulating domains include co-inhibitory domains.

A modulating domain suitable for inclusion in the library elements of the present invention may be any polypeptide functional unit that is equal in length to the 3 amino acid motif and is equal in length to the full-length protein, with the modulating domain being limited in size only by the fact that the domain must be large enough to retain its function and small enough to be compatible with the desired assembly method. Accordingly, the modulating domain may range in size from 3 to 1000 amino acids or more, and, in some cases, may be from about 30 to about 70 amino acids (a.a.) in length, for example, the modulating domain may be from about 30 to about 35 a.a., from about 35 to about 40 a.a., from about 40 to about 45 a.a., from about 45 to about 50 a.a., from about 50 to about 55 a.a. k., from about 55 to about 60 a.k., from about 60 to about 65 a.k. or from about 65 to about 70 a.k. In other cases, the modulating domain may be from about 70 to about 100 aa, from about 100 to about 200 aa in length. or more than 200 a.c.

In some cases, co-stimulatory domains can be used in individual elements of the libraries of the present invention. Co-stimulation generally refers to a secondary non-specific activation mechanism through which the primary specific stimulation is propagated. Examples of co-stimulation include non-antigen specific T cell stimulation following antigen specific T cell receptor signaling and non antigen specific B cell co stimulation following B cell receptor signaling. Co-stimulation, such as co-stimulation of T-cells, and related factors have been described by Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42, the contents of which are hereby incorporated by reference in their entirety. Co-stimulatory domains are usually polypeptides derived from receptors. In some embodiments of the present invention, the co-stimulatory domains are homodimerized. The co-stimulatory domain of the present invention may be an intracellular portion of a transmembrane protein (i.e., braid

- 19 041085 the stimulating domain can be derived from a transmembrane protein). Non-limiting examples of suitable costimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In some cases, the co-stimulatory domain, such as that used in the library element of the present invention, may include the co-stimulatory domain listed in Table. 1. In some cases, the co-stimulatory domain of an individual library element contains an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the amino acid sequence of the costimulatory domain described herein.

In some cases, co-inhibitory domains can be used in individual elements of the libraries according to the present invention. Suitable co-inhibitory domains are typically receptor-derived polypeptides. Co-inhibition generally refers to secondary inhibition of primary antigen-specific activation mechanisms that prevents co-stimulation. Co-inhibition, eg, co-inhibition of T cells, and related factors have been described by Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12, the contents of which are incorporated herein by reference in their entirety. In some embodiments of the present invention, the co-inhibitory domains are homodimerized. The co-inhibitory domain of the present invention may be an intracellular portion of a transmembrane protein (ie, the co-inhibitory domain may be derived from a transmembrane protein). Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1. In some instances, a co-inhibitory domain, such as that used in a library element of the present invention, may include the co-inhibitory domain listed in Table 1. 1. In some cases, the co-stimulatory domain of an individual library element contains an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the amino acid sequence of the costimulatory domain described herein.

In some instances, individual library elements from a library of synthetic modular polypeptides may include an intracellular signaling domain module. Suitable intracellular signaling domains for use as modules of the library of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by a cell; alteration in target gene transcription; alteration in protein activity; alteration in behavior cells, such as cell death, cell proliferation, cell differentiation, cell survival, modulation of cell signaling responses, etc.) in response to activation of a single element of the library. The intracellular signaling domain may or may not be covalently attached to individual elements of the library, for example, in cases where all elements of the library use a common intracellular signaling domain, the intracellular signaling domain may not be associated with elements of the library, for example, it may diffuse into cytoplasm.

In some cases, individual elements of the library according to the present invention will include a transmembrane domain for insertion into the membrane of eukaryotic cells. The transmembrane domain may be present at any convenient location within the library elements, e.g., at the N-terminus for modular library components, at the C-terminus for modular library components, may be located between at least two modular library components (e.g., between the antigen-binding domain and the costimulatory domain). elements of the CAR modular library), etc.

Any transmembrane (TM) domain that allows the introduction of the polypeptide into the membrane of eukaryotic cells (eg, mammalian) is suitable for use. As one non-limiting example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 1) is suitable for use. Additional non-limiting examples of suitable TM sequences include:

a) sequences derived from CD8-beta: LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 2);

b) sequences derived from CD4: ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO: 3);

c) sequences derived from CD3-zeta: LCYLLDGILFIYGVILTALFLRV (SEQ ID NO: 4);

d) sequences derived from CD28: WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 5);

e) sequences derived from CD134 (OX40): VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO: 6); And

f) sequences derived from CD7: ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 7).

- 20 041085

In some cases, individual elements of libraries of synthetic modular polypeptides can be configured to contain one or more additional modular elements. For example, where the library is a chimeric antigen receptor (CAR) library, individual elements of the library can be configured to contain one or more additional components as described, for example, in PCT patent application publication WO 2014/127261, the contents of which are incorporated herein by reference in their entirety.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more multidomain backbone protein modules. In this application, the term scaffold proteins includes anchor proteins and adapter proteins. These proteins contain several binding domains, each of which attracts or anchors certain elements of the signaling pathway, for example, anchors them in complexes, localizes them in the cell, or modulates the signaling process (for example, controls positive and/or negative feedback, stabilizes activated signaling components from inactivation, etc.). Therefore, the domains of multidomain scaffold proteins, multidomain anchor proteins, and multidomain adapter proteins typically comprise the binding domains of signaling pathway members.

Non-limiting examples of scaffold proteins and the signaling pathways in which they function include, for example, scaffold protein Ste5 of the mitogen-activated protein kinase (MAPK) pathway; A-kinase anchor proteins (AKAP) signaling pathways involving protein kinase A (PKA); kinase suppressor Ras 1 (KSR) signaling pathway involving MAPK; B-cell lymphoma protein 10 (BCL-10) of the JUN N-terminal kinase (JNK) signaling pathway and the MAPK signaling pathway; mitogen-activated protein kinase kinase kinase 1 (MEKK1) of the JNK signaling pathway and the MAPK signaling pathway; AHNAK-1 calcium-dependent signaling pathway; HOMER calcium-dependent signaling pathway; Pellino proteins of the signaling pathway of the innate immune system; the NLR family containing pyrine domains (NLRP) proteins of the innate immune signaling pathway; a Disks large 1 (DLG1) homologue of the T cell receptor signaling pathway; spinophilin dendritic cell signaling pathway; and so on. Scaffold proteins also include, but are not limited to, for example those described by Buday & Tompa. FEBS Journal (2010) 277:4348-4355; the contents of which are incorporated herein by reference in their entirety. In some cases, a scaffold protein may be a protein associated with the gene ontology (GO) terms scaffold protein complex (GO: 0032947) and synonymous terms that can be used to obtain information related to protein kinases, including sequences such as those provided on the website www.ebi.ac.uk/QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more protein kinase modules. Protein kinases are proteins that function by adding phosphate groups to substrate proteins to control substrate protein activity, association with other proteins, and/or localization. Protein kinases may include proteins that are associated with the terms GO protein phosphorylation (GO: 0006468), protein kinase activity (GO: 0004672), serine/threonine protein kinase activity (GO: 0004674), kinase activity (GO: 0016301), and synonymous terms that may be used to obtain information relating to protein kinases, including sequences such as those provided at www.ebi.ac.uk/QuickGO. Protein kinases contain one or more kinase domains that perform the catalytic function of the protein kinase. The protein kinase domains are associated with the Pfam identifier PF00069, which can be used to obtain information about the protein kinase domains of those proteins that contain protein kinase domains, including sequences and structures presented, for example, on the pfam.xfam.org website.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more protein phosphatase modules. Protein phosphatases are proteins that function by removing a phosphate group from a phosphorylated amino acid residue of their substrate protein, resulting in dephosphorylation to control the activity of the substrate protein, and act inversely to those of protein kinases. Protein phosphatases are grouped into three classes: phosphoprotein phosphatases (for example, PPP1CA, PPP1CB, PPP1CC, PPP2CA, PPP2CB, PPP3CA, PPP3CB, PPP3CC, PPP4C, PPP5C, PPP6C, etc.), protein tyrosine phosphatases (for example, CDC14A, CDC14B, CDC14C, CDKN3 , PTEN, SSH1, SSH2, SSH3, etc.) and dual specific protein phosphatases (e.g., DUSP1, DUSP2, DUSP3, DUSP4, DUSP5, DUSP6, DUSP7, DUSP8, DUSP9, DUSP10, DUSP11, DUSP12, DUSP13, DUSP14, DUSP15, DUSP16, DUSP18, DUSP19, DUSP21, DUSP22, DUSP23, DUSP26, DUSP27, DUSP28, etc.); however, some protein phosphatases remain unclassified. Protein phosphatases may include proteins that are associated with the term GO phosphatase activity (GO: 0016791) and synonymous terms that can be used to obtain information related to protein phosphatases,

- 21 041085 including sequences such as those provided on the website www.ebi.ac.uk/QuickGO. Protein phosphatases contain one or more phosphatase domains that perform the function of dephosphorylation of the protein phosphatase. The protein phosphatase domains are associated with the Pfam identifier PF15698, which can be used to obtain information on the protein phosphatase domains of those proteins that contain protein phosphatase domains, including sequences such as those presented on the pfam.xfam.org website.

In some instances, individual elements of a synthetic modular polypeptide library may be configured to contain one or more receptor tyrosine kinase protein modules, also referred to as receptor tyrosine kinases (RTKs). RTKs are membrane-bound cell surface receptors. There is a subclass of tyrosine kinases, RTKs, which function by binding an extracellular ligand and subsequent phosphorylation of the cytoplasmic portion of the protein. RTKs can be divided into families including, for example, the epidermal growth factor receptor family, the fibroblast growth factor receptor (FGFR) family, the vascular endothelial growth factor receptor (VEGFR) family, the RET receptor family, and the discoidin domain receptor (DDR) family. RTKs may include proteins that are associated with the terms GO transmembrane receptor tyrosine kinase activity (GO: 0004714), transmembrane receptor tyrosine kinase signaling pathway (GO: 0007169) and synonymous terms that can be used to obtain information related to RTK, including sequences such as those provided on the website www.ebi.ac.uk/QuickGO. RTKs contain one or more tyrosine kinase domains that carry out the RTK kinase function. The RTK kinase domains are linked to the Pfam identifier PF07714, which can be used to obtain information about RTK kinase domains and proteins containing RTK kinase domains, including sequences and structures, such as those provided at the pfam.xfam.org website.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more lipid kinase protein modules. Lipid kinase proteins phosphorylate cellular lipids, resulting in modulation of lipid reactivity, signal transduction, and/or lipid localization. Lipid kinases can be divided into families including, for example, phosphatidylinositol kinases and sphingosine kinases. Lipid kinases may include proteins that are associated with the terms GO lipid kinase activity (GO: 0001727), lipid phosphorylation (GO: 0046834) and synonymous terms that can be used to obtain information related to lipid kinases, including sequences, for example, presented on the web. www.ebi.ac.uk/QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more lipid phosphatase protein modules. Lipid phosphatase proteins dephosphorylate cellular lipids and their activity is opposite to that of lipid kinases, which results in modulation of lipid reactivity, signal transduction and/or lipid localization. Lipid phosphatases may include proteins that are related to the terms GO lipid phosphatase activity (GO: 0042577), phospholipid dephosphorylation (GO: 0046839) and synonymous terms that can be used to obtain information related to lipid phosphatases, including sequences such as those shown on the web. www.ebi.ac.uk/QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more ubiquitinylase protein modules. Ubiquitinylase proteins are proteins that mediate the post-translational modification of ubiquitination, the addition of ubiquitin to a substrate protein. Ubiquitination of a substrate protein can lead to degradation of the substrate protein, translocation of the substrate protein, modulation of the activity of the substrate protein, modulation of protein-protein interaction of the substrate protein, etc. Ubiquitinylases may include proteins associated with the terms GO protein ubiquitination (GO: 0016567), ubiquitin protein transferase activity (GO: 0004842) and synonymous terms that can be used to obtain information related to ubiquitinylases, including sequences shown, for example, in website www.ebi.ac.uk/QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more deubiquitinylase protein modules. Deubiquitinylase proteins are proteins that mediate the reversal of ubiquitination, the removal of ubiquitin from a substrate protein. Deubiquitination of the substrate protein can reverse the action of ubiquitinases and prevent degradation of the substrate protein, reverse ubiquitin-related substrate protein trafficking, reverse ubiquitin-related modulation of substrate protein activity, reverse ubiquitin-related modulation of protein-protein interaction of the substrate protein, etc. Deubiquitinylases may include proteins that are associated with the term GO protein deubiquitination (GO: 0016579) and synonymous terms that can be used to obtain information related to deubiquitinylases, including sequences such as those provided on the website www.ebi.ac.uk/ QuickGO.

In some cases, individual elements of libraries of synthetic modular polypeptides can

- 22 041085 can be configured to contain one or more SUMOylase protein modules. SUMOylase proteins are proteins that mediate post-translational modification of SUMOylation, the addition of a small ubiquitin-like modifying protein (SUMO) to a substrate protein. SUMOylation can modulate various protein functions, including protein stability, nuclear-cytosolic transport, transcriptional regulation, and so on. SUMOylases may include proteins associated with the term GO protein SUMOylation (GO: 0016925) and synonymous terms that can be used to obtain information related to SUMOylases, including sequences such as those provided on the website www.ebi.ac.uk /QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more acetylase protein modules, also referred to as acetyltransferases. Acetyltransferases are transferase enzymes that catalyze the transfer of an acetyl group to a protein substrate and are involved in epigenetic and transcriptional modulation. Acetyltransferases can be classified into groups including, for example, histone acetyltransferases, choline acetyltransferases, chloramphenicol acetyltransferases, serotonin K acetyltransferases, NatA acetyltransferases, NatB acetyltransferases. Acetyltransferases may include proteins that are associated with the term GO acetyltransferase activity (GO: 0016407) and synonymous terms that can be used to obtain information related to acetyltransferases, including sequences such as those provided on the website www.ebi.ac.uk /QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more deacetylase protein modules. Deacetylase proteins reverse the action of acetyltransferases and remove acetyl groups transferred to the substrate protein and hence also participate in epigenetic and transcriptional modulation. Deacetylases include, for example, histone deacetylases and sirtuins. Deacetylases may include proteins that are associated with the term GO deacetylase activity (GO: 0019213) and synonymous terms that can be used to obtain information related to deacetylases, including sequences such as those provided on the website www.ebi.ac.uk /QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries can be configured to contain one or more methylase protein modules, also referred to as methyltransferases. Methyl transferases alkylate substrates (including substrates such as proteins and nucleic acids) by transferring a methyl group to the substrate and are involved in epigenetic and transcriptional modulation. Methyltransferases can be divided into classes based on their structure, including, for example, class I, class II, class III, and can be grouped according to their substrates or mode of methylation, including, for example, protein methyltransferases, DNA methyltransferases, natural product methyltransferases, and SAM-independent methyltransferases. Methyltransferases may include proteins that are associated with the terms GO DNA methyltransferase activity (GO: 0009008), histone methyltransferase activity (GO: 0042054) and synonymous terms that can be used to obtain information related to methyltransferases, including sequences, for example, presented on the website www .ebi.ac.uk/QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more demethylase protein modules. Demethylases reverse the action of methyltransferases and catalyze the removal of methyl groups from substrates (including substrates such as proteins and nucleic acids) and hence are also involved in epigenetic and transcriptional modulation. Demethylases may include proteins that are associated with the terms GO demethylase activity (GO: 0032451), DNA demethylase activity (GO: 0035514), histone demethylase activity (GO: 0032452) and synonymous terms that can be used to obtain information related to demethylases , including sequences such as those provided on the website www.ebi.ac.uk/QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more nuclease protein modules. Nucleases catalyze the cleavage of phosphodiester bonds between the nucleotide subunits of nucleic acid substrates. Nucleases can be classified into non-mutually exclusive categories such as endonucleases and exonucleases. Nucleases may include proteins that are associated with the terms GO nuclease activity (GO: 0004518), deoxyribonuclease I activity (GO: 0004530), RNA-DNA fusion ribonuclease activity (GO: 0004523), nucleic acid phosphodiester bond hydrolysis (GO: 0090305 ), endonuclease activity (GO: 0004519), exonuclease activity (GO: 0004527), and synonymous terms that can be used to obtain information related to nucleases, including sequences such as those provided on the website www.ebi.ac.uk /QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more recombinase protein modules. rekom

- 23 041085 binases catalyze direction-sensitive nucleic acid exchange reactions between target site sequences specific for each recombinase, resulting in excision/insertion, inversion, translocation and exchange of nucleic acid fragments. Examples of recombinases include, but are not limited to, Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, and the like. Recombinases may include proteins that are associated with the terms GO recombinase activity (GO: 0000150), DNA recombination (GO: 0006310) and synonymous terms that can be used to obtain information related to recombinases, including sequences such as those presented on the web. www.ebi.ac.uk/QuickGO.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more transcription factor protein modules. Transcription factors are proteins that bind to specific DNA sequences and control transcription. Transcription factors can be activators that increase transcription or repressors that repress transcription. Transcription factors can be classified into superclasses based on the structure of their DNA-binding domains, including, for example, core domain-containing transcription factors, transcription factors containing a zinc-coordinating DNA-binding domain, helix-turn-helix transcription factors, transcription factors containing beta- framework factors, and those transcription factors that are not included in any of the above superclasses (see, for example, Stegmaier et al. Genome Inform (2004) 15(2):276-86). Transcription factors contain one or more DNA binding domains. Transcription factor DNA binding domains can be one or more DNA binding domains associated with Pfam identifiers PF00010, PF00170, PF00172, PF00046, PF00319, PF08279, PF00096, PF00105, etc., which can be used to obtain information about sequences and domain structure, such as the pfam.xfam.org website. Transcription factors may also contain one or more trans-activating domains, one or more signaling domains. Transcription factors may include proteins associated with the term GO transcription factor complex (GO: 0005667) and synonymous and related terms that can be used to obtain information related to transcription factors, including sequences, for example, presented on the website www. ebi.ac.uk/QuickGO.

Other DNA binding domains, such as DNA binding domains not derived from transcription factors or DNA binding domains not belonging to transcription factors, can also be used in some instances in one or more individual element modules of the synthetic modular polypeptide libraries described herein. document. Suitable DNA-binding domains derived from non-transcription factors include natural and synthetic polypeptide domains that bind non-specifically to DNA or bind to specific DNA sequences. DNA-binding domains derived from non-transcription factors may include, but are not limited to, for example, a natural or modified DNA-binding domain from a zinc finger endonuclease polypeptide, a natural or modified DNA-binding domain from a transcription activator-like effector nuclease polypeptide ( TALEN), a natural or modified DNA-binding domain of a Cas9 polypeptide (including, for example, Cas9 with complete nuclease inactivation (dCas9) and the like), etc.

In some instances, individual elements of synthetic modular polypeptide libraries may be configured to contain one or more of the modules described in PCT application US 2004/019778, the contents of which are hereby incorporated by reference in their entirety.

The assembly of the modules into the synthetic modular polypeptides described herein will result in a library containing a plurality of individual modular functional proteins, which may, but need not, share a common function. For example, individual library elements may contain or may consist of synthetic modular polypeptides that are modular scaffold proteins, synthetic modular polypeptides that are modular receptor proteins, synthetic modular polypeptides that are protein kinase or protein phosphatase modular proteins, synthetic modular polypeptides, which are modular transcriptional regulatory proteins, synthetic modular polypeptides which are modular epigenetic regulatory proteins, synthetic modular polypeptides which are modular proteins of recombinases or nucleases, etc. Suitable libraries can be screened for the desired phenotype according to the methods described herein.

Reporters.

The libraries described herein include detectable signal proteins whose expression is encoded by the nucleic acid sequences of the libraries. The specific detectable signal proteins used in the library systems described herein will vary and depend in part on the preferred method for detecting the resulting

- 24 041085 signal. For example, if the signal is detected by optical methods, such as using fluorescence microscopy or flow cytometry (including fluorescence-activated cell sorting (FACS)), then a fluorescent reporter is used.

Suitable detectable signal proteins include, for example, fluorescent proteins; enzymes that catalyze a reaction that results in a detectable signal as a product; epitope marks, surface markers, and the like. Detectable signaling proteins can be detected directly or indirectly. For example, if a fluorescent reporter is used, then reporter fluorescence can be detected directly. In some cases, if an epitope tag or surface marker is used, then the epitope tag or surface marker can be detected indirectly, for example, with a detectable binding agent that specifically binds to the epitope tag or surface marker, for example, with a fluorescently labeled antibody that specifically binds with epitope tag or surface marker. In some cases, a reporter that is normally detected indirectly, such as an epitope tag or surface marker, can be detected directly, or a reporter that is normally detected directly can be detected indirectly, such as by using a detectable antibody that specifically binds to a fluorescent reporter.

Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, GFP blue fluorescent variant (BFP), GFP blue fluorescent variant (CFP), GFP yellow fluorescent variant (YFP), GFP enhanced (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean , T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, monomeric DsRed, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, protein Kaede and photoactivated protein, phycobiliproteins and phycobiliprotein conjugates including B-phycoerythrin, R-phycoerythrin and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909) and the like.

Any of the many fluorescent and stained proteins from Anthozoa species described, for example, by Matz et al. (1999) Nature Biotechnol. 17:969-973, suitable for use.

Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-Nacetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO) etc.

Linkers and connections.

Links between modular components encoding a single polypeptide within individual library elements typically maintain a reading frame, i.e. the open reading frame of codons of a multi-module coding sequence is maintained from one module to the next. These compounds may be referred to herein as open reading frame compounds and/or open reading frame bonds. Linkers between the modular components of multimodular polypeptides will typically be flexible and/or will not contain amino acid residues that interfere with the function of the modular domains.

Suitable open reading frame connections can be achieved, as described in more detail below, by any convenient method of arranging a coding sequence and nesting modular components to include an open reading frame linker.

Suitable linkers can be readily selected and can be of any suitable length, for example, from 1 amino acid (e.g. Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may contain 1, 2, 3, 4, 5, 6, or 7 amino acids.

Typical linkers include glycine polymers (G) _n , glycine-serine polymers (including, for example, (GS) _n , (GSGGS) _n (SEQ ID NO: 8) and (GGGS) _n (SEQ ID NO: 9), where n is an integer, at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Suitable for use are glycine polymers and glycine-serine polymers; Gly and Ser are relatively unstructured and therefore can serve as a neutral linker between components. Suitable polymers are glycine; glycine receives significantly more φ-ψ conformational space, even compared to alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Typical linkers may contain amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 10), GGSGG (SEQ ID NO: 11), GSGSG

- 25 041085 (SEQ ID NO: 12), GSGGG (SEQ ID NO: 13), GGGSG (SEQ ID NO: 14), GSSSG (SEQ ID NO: 15) and the like.

In some instances, the linker contains the BamHI restriction enzyme recognition site sequence for cloning purposes and as part of the linker because the BamHI site encodes GS.

In some cases, linker sequences can be eliminated by one or more cloning steps (eg, using restriction endonucleases type IIS or homologous recombination) or by direct cleavage (eg, cleavage with BamHI restriction enzyme).

In some cases, an open reading frame connection is achieved by the absence of a linker at the junction of the two modular components. Therefore, an open reading frame compound may, in a sense, not contain an intermediate nucleic acid encoding amino acids between the modular coding sequences. In some instances, an open reading frame compound may contain two or fewer intermediate nucleobase pairs between the modular coding sequences. In some instances, an open reading frame compound may contain one or more intermediate nucleobase pairs between the modular coding sequences. In some cases, an open reading frame compound may not contain intermediate nucleobase pairs between the modular coding sequences.

Nucleic sequences-barcodes.

Nucleic barcode sequences are specific, unique nucleic acid sequences that can be identified by any suitable nucleic acid sequence identification method, including but not limited to, for example, hybridization-based identification (i.e., in situ hybridization), identification by amplification-based (i.e. PCR-based identification) and nucleic acid sequencing. The barcode nucleic acid sequences of the present invention may be module specific barcode nucleic acid sequences, meaning that each unique module of the multi-module library is correlated with a particular unique barcode nucleic acid sequence such that identification of a particular barcode sequence is equivalent to a positive identification of the corresponding sequence, encoding module.

The module-specific nucleic sequence-barcode described herein will be limited by the method used to assemble the library. For example, if nested assembly of multimodule constructs is performed using a restriction enzyme method, the barcode nucleic acid sequences will exclude any sequence that represents a restriction enzyme recognition site for the restriction enzymes used in the assembly. Therefore, in some cases, barcode sequences will not contain sequences recognized by restriction enzymes. In some cases, the barcode sequences will not contain sequences recognized by restriction enzyme type IIS.

Where barcode nucleic acid sequence identification and/or quantification is performed by sequencing, including, for example, next generation sequencing methods, standard criteria for barcode nucleic acid sequences detected by sequencing will apply. In some instances, commercially available barcode nucleotide sequences and/or kits containing barcode nucleotide sequences and/or barcode nucleotide sequence adapters may be used or modified for use in the methods described herein, including, for example, barcode nucleotide sequences and /or barcode nucleic sequence adapter kits commercially available from vendors such as, but not limited to, New England Biolabs (Ipswich, MA, USA), Illumina, Inc. (Hayward, California, USA), Life Technologies, Inc. (Grand Island, New York, USA), Bioo Scientific Corporation (Austin, Texas, USA) and the like, or can be custom made, for example, as available from, for example, Integrated DNA Technologies, Inc. (Coralville, Iowa, USA).

The length of the nucleic barcode sequence will vary and will depend on the complexity of the library and the barcode sequence detection method used. Since nucleic barcode sequences (eg, DNA barcode sequences) are well known, the design, synthesis and use of nucleic barcode sequences is within the skill of the art.

In some cases, the length of the barcode nucleic sequences used will also depend on the likelihood that a particular barcode nucleic sequence will accidentally appear in some other component of the library, including, but not limited to, for example, a coding sequence, a vector, a vector component, etc. .d., or at the junction of two links of nucleic barcode sequences. For example, in some cases, if there is a significant possibility that a sequence that is not related to the nucleic 26 041085 sti-barcode sequence can be accidentally detected as a barcode sequence, for example, as is the case in very complex libraries, there may be links of nucleic sequences-barcodes of greater length were used. In some cases, the barcode nucleic acid sequence detection method may be taken into account when determining the required barcode sequence length, for example, in cases where the barcode nucleic acid sequence is detected by hybridization, longer barcode nucleic sequences can be used compared to that case. when sequencing is applied using specific sequencing primers.

As used herein, the term barcode nucleic sequence region, as applied to individual elements of a nucleic acid library, refers to the region of each nucleic acid that contains a nucleic acid sequence specific for the variable modular portion of a synthetic polypeptide. In some cases, the nucleic sequence-barcode region can be used to specifically identify the variable module or modules present in the coding region of a particular library element. In some cases, the nucleic sequence-barcode region can be used to specifically identify the variable module(s) and identify the order of the variable modules present in the coding region of a particular library element (ie, architecture). In some instances, the barcode nucleic sequence region can be used to quantify, eg, semi-quantify, the frequency of a particular library element or the frequency of a particular module in a population containing multiple library elements.

Within the synthetic modular barcode nucleic acid sequence library described herein, the barcode nucleic acid sequence units in the barcode sequence region will be in reverse orientation with respect to the sequences encoding the modules. In addition, the units of nucleic barcode sequences in the area of barcode sequences will be located in reverse order with respect to their corresponding sequence encoding the module. However, with respect to the encoded polypeptide, the region of nucleic acid sequences-barcodes can be located in the direction of the N-terminus or C-terminus relative to the nucleic acid sequence encoding the synthetic modular polypeptide.

Vector-specific elements.

By vector-specific elements is meant elements that are used in the production, construction, distribution, maintenance and/or quantification of a vector before, during or after its construction. Suitable vector-specific elements include, but are not limited to, for example, vector elements necessary for propagation, cloning, and selection of the vector during use, and may include, but are not limited to, for example, replication origin, multiple cloning site, prokaryotic a promoter, a phage promoter, a selectable marker (eg, an antibiotic resistance gene, an encoded enzyme protein, an encoded fluorescent or chromogenic protein, etc.), and the like. In the vectors described herein, any suitable vector-specific elements can be used, if desired.

Suitable promoter and enhancer elements to be used as vector-specific elements are known in the art. Promoters suitable for expression in a bacterial cell include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. Promoters suitable for expression in a eukaryotic cell include, but are not limited to, the promoter and enhancer elements of the immunoglobulin light and/or heavy chain gene; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; SV40 early and late promoters; a promoter present in the long terminal repeats of the retrovirus; mouse metallothionein-1 promoter; and various tissue-specific promoters known in the art.

Suitable reversible promoters, including reversible inducible promoters, are known in the art. Suitable reversible promoters can be isolated and obtained from many organisms, such as eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, eg, the first organism is a prokaryote and the second organism is a eukaryote, the first organism is a eukaryote and the second organism is a prokaryote, etc., is well known in the art. Suitable reversible promoters and systems based on suitable reversible promoters, while also containing additional control proteins, include, but are not limited to, alcohol-regulated promoters (e.g., the alcohol dehydrogenase I (alcA) gene promoter, alcohol-transactivated protein-responsive promoters). (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid-regulated promoter systems, thyroid promoter systems

- 27 041085, ecdysone-regulated promoter systems, mifepristone-regulated promoter systems, etc.), metal-regulated promoters (for example, metallothionein promoter systems, etc.), pathogenesis-regulated promoters (for example, salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock protein promoter, etc.), light promoters, synthetic inducible promoters, and the like.

In some cases, the locus or construct or transgene containing a suitable promoter is irreversibly switched by inducing an inducible system. Suitable systems for inducing an irreversible switch are well known in the art, for example, induction of an irreversible switch can be based on the use of Cre-lox mediated recombination (see e.g. Fuhrmann-Benzakein, et al., PNAS (2000) 28: e99, the contents of which are incorporated herein by reference in their entirety). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known in the art can be used to create an irreversible switch promoter. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein can be used to generate irreversible switch promoters and are well known in the art, see, for example, Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), the contents of which are incorporated herein by reference.

Methods for obtaining libraries.

The present invention provides methods for obtaining nucleic acid libraries encoding synthetic modular polypeptides, each nucleic acid of the library containing a multi-link nucleic barcode sequence that identifies the variable module(s) of the modular polypeptide and, if multiple variable modules are present, the orientation of the modules relative to each other. In numerous embodiments of the present invention, methods are provided for stepwise combinatorial assembly of synthetic modular polypeptides from nucleic acids encoding modules containing barcode nucleic sequences, such that each of the resulting nucleic acids encoding synthetic modular polypeptides contains a region encoding modules inserted while maintaining an open reading frame. , and a multi-link nucleic sequence-barcode, while the arrangement of the nucleic sequence-barcode units corresponds to the arrangement of modules built-in while maintaining an open reading frame. In general, where multiple variable modules are included in each library element, the co-assembled multi-unit barcode nucleic acid sequence provides characterization of the assembly of the variable modules of each library element.

Without wishing to be bound by any theory, the present inventors believe that the stepwise combinatorial assembly of the synthetic modular polypeptides provided herein allows for the construction of larger and more complex libraries than would be possible or feasible using standard construction methods based on individual (ie, one at a time) polypeptides. The authors of the present invention have found that standard methods for constructing synthetic modular polypeptides represent a significant technical obstacle to high-throughput screening of synthetic modular polypeptides. The coordinated assembly of each multi-module synthetic polypeptide and the corresponding multi-unit barcode nucleic acid sequence provided herein overcomes this hurdle and allows assembly in a single reactor of a plurality of unique nucleic acids encoding suitable synthetic modular polypeptides for screening. Said high-throughput, single-pot assembly cannot be accomplished using standard polypeptide construction techniques.

The authors of the present invention also found that standard, physically grouped, synthetic polypeptide libraries also represent serious technical barriers to high-throughput screening. Particularly when screening large libraries, the number of physically separated reaction chambers and the variability of assay conditions between each chamber present significant technical hurdles when attempting to screen an entire complex large library and analyze the resulting data. The present inventors have found that pooled library screening can solve this problem, but has other significant hurdles, such as the difficulty or inappropriateness of identifying and/or quantifying individual polypeptides that provide the desired phenotype when screened from a complex mixed pool of unique modular polypeptides. The use of a short multi-link barcode nucleic acid sequence eliminates this problem by providing a posteriori efficient positive identification and/or quantification of individual unique synthetic modular polypeptides that provide the desired phenotype in a complex pool,

- 28 041085 by sequencing only the multilink nucleic barcode sequence.

Cloning strategies.

In general, the present invention provides a method for obtaining the libraries described herein by nesting nucleic acids encoding polypeptide modules containing barcode nucleic sequences. For example, as shown in FIG. 12, a nucleic acid vector (100) containing a sequence encoding a polypeptide module (101) linked to a module-specific barcode nucleic sequence (102) is linearized (103) by cleaving the link between the sequence encoding the polypeptide module and the module-specific nucleic sequence-barcode. After linearization of the vector containing the first coding module sequence and the first module-specific barcode sequence, the nucleic acid containing the second module-coding sequence (104) and the second module-specific nucleic acid sequence (105) is inserted (i.e. . embed) between the first coding module sequence and the first nucleic sequence-barcode (106). The assembled nucleic acid contains a coding region encoding a synthetic modular polypeptide and a nucleic barcode sequence region that contains a multi-link nucleic barcode sequence (ie barcode nucleic acid sequences (BCs)). In some cases, sequences encoding modules are assembled so that they are joined by a sequence encoding the desired linker described herein. In some cases, sequences encoding modules are assembled so that they are connected without the use of a linker sequence, for example, without a linker sequence encoding one or more linker amino acids, without any intervening non-coding nucleotides between the first and second sequences encoding modules, etc. d. Therefore, the designation of a desired linker sequence or linker, particularly as used in the drawings, includes the complete absence of any linker or linker sequence and the direct connection of polypeptide modules and sequences encoding modules.

Linearization of the vector sequence by splitting the region between the first module-encoding sequence and the first module-specific nucleic barcode sequence can be achieved by any convenient and appropriate means. For example, a polynucleotide comprising a module-coding sequence and a barcode nucleic sequence can be configured to contain a restriction enzyme (ie, restriction endonuclease) cleavage site between the module-coding sequence and the barcode nucleic acid sequence. The cleavage site may be a type II restriction enzyme cleavage site, and more specifically, may be a cleavage site that is contained in a sequence recognized by the type II restriction enzyme used. Any convenient type II restriction enzyme that cleaves within its recognition sequence can be used for the described purpose, including type II restriction enzymes that cleave within its recognition sequences, which are known in the art. In some cases, the cleavage site may be a BamHI cleavage site that has a 5'GGATCC-3' recognition sequence and cleaves strands after the first G recognition sequence on both strands, leaving a 5'-GATC-3' sticky end. Other restriction enzymes that can be used for this purpose include, but are not limited to, for example.

In some cases, nested assembly is performed by using sites of two restriction enzymes (RE1 and RE2) located between the first coding module sequence and the first module-specific barcode nucleic sequence and flanking the second module coding sequence containing the barcode nucleic sequence (Fig. 13). In the case of an assembly strategy based on the use of two restriction enzymes, cleavage at the two sites RE1 and RE2 results in linearization of the vector and release of a fragment containing a second coding module sequence containing a nucleic barcode sequence. After ligation of the linearized vector and the released fragment, the desired linker sequence is present between the first and second module coding sequences, and the first and second barcode nucleic sequences are in reverse orientation with respect to the first and second module coding sequences.

In some cases, nested assembly is carried out by using the sites of four restriction enzymes (RE1, RE2, RE3 and RE4), with RE1 and RE2 located between both coding modules sequences and their respective nucleic barcode sequences, while RE3 and RE4 flank the second coding module sequence containing nucleic sequence-barcode (Fig. 14). In the case of an assembly strategy using four restriction enzymes, separate cleavage of the first vector at the RE1 and RE2 sites and the second vector at the RE3 and RE4 sites results in the linearization of the vector and the release of a fragment containing a second coding module sequence containing a nucleic sequence-barcode. After ligation of the linearized vector and the released fragment, the desired linker sequence is present between the first and second module encoding sequences, while the first and second nucleic barcode sequences are in reverse orientation with respect to the first and second module encoding sequences.

In some cases, the restriction enzymes are deliberately chosen such that, after ligation, the junctions between the first and second coding modules sequences and the first and second nucleic barcode sequences do not contain restriction enzyme recognition sequences RE1, RE2, RE3 or RE4. In general, according to this strategy, the RE1, RE2, RE3 and RE4 sites used are inactivated after ligation so that the resulting vector contains only active RE1 and RE2 sites between the second coding module sequence and its corresponding nucleic barcode sequence. Therefore, this strategy allows sequential nesting of more than just a two-dimensional construct by re-linearizing the resulting vector with restriction enzyme digestion of RE1 and RE2 of the module coding sequence containing the barcode nucleic acid sequence that was inserted last.

In the case of the assembly method using four restriction enzymes, RE1 is chosen so that after cleavage, the resulting end of the linearized vector is compatible (ie, capable of ligation) with the end of the released fragment obtained by cleavage with the RE3 restriction enzyme. RE2 is chosen such that, after digestion, the resulting end of the linearized vector is compatible (i.e., can be ligated and/or is capable of full or at least partial hybridization) with the end of the released fragment resulting from digestion with the restriction enzyme RE4 . In some cases, one or more of the ends resulting from cleavage with RE1, RE2, RE3, or RE4 may be modified, for example, by adding or removing one or more nucleotides or other chemical modification to obtain ends that are compatible between RE1 and RE3 or RE2 and RE4. Any convenient end modification method can be used to obtain compatible ends, including end modification methods that are well known in the art, including, but not limited to, blunt end generation, phosphorylation, dephosphorylation, and the like.

In some cases, nested assembly is performed by using an IIS restriction enzyme recognition sequence (RE1), with two RE1 sites present between the first coding module sequence and its corresponding barcode nucleic sequence, and two RE1 sites flanking the second coding module sequence containing the nucleic barcode sequence ( Fig. 15). In the case of this IIS-type restriction enzyme-mediated strategy, cleavage at sites adjacent to the RE1 recognition sites results in linearization of the vector and release of a fragment containing a second coding module sequence containing a barcode nucleic sequence. The cleavage sites adjacent to the RE1 recognition sites are configured such that, after cleavage, the 3' end of the vector is compatible with the 5' end of the fragment and the 5' end of the vector is compatible with the 3' end of the fragment. In some cases, these compatible ends may be referred to as compatible sticky ends. Therefore, when the linearized vector and the released fragment are ligated, the desired linker sequence is present between the first and second module coding sequences, while the first and second nucleic barcode sequences are in reverse orientation with respect to the first and second module coding sequences.

Any convenient type IIS restriction enzyme can be used in assembly strategies using the indicated enzymes described herein, including, but not limited to, for example, AceIII, AcuI, AlwI, AarI, BbsI, BbvI, BbvII, BccI, Bce83I, BceAI, Bcefl, BciVI, BfuAI, BmrI, BmuI, BpmI, BpuEI, BsaI, BsbI, BseRI, BsgI, BslFI, BsmAI, BsmFI, BsoMAI, BspCNI, BspGI, BspMI, BspNCI, BspQI, BsrDI, Bst71I, BtgZI, BtsCI, BtsI, BveI, DrdII, EarI, EciI, FaqI, FinI, FokI, HgaI, Hin4II, HphI, HpyAV, LguI, MboII, MmeI, MnlI, NmeAIII, PleI, SapI, SfaNI, SgeI, etc.

In some instances, the restriction enzyme used, for example, in linearizing the vector and/or releasing the fragment containing the second module, is a restriction enzyme that cleaves strands on either side of its recognition site. Any convenient restriction enzyme having this activity can be used in the assembly methods described herein, including, but not limited to, for example, BcgI.

In some cases, nesting is accomplished by using multiple IIS restriction enzyme recognition sequences, for example two IIS restriction enzyme recognition sequences (RE1 and RE2), where two RE1 sites are present between the module-coding sequences and their corresponding barcode nucleic acid sequences, and two RE2 sites are flanked a second coding module sequence containing a nucleic barcode sequence (Fig. 16). In the case of an assembly strategy using two type IIS restriction enzymes, separate cleavage of the first vector at the RE1 sites and the second vector at the RE2 sites results in vector linearization and release of a fragment containing a second coding module sequence containing a barcode nucleic sequence. After ligation of the linearized vector and the released fragment, the desired linker sequence is present between the first and second module coding sequences, while the first and second nucleic barcode sequences are in reverse orientation with respect to the first and second module coding sequences.

In general, according to this strategy, the RE1 and RE2 sites used in the first round of linearization and fragment release are not retained in the vector and inserted so that the resulting vector contains only active RE1 sites between the second coding module sequence and its corresponding nucleic barcode sequence. . Therefore, this strategy allows sequential nesting of more than just a two-dimensional construct by re-linearizing the resulting vector with restriction enzyme cleavage of the RE1 recognition sites in the module coding sequence containing the barcode nucleic acid sequence that was inserted last.

In this type IIS assembly method, cleavage sites adjacent to the RE1 and RE2 recognition sequences are configured such that, after cleavage, the resulting ends of the linearized vector are compatible (i.e., capable of ligation) with the ends of the released fragment. In some cases, one or more of the ends resulting from RE1 and/or RE2 cleavage may be modified, for example, by adding or removing one or more nucleotides or other chemical modification to obtain compatible ends. Any convenient end modification method can be used to obtain compatible ends, including end modification methods that are well known in the art, including, but not limited to, for example, blunt end preparation, phosphorylation, dephosphorylation, and so on.

In some cases, compatible ends can be obtained using an enzyme with exonuclease activity, such as exonuclease. For example, in some embodiments, the first vector and the second vector or nucleic acid fragment are configured such that when the first vector or nucleic acid is cleaved with a first restriction enzyme (RE1) and the second vector or nucleic acid with a second restriction enzyme (RE2), the resulting new ends are compatible for ligation after use of the enzyme. with exonuclease activity. Methods for producing compatible ends using an enzyme with exonuclease activity are well known in the art and include, but are not limited to, for example, In-Fusion® system cloning, Gibson assembly, and the like.

In some cases, the assembly of the first vector cleaved with the restriction enzyme and the second nucleic acid, such as the second vector containing the nucleic acid fragment, is carried out using the In-Fusion® reaction, and, in such cases, the compatible ends resulting from the use of the enzyme with exonuclease activity can be referred to as In-Fusion® sticky ends (IF sticky ends) (FIG. 17). The connection of the two sticky ends of the IF can be configured to obtain the desired linkers and/or the desired linker sequences between the two joined ends of the nucleic acid. For example, the connected IF sticky ends between the first coding module sequence and the second coding module sequence can be configured such that the first and second modules are arranged in an open reading frame. As described in more detail herein, coding sequence modules that are arranged in an open reading frame may or may not be separated by a sequence encoding one or more linker amino acids. For example, as shown in FIG. 17, cleavage of the first vector at the recognition site (RE1) of the first restriction enzyme results in ends that can be In-Fusion® fused with the ends obtained by cleaving the second vector or nucleic acid fragment with the type IIS restriction enzyme at the cleavage sites defined recognition sites (RE2) present on the second vector or nucleic acid fragment. The compatible ends are ligated via an In-Fusion® reaction resulting in the desired linker sequence between the first and second module-encoding sequences and a multi-unit barcode nucleic sequence containing a first unit and a second unit of barcode nucleic sequences that are in reverse orientation to the first. and a second coding module sequences. In some cases, after In-Fusion® assembly of the first and second coding module sequences and the multi-unit barcode nucleic sequence, a restriction enzyme site is present between the coding region and the barcode sequence region to allow the introduction of additional coding module sequences and barcode nucleic sequence units.

In some cases, after ligation of a nucleic acid containing a first coding module sequence with a second nucleic acid containing a second coding module

- 31 041085 sequence, the first and second coding sequence modules are connected so that between the first and second coding sequences there is no linker and intermediate non-coding nucleotides. In one non-limiting example, a first vector containing a first coding module sequence and its corresponding barcode nucleic sequence is connected without a linker or intermediate non-coding nucleotides to a second vector or nucleic acid fragment containing a second coding module sequence and its corresponding barcode nucleic sequence by enzyme digestion. restriction (Fig. 18). The first vector and the second vector or nucleic acid fragment are cleaved using two different type IIS restriction enzymes having two different recognition sites (RE1 and RE2), wherein the first restriction enzyme cleaves both nucleic acid strands at the same position some distance from its own. recognition site (RE1), leaving a blunt end, while the second restriction enzyme cleaves both strands of nucleic acid at different positions some distance from its recognition site (RE2), leaving an overhang or sticky end. The parent nucleic acids are configured such that after cleavage of the first vector and the second vector with a second restriction enzyme, the resulting sticky ends are compatible for ligation. Therefore, after ligation of the resulting blunt ends and sticky ends, the resulting vector contains the first coding module sequence hybridized directly, without a linker or intermediate non-coding nucleic acids, with the second coding module sequence and a barcode multilink nucleic sequence containing the nucleic sequence-barcode of the first coding module sequence and the nucleic acid a barcode sequence of the second coding module sequence in reverse orientation with respect to the first and second coding module sequences.

Any convenient type IIS restriction enzyme that results in blunt ends upon digestion can be used in the methods described herein using blunt end ligation, including enzymes that are well known in the art, including, but not limited to, e.g. SlyI, MlyI, etc. In addition, methods for producing blunt ends after digestion with a restriction endonuclease that does not form blunt ends, i. blunting may be used, as appropriate, including, but not limited to, terminating with a DNA polymerase such as, for example, a large fragment of DNA polymerase I (e.g., a Klenow fragment), T4 DNA polymerase, mung bean nuclease, etc. or the terminal mismatch can be removed by an enzyme with exonuclease activity.

In some cases, where a sequence compatible with a non-type IIS restriction enzyme is present at the end of the module-coding sequence, cleavage can be performed with a non-type IIS restriction enzyme, such as a type II restriction enzyme that cleaves the strands within its recognition sequence. In such cases, a restriction enzyme that forms a blunt end at the end of the coding sequence may be used if the coding sequence contains all or part of the restriction enzyme recognition sequence at its 3' or 5' end. In some cases, where a coding module sequence contains all or part of a restriction enzyme recognition sequence that cuts strands within its recognition sequence and does not form a blunt end, a non-blunt restriction enzyme can be used and the resulting sticky ends can be blunted, e.g. , in any convenient way, including, but not limited to, for example, the methods described herein.

Since the sequences at the ends of the module-coding sequences will be limited to the terminal amino acids of the module, where suitable restriction enzyme recognition sequences are present in a convenient position, or can be modified to do so, at one or more ends of the module-coding sequence to allow for proper cleavage and/or blunt end formation may be rare depending on the specific module used. Therefore, in many cases, efficient production of a large or multidimensional library of synthetic modular polypeptides will depend on enzyme recognition sites that are present outside the module-coding sequence. Therefore, in some cases, one or more, including all, of the restriction enzyme recognition sequences used in library assembly will be present outside of the module-coding sequence. In many cases, one or more, including all, of the restriction enzyme recognition sequences used in library assembly will be present outside of the barcode nucleic sequence.

In some instances, a synthetic modular polypeptide is prepared in which the coding sequence modules are readily fused to the ends of the linker domain such that there is no intervening sequence between the module coding sequences and their corresponding linker domain compounds. In one non-limiting example, said continuous

- 32 041085 The specific connection of module-coding sequences to a linker domain is facilitated by a vector or nucleic acid fragment configured to contain a portion of the desired linker sequence readily attached to either end of the module-coding sequence. For example, a first vector configured to contain a first coding module sequence fused to a first linker domain portion is ligated to a second vector or nucleic acid fragment configured to contain a second linker domain portion fused sequentially to a second coding module sequence ( Fig. 19). The first vector can be configured to contain two recognition sites for the first IIS type restriction enzyme (RE1) between the module coding sequence and its corresponding barcode nucleic sequence. The second vector, or nucleic acid fragment, can be configured to contain two second type IIS restriction enzyme (RE2) recognition sites flanking a second coding module sequence containing a nucleic barcode sequence. A third vector or nucleic acid fragment may be used containing the remainder, for example, the middle portion of the linker domain, which is flanked by two recognition sites of a second type IIS restriction enzyme (RE2). The sequence of pre-cleaved vectors and/or nucleic acid fragments is configured so that, after cleavage, compatible ends are obtained between the first part of the linker domain and the middle part of the linker domain, the second part of the linker domain and the middle part of the linker domain and two nucleic barcode sequences. After cleavage and ligation, the resulting vector contains a first coding module sequence connected in series with a linker domain connected in series with a second coding module sequence connected to a region of a nucleic barcode sequence containing the first and second nucleic barcode sequences in reverse orientation with respect to the first and to the second coding sequences (FIG. 19). Sequential assembly, eg between modules and linker domains, can be achieved with or without exonuclease mediated assembly (eg In-Fusion® system cloning, Gibson assembly, etc.).

The above-described cleavage-based assembly strategies may, in some instances, be combined, in whole or in part, in any convenient and appropriate way to provide a method suitable for preparing the library of synthetic modular polypeptides described herein. In addition, where alternative cleavage-based assembly methods are known in the art and are compatible with the methods described herein, suitable alternative methods can be used in assembling the synthetic modular polypeptide library described herein. In some instances, the cleavage strategies described can be combined, in whole or in part, with non-cleavage methods to assemble nucleic acids.

The assembly of nucleic acids encoding the synthetic modular polypeptide library described herein is not limited to cleavage-based assembly strategies, i. assembly based on restriction enzymes. In some instances, non-cleavage methods may be used in the assembly of the library described herein, including but not limited to, for example, amplification-based strategies, recombination-based strategies, and so on. Non-splitting methods are suitable for use instead of split-based strategies, ie. the assembly strategy as a whole does not involve restriction enzyme digestion, or can be used in combination with cleavage-based strategies, i.e., such that the assembly strategy as a whole includes both restriction enzyme-based methods and methods that do not associated with cleavage by restriction enzymes.

In some instances, the synthetic modular polypeptide library described herein may be assembled in whole or in part using an amplification-based assembly method including, but not limited to, e.g., PCR cloning, TA cloning, PCR end extension, and the like. . Suitable strategies based on amplification will vary, but will usually be based on the use of multiple primer binding sites in the original vectors and/or nucleic acid fragments. Suitable primer binding sites, depending on the desired end product, can be specifically added to the vectors and/or nucleic acid fragments, or a pre-existing sequence present in the vector or nucleic acid fragment can be used as the primer binding site, according to various assembly strategies based on amplification. The primer binding sites may be located in any convenient configuration and/or orientation sufficient to produce the desired cloned product, including but not limited to: location between the module coding sequence and its corresponding nucleic barcode sequence in the 5' to 3 direction '-end relative to the sequence encoding the module; the location between the coding sequence of the module and its corresponding nucleic sequence-barcode in the direction from the 5'-end to the 3'-end relative to

- 33 041085 barcode sequence; position before (ie, in the direction of the 5'-end) encoding the module sequence in the direction from the 5'-end to the 3'-end relative to the encoding module sequence; the location after (ie, in the direction of the 3'-end) encoding the module sequence in the direction from the 5'-end to the 3'-end relative to the coding module sequence containing the nucleic sequence-barcode; and so on. Primer binding site sequences can be configured such that after amplification-based cloning, including after one round of amplification-based cloning, one or more desired linker sequences are present between assembled elements, including, for example, assembled sequences, coding modules, assembled nucleic barcode sequences, etc.

As a non-limiting example of amplification-based assembly strategies, the PCR sticky-end amplification strategy (FIG. 20) can be used, in which the first vector contains a first primer binding site (PBS1) and a second primer binding site (PBS2) located, in the orientation opposite orientation from the 5'-end to the 3'-end, between the coding sequence of the module and its corresponding nucleic sequence-barcode. A second vector or nucleic acid fragment can be used containing a second coding module sequence containing a nucleic barcode sequence flanked by first and second primer binding sites (PBS1 and PBS2). After extension and amplification by sticky-end extension by PCR using primers that specifically hybridize to the PBS1 and PBS2 sites, an assembled product is synthesized in which the first coding module sequence is connected via the desired linker to the second coding module sequence, which is connected to the multi-nucleotide nucleic acid a barcode sequence containing the first and second nucleic barcode sequences in reverse orientation with respect to their respective coding modules sequences (FIG. 20).

In view of the above assembly strategies, one skilled in the art will readily understand how any of the above strategies can be combined, in whole or in part, to produce the desired result and/or provide the greatest advantages and/or minimize the disadvantages of specific assembly-specific cloning techniques. using the desired library and/or library component. As a non-limiting example, amplification-based strategies can be combined with cleavage-based strategies when the combination of these strategies results in the assembly of the desired library and/or library components. For example, as shown in FIG. 21, restriction enzyme digestion of the first vector according to the restriction enzyme recognition site (RE1) located between the first coding module sequence and its corresponding nucleic barcode sequence can be combined with PCR-based amplification using primer binding sites flanking the second coding module sequence. , containing the nucleic barcode sequence contained within the second vector or nucleic acid fragment, to provide a nested assembly. After assembly using the described hybrid assembly strategy, an assembled product can be obtained in which the first coding module sequence is connected via the desired linker to a second coding module sequence that is connected to a multi-link barcode nucleic sequence containing the first and second nucleic acid barcode sequences in reverse order. orientations with respect to their respective module coding sequences (FIG. 21).

Hybrid strategies are not limited to combining cleavage and amplification based strategies and may include other cloning and/or synthetic biology techniques, in whole or in part, including but not limited to, for example, recombination based cloning strategies (including but not limited to eg Gateway cloning strategies, Cre/Flp recombinase cloning (including strategies in which the site is inactivated by recombination), etc.), de novo sequence assembly, de novo nucleic acid synthesis, etc. . In some cases, cloning strategies based on recombination, de novo sequence assembly, de novo nucleic acid synthesis, and the like. can be used independently, i.e. separately as an independent cloning strategy, not as part of a hybrid cloning strategy.

In some cases, where a particular cloning strategy used results in the presence of an unwanted intermediate sequence between two cloned elements, also known as cloning scars or sutures, such cloning scars can be reduced and/or removed by unimolecular cleavage and religation of the vector containing the scar. Monomolecular cleavage and religation can be performed in any convenient manner, including, but not limited to, for example, restriction enzyme mediated unimolecular cleavage and religation, such as, for example, IIS type unimolecular cleavage and religation. For example, in some cases, the seam,

- 34 041085 which contains a complete recombination site or part thereof, as a result of the assembly based on recombination, can be partially or completely removed by monomolecular cleavage and re-ligation. In some instances, a cleavage scar that contains all or part of the restriction enzyme recognition site as a result of a cleavage strategy may be partially or completely removed by unimolecular cleavage and religation. In some cases, a suture that contains all or part of the primer binding site as a result of amplification-based assembly can be partially or completely removed by unimolecular cleavage and religation.

In one non-limiting embodiment, a combinatorial library is generated by iteratively cloning the library components of each dimension type via split-based cloning and In-Fusion® cloning. For example, as shown in FIG. 22, in a first step, a parent nucleic acid (eg, vector) containing the scFv coding sequence and an adjacent Gly/Ser linker is cleaved at a BamHI site within or adjacent to the Gly/Ser linker. In a second step, a nucleic acid fragment containing a first coding module sequence linked to a second Gly/Ser linker hybridized to the first module-specific barcode nucleic sequence is cloned into a cleaved BamHI site by cloning with an In-Fusion® system. After ligation by cloning with the In-Fusion® system, the Gly/Ser linker between the scFv and the first coding module sequence is retained. In the third step, the nucleic acid assembled in step 2 is cleaved at the BamHI site imported within or adjacent to the Gly/Ser linker of the first nucleic acid coding module fragment, and cloning is repeated using the In-Fusion® system with the second nucleic acid fragment containing a second module-encoding sequence linked to a third Gly/Ser linker hybridized to a second module-specific barcode nucleic acid sequence. As in step 2, after ligation by cloning with the In-Fusion® system, the Gly/Ser linker between the first and second coding sequences is preserved. If larger library elements are needed, step 3 can be repeated iteratively. In a fourth step, cleavage at a BamHI site imported within or adjacent to the Gly/Ser linker encoding a module of the last added nucleic acid sequence allows In-Fusion® cloning of the terminal reporter sequence (e.g., the sequence encoding GFP that also contains a stop signal sequence (eg, a stop codon)) between the finished module coding sequence and the nucleic barcode sequence located after it. In this implementation, each completed library element contains a combinatorial CAR embedded in an open reading frame and a combinatorial nucleic barcode sequence describing the architecture of a combinatorial CAR embedded in an open reading frame.

United Libraries.

The present invention provides methods for preparing pooled libraries of synthetic modular polypeptides containing nucleic barcode sequences. It is understood that in a pooled library, library elements are present in a common container and/or common solution, and individual library elements do not need to be physically separated in space, for example, individual library elements can be combined during library construction (for example, as when assembled in a single reactor ) or after creating the individual elements of the library. For example, in the case of a combined library of synthetic modular polypeptides constructed by combinatorial nesting, library components can be combined during assembly of library elements, before assembly of library elements is completed, and/or after assembly of library elements is completed, and so on. In the present application, pooled libraries are not limited to libraries of only synthetic modular polypeptides, but also include pooled libraries of cells expressing synthetic modular polypeptides, pooled libraries of nucleic acids encoding synthetic modular polypeptides, and the like.

Accordingly, in some cases, individual nucleic acid components used to assemble library elements may be combined prior to assembly and may remain combined during assembly of the library elements. In other cases, the assembled library elements may be mixed after assembly to form a combined library.

Because the libraries and library assembly methods described herein involve generating library elements that can be identified by sequencing the appropriate region of the nucleic barcode sequence, individual nucleic acids can be combined at any time before, during, or after assembly while maintaining the possibility of subsequent identification and quantification of individual elements of the library in quantitative studies. In some cases, the final dimension of a library can be controlled by combining library components at certain points during a build. For example, a mixed dimensional library can be obtained by separately assembling different sized partial libraries and then combining the partial libraries (eg, using split-and-merge assembly). In some cases, for example, after combining partial libraries of different dimensions, a subsequent assembly may be performed, including the addition of additional variable domains.

In one non-limiting embodiment, as shown in FIG. 23, a pooled library of nucleic acids encoding synthetic modular CAR polypeptides is transformed into a total population of primary human T cells to generate primary human T cells expressing the synthetic modular CAR polypeptide. Optionally, if the encoded elements of the synthetic modular CAR polypeptide library contain one or more reporter modules, the transformed T cells can be sorted based on their expression of the reporter module (which is indicative of the expression of the synthetic modular CAR polypeptide) to isolate transformed cells with similar element expression levels. libraries of synthetic modular CAR polypeptides. These sorted transformed T cells with the same levels of expression represent a pooled library of T cells expressing synthetic modular CAR polypeptides.

In some cases, modulate the transformational efficiency of nucleic acids and/or primary human T cells expressing synthetic modular CAR polypeptides. Such modulation may be performed for various practical reasons, such as to control the probability of expression of each element of the library and/or to control the probability of each cell expressing no more than one element of the library. Said modulation may be achieved by any convenient means, including, but not limited to, for example, modulating the initial amount of nucleic acid encoding the synthetic modular polypeptide present during transformation, controlling the initial amount of primary T cells present during transformation, controlling the ratio of coding nucleic acid and primary T cells during transformation, and the like. Therefore, in some cases, transformation efficiency is modulated such that essentially each transduced T cell expresses one unique synthetic modular CAR polypeptide. In some instances, pooled libraries of cells expressing unique synthetic modular CAR polypeptides can be cultured, expanded, stored, and so forth. in accordance with the requirements of subsequent quantitative studies.

Suitable pooled libraries, whether containing pooled nucleic acids, pooled modular polypeptides, pooled transduced cells, etc., are not limited to nucleic acids encoding synthetic CAR modular polypeptides, synthetic CAR modular polypeptides, or cells (e.g., T cells). ) expressing synthetic modular CAR polypeptides. Any library of nucleic acids encoding modular polypeptides, modular polypeptides and/or corresponding transduced cells expressing modular polypeptides obtained in accordance with the methods described herein can be combined in a similar manner to obtain a combined library that can be used, for example, in screening studies.

The individual elements of the pooled libraries described herein can be positively identified by the specific multi-merged nucleic acid barcode sequence associated with each member of the library. Due to the specific combinatorial nesting that results in a predictable positional relationship between each module coding sequence and its corresponding barcode nucleic acid sequence, the identity and architecture of each synthetic modular polypeptide can be established by simple sequencing of the corresponding multi-unit barcode nucleic sequence. Therefore, in some cases, the identity and/or architecture of individual elements of a library of synthetic modular polypeptides can be determined based on the sequence data associated with the nucleic acid barcode regions of the library elements. For example, in some cases, all elements and/or each individual element of the combined library described herein may be determined, for example, after the library has been constructed, after the library has been used in a particular assay, and so on.

Compartmentalized Library Components.

Individual nucleic acid elements containing a coding region and a nucleic barcode sequence region can, in some cases, be compartmentalized. Bundled libraries and libraries with compartmentalized components need not be mutually exclusive, and for example, in some cases a library may be combined and may contain compartmentalized components at different times, for example, a library may be constructed, for example, library elements may be assembled, in the form the pool, for example, as when assembled in a single reactor, and then can be subsequently compartmentalized, for example, by transfection of nucleic acid library elements into single cells or non-cellular compartments. In other cases, library elements may be compartmentalized during their assembly and then combined for subsequent methods, including, but not limited to, for example, further assembly or screening. In general, nucleic acids encoding synthetic barcode nucleic acid sequence polypeptides assembled according to the nested assembly methods and cloning strategies described herein are pooled and may subsequently be compartmentalized or may be left unchanged.

Compartmentalization of assembled nucleic acids encoding synthetic polypeptides containing nucleic barcode sequences can be achieved by any suitable method that allows the transcription and translation of individual elements of the nucleic acid libraries so that each element of the nucleic acid library remains associated with the product that it encodes. In some cases, as described in more detail below, compartmentalization can be achieved by creating cell libraries in which individual cells serve as a compartment and provide translation and transcription of nucleic acid library elements.

In some cases, compartmentalization can be achieved by creating non-cellular libraries, such as libraries based on encapsulation. Encapsulation-based cell-free libraries typically contain an emulsion of two or more immiscible liquids, wherein the nucleic acid library elements are soluble in a first liquid, such as an aqueous liquid, such as water or an aqueous buffer, and insoluble in a second liquid, such as an oil or other organic solvent, so that the first liquid forms compartments, such as drops, containing the individual elements of the library. The library-containing emulsion may be configured such that each compartment contains any number of individual library elements, including, but not limited to, no more than one element per compartment. Nucleic acid elements from encapsulated libraries can be transcribed (eg, in vitro conditions) and translated (eg, in vitro conditions) under conditions in which transcription and translation products remain bound, i. remain within the compartment, with a separate element of the nucleic acid library encoding them. Any convenient and suitable method for obtaining an encapsulated library of polypeptides encoded by nucleic acids can be used in the methods described herein, including, but not limited to, for example, the methods described in Bernath et al. Anal Biochem. (2004) 325(1): 151-7; the contents of which are incorporated herein by reference in their entirety.

Upon receipt of the encoded product of a library element within a compartment, the nucleic acid library element and the encoded synthetic modular polypeptide may optionally be physically linked. For example, in some cases, the nucleic acid library element and the encoded product may be joined, for example, by any convenient and appropriate method, including chemical bonding or conjugation (i.e., by obtaining a covalent bond between the nucleic acid library element and the encoded synthetic modular polypeptide) or via molecular linkage (e.g., direct linkage of a library element to an encoded synthetic modular polypeptide, or indirect linkage of a library element to an encoded product that is mediated by one or more binding intermediates (e.g., binding partners, substrates, etc.). .) Any convenient and appropriate method for linking compartmentalized encoded polypeptides to nucleic acid library elements can be used in the methods described herein, including, but not limited to, using, for example, a substrate containing an attached An epitope tag binding agent that specifically binds to an epitope tag encoded by a nucleic acid library element, for example, as described in Griffiths & Tawfik. EMBO J (20030 22(1):24-35, the contents of which are incorporated herein by reference in their entirety. In some cases, once linked, elements of nucleic acid libraries can be compartmentalized, such as pooled, and then quantified as a pooled library.

In some cases, the nucleic acid library element and the encoded product remain sufficiently linked without being physically connected, for example, by compartmentalization within a compartment, including cellular and non-cellular compartments.

Compartmentalization, both cellular and non-cellular, typically identifies a synthetic modular polypeptide, or a portion thereof, that correlates with a detected phenotype by sequencing the barcode region of a single element of a nucleic acid library encoding the synthetic modular polypeptide that remains linked to the synthetic modular polypeptide for on the basis of their compartmentalization or as a result of the physical connection formed during their compartmentalization.

Cell Libraries.

As described in more detail above, in the present application, libraries include cell libraries in which the cells of the library express synthetic modular polypeptides. Transform

- 37 041085 Nucleic acids encoding synthetic modular polypeptides can be carried out by any convenient method, including, but not limited to, for example, viral transfection, electroporation, lipofection, bombardment, chemical transformation, use of a carrier suitable for transduction (for example, suitable for carrier protein transduction), and the like. In some cases, the cell into which the nucleic acid encoding the synthetic modular polypeptide is transformed is referred to herein as the host cell.

Host cells may express a single nucleic acid containing a nucleic acid barcode sequence encoding a unique synthetic modular polypeptide, or may express multiple, including two or more, individual nucleic acids containing a nucleic acid barcode sequence encoding unique synthetic modular polypeptides. It should be understood that the amount of individual nucleic acids containing nucleic barcode sequences that the host cell expresses can be controlled, for example, by controlling the frequency or likelihood of delivery of the nucleic acids in question to the host cell, for example, by modulating delivery method parameters. In some cases, the final number of single nucleic acids containing barcode nucleic sequences that encode unique synthetic modular polypeptides present in a host cell may be referred to as the multiplicity of infection (MOI) and may be defined as the ratio of nucleic acids and host cells before or after delivery. Standard methods for modulating MOI, for example by increasing or decreasing the ratio of nucleic acids to host cells prior to delivery, can be used to produce the desired final amount of single nucleic acids containing barcode nucleic acid sequences encoding unique synthetic modular polypeptides per host cell after delivery.

Nucleic acids encoding synthetic modular polypeptides can be transformed into any suitable host cell or cell line, including, for example, prokaryotic and eukaryotic cells. The choice of host cell type will depend on a number of factors, including the type of synthetic modular polypeptide library to be screened and the particular screening assay. In some cases, the host cell may be a prokaryotic cell, including, but not limited to, Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres , Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae and Verrucomicrobia. In some embodiments of the present invention, the host cell may be a bacterial cell, such as E. coli. In some cases, a standard bacterial strain may be used, including but not limited to, for example, commercially available strains from vendors such as the American Type Culture Collection (ATCC) (Manassas, VA, USA), Life Technologies, Inc. (Grand Island, New York, USA), etc.

Suitable eukaryotic cells include primary cells and cultured cells originally derived from an animal host, including but not limited to, for example, mammals (including, for example, humans, primates, great apes, ungulates, canines, felines, rabbits, rodents, etc.). .d.), reptiles, amphibians (e.g. clawed frogs, salamander, newt, etc.), fish (e.g. zebrafish, etc.), birds (e.g. chicken, etc.), invertebrates ( e.g. insects (e.g. fruit fly etc.)), worms (e.g. nematodes etc.), marine invertebrates (e.g. sea urchin etc.) etc.), yeasts and etc. In some embodiments of the present invention, the cells may be primary rodent cells or cultured rodent cells derived from a mouse or rat. In other embodiments, the cells may be primary human cells or cultured human cells. Any suitable eukaryotic cell may be used as the host cell depending on the particular library to be screened and the particular screening assay, however, in some cases, suitable eukaryotic cell lines may be used, including but not limited to, for example, commercially available from suppliers such as the American Type Culture Collection (ATCC) (Manassas, VA, USA), Life Technologies, Inc. (Grand Island, New York, USA), etc.

In some cases, the cells of the cell library are primary cells (e.g., primary monocytes, primary lymphocytes (e.g., primary T cells, primary B cells, primary NK cells, etc.), primary dendritic cells, etc.). ), primary endothelial cells, primary epithelial cells, primary fibroblasts, primary hematopoietic stem cells, primary keratinocytes, primary melanocytes, primary mesenchymal stem cells, primary preadipocytes, primary muscle cells (e.g., primary smooth muscle cells, primary skeletal muscle cells, etc.). etc.), etc. In some cases, the cells of the cell library are known cell lines (eg, Jurkat cells, etc.). In some cases, cells in the cell library are patient-specific cells (patient-specific immune cells (e.g., primary T cells, etc.), patient-specific stem cells (e.g., hematopoietic stem cells, mesenchymal stem cells). cells, adipose-derived stem cells, etc.), patient-specific cancer cells (eg, tumor cells, blood cancer cells, etc.).

Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (eg, mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), HEK293 cells (e.g., ATCC No. CrL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC # CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC # CCL10), PC12 cells (ATCC # CRL1721), COS cells, COS- 7 (ATCC # CRL1651), RAT1 cells, mouse L cells (ATCC # CCLI.3), human mesonephros (HEK) cells (ATCC # CRL1573), HLHepG2 cell lines, Hut-78, Jurkat, HL-60, NK ( e.g. NKL, NK92 and YTS), etc.

In some cases, the cell is not from an immortalized cell line, but instead is a cell (eg, a primary cell) derived from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T-lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem or progenitor cell derived from an individual.

After transforming a plurality of nucleic acids encoding the synthetic modular polypeptides of the library, the transformed host cells can be sorted based on their expression of the synthetic modular polypeptides, for example, to remove cells that do not express the synthetic modular polypeptides from the library to isolate only those cells that expressing synthetic modular polypeptides at levels above a certain expression threshold, to isolate only those cells that express synthetic modular polypeptides at levels below a certain expression threshold, to isolate only those cells that express synthetic modular polypeptides in a certain range of expression levels, and t .d. In some cases, sorting of transformed cells based on expression level can be performed to isolate only those cells in the library that have the same level of expression, the same level of expression may vary depending on specific applications and in some cases can be defined as the level of expression within a certain range, which is above and below the average expression level of the population.

In some embodiments of the present invention, sorting transformed cells based on their level of expression of library elements, such as sorting cells that express or have approximately the same level of expression of library elements, improves the assessment of the influence of the library element and / or modules included in it. For example, by isolating only those cells that express library elements in a certain range of levels, the identification of library elements that affect a particular phenotype will be based on the actual function of the library elements and those modules that are part of it, and not on the efficiency of expression of a particular element. libraries. According to some embodiments of the present invention, for example, when performing a semi-quantitative study of the frequency of expression of individual elements of the library and/or its modules, sorting cells expressing library elements within a predetermined range of levels provides more accurate quantification and quantification of library elements and/or modules on the basis of their influence on a certain phenotype, and not on the basis of their relative level of expression.

In addition, sorting allows the identification of library elements and modules whose functioning creates or influences a particular phenotype when expressed within a certain range of expression levels or above or below a certain threshold, including, for example, low expression level or high expression level.

Library normalization.

Libraries of the present invention may or may not be standardized depending on the situation in which the library is created and/or the intended final use of the library. In this application, the term normalized, in relation to the libraries described, means that the relative amounts of each element of the library are adjusted to at least equalize their amounts, compared with the relative amounts of each element of the library before adjustment. In some cases, library normalization results in the library having a smaller range between the number of the most represented library items and the number of the least represented library items. In some cases, normalization leads to an increase in the number of the least represented element(s) of the library. In some cases, normalization results in a reduction in the number of the most represented element(s) of the library.

In some cases, library normalization may involve quantifying all or most or a representative sample (or all or most of a representative sample) of library items to determine the relative amount of each library item in the library. After quantification, an adjustment is made based on the quantification to equalize the relative presence of each library element in the library. Depending on the situation, said adjustment can be performed directly on the library already obtained, so that the library is subjected to direct normalization. In another embodiment, the adjustment may be performed as part of the library acquisition process such that the next library obtained will be normalized.

Any library of the present invention may be normalized, including but not limited to, for example, nucleic acid libraries, polypeptide libraries, non-cellular encapsulated libraries, cell libraries, etc., and depending on the library being normalized, various ways. For example, depending on the type of library being normalized, different methods can be used to quantify the relative amounts of library elements. Various methods for quantifying elements of a nucleic acid library that can be used include, but are not limited to, e.g., quantitative sequencing (e.g., next generation sequencing), quantitative PCR, quantitative hybridization (e.g., microarray), and the like. Various methods can be used to quantify elements of a polypeptide library, including but not limited to, for example, quantitative mass spectrometry, ELISA, and the like. Various methods for quantifying cell library elements can be used, including, but not limited to, flow cytometry, immunohistochemistry, quantitative sequencing (e.g., next generation sequencing), quantitative PCR, quantitative hybridization (e.g., microarray), and the like.

In some cases, when library elements are quantified, the adjustment(s) required for normalization can be calculated. Any convenient and appropriate method for calculating normalization may be used depending on the type of library and/or the size of the library. In some cases, a linear equation may be used, including, but not limited to, the linear equation shown in FIG. 28.

In some cases, when the normalization is calculated for each element of the library, the library can be adjusted. Various methods of directly updating the library can be used. For example, in some cases, a cell library may be normalized using FACS to sort an equal number of cells representing each element of the library into a pool or individually accessible compartments. In some cases where the library is already compartmentalized, the library can be normalized by adjusting the volume of each compartment, including, for example, those cases where different concentrations of library elements in each compartment are normalized by adding a certain volume of liquid to each compartment, sufficient to equalize the concentrations.

In some cases, normalization of the pooled nucleic acid library may be performed. Pooled nucleic acid libraries can be normalized for a variety of reasons. In one embodiment, a pooled nucleic acid library may be normalized to compensate for over- or under-representation of individual library elements in the library, for example, due to more or less efficient incorporation of individual nucleic acid modules into library elements during combinatorial assembly.

In some instances, the nucleic acid library elements and/or nucleic acid modules that make up the library elements can be quantified (eg, by quantitative sequencing). After such a quantitative assessment, the adjustment of each element necessary for normalization is calculated. In one embodiment, the calculated adjustment may be applied to the next combinatorial assembly of the library, for example, the amount of each nucleic acid module used to assemble the nucleic acid library may be adjusted based on the relative abundance of that module in the quantified library. Therefore, the completed combinatorial library will be normalized by adjusting the initial number of nucleic acid modules before the next assembly of the library. Accordingly, in some instances, normalizing a library described herein may involve assembling the library, followed by quantification of the assembled library, and reassembly of a normalized version of the library that is based on the quantification.

Screening methods.

The present invention provides methods for screening libraries of synthetic modular polypeptides, including, but not limited to, for example, in vitro screening methods and

- 40 041085 in vivo screening methods. By in vivo screening is usually meant that a library containing a plurality of unique synthetic modular polypeptides is quantitatively assayed in the biological environment of a living organism. Living organisms that can be quantitatively tested under in vivo conditions in accordance with the methods described herein include unicellular and multicellular organisms.

In vivo screening of a single celled organism typically involves contacting the single celled organism with a library of synthetic modular polypeptides, said library of synthetic modular polypeptides may be a library of polypeptides or a library of cells expressing synthetic modular polypeptides, and detecting the phenotype in the single celled organism. In other instances, in vivo screening of a single-celled organism may involve bringing the single-celled organism, or a plurality of single-celled organisms, into contact with a library of nucleic acids encoding synthetic modular polypeptides under conditions sufficient to allow expression of the encoded synthetic modular polypeptides by single-celled organisms.

In vivo screening of a multicellular organism typically involves contacting the multicellular organism with a library of synthetic modular polypeptides, said library of synthetic modular polypeptides may be a library of polypeptides or a library of cells expressing synthetic modular polypeptides, and detecting the phenotype in the multicellular organism. In other instances, in vivo screening of a multicellular organism may involve bringing the multicellular organism or a plurality of multicellular organisms into contact with a library of nucleic acids encoding synthetic modular polypeptides under conditions sufficient for expression of the encoded synthetic modular polypeptides by the multicellular organism(s). Any suitable multicellular organism may be used in the in vivo screening of the library described herein, depending on the particular library to be screened and the particular in vivo assay used, with particular multicellular organisms including, but not limited to, for example, mammals (eg, mice, rats, etc.).

The term in vitro screening generally means that a library containing a plurality of unique synthetic modular polypeptides is quantified outside the normal biological environment, such as the library polypeptide modules, the biological material used in the screening, or the phenotype being screened. For example, in some cases, in vitro screening can be performed using an artificial or synthetic experimental environment, including, but not limited to, for example, an isolated sample, an isolated cell, a cell culture, an isolated or excised tissue, a specific sample, a specific medium. , artificial tissue, artificial organ, cell extract, tissue extract, many samples, etc. In vitro screening can be performed in any convenient and suitable vessel, including but not limited to, for example, a reaction vessel, a reaction chamber, a test tube, a vial, a plate, a flask, a dish, a glass slide, and the like.

In vitro screening of a sample, including a cellular sample or a non-cellular sample, typically involves contacting the sample with a library of synthetic modular polypeptides, said library of synthetic modular polypeptides may be a library of polypeptides or a library of cells expressing synthetic modular polypeptides, and detecting the cellular phenotype or another reaction or molecular phenotype. Any suitable sample can be used in in vitro screening of the library described herein depending on the particular library to be screened and the particular in vitro assay used, and specific samples include, but are not limited to, for example, biological samples, cellular samples, polypeptide samples, nucleic acid samples, chemical samples, and the like.

In some cases, cell-free libraries of synthetic modular polypeptides can be screened in vitro. For example, a compartmentalized library of synthetic modular polypeptides can be screened for phenotype by contacting the compartmentalized library of synthetic modular polypeptides with one or more agents with which certain members of the library are predicted to react. Any suitable methods for screening cell-free libraries of polypeptides, whether encapsulated or pooled, can be used in the methods described herein, including, but not limited to, for example, flow cytometry detection or phenotype detection by fluorescence-activated cell sorting in cell-free quantitative assays. encapsulation based studies such as those described in Griffiths & Tawfik. EMBO J (2003) 22(1):24-35 and Bernath et al. Anal Biochem (2004) 325(1): 151-7; the contents of which are incorporated herein by reference in their entirety.

Phenotypes and methods of identification.

Screening methods, either in vivo or in vitro, typically include detection

- 41 041085 phenotype and identification of one or more elements of the library associated with the phenotype. As used herein, the term phenotype generally refers to the characteristic of a molecule, cell, tissue, organ, or organism that is detected in a particular assay, and therefore may include, but is not limited to, e.g., molecular phenotypes, cellular phenotypes, organism phenotypes, phenotypes tissues, organ phenotypes, organism phenotypes, etc. The phenotype detected in a particular assay may be a predetermined phenotype, such as a known or expected phenotype (eg, including a known or expected level of a certain characteristic, the presence or absence of a known or expected level characteristic, etc.) or may be identified in time of assay, eg, a newly detected or previously undetermined phenotype (eg, including a newly detected or previously undetermined level of a particular characteristic, the presence or absence of a first-detected or previously undetermined characteristic, etc.). Any suitable assay method for detecting the phenotype associated with the library of synthetic modular polypeptides described herein can be used in screening said libraries.

Screening of a library of synthetic modular polypeptides or nucleic acids encoding a library of synthetic modular polypeptides allows the identification of polypeptides and/or modules thereof that effectively provide the desired phenotype. Accordingly, polypeptides identified by screening the libraries described herein are generally included within the scope of the present invention.

In some cases, a cell phenotype is detected after a population of cells is brought into contact with the library described herein. Cellular phenotypes may include, but are not limited to, for example, cell behavior (including but not limited to cell viability, cell proliferation, cell activation, cell morphology, cell migration, cell adhesion, cell differentiation, cell pluripotency, etc. ), cellular expression (including, but not limited to, gene expression, protein expression, non-coding RNA expression, gene activation, gene repression, etc.), reporter expression (including, but not limited to, e.g., transgenic reporter expression, marker expression), and the like.

In some cases, the phenotype of a tissue, organ, or organism is detected after the tissue, organ, or organism is brought into contact with the library described herein. Tissue phenotypes include, but are not limited to, for example, tissue viability, tissue morphology, tissue physical characteristics (including, but not limited to, protective function, mechanical strength, elasticity, etc.), tissue expression (including, but not limited to limited to, e.g., tissue gene expression, tissue protein expression, etc.), reporter expression in tissue (including, but not limited to, e.g., transgenic reporter expression, marker expression), and the like. Phenotypes of an organ include, but are not limited to, for example, organ appearance, organ viability, organ morphology, organ function (including, but not limited to, production of a biomolecule (e.g., enzyme, metabolite, protein, etc.), filtration, mechanical function, etc.). The phenotypes of an organism include, but are not limited to, for example, the appearance of the organism, the viability of the organism (eg, lifespan), the physiology of the organism, the fertility of the organism/fertility, the behavior of the organism, and so on.

In some instances, a phenotype may be quantified in relation to a pathological condition, wherein said pathological condition may be a simulated pathological condition (e.g., a cell, tissue, or organism that has been altered or manipulated to exhibit characteristics of a particular disease) or may be a clinical pathological condition. a condition (eg, an organism exhibiting the characteristics of a disease or diagnosed with a disease, or cells or tissues derived therefrom). Disease-associated phenotypes can be quantified at any convenient level, including, but not limited to, eg, cellular level, tissue level, organ level, organism level. In some cases, the quantified disease phenotypes may be those of the pathogen itself, including but not limited to, for example, tumor phenotypes, cancer cell phenotypes, autoimmune cell phenotypes, infectious agent phenotypes (bacterial, viral, etc.), etc. .d. In other cases, quantified disease phenotypes may be phenotypes of a cell, tissue, or organism affected by said disease, or associated with the disease being modeled, that provide information about the presence and/or progression of the disease, including, but not limited to, for example, cell activation ( e.g. immune cell activation), disease response (e.g. immune response), biomarkers, cell counts, body responses, clinical outcomes, etc.

In some cases, the phenotype assessment may be carried out at the population level, for example, the assessment of a population of cells, an assessment of a population of organisms, etc. can be carried out. In some cases, in a population phenotype assessment, the effect of a particular library element on the presence or absence of a population phenotype can be measured. For example, the effect of a particular library element on the cellular phenotype of a population of cells can be assessed. In other cases, the effect of a particular library element on the phenotype of organisms in a population of organisms can be assessed.

According to some embodiments of the present invention, the phenotype is assessed in response to the applied stimulus, and the application of the stimulus includes, but is not limited to, for example, bringing cells into contact with a stimulus, bringing a tissue into contact with a stimulus, bringing an organ into contact with a stimulus, bringing an organism into contact with a stimulus, etc. As such, the test sample or test subject may be contacted with a stimulus under in vitro or in vivo conditions, depending on the quantitative assay method used, depending on the stimulus, and depending on the particular library being screened. Various stimuli may be used singly or in combination. The stimulus may be a free stimulus (eg, a soluble stimulus, a free ligand, etc.), an associated stimulus (eg, associated with a solid carrier), a cellularly expressed stimulus (eg, an expressed costimulatory molecule, an expressed antigen, an expressed cellular ligand, etc.). .d.), etc.

In some embodiments of the present invention, a population of T cells expressing a synthetic modular CAR library is contacted in vitro with a stimulus and the resulting phenotype is detected. In vitro stimuli suitable for screening a cell library expressing synthetic modular CARs will typically include antigens including, for example, free antigen, bound antigen, cell-expressed antigen (e.g., expressed on an antigen-presenting cell, expressed on a target cell, etc.). .d.), etc. Suitable antigens will vary depending on the particular CAR library to be screened and the desired screening result. Non-limiting examples of antigens include, but are not limited to, e.g., soluble antigen, solid support antigen (e.g., plate-bound antigen, bead-bound antigen, slide-bound antigen, etc.), expressed antigen (e.g., , transgenic cell expressing antigen, cell expressing antigen naturally (e.g., cell expressing native antigen, cancer cell expressing cancer antigen, etc.) Cells expressing native antigen that can be used to screen the library under conditions in vitro will vary and may include, but are not limited to, for example, naive tumor cells (eg obtained from tumor biopsy).

In some embodiments of the present invention, a population of T cells expressing a synthetic modular CAR library is contacted in vivo with a stimulus and the resulting phenotype is detected. In vivo screening situations for a library of synthetic modular CARs will vary greatly and may include, but are not limited to, animal models. In some instances, in vivo screening may be performed in small animal models such as, for example, rodent models, including but not limited to, for example, mouse models, rat models, and the like. In some instances, in vivo screening is performed in mouse tumor models, including transgenic and non-transgenic mouse tumor models.

In some cases, the model used may be a xenograft model. For example, the model used may be a humanized model, said humanized models being defined as having one or more components of human origin, such as a humanized immune system, humanized T cells expressing a human protein, harboring human cancer cells, etc. In this regard, humanized models can be fully or partially humanized. In other cases, the model may not be fully or partially humanized, but may instead be simply administered with human cells or human tissue via injection or transplantation. For example, in some cases, human cancer cells or cells from human cancer cell lines are introduced into an animal model. Any convenient human tumor cell or human tumor cell line can be used in these models, including, but not limited to, K562 cells, Dowdy's lymphoma cells, etc., for example.

Animal models and/or cells or tissues introduced into animal models may be transgenic or non-transgenic, eg, modified to express one or more transgenes. For example, in some instances, an animal model can be transgenetically modified to express a heterologous gene, e.g., a reporter gene (e.g., to identify cells of a host animal), a target gene (e.g., a gene encoding a gene product to be screened in in vivo conditions). In some instances, a cell introduced into an animal model may be transgenically modified to express a heterologous gene, e.g., a reporter gene (e.g., to identify the introduced cell), a target gene (e.g., a gene encoding a gene product to be screened in in vivo conditions). As a non-limiting example, a mouse tumor model can be screened according to the methods described herein, with mice injected with human tumor cells expressing a target cancer transgene (eg, CD19, mesothelin, etc.).

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Library elements introduced into in vivo systems may be screened for any suitable phenotype, which phenotype may be dependent on the particular library being screened, the particular in vivo situation (e.g. animal model), etc. In some cases, for example, if the in vivo system is an animal model of a tumor, the library can be screened for phenotypes associated with the selectivity of the library elements for tumors, for example, by introducing the library into an animal model containing two different tumors, by introducing the library into an animal model or several animal models containing tumors expressing different levels of tumor antigen, etc. Any convenient method for quantifying phenotype, including the cellular and biochemical/molecular methods described herein, can be used in evaluating in vivo systems, and such evaluations typically involve obtaining a biological sample from an animal model. In some cases, a biological sample suitable for in vivo model evaluation may include a tissue sample (eg, blood, tumors, etc.) or an organ sample (eg, spleen).

In some cases, the library may be screened in vitro or in vivo according to the T cell phenotype. T cell phenotypes will vary and will include stimulated T cell phenotypes, ie. response to an antigen. Non-limiting examples of T cell phenotypes include, but are not limited to, e.g., T cell proliferation, cytokine production (e.g., IL-2, IFN-γ, TNF, LT-α, IL-4, IL-5, IL-6 , IL-13, IL-9, IL-10, IL-17A, IL17F, IL-21, IL-22, IL-26, CCL20, IL-21, FRO-β, etc.), expression of surface markers T cells (eg, CD3, CD4, CD8, etc.), T cell activation markers (eg, CD69, etc.), intracellular signaling markers (eg, phosphorylated form of ERK1/2, phosphorylated form of p38MAPK, and etc.), etc.

T cell phenotypes can be quantified under in vitro and in vivo conditions and can be detected by any convenient method. In some instances, a cell counting device or flow cytometer can be used to quantify T cell phenotype, including, for example, T cell proliferation and/or T cell quantification. For example, T cell proliferation can be quantified by flow cytometry by diluting the cell labeling dye. In some cases, the expression of cell surface markers can also be quantified using flow cytometry. Expression of intracellular markers can be determined by cellular methods (e.g., flow cytometry, flow cytometry using phospho-specific antibodies, flow cytometry of intracellular markers, immunofluorimetry, in situ hybridization, fluorescence in situ hybridization, etc.) or can be quantified investigated by molecular and/or biochemical methods (e.g., ELISA, cytokine capture, amplification-based methods (e.g., quantitative PCR), sequencing-based methods (e.g., quantitative sequencing), quantitative mass spectrometry, etc. .).

In some cases, T cells can be quantified to determine the natural killer activation phenotype. Any suitable method for assessing natural killer activation can be used in these assays. For example, T cells can be quantified to determine the expression of CD107a/b, for example, by flow cytometry.

In some cases, T cells can be quantified to determine one or more differentiation phenotypes. Any suitable method for assessing T cell differentiation can be used in these assays. For example, memory T cell differentiation can be determined, for example, by quantifying memory T cell markers (eg, Th1, Th2, Th17, Treg, etc.) using any suitable cellular or molecular biochemical method. In some cases, expression of one or more intracellular transcription factors indicative of differentiation into memory T cells (e.g., Gata3, Tbet, RORyt, FoxP3, Bcl-6, CCR7, CD45RO, CD45RA, CD69, etc.) can be assessed. ).

Screening of a library of synthetic modular CAR polypeptides or nucleic acids encoding a library of synthetic modular CAR polypeptides allows the identification of CARs and/or portions thereof (e.g., antigen-binding domains, primary signaling domains, comodulatory domains, etc.) that are effective in generating the desired phenotype of T cells. Accordingly, CARs identified by screening the libraries described herein as well as nucleic acids encoding said CARs are included within the scope of the present invention. Also included within the scope of the present invention are CARs containing suitable CAR modules (eg, antigen binding domains, primary signaling domains, comodulatory domains, etc.) identified by screening the libraries described herein as well as nucleic acids encoding said CARs.

In some cases, the CAR of the present invention may contain one or more comodulatory domains identified as stimulatory T cells or inhibitory T

- 44 041085 cell domains when screening a library of synthetic modular CAR polypeptides or nucleic acids encoding synthetic modular CAR polypeptides described herein. Accordingly, the overall T cell phenotype of CAR may be to mimic T cell activity or to inhibit T cell activity. T cell activities that may be stimulated or inhibited include, but are not limited to, for example, the T cell activities described herein.

In some instances, a CAR identified by library screening may contain at least one comodulatory domain listed in Table 1. 3 or 4, including, but not limited to, for example, comodulatory domains containing an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% identical to the listed domain sequence. In some instances, a CAR identified by library screening may contain two or more of the co-modulating domains listed in Table 1. 3 and 4, including, but not limited to, comodulatory domains containing an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90% , at least about 95%, at least about 98% identical to the listed domain sequence. In some instances, the CAR of the present invention may contain the co-modulating domain identified in the screening described herein as the co-stimulatory domain, including but not limited to, for example, those listed in Table 1. 3, including, but not limited to, comodulatory domains containing an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% identical to the listed domain sequence. In some instances, the CAR of the present invention may contain a co-modulating domain identified in the screening described herein as a co-inhibitory domain, including but not limited to, for example, those listed in Table 1. 4, including, but not limited to, comodulatory domains containing an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% identical to the listed domain sequence. In some cases, a CAR that contains one or more coinhibitory domains may be an iCAR.

In some cases, a CAR containing two or more co-modulating domains may contain two co-stimulatory domains, including, but not limited to, for example, two or more co-stimulatory domains listed in table. 3. In some cases, a CAR containing two or more co-inhibitory domains may contain two co-inhibitory domains, including, but not limited to, for example, two or more co-inhibitory domains listed in table. 4. In some cases, a CAR containing two or more co-modulating domains may include a mixture of co-stimulatory and co-inhibitory domains, including, but not limited to, at least one co-stimulatory domain listed in table. 3, and at least one coinhibitory domain listed in table. 4.

Table 3. Comodulatory domains with a stimulatory function (costimulatory domains)

Domain Subsequence SEQID NO: 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 26 CD7 ARTQIKKLCSWRDKNSAACWYEDMSHSRCNTLSSPNQYQ 25 2B4 WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQS QSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNP ARLSRKELENFDVYS 41 HVEM MEESWRPSVFWDGQTDIPFTRLGRSHRRQSCSV 23 CRTAM KLRKAHVIWKKENEVSEHTLESYRSRSNNEETSSEEKNGQSSHPMRCMNYI TKLYSEAKTKRKENVQHSKLEEKHIQVPESIV 35 CD30 RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEE RGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTN NKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQ ETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 42 TLT2 KKRHMASYSMCSDPSTRDPPGRPEPYVEVYLI 21 CD27 HQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP 28 CTLA4 SLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN 24

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Table 4. Co-modulating domains with inhibitory function (co-inhibitory domains)

Domain Subsequence SEQID NO: DNAM-1 NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRR PKTRV 31 CD80 RCRERRRNERLRRESVRPV 19 PD-1 ICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQT EYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL 36 TIM-3 KWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYY CYVSSRQQPSQPLGCRFAMP 33 BTLA CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPD LCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICV RS 39 CD40 KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRI SVQERQ 32 ICOS TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL 22 LAG3 RRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL 29 GITR HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV thirty TIGIT RKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDC AELHDYFNVLSYRSLGNCSFFTETG 34 PD-L1 RKGRMMDVKKCGIQDTNSKKQSDTHLEET 20 CD28 WVRSKRSRLLHSDYMNMTPRRPPGPTRKHYQPYAPPRDFAAYRS 27 LAIR1 HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSAL AAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH 37 CAR CCRKKRREEKYEKEVHHDIREDVPPPKSRTSTARSYIGSNHSSLGSMSPSNMEGYS KTQYNQVPSEDFERTPQSPTLPPAKVAAPNLSRMGAIPVMIPAQSKDGSIV 38 CD2 TKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRS QAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSL SPSSN 40

A CAR identified by screening a library of synthetic CAR modular polypeptides or a library of nucleic acids encoding synthetic CAR polypeptides may contain any suitable antigen-binding domain, including, but not limited to, those used clinically in various CAR constructs, including, for example, BCMA specific antigen binding domain, CD123 specific antigen binding domain, CD138 specific antigen binding domain, CD171 specific antigen binding domain, CD19 specific antigen binding domain, CD22 specific antigen binding domain, CD30 specific antigen binding domain, CD33 specific antigen binding domain, CD7 specific antigen binding domain, CD70 specific antigen binding domain, CEA-specific antigen-binding domain, EGFRvIII-specific antigen-binding domain, EPCAM-specific antigen-binding domain, EphA2-specific antigen-binding domain, ErbB-specific antigen-binding domain n, FAP-specific antigen-binding domain, GD2-specific antigen-binding domain, GPC3-specific antigen-binding domain, HER2-specific antigen-binding domain, IL1RAP-specific antigen-binding domain, kappa-specific antigen-binding domain, LeY-specific antigen-binding domain, Meso-specific antigen-binding domain, MG7-specific antigen-binding domain, MUC1-specific antigen-binding domain domain, NKG2D specific antigen binding domain, PSCA specific antigen binding domain, ROR1 specific antigen binding domain, and the like.

A CAR identified by screening a library of synthetic modular CAR polypeptides or a library of nucleic acids encoding synthetic modular CAR polypeptides may contain any suitable primary signaling domain (also referred to herein as an intracellular signaling domain), including, but not limited to, for example, those which contain one or more immunoreceptor tyrosine activating motifs (ITAM).

A suitable intracellular signaling domain may be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain may be an ITAM motif containing domain from any ITAM motif containing protein. Therefore, a suitable intracellular signaling domain does not necessarily contain the complete sequence of the entire protein from which it is derived. Examples of suitable polypeptides containing the ITAM motif include, but are not limited to: DAP12; FCER1G (gamma chain of Fcε I receptor); CD3D (CD3 delta); CD3E (CD3-epsilon); CD3G (CD3-gammaα); CD3Z (CD3-zeta); and CD79A (alpha chain protein associated with the antigen recognition receptor complex).

In some cases, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO tyrosine kinase binding protein, KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; tyrosine kinase-binding protein TYRO; killer-activating receptor-associated protein; activating killers receptor-associated protein, etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid

- 46 041085 a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% identical to any of the following amino acid sequences (4 isoforms):

MGGLEPCSRLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMMGDLVLTVLIAL AVYFLGRLVPRGRGAAEAATRKORITETESPYQELOGORSDVYSDLNTORPYYK (SEQ ID NO: 107);

MGGLEPCSRLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLT

VLIALAVYFLGRLVPRGRGAAEATRKORITETESPYQELOGORSDVYSDLNTORPYYK (SEQ ID NO: 108);

MGGLEPCSRLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGR

LVPRGRGAAEAATRKORITETESPYQELQGORSDVYSDLNTORPYYK (SEQ ID NO: 109); or

MGGLEPCSRLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGR

LVPRGRGAAEATRKORITETESPYQELQGORSDVYSDLNTQRPYYK (SEQ ID NO: 110) in which the ITAM motifs are bold and underlined.

Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion from the full length amino acid sequence of DAP12. Therefore, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence:

ESPYQELQGORSDVYSDLNTO (SEQ ID NO: 111), in which the ITAM motifs are bold and underlined.

In some cases, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fce I receptor gamma chain; Fc receptor gamma chain; Fc epsilon RI-gamma; FcR-gamma; FcaRI gamma; high-affinity immunoglobulin epsilon receptor gamma subunit , immunoglobulin E receptor, high affinity gamma chain, etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence:

MIPAVVLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKS

DGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 112), in which the ITAM motifs are bold and underlined.

Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion from the full length amino acid sequence of FCER1G. Therefore, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence:

DGVYTGLSTRNQETYETLKHE (SEQ ID NO: 113), in which the ITAM motifs are bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell CD3 surface glycoprotein delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen; delta subunit; CD3 delta; CD3d antigen; delta polypeptide (TiT3 complex); OKT3, delta chain, delta chain of T-cell receptor T3, delta chain of CD3 surface glycoprotein of T-cells, etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a. .k., from about 120 to about 130 a.k., from about 130 to about 140 a.k., from about 140 to about 150 a.k. or from about 150 to about 170 a.c. any of the following amino acid sequences (2 isoforms):

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MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG KRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALG VFCFAGHETGRLSGAADTOALLRNDOVYQPLRDRDDAOYSHLGGNWARNK (SEQ ID NO: 114) or

MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG KRILDPRGIYRCNGTDIYKDKESTVOVHYRTADTOALLRNDOVYQPLRDRDDAQYSHL GGNWARNK (SEQ ID NO: 115), in which the ITAM motifs are in bold and underlined.

Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion from the full-length CD3 delta amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence:

DQVYQPLRDRDDAOYSHLGGN (SEQ ID NO; 116), in which the ITAM motifs are in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell CD3 surface glycoprotein epsilon chain (also known as CD3ε; T cell surface antigen epsilon T3/Leu-4; T cell surface glycoprotein epsilon CD3; AI504783; CD3 ; CD3epsilon; T3ε, etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a. .k., from about 120 to about 130 a.k., from about 130 to about 140 a.k., from about 140 to about 150 a.k. or from about 150 to about 205 a.a. following amino acid sequence:

MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTV1LTCPQYPGSEILW QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARV CENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQ NKERPPPVPNPDYEPIRKGORDLYSGLNORRI (SEQ ID NO: 117), в которой мотивы ITAM выделены жирным шрифтом и подчеркнуты.

Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion from the full-length amino acid sequence of CD3 epsilon. Therefore, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence:

NPDYEPIRKGQRDLYSGLNOR (SEQ ID NO: 118), in which the ITAM motifs are bold and underlined.

In some cases, the intracellular signaling domain is derived from the gamma chain of T cell surface glycoprotein CD3 (also known as CD3G; T3 cell receptor gamma chain; CD3gamma; T3G; gamma polypeptide (TiT3 complex), etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a. .k., from about 120 to about 130 a.k., from about 130 to about 140 a.k., from about 140 to about 150 a.k. or from about 150 to about 180 a.c. following amino acid sequence:

MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDG KMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS GFLFAEIVSIFVLAVGVYFIAGODGVROSRASDKOTLLPNDOLYOPLKDREDDOYSHLO GNQLRRN (SEQ ID NO: 1111 is underlined) in which the motifs are underlined.

- 48 041085

Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion from the full-length CD3gamma amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence:

DQLYQPLKDREDDOYSHLOGN (SEQ ID NO: 120), in which the ITAM motifs are in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z; T-cell receptor T3 zeta chain; CD247; CD3-zeta; CD3H; CD3Q; T3Z; TCRZ, etc. ). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a. .k., from about 120 to about 130 a.k., from about 130 to about 140 a.k., from about 140 to about 150 a.k. or from about 150 to about 160 a. to. any of the following amino acid sequences (2 isoforms):

MKWKALFTAAILQAQLP1TEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADA PAYQQGONOLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELOKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMOALPPR (SEQ ID NO: 121) or

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADA PAYQOGONOLYNELNLGRREEYDVLDKRRGRDPEMGGKPORRKNPOEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMOALPPR (SEQ ID NO: 122), in which the ITAM motifs are in bold and underlined.

Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion from the full-length amino acid sequence of CD3zeta. Therefore, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to any of the following amino acid sequences:

RVKFSRSADAPAYOOGONOLYNELNLGRREEYDVLDKRRGRRDPEMGGKPRRKNPOEG

LYNELOKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMOALPPR (SEQ ID NO: 123);

NQLYNELNLGRREEYDVLDKR (SEQ ID NO: 124); EGLYNELQKDKMAEAYSEIGMK (SEQ ID NO: 125); or DGLYQGLSTATKDTYDALHMO (SEQ ID NO: 126) in which the ITAM motifs are bold and underlined.

In some cases, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha chain); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein, surface IgM-associated protein, etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a. .k., from about 120 to about 130 a.k., from about 130 to about 150 a.k., from about 150 to about 200 a.k. or from about 200 to about 220 a.c. any of the following amino acid sequences (2 isoforms):

- 49 041085

MPGGPGVLQALPAT1FLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNS SNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQ QSCGTYLRVRQPPPRPFLDMGEGTKNR1ITAEGIILLFCAVVPGTLLLFRKRWQNEKLGL DAGDEYEDENLYEGLNLDDCSMYEDISRGODTY2; or MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNS SNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCA VVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDV GSLNIGDVQLEKP (SEQ8 ID NO: 12 ITAM) in which the motive fonts are underlined.

Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif containing portion from the full length amino acid sequence of CD79A. Therefore, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence: ENLYEGLNLDDCSMYEDISRG (SEQ ID NO: 129), in which the ITAM motifs are in bold and underlined. Intracellular signaling domains suitable for use in the CARs of the present invention comprise a DAP10/CD28 type signal chain.

An example of a DAP10 signal chain is the amino acid sequence: RPRRSPAODGKVYINMPGRG (SEQ ID NO: 130).

According to some embodiments of the present invention, a suitable intracellular signaling domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least approximately 99% identical throughout the amino acid sequence

RPRRSPAODGKVYINMPGRG (SEQ ID NO: 130).

An example of a CD28 signal chain is the amino acid sequence:

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA

PPRDFAAYRS (SEQ ID NO: 131).

According to some embodiments of the present invention, a suitable intracellular signaling domain comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least approximately 99% identical throughout the amino acid sequence

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRS (SEQ ID NO: 131).

Intracellular signaling domains suitable for use in the CARs of the present invention include a ZAP70 polypeptide, e.g., a polypeptide containing an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to a contiguous stretch of amino acid sequence of about 300 to about 400 amino acids, about 400 to about 500 amino acids, or about 500 to 619 amino acids of the following amino acid sequence:

MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHF PIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLR DAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKL YSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLV EYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEP ARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGV YRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLV MEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLV NRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVT MWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPD FLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA (SEQ ID NO: 132).

In some cases, a CAR identified by screening a library of synthetic mo

- 50 041085 muzzle CAR polypeptides or nucleic acid libraries encoding synthetic modular CAR polypeptides, including CAR containing at least one or two or more co-modulating domains listed in table. 3 and 4 can be separated into two polypeptide chains capable of joining, in the presence of a dimerizer, by means of a dimerization domain present in each chain. Said cleaved CARs are conditionally active and pharmacologically inducible/suppressed, such as those described in PCT patent application publication WO 2014/127261, the contents of which are incorporated herein by reference in their entirety.

Accordingly, in some cases, each CAR cleavage variant polypeptide of a CAR identified by screening the library described herein may contain one half of a dimerization pair (also referred to as a dimerizer-binding pair). Non-limiting examples of suitable dimers (eg, dimerizer-binding pairs) include, but are not limited to: a) FK506 binding protein (FKBP) and FKBP; b) FKBP and catalytic subunit of calcineurin A (CnA); c) FKBP and cyclophilin; d) FKBP and FKBP-rapamycin-associated protein (FRB); f) gyrase B (GyrB) and GyrB; f) dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB; h) PYL and ABI; i) Cry2 and CIB1; and j) GAI and GID1.

In some instances, the dimer element (eg, dimerizer-binding pair) of the CAR in question is derived from FKBP. For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least approximately 95%, at least approximately 98%, or 100% identical to the following amino acid sequence:

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIR

GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 78).

In some cases, the member of the dimerizer-binding pair of the CAR in question is derived from the calcineurin A catalytic subunit (also known as PPP3CA; CALN; CALNA; CALNA1; CCN1; CNA1; PPP2B; CAM-PRP catalytic subunit; calcineurin A alpha; calmodulin-dependent isoform alpha calcineurin A subunits, protein phosphatase 2B, catalytic subunit, alpha isoform, etc.). For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence (PP2Ac domain):

LEESVALRIITEGASILRQEKNLLDIDAPVTVCGDIHGQFFDLMKLFEVGGSPANTRYLFL GDYVDRGYFSIECVLYLWALKILYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVY DACMDAFDCLPLAALMNQQFLCVHGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSD PLEDFGNEKTQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYR KSQTTGFPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFM (SEQ ID NO: 79).

In some cases, the dimer element (eg, dimerizer-binding pair) is derived from cyclophilin (also known as cyclophilin A; PPIA; CYPA; CYPH; PPIase A, etc.). For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least approximately 95%, at least approximately 98%, or 100% identical to the following amino acid sequence:

MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGF

MCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTE

WLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE (SEQ ID NO: 80).

In some cases, a dimer element (e.g., a dimerizer-binding pair) is derived from MTOR (also known as FKBP-rapamycin-associated protein; FK506-binding protein 12-rapamycin-associated protein 1; FK506-binding protein 12-rapamycin-associated protein 2; FK506-binding protein 12-associated rapamycin complex protein 1; FRAP; FRAP1; FRAP2; RAFT1; and RAPT1). For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to the following amino acid sequence (also known as Frb: Fbbp-rapamycin binding domain):

MILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR

DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 81).

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In some cases, a dimer element (eg, a dimerizer-binding pair) is derived from GyrB (also known as the B subunit of DNA gyrase). For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 200 amino acids (a.a.), from about 200 to about 300 a.a., from about 300 to about 400 a.a., from about 400 to about 500 a.a., from about 500 to about 600 a.a., from about 600 to about 700 a.a. or from about 700 to about 800 a.c. the following amino acid sequence of GyrB from Escherichia coli (or the sequence of subunit B of DNA gyrase from any organism):

MSNSYDSSSIKVLKGLDAVRKRPGMYIGDTDDGTGLHHMVFEVVDNAIDEALAGHCKE IIVTIHADNSVSVQDDGRGIPTGIHPEEGVSAAEVIMTVLHAGGKFDDNSYKVSGGLHGV GVSVVNALSQKLELVIQREGKIHRQIYEHGVPQAPLAVTGETEKTGTMVRFWPSLETFT NVTEFEYEILAKRLRELSFLNSGVSIRLRDKRDGKEDHFHYEGGIKAFVEYLNKNKTPIH PNIFYFSTEKDGIGVEVALQWNDGFQENIYCFTNNIPQRDGGTHLAGFRAAMTRTLNAY MDKEGYSKKAKVSATGDDAREGLIAVVSVKVPDPKFSSQTKDKLVSSEVKSAVEQQM NELLAEYLLENPTDAKIVVGKIIDAARAREAARRAREMTRRKGALDLAGLPGKLADCQ ERDPALSELYLVEGDSAGGSAKQGRNRKNQAILPLKGKILNVEKARFDKMLSSQEVATL ITALGCGIGRDEYNPDKLRYHSIIIMTDADVDGSHIRTLLLTFFYRQMPEIVERGHVYIAQ PPLYKVKKGKQEQYIKDDEAMDQYQISIALDGATLHTNASAPALAGEALEKLVSEYNA TQKMINRMERRYPKAMLKELIYQPTLTEADLSDEQTVTRWVNALVSELNDKEQHGSQ WKFDVHTNAEQNLFEPIVRVRTHGVDTDYPLDHEFITGGEYRRICTLGEKLRGLLEEDA FIERGERRQPVASFEQALDWLVKESRRGLSIQRYKGLGEMNPEQLWETTMDPESRRML RVTVKDAIAADQLFTTLMGDAVEPRRAFIEENALKAANIDI (SEQ ID NO: 82).

In some instances, an element of a dimerizer-binding pair contains an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identical to amino acids 1-220 of the above GyrB amino acid sequence from Escherichia coli.

In some cases, the dimer element (eg, the dimerizer binding pair) is derived from DHFR (also known as dihydrofolate reductase, DHFRP1 and DYR). For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least approximately 95%, at least approximately 98%, or 100% identical to the following amino acid sequence:

(SEQ ID NO: 83).

In some cases, a dimer element (eg, a dimerizer-binding pair) is derived from a DmrB-binding domain (ie, a DmrB homodimerization domain). For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least approximately 95%, at least approximately 98%, or 100% identical to the following amino acid sequence:

MASRGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQ EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 84).

In some cases, a dimer element (eg, a dimerizer-binding pair) is derived from the PYL protein (also known as abscisic acid receptor and RCAR). For example, an element of the dimerizer-binding pair in question can be derived from proteins, such as Arabidopsis thaliana proteins: PYR1, RCAR1(PYL9), PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8 (RCAR3), PYL10, PYL11, PYL12, PYL13. For example, a suitable element of a pair that binds a dimerizer may contain an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90% is at least about 95%, at least about 98%, or 100% identical to any of the following amino acid sequences:

PYL10:

MNGDETKKVESEYIKKHHRHELVESQCSSTLVKHIKAPLHLVWSIVRRFDEPQKYKPFIS RCVVQGKKLEVGSVREVDLKSGLPATKSTEVLEILDDNEHILGIRIVGGDHRLKNYSSTI SLHSETIDGKTGTLAIESFVVDVPEGNTKEETCFFVEALIQCNLNSLADVTERLQAESME KKI(SEQIDNO: 85).

PYL11:

(SEQ ID NO: 86)

PYL12:

(SEQ ID NO: 87)

PYL13:

MES SKQKRCRS S VVETIEAPLPLVWSILRSFDKPQ AYQRF VKSCTMRSGGGGGK GGEGKGSVRDVTLVSGFPADFSTERLEELDDESHVMVVSIIGGNHRLVNYKSKTKVVAS PEDMAKKTVVVESYVVDVPEGTSEEDTIFFVDNIIRYNLTSLAKLTKKMMK (SEQ ID NO: 88).

PYL1:

MANSESSSSPVNEEENSQRISTLHQTMPSDLTQDEFTQLSQSIAEFHTYQLGNGR CSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPA NTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVV DVPEGNSEEDTRLFADTVIRLNLQKLASITEAMNRSENNQVR: NONNQ8.

PYL2:

MSSSPAVKGLTDEEQKTLEPVIKTYHQFEPDPTTCTSLITQRIHAPASVVWPLIRRF DNPERYKHFVKRCRLISGDGDVGSVREVTVISGLPASTSTERLEFVDDDHRVLSFRVVG GEHRLKNYKSVTSVNEFLNQDSGKVYTVVLESYTVDIPEGNTEEDTKMFVDTVVKLNL QKLGVAATSAPMHDDE (SEQ ID: 90).

PYL3:

MNLAPIHDPSSSSTTTTSSSTPYGLTKDEFSTLDSIIRTHHTFPRSPNTCTSLIAHRV DAPAHAIWRFVRDFANPNKYKHFIKSCTIRVNGNGIKEIKVGTIREVSVVSGLPASTSVEI LEVLDEEKRILSFRVLGGEHRLNNYRSVTSVNEFVVLEKDKKKRVYSVVLESYIVDIPQG NTEEDTRMFVDTVVKSNLQNLAVIST

PYL4:

MLAVHRPSSAVSDGDSVQIPMMIASFQKRFPSLSRDSTAARFHTHEVGPNQCCSA VIQEISAPISTVWSVVRRFDNPQAYKHFLKSCSVIGGDGDNVGSLRQVHVVSGLPAASST

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ERLDILDDERHVISFSVVGGDHRLSNYRSVTTLHPSPISGTVVVESYVVDVPPGNTKEET CDFVDVIVRCNLQSLAKIAENTAAESKKKMSL (SEQ ID NO: 92).

PYL5:

MRSPVQLQHGSDATNGFHTLQPHDQTDGPIKRVCLTRGMHVPEHVAMHHTHD VGPDQCCSSVVQMIHAPPESVWALVRRFDNPKVYKNFIRQCRIVQGDGLHVGDLREVM VVSGLPAVSSTERLEILDEERHVISFSVVGGDHRLKNYRSVTTLHASDDEGTVVVESYIV DVPPGNTEEETLSFVDTIVRCNLQSENRQ3.

PYL6:

MPTSIQFQRSSTAAEAANATVRNYPHHHQKQVQKVSLTRGMADVPEHVELSHT HVVGPSQCFSVVVQDVEAPVSTVWSILSRFEHPQAYKHFVKSCHVVIGDGREVGSVREV RVVSGLPAAFSLERLEIMDDDRHVISFSVVGGDHRLMNYKSVTTVQHESEEDSDGKKRTR VVESYVVDVPAGNDKETSLFADTIVRCN4.

PYL7:

MEMIGGDDTDTEMYGALVTAQSLRHLHHCRENQCTSVLVKYIQAPVHLVWS LVRRFDQPQKYKPFISRCTVNGDPEIGCLREVNVKSGLPATTSTERLEQLDDEEHILGINII GGDHRLKNYSSILTVHPEMIDGRSGTMVMESFVVDVPQGNTKDTCYFVESLIKCNLKS LACVSERLAAQDITNSIATFCNASNGYREKNHTETN 5.

PYL8:

MEANGIENLTNPNQEREFIRRHHKHELVDNQCSSTLVKHINAPVHIVWSLVRRFD QPQKYKPFISRCVVKGNMEIGTVREVDVKSGLPATRSTERLELLDDNEHILSIRIVGGDH RLKNYSSIISLHPETIEGRIGTLVIESFVVDVPEGNTKDETCYFVEALIKCNLKSLADISERL AVQDTTESRV (SEQ ID NO: 96).

PYL9:

MMDGVEGGTAMYGGLETVQYVRTHHQHLCRENQCTSALVKHIKAPLHLVWSL VRRFDQPQKYKPFVSRCTVIGDPEIGSLREVNVKSGLPATTSTERLELLDDEEHILGIKIIG GDHRLKNYSSILTVHPEIIEGRAGTMVIESFVVDVPQGNTKDETCYFVEALIRCNLKSLA DVSERLASQDITQ (SEQ ID NO: 97).

PYRL:

MPSELTPEERSELKNSIAEFHTYQLDPGSCSSLHAQRIHAPPELVWSIVRRFDKPQ TYKHFIKSCSVEQNFEMRVGCTRDVIVISGLPANTSTERLDILDDERRVTGFSIIGGEHRL TNYKSVTTVHRFEKENRIWTVVLESYVVDMPEGNSEDDTRMFADTVVKLNLQKLATV AEAMARNSGDGSGSQVT (SEQ ID NO: 98).

In some cases, a dimer element (eg, a dimerizer-binding pair) is derived from an ABI protein (also known as an abscisic acid insensitive protein). For example, an element of the dimerizer binding pair in question can be derived from proteins such as the Arabidopsis thaliana proteins: ABI1 (also known as abscisic acid insensitive protein 1, protein phosphatase 2C56, AtPP2C56, P2C56 and PP2C ABI1) and/or ABI2 (also known as P2C77, protein phosphatase 2C 77, AtPP2C77, abscisic acid insensitive protein 2, protein phosphatase 2C ABI2 and PP2C ABI2). For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a., from about 120 to about 130 a.a., from about 130 to about 140 a.a., from about 140 to about 150 a.a., from about 150 to about 160 a.a., about 160 to about 170 a.a., from about 170 to about 180 a.a., from about 180 to about 190 a.a. or from about 190 to about 200 a.c. any of the following amino acid sequences:

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ABIl

MEEVSPAIAGPFRPFSETQMDFTGIRLGKGYCNNQYSNQDSENGDLMVSLPETSSCSVSG SHGSESRKVLISRINSPNLNMKESAAADIVVVDISAGDEINGSDITSEKKMISRTESRSLFE

FKSVPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGS QVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETV GSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQ WNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEE ACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDN ISVVVVDLKPRRKLKSKPLN (SEQ ID NO: 99).

ABi2:

MDEVSPAVAVPFRPFTDPHAGLRGYCNGESRVTLPESSCSGDGAMKDSSFEINTRQDSL T S S S S AMAGVDIS AGDEINGSDEFDPRSMNQ SEKKVL SRTESRSLFEFKC VPL YGVT SICG RRPEMEDS VSTIPRFLQ VS S S SLLDGRVTNGFNPHLS AHFFGVYDGHGGSQ VANYCRER MHLALTEEIVKEKPEFCDGDTWQEKWKKALFNSFMRVDSEIETVAHAPETVGSTSVVA VVFPTHIFVANCGDSRAVLCRGKTPLALSVDHKPDRDDEAARIEAAGGKVIRWNGARV

FGVLAMSRSIGDRYLKPSVIPDPEVTSVRRVKEDDCLILASDGLWDVMTNEEVCDLARK

RILLWHKKNAMAGEALLPAEKRGEGKDPAAMSAAEYLSKMALQKGSKDNISVVVVDL KGIRKFKSKSLN (SEQ ID NO: 100).

In some cases, a dimer element (eg, a dimerizer-binding pair) is derived from the Cry2 protein (also known as cryptochrome 2). For example, a member of the dimer in question (eg, a dimerizer-binding pair) can be derived from Cry2 proteins from any organism (eg, plant), such as, but not limited to, Arabidopsis thaliana. For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a., from about 120 to about 130 a.a., from about 130 to about 140 a.a., from about 140 to about 150 a.a., from about 150 to about 160 a.a., about 160 to about 170 a.a., from about 170 to about 180 a.a., from about 180 to about 190 a.a. or from about 190 to about 200 a.c. any of the following amino acid sequences:

Cry2 (Arabidopsis thaliana)

MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMK

QSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKE KLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMP ITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNS KKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREY SRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGW MHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDR LDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPI VDIDTARELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVP

SAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNL

EGIQDSSDQITTSLGKNGCK (SEQ ID NO: 101)

In some cases, the element of the dimer (eg, the dimerizer-binding pair) is derived from the Arabidopsis thaliana CIB1 protein (also known as the bHLH63 transcription factor). For example, a suitable dimer element (e.g., a pair that binds a dimerizer) may contain an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90% , at least about 95%, at least about 98%, or 100% identical to a contiguous stretch of amino acid sequence from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a., from about 120 to about 130 a.a., from about 130 to about 140 a.a., from about 140 to about 150 a.a., from about 150 to about 160 a.a. .k., from about 160 to about 170 a.k., from about 170 to about 180 a.k., from about 180 to about 190 a.k. or from about 190 to about 200 a.c. following amino acid sequence:

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MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTA GLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFD TETKD CNEKKKKMTMNRDDL VEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARR GQATDSHSIAERVRREKISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLS MKLAIVNPRPDFDMDDIFAKEVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGY

LHVNPMQQVNTSSDPLSCFNNGEAPSMWDSHVQNLYGNLGV (SEQ ID NO: 102).

In some cases, a dimer element (eg, a dimerizer-binding pair) is derived from the GAI protein of Arabidopsis thaliana (also known as the gibberellic acid-insensitive protein and the GAI protein of the DELLA family). For example, a suitable member of a dimerizer-binding pair may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% contiguous amino acid sequence identity from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a., from about 120 to about 130 a.a., from about 130 to about 140 a.a., from about 140 to about 150 a.a., from about 150 to about 160 a.a., about 160 to about 170 a.a., from about 170 to about 180 a.a., from about 180 to about 190 a.a. or from about 190 to about 200 a.c. following amino acid sequence:

MKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEV

MMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNPPSSNAEYDLKAIPGDAILNQ FAIDSASSSNQGGGGDTYTTNKRLKCSNGVVETTTATAESTRHVVLVDSQENGVRLVH ALLACAEAVQKENLTVAEALVKQIGFLAVSQIGAMRKVATYFAEALARRIYRLSPSQSPI DHSLSDTLQMHFYETCPYLKFAHFTANQAILEAFQGKKRVHVIDFSMSQGLQWPALMQ ALALRPGGPPVFRLTGIGPPAPDNFDYLHEVGCKLAHLAEAIHVEFEYRGFVANTLADL DASMLELRPSEIESVAVNSVFELHKLLGRPGAIDKVLGVVNQIKPEIFTVVEQESNHNSPI FLDRFTESLHYYSTLFDSLEGVPSGQDKVMSEVYLGKQICNVVACDGPDRVERHETLSQ WRNRFGSAGFAAAHIGSNAFKQASMLLALFNGGEGYRVEESDGCLMLGWHTRPLIATS AWKLSTN (SEQ ID NO: 103).

In some instances, the dimer element (eg, the dimerizer-binding pair) is derived from the Arabidopsis thaliana GID1 protein (also known as the GID1 gibberellic acid receptor). For example, a suitable dimer element may contain an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , at least about 98% or 100% identical to a contiguous stretch of amino acid sequence from about 100 to about 110 amino acids (a.a.), from about 110 to about 115 a.a., from about 115 to about 120 a.a. ., from approx.

120 to about 130 a.a., from about 130 to about 140 a.a., from about

140 to about 150 a.a., from about 150 to about 160 a.a., from about

160 to about 170 a.a., from about 170 to about 180 a.a., from about

180 to about 190 a.a. or from about 190 to about 200 a.c. any of the following amino acid sequences:

GID1A:

MAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANP VDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHS SANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKS KKDSKVHIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLDGK YFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGLDLIRDW QLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAEC (SEQ ID NO: 104).

- 56 041085

GIDIB:

MAGGNEVNLNECKRIVPLNTWVLISNFKLAYKVLRRPPDGSFNRDLAEFLDRKVPANSFP

LDGVFSFDHVDSTTNLLTRIYQPASLLHQTRHGTLELTKPLSTTEIVPVLIFFHGGSFTHSS ANSAIYDTFCRRLVTICGVVVVSVDYRRSPEHRYPCAYDDGWNALNWVKSRVWLQSG KDSNVYVYLAGDSSGGNIAHNVAVRATNEGVKVLGNILLHPMFGGQERTQSEKTLDGK YFVTIQDRDWYWRAYLPEGEDRDHPACNPFGPRGQSLKGVNFPKSLVVVAGLDLVQD WQLAYVDGLKKTGLEVNLLYLKQATIGFYFLPNNDHFHCLMEELNKFVHSIEDSQSKSS PVLLTP (SEQ ID NO: 105).

GID1C:

MAGSEEVNLIESKTVVPLNTWVLISNFKLAYNLLRRPDGTFNRHLAEFLDRKVPANANP

VNGVFSFDVIIDRQTNLLSRVYRPADAGTSPSITDLQNPVDGEIVPVIVFFHGGSFAHSSA

NS AI YDTLC RRLVGLCG A V V VS VN YRR APENR YPC A YDDG W A VLK W VNS S SWLRSKK DSKVRIFLAGDSSGGNIVHNVAVRAVESRIDVLGNILLNPMFGGTERTESEKRLDGKYFV TVRDRDWYWRAFLPEGEDREHPACSPFGPRSKSLEGLSFPKSLVVVAGLDLIQDWQLK YAEGLKKAGQEVKLLYLEQATIGFYLLPNNNHFHTVMDEIAAFVNAECQ (SEQ ID NO: 106).

As is readily understood, a CAR identified by screening a nucleic acid library encoding synthetic CAR modular polypeptides or a library of synthetic CAR modular polypeptides can be modified, for example, by adding one or more domains (e.g., comodulatory domains), by deleting one or more domains (eg, by removing the fluorescent reporter used in the screening process), by splitting the CAR into two polypeptides (and adding dimerization domains), by rearranging the domains, and so on.

In some cases, the library may be screened to determine the phenotype associated with a cellular response to a particular cellular environment. In this regard, the cellular phenotype can be determined by the response of the cell (for example, based on activation or inhibition) to the impact of a particular environment. For example, the response of T cells can be assessed in response to exposure to a particular cellular environment. In some embodiments of the present invention, T cell inhibition can be assessed in response to exposure to the tumor microenvironment.

In some cases, the library may be screened to determine the phenotype associated with the localization of cells, for example, under the influence of homing or targeting the cell. Therefore, screening can be performed to determine the effect of library elements on cell targeting. For example, a cell library can be introduced into a host organism, and the cells of the library can be isolated from the target position of the host organism after some time to assess which cells were successfully directed to the target position. In some cases, T cells can be quantified to determine their tumor targeting in vivo.

In some cases, the library may be screened for a phenotype specific to the patient's condition. Patient-specific conditions screened in this manner will vary greatly and may include conditions associated with a particular pathological condition of the patient and may include library screening to identify specific library element(s) exhibiting an enhanced or optimal phenotype specific to the patient's condition. In some cases, the localization of elements of the cell library can be assessed after contact with the explant or xenograft obtained from the patient. For example, T cell localization of a CAR expressing T cell library can be assessed upon contact with a patient-specific tumor xenograft. In some cases, the proliferation of cell library elements can be assessed after contact with an explant or xenograft obtained from a patient. For example, T cell proliferation of a CAR expressing T cell library can be assessed upon contact with a patient-specific tumor xenograft. In some cases, a patient-specific explant or xenograft can be quantitatively examined to determine the increase or decrease in viability after contact with the cell library or specific elements of the cell library. For example, T cell death of a CAR expressing T cell library can be assessed upon contact with a patient-specific tumor xenograft. In this regard, the library can be screened to identify the optimal element(s) of the library for the treatment of a particular patient.

In some cases, phenotypes can be quantified in vitro using dynamic antigen immunization. By dynamic immunization with an antigen, it is meant that the phenotype is not only assessed to determine the presence or absence of an antigen, and therefore the antigen can vary dynamically, for example, dynamically vary over a range of concentrations, dynamically vary over a certain period of time, etc. For example,

- 57 041085 antigen levels can be titrated (eg using different concentrations) to assess the screened phenotype at different doses, ie. to evaluate response in a dose-dependent manner. The antigen can be presented at different concentrations in any convenient way. As a non-limiting example, different amounts of antigen can be presented using cells expressing the antigen at different levels, including a range of levels. In some cases, the time of application of the antigen can be varied dynamically, for example, to assess the phenotype in a time-dependent assay, or to assess the kinetics of the phenotype being screened. In some cases, the library may be screened for a distinctive phenotype. In this application, the term distinctive phenotype generally refers to a combination of distinct phenotypes. For example, in some cases, a cell may have a distinctive phenotype that includes a certain morphology in combination with the expression of a particular marker. A phenotypic feature may combine phenotypes with similar or different phenotypic categories, for example, a phenotypic feature may include the expression of two related but different cell surface markers, or a phenotype feature may include the expression of a cell surface marker and a cell proliferation marker, or a phenotype feature may include the expression of a cell surface marker and a specific secreted marker (eg, a cytokine), or a distinctive phenotype may include the expression of two different cytokines, etc. Any suitable phenotype, including that described herein, can be used as a component of a distinctive phenotype.

Identification of Synthetic Modular Polypeptides Associated with a Phenotype The present invention provides methods for identifying library elements that are associated with a particular detectable phenotype. Without wishing to be bound by any theory, the present inventors believe that the coordinated assembly of each multimodular synthetic polypeptide along with each corresponding multi-unit barcode nucleic sequence ensures the assembly and subsequent identification of each unique synthetic modular polypeptide. As described above, the barcode nucleic sequence region of each nucleic acid encoding a synthetic modular polypeptide provides not only the identity of the individual modules that make up each synthetic modular polypeptide, but also the specific arrangement (referred to herein as the architecture) of the modules. Therefore, the identity and architecture of each library element can be determined by sequencing a region of the nucleic barcode sequence.

Accordingly, library screening need not be performed using physically separated library elements, and library elements may be combined and screened at the same time. The combination of library elements can be performed in vitro, for example, in a test tube or in a sample of cells isolated from the corresponding organism, or in vivo, in an animal or tissue. Following simultaneous screening, library elements and/or their phenotype-associated modules can be identified by identifying and/or quantifying the associated region of the nucleic barcode sequence. In some cases, pooled screening allows a large number of unique library elements to be screened, which is not feasible with standard serial or parallel screening. The number of unique library elements that can be screened for phenotype will depend on the size and complexity of the library and can therefore range from 96 or less to millions or more, including but not limited to, for example, 100 or more , 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 60,000 or more, 70,000 or more, 80,000 or more, 90,000 or more, 100,000 or more, etc.

In some cases, the amount or frequency of a particular barcode nucleic sequence can be measured to identify the most represented module. For example, the frequency of each barcode nucleic acid sequence can be quantified in a pooled sample containing library elements and its associated nucleic acids encoding library elements, such that the barcode nucleic acid sequences with the highest frequency will identify those modules that are most represented in the sample. . According to some embodiments of the present invention, such samples may be cellular samples.

In some instances, the number or frequency of a particular multi-merged nucleic barcode sequence (eg, regions of the barcode sequence) can be measured to identify the most representative modular polypeptide. For example, the frequency of each nucleotide barcode sequence can be quantified in a pooled sample containing library elements and associated nucleic acids encoding the library elements such that the most frequently occurring nucleotide barcode sequences provide identification of said modular elements. polypeptides, the most represented in the sample. According to some variants of implementation of the present invention, these samples may be cellular samples.

In some cases, phenotype detection and identification of library elements and their components may be performed as part of an integrated method. For example, in some cases, the phenotype can be detected by flow cytometry and library elements can be identified by sequencing. These integrated methods can be performed in combination with in vitro and/or in vivo assays, for example as shown in FIG. 24, where FLOW-Seq is used as a non-limiting example of an integrated method.

As used herein, the term FLOW-seq generally refers to the combination of flow cytometry (eg, FACS) sorting methods with sequencing (eg, next generation sequencing) methods in one related workflow. Any convenient and suitable flow cytometry sorting method and any convenient and suitable sequencing method can be used in said FLOW-Seq method. For example, in some cases, a cell library expressing the synthetic modular polypeptides containing the barcode nucleic acid sequences described herein can be quantitatively assayed for phenotype determination by flow cytometry, and those cells that have a particular phenotype can be sorted, and their nucleic barcode sequences are subsequently sequenced to identify specific library elements and/or to quantify the frequency of individual library elements and/or their modules expressed in sorted cells. Sorting may be performed in any convenient and suitable manner, including, for example, sorting into one or more groups based on the phenotype detected by flow cytometry. After sorting, sequencing can be performed directly on the sorted sample and/or sorted cell, or the sorted sample and/or sorted cell can be expanded and/or cultured before sorting, for example, to increase the copy number of nucleic acids encoding library elements. FLOW-seq methods have been used, for example, for phenotypic measurement of protein levels and identification of related genetic elements in bacteria (see, for example, Kosuri et al. Proc Natl Acad Sci USA (2013) 110(34): 14024-9 and Goodman et al. Science (2013) 342(6157):475-479), in addition, sequencing was combined with flow cytometry to correlate T cells sorted based on their function with their corresponding sequenced T cell receptor genes (see, e.g., Han et al Nature Biotechnology (2014) 32:684-692).

In some instances, identifying a synthetic modular polypeptide associated with a particular phenotype may involve surgically isolating a tissue or organ from an in vivo model into which the library has been introduced. For example, in some cases, after a period of time sufficient to conduct a quantitative study, the organ or tissue can be isolated from the animal host, and the nucleic acids present in the organ or tissue can be sequenced to identify individual library elements present in the organ or tissue. In other cases, nucleic acid isolated from an organ or tissue or host animal may be quantified, including semi-quantitative evaluation, sequenced to quantify the relative frequency or presence of a particular library element in the organ or tissue. In other instances, nucleic acid isolated from an organ or tissue or host animal may be quantified, including semi-quantification, sequenced to quantify the relative frequency or presence of a particular module in the organ or tissue.

In some cases, for example, when a particular module is highly represented after a semi-quantitative evaluation, or a particular module is identified as contributing to a desired phenotype, a next round of new library assembly may be performed in which the identified module is included in all newly created library elements (i.e., the identified variable the module is subsequently used as a non-variable module), and the newly created library is screened to identify additional modules that affect the phenotype in conjunction with the originally identified module. One of skill in the art will readily appreciate in which cases iterative assembly and library screening can be performed to develop libraries and individual library elements with desired phenotypes.

Examples

The following examples are provided to provide those skilled in the art with a complete description of the present invention, as well as a description of the methods for making and using the present invention, and are not intended to limit the scope of the present invention according to the present inventors, nor do the present inventors imply that experiments, below represent all or the only experiments that have been performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg amounts, temperatures, etc.), however, some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or about atmospheric pressure. Standard abbreviations may be used, eg b.p., base pair(s); thousand bp, one thousand (thousand) base pairs; pl, picoliter (picolitres); s or sec., second (seconds); min, minute(s); h or hour, hours; a.k., amino acid(s); thousand bp, one thousand (thousand) base pairs; p.o., a pair (pairs) of bases; nuc., nucleotide(s); in / m, intramuscular (intramuscularly); in / b, intraperitoneal (intraperitoneally); s / c, subcutaneous (subcutaneous); and so on.

Example 1 Construction and screening of modular libraries of co-modulating domains.

To obtain nucleic acid fragments encoding modules containing nucleic barcode sequences that contain the necessary elements for constructing a library, nucleic acids encoding polypeptide modules (i.e. co-modulating domains (i.e. co-stimulatory or co-inhibitory domains)) were subcloned a cloning vector suitable for sequencing and digestion with type IIS restriction enzymes. After subcloning, the vector contained: the sequence encoding the module, the optimal Gly/Ser linker sequences, flanking the sequence encoding the module, the module-specific nucleic barcode sequence, suitable for cloning, the 3'-terminal homologous arms flanking the module-specific barcode sequence, the site BamHI restriction between the module-coding sequence and the module-specific barcode sequence and type IIS restriction enzyme sites at the 5'end of the module-coding sequence and at the 3'end of the module-specific barcode sequence (FIG. 1). Subcloned vector inserts were sequenced to confirm the identity of the inserted comodulatory domains. The co-modulating domains used in this combinatorial library and their corresponding protein sequences are shown in Table 1. 1.

Cloning vectors containing a coding module sequence containing a nucleic barcode sequence were digested using a type IIS restriction enzyme to release nucleic acids with the optimal sequence encoding each polypeptide module (FIG. 2). An example of a cloning vector portion containing the described elements and the CD28 co-stimulatory domain coding sequence before and after IIS digestion (FIG. 3) is shown, which represents the general configuration of each modular plasmid/fragment used in the construction of the library.

An expression vector (ie pHR lentiviral packaging vector (also referred to as a recipient vector)) was generated by cleaving the 3' end of the spleen necrosis virus (pSFFV) promoter with a restriction enzyme at the BamHI site. The library element designs were assembled step by step (FIG. 5). In-Fusion® cloning was first used to insert into an expression vector a sequence encoding a single chain variable fragment (scFv) and transmembrane (TM) domains (scFVTM) common to all library elements. The In-Fusion® reaction mixture was transformed into competent E. coli cells. The transformed cells were planted. Colonies were selected and DNA extraction minipreparations were prepared to isolate the engineered plasmid. After successfully constructing an expression plasmid containing scFV-TM, each successive component of the multimodule polypeptide, including the EGFP reporter, was inserted 3' from the previous component in separate steps according to FIG. 5. Each step included BamHI digestion and In-Fusion® assembly followed by transfection of the InFusion® reaction mixture, colony selection and plasmid purification.

As shown in FIG. 5, each final element of the In-Fusion® post reaction library contained a generic scFV-TM coupled to an element-specific combination of two co-modulating domains coupled to a common reporter (CD3z-EGFP) and a pair of module-specific nucleic sequence-barcodes in reverse orientation. with respect to comodulating domains. Nucleic barcode sequences were flanked by primer binding sites allowing amplification and/or sequencing of a specific combination of nucleic barcode sequences corresponding to the combination of comodulatory domains specific to library elements.

After construction of the last plasmid for the library was completed, individual elements of the library were transfected into HEK-293 cells and lentiviral particles were obtained by standard methods. The obtained lentivirus was used to infect T cells to obtain modified immune cells expressing multimodular polypeptides.

The modified immune cells were sorted by flow cytometry based on the expression of the EGFP reporter. Sorting was performed to isolate a population of modified cells with the same expression levels. Sorted cells with the same levels of expression of multimodular polypeptides were used for subsequent stages of functional screening.

This general strategy was used to create four separate libraries. One-dimensional libraries (ie, each element of the library contained one co-modulating domain) and two-dimensional libraries (ie, each element of the library contained two co-modulating domains) were constructed.

Elements of the 2D libraries were assembled according to the general scheme shown in FIG. 6.

The four libraries and their respective screening steps were as follows.

1) A one-dimensional library of 20 elements was constructed and used to test the feasibility of quantifying T cell function with FLOWseq. Each element of the library was configured to contain a CD8 domain fused to a comodulatory domain module fused to a CD3Z domain. The modified T cells were grouped according to their reporter expression by flow cytometry, and the library was screened to measure the dose-dependent response of T cell activation (CD69) to plate-bound antigens.

2) A one-dimensional CD19-specific library of 62 elements was constructed and screened in a mouse tumor model in vivo. Each element of the library was configured to contain a CD19-specific domain fused to a comodulatory domain module fused to a CD3Z domain that was fused to an EGFP reporter.

3) Designed a two-dimensional library consisting of 62x62 elements. Each library element contained a CD19-specific domain fused to a first comodulatory domain module fused to a second comodulatory domain module fused to a CD3Z domain fused to an EGFP reporter.

4) Designed one-dimensional mesothelin-specific library of 62 elements. Each element of the library contained a mesothelin-specific domain hybridized to a comodulatory module domain hybridized to a CD3Z domain hybridized to an EGFP reporter.

The pooled one-dimensional CD19-specific library of 62 elements was subjected to functional screening to identify alternative comodulatory sequences in chimeric antigen receptors (CARs). Each element of the library contained a CD19-specific scFv that points to the target cancer cell antigen, one of the comodulatory domain modules listed in Table 1. 1 and the primary signaling domain of CD3Z (FIG. 7).

After antigen challenge (at 32, 125 and 1000 ng/ml antigen) of the pooled library modified T cells, T cells were functionally sorted by flow cytometry into high or low challenge groups based on CD69 expression (FIG. 8). The relative enrichment of specific barcode sequences corresponding to individual comodulatory domains was quantified in high and low stimulated cells by sequencing (at an antigen concentration of 1000 ng/mL) (FIG. 9). This approach made it possible to compare the stimulatory and inhibitory effects of each comodulatory domain in CD19-CD3Z-specific CARs and in relation to the specific antigen stimulation used. For example, screening results were further analyzed at different levels of applied antigen, allowing a rapid assessment of the dose-dependent response of individual comodulatory domains (FIG. 10).

Example 2. Confirmation of the complete assembly of a two-dimensional library of 61x61 elements.

Sixty-one CAR variable modules (Table 2 shown in FIG. 25) were assembled into a nucleic acid library containing barcode nucleic acid sequences encoding two-dimensional (2D) synthetic modular CAR polypeptides using a nested assembly. The library was deeply sequenced by sequencing by synthesis (SBS) using the MiSeq system (Illumina Inc., Hayward, CA, USA) and the number of reads of each element of the assembled library was determined (FIG. 26).

Out of a total of 3721 possible library elements (ie CAR variants), all possible library elements were detected by sequencing at a maximum of 1216 reads and a minimum of 2 reads. The mean number of reads across the entire library was 333 with a median of 311 reads and a standard deviation of 140. 90% of library items were represented by reads that were within two times the median value, and 98% of library items were represented by reads that were within three times the median value. medians.

Therefore, the sequencing results confirmed that the nested assembly method is capable of providing a 2D library containing library elements that represent all possible combinations of variable modules.

Example 3. Rationing parts of the library.

A method has been developed to increase the distribution of clones in a combinatorial library using normalization of parts of the library.

In any combinatorial assembly reaction, some parts (such as modules) are more efficiently integrated into the assembled products than others. As a consequence, assembled products (eg, protein variants) that contain parts that integrate relatively inefficiently are under-represented or absent from the finished combinatorial library. For similar

-

Claims (11)

For other reasons, other assembled products (eg, those protein variants that contain parts that integrate efficiently) are overrepresented and therefore over-reported in all subsequent assays. To solve this problem, a method was developed to improve the assembly of libraries. Said method involves conducting an initial assembly using equal volumes of each part (DNA insert). For this purpose, a total reaction mixture was created containing 1 μl of each part. After completion of the combinatorial assembly, the mixture of products was determined using an appropriate quantification method. For example, next generation sequencing was used in this example (see FIG. 27) and the proportion of each part in the total library (denoted as d, e ₂ ... in the linear equation shown in FIG. 28) was calculated. Linear algebra was used to simultaneously calculate a new optimized volume for each part (in the total reaction mixture), and then the library was resynthesized to achieve a more even distribution of variants. This approach was validated by synthesizing a combinatorial CAR library in which each CAR contains two of 61 different co-stimulatory domains linked in tandem (612 for 3721 protein variants). Taking advantage of the simple, linear relationship between potency, concentration, and abundance in the assembled combinatorial library, it has been demonstrated that this method can be used to quantify the relative frequency of individual moieties. As shown in FIG. 29, as predicted (black bars), a 10-fold change in the relative concentration of a set of parts in the main assembly reaction mixture will result in a 10-fold change in the representation of parts in the final composition of the combinatorial library. When measuring the mean frequency of 5 parts in the assembled combinatorial library (white bars), the predicted effect of the 10-fold change was indeed observed. The resulting normalized large combinatorial library (as shown in FIG. 26) had a significantly improved distribution of variants in the library (eg, compared to the distribution of variants in the library before normalization (FIG. 27). While the present invention has been described with reference to specific embodiments, those skilled in the art will appreciate that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt a particular situation, material, formulation, method, process step or steps to achieve the purpose, spirit and scope of the present invention. All such modifications are intended to be included in the appended claims. CLAIM 1. A method for ex vivo/in vitro identification in a cell of a selected phenotype associated with a synthetic modular polypeptide, comprising: a) introducing a barcoded nucleic acid library into host cells to obtain a heterogeneous population of genetically modified host cells, wherein said barcoded nucleic acid library contains a plurality of members, each of which contains a nucleotide sequence encoding a different synthetic modular polypeptide, wherein said nucleotide sequence contains an encoding region encoding two or more variable modules and a barcode region, where each variable module corresponds to a specific unique barcode in this barcode region; b) identifying, within said heterogeneous population, a genetically modified host cell that exhibits the selected phenotype in response to the stimulus, and c) identifying and/or calculating by sequencing a barcode region from said host cell identified in step (b), thereby identifying the synthetic modular polypeptide and/or module thereof produced in the selected phenotype. 2. The method of claim 1, wherein said synthetic modular polypeptide is: a) a modular receptor polypeptide, optionally a chimeric antigen receptor (CAR) polypeptide or a chimeric Notch receptor polypeptide; b) modular scaffold protein; c) a protein kinase or protein phosphatase modular protein; d) a modular protein that regulates transcription; e) modular protein of epigenetic regulation; or f) a recombinase or nuclease modular protein. 3. The method according to claim 2, characterized in that said synthetic modular polypeptide is a CAR, and the stimulus is an antigen-presenting cell that presents on its surface an antigen that binds to the CAR. - 62 041085 4. A method according to any one of the preceding claims, characterized in that said phenotype is one or more of the following: a) proliferation; b) cytokine production; c) expression of a cell surface marker; d) reporter protein expression. 5. The method according to any of the preceding claims, characterized in that said plurality of members comprises a nucleotide sequence encoding a synthetic modular polypeptide containing a modular domain selected from the group consisting of: an antigen-binding domain, a specific binding domain, or a protein that is a specific partner for binding, co-stimulatory domain, co-inhibitory domain, intracellular signaling domain, transmembrane domain, scaffold protein domain, protein kinase protein domain, protein phosphatase protein domain, receptor tyrosine kinase protein domain, lipid kinase protein domain, lipid phosphatase protein domain, ubiquitinylase protein domain, deubiquitinylase protein domain, protein domain SUMOulase, acetylase protein domain, deacetylase protein domain, methylase protein domain, demethylase protein domain, nuclease protein domain, recombinase protein domain, transcription factor protein domain, and combinations thereof. 6. Library of nucleic acids with barcodes, containing: a plurality of unique polynucleotides, each containing a nucleotide sequence encoding a unique synthetic modular polypeptide, each unique polynucleotide containing: i) a coding region encoding a unique synthetic modular polypeptide containing a first coding sequence encoding a first module connected in an open reading frame to a second coding sequence encoding a second module, ii) a multi-link barcode region containing a first barcode specific for the first coding sequence connected to a second barcode specific to the second coding sequence, wherein said first and second barcodes are in reverse order from 5' to 3' with respect to the first and second coding sequences; and while sequencing each multi-link barcode region allows you to identify each unique synthetic modular polypeptide. 7. The library according to claim 6, characterized in that said first and second coding sequences are linked directly without any intermediate non-coding nucleotides. 8. The library according to claim 6 or 7, characterized in that said unique synthetic modular polypeptide contains a costimulatory domain, optionally said first and second modules contain different costimulatory domains. 9. The library according to claims 6-8, characterized in that said unique synthetic modular polypeptides, each encoded by a unique polynucleotide from said plurality, are one or more of the following: a) a modular receptor polypeptide, optionally a chimeric antigen receptor (CAR) polypeptide or a chimeric Notch receptor polypeptide; b) modular scaffold protein; c) a protein kinase or protein phosphatase modular protein; d) a modular protein that regulates transcription; e) modular protein of epigenetic regulation; or f) a recombinase or nuclease modular protein. 10. The library according to any one of claims 6-9, characterized in that said unique synthetic modular polypeptides, each of which is encoded by a unique polynucleotide from the specified set, contain a modular domain selected from the group consisting of: an antigen-binding domain, a specific binding domain, or a protein , which is a specific binding partner, costimulatory domain, coinhibitory domain, intracellular signaling domain, transmembrane domain, scaffold protein domain, protein kinase protein domain, protein phosphatase protein domain, receptor tyrosine kinase protein domain, lipid kinase protein domain, lipid phosphatase protein domain, ubiquitinylase protein domain, a deubiquitinylase protein domain, a SUMOylase protein domain, an acetylase protein domain, a deacetylase protein domain, a methylase protein domain, a demethylase protein domain, a nuclease protein domain, a recombinase protein domain, a transcription factor protein domain, and combinations thereof. 11. Cell library containing: a plurality of cells, each containing a unique polynucleotide of the barcoded nucleic acid library according to any one of claims 6-10, wherein sequencing of each barcode region allows each unique synthetic modular polypeptide of each cell from the library to be identified. -